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    1. Certificate retrieval, supported verification tooling, and example verification commands see the signing documentation. For example, you can verify a signed skill locally. To do so, follow these steps: Download the NVIDIA Agentic Capabilities root certificate as nv-agent-root-cert.pem Install an OpenSSF Model Signing (OMS) verifier, such as pip install model-signing Execute the following command to verify the skill signature

      行动建议:按照文中提供的步骤下载NVIDIA代理能力根证书,安装OpenSSF模型签名验证器,并使用提供的命令验证技能签名。这种实践可以确保您下载的技能是真实的且未被篡改,增强对AI代理能力的信任。

    2. To get started with the cuOpt verified skill, for example, follow these steps: 1. Pull the cuOpt verified skill from the catalog: git clone github.com/nvidia/skills && cd skills/skills/cuopt 2. Verify the signature: model_signing verify certificate. --signature skill.oms.sig --certificate-chain nv-agent-root-cert.pem --ignore-unsigned-files 3. Open SKILLCARD.yaml to see ownership, dependencies, license, and verification status.

      行动建议:按照文中提供的具体步骤,克隆并验证NVIDIA的cuOpt技能,查看技能卡片以了解所有权、依赖关系、许可证和验证状态。这种实践可以确保您使用的技能是经过验证的,并且可以安全地集成到您的AI代理工作流中。

    1. Over the past six months, OpenAI forward deployed engineers and researchers along with Thrive Holdings' engineers collaborated to build Tax AI

      六个月的开发周期表明这是一个长期、复杂的项目。'forward deployed engineers'表明OpenAI团队采用了嵌入式工作方式,这有助于更好地理解实际业务需求。这种跨公司合作模式可能成为AI专业领域应用的标准开发方式。

    1. The best agent businesses are going to need to execute like hedge funds — winning on alpha measured in customer P&L, not in benchmark scores.

      这句话用对冲基金作为比喻,生动地描述了优秀AI应用公司的成功标准。作者指出,这些公司需要在客户的实际业务成果(P&L)上获得超额收益(alpha),而不是在通用基准测试上获得高分。这个洞见强调了AI应用公司应该以客户的实际业务价值为中心,而不是技术指标。

    2. The model is fungible underneath; the system of work is not.

      这句话简洁而深刻地指出了AI应用层的本质区别。作者认为,底层的AI模型是可以互换的,但工作的系统(system of work)却是独特的。这个洞见揭示了为什么专注于构建特定工作系统的公司能够长期保持竞争优势,而仅仅依赖通用模型的公司则难以建立持久的业务。

    3. The workflow you ship on day one is not the moat. The loop that production usage creates over time is.

      这句话深刻地揭示了AI应用公司的真正护城河所在。作者指出,初始的工作流程不是竞争壁垒,而是在生产环境中持续使用、学习和改进所形成的循环才是真正的护城河。这个洞见强调了实践经验、数据积累和持续迭代的重要性,对于理解AI应用公司的长期价值至关重要。

    4. You can be everywhere at once, or you can be great at one thing. Not both.

      这句话简洁有力地表达了大型实验室与专注应用公司之间的核心区别和战略选择。它揭示了为什么大型AI实验室无法深入解决特定垂直领域的复杂问题,为什么专注的垂直应用公司有机会在这些领域建立竞争优势。这个结论句为创业者提供了清晰的战略指导。

    5. The labs really are coming for a huge swath of the application surface. But 'the application layer' isn't just one homogenous opportunity.

      这句话精准地捕捉了AI应用层的复杂性和多样性。作者指出大型AI实验室确实会覆盖大量应用领域,但这并不意味着所有应用机会都是同质的。这个洞见反驳了'AI将杀死所有应用层'的简单化观点,为创业者指明了在特定垂直领域寻找机会的方向。

    6. The Yellow Brick Road is our shorthand for the path the labs are walking, where they're committing extraordinary resources.

      这句话用《绿野仙踪》中的黄砖路作为比喻,形象地描述了大型AI实验室正在走的道路。这个比喻生动地表达了这些实验室拥有巨大资源,正在构建一条明显可见的发展路径。这个洞见帮助读者理解AI应用生态中的不同发展方向,以及为什么有些领域竞争激烈而有些领域则存在机会。

    1. Model Labs are increasingly also building Agents as the product

      大多数人认为模型实验室应该专注于提升基础模型的能力,但作者认为这些实验室现在正转变为代理实验室。这一观点挑战了AI行业的基础假设,即模型本身是产品,而不是模型只是更大代理系统的一部分。这标志着AI行业从'模型即产品'向'代理即产品'的根本性转变。

    2. if you can effectively posttrain a model to only meaningfully perform with your closed source agent, then you get to funnel the majority of users to your agent at the expense of your model/API co-opetition

      大多数人认为开源模型会促进竞争和开放生态,但作者认为模型与代理的协同可能导致更封闭的生态系统。这一反直觉观点指出,企业可能通过训练模型使其仅在特定代理环境中有效工作,从而将用户锁定在自己的代理产品中,这与开源社区期望的开放性背道而驰。

    3. The quote is a big reversal of stance from a position ~uniformly held by anyone who worked at **Team Big Model**, including his previous head of OpenAI Labs

      大多数人认为大型模型实验室会继续专注于基础模型研发,但作者认为这是一个立场的重大转变,因为连OpenAI前高管都开始转向代理产品。这挑战了AI行业长期以来的'模型优先'共识,表明即使是Big Model团队也开始认可代理产品的价值。

    1. discussing

      In-class exercises can include demonstrating how AI-generated citations can look authentic to instruct students on both the pitfalls of relying too heavily on AI and reinforcing what should be included in a source citation.

      Honestly, another application could be to generate sources and citations--teacher verified--to use in curriculum when teaching these research skills. This can be a huge timesaver for teachers.

    1. Very beautiful work!

      As far as I'm aware there aren't any structures of DksA bound to an RNAP elongation complex. Have you thought about how this interaction would impact your model of ppGpp-binding and RNAP swiveling? Would DksA prevent swiveling given its binding location, or the structural states be in competition? Would be curious to see impact of DksA in vitro as well as what your in vivo results would look like in a ∆dksA background

    1. The Strange Melancholy of Slaying Monsters
      • Video games have traditionally used a "player-versus-environment" model of monster slaying for accomplishment, but many titles subvert this ritual to introduce ethical dilemmas and an elegiac tone.
      • In the Western tradition, the concept of the "tragic monster killer" dates back to J.R.R. Tolkien’s analysis of Beowulf, which rejects the notion of martial heroism as its own end and acknowledges the inevitable ruin of the warrior.
      • Games like Shadow of the Colossus highlight this moral complexity by forcing players to slay peaceful, majestic creatures; the game lacks regular enemies and presents the colossi's deaths with agonizing visual effects and mournful music rather than a celebratory fanfare.
      • Titles such as Dark Souls and Bloodborne reinforce a melancholic atmosphere by designing bosses characterized by deep sorrow and tragic descents into ruin, mirroring Friedrich Nietzsche's warning about becoming a monster when fighting them.
      • Mainstream titles like BioShock, Spec Ops: The Line, and God of War incorporate the "false hero" trope, forcing players to confront their complicity in violence or show resignation toward inescapable gaming conventions.
      • The indie game Undertale subverts RPG norms by humanizing its quirky monsters and allowing players to spare them through non-violent negotiation, ultimately revealing that classic progression mechanics like EXP and LV stand for "execution points" and "level of violence."

      Hacker News Discussion

      • Personal Experiences of Disenchantment: Several commenters shared specific gameplay moments where accidentally humanizing a virtual opponent permanently altered their perception of video game violence, including a player who quit Skyrim after realizing they had slaughtered a homeless bandit family for meaningless loot.
      • The Psychology of Fiction and Reality: A discussion developed around how players reconcile virtual actions; while most understand that video game enemies are just code, the introduction of narrative texture and realistic consequences can pierce the layer of abstraction and invoke genuine guilt or melancholy.
      • Military Shooters and Propaganda: Some users recalled playing tactical shooters like Operation Flashpoint, where the sudden realization of the geopolitical absurdity or human cost behind a simulated conflict broke their immersion and temporarily ruined first-person shooters for them.
      • Intentional Game Design: Participants praised developers who deliberately use ludonarrative resonance—aligning gameplay mechanics with the narrative—to challenge the mindless power fantasies common to the medium.
    1. Tech CEOs are apparently suffering from AI psychosis
      • Box founder Aaron Levie coined the phrase "AI psychosis" to describe tech executives who suffer from delusions of AI grandeur due to being too distant from the actual day-to-day operations where value is generated.
      • Because CEOs only interact with high-level prototypes, they mistakenly leap to the conclusion that AI agents can effortlessly handle full workloads without realizing the heavy human labor required to review code, patch bugs, catch hallucinations, and train models.
      • This executive delusion has real-world consequences, driving severe workforce reductions; in the first five months of 2026, over 115,000 tech workers were laid off—nearly matching the total for all of 2025—with AI cited as a primary justification.
      • High-profile actions, such as ClickUp CEO Zeb Evans laying off 22% of his workforce after deploying 3,000 AI agents, are framed as shifting humans into "manager and verifier" roles for AI outputs.
      • Empirical data from UC Berkeley, NBER, and MIT refutes these massive productivity assumptions, demonstrating no robust link between current AI adoption and aggregate productivity gains, with MIT predicting baseline competence on text tasks will not materialize until 2029.
      • A Harvard Business Review study warns that flooding an organization with unverified AI output merely shifts bottlenecks onto executives, risking widespread structural and operational chaos if human oversight fails to scale.

      Hacker News Discussion

      • Distance from Reality: Commenters strongly agreed with the premise that executives live in a bubble, noting that they deal primarily with administrative assistants, sycophants, and curated, "happy path" demos that look like magic, making them blind to edge cases and errors.
      • The "Yes-Man" Nature of AI: Multiple users pointed out that AI agents behave like the ultimate corporate sycophants—they work 24/7, lack internal moral conflict, and never say no—making them highly attractive to authoritative executives who dislike pushback from human workers.
      • Absence of Self-Preservation: A key distinction raised in the comments is that unlike human employees, AI lacks "self-preservation," a sense of reputation, or a fear of consequences, meaning an agent will confidently delete a production database or kill its own server processes without hesitation.
      • Misuse of the Term: Some participants criticized the article's title as clickbait, arguing that "AI psychosis" should describe literal psychological delusions in individuals interacting with AI rather than standard corporate incompetence or unrealistic executive expectations.
      • Projection of Executive Work: A popular theory suggested that CEOs assume AI can replace everyone's job because it can easily replicate their own daily tasks, such as generating slide decks, sending emails, and attending high-level meetings.
    1. Can we have the day off?
      • The author questions why the promised 10x productivity gains from AI do not result in more time off for workers, such as a four-day work week.
      • If AI can allow a worker to complete a week's worth of output by Monday afternoon, Friday could theoretically be declared an "AI workers' day" where agents handle the workload.
      • This extra day off would benefit everyone, including the C-suite and boards of directors, who could spend the time leisure-seeking rather than being at the office.
      • Despite entering a revolution across every sector of human productivity, the fundamental structure of the five-day work week remains unchanged.
      • The high cost of living and childcare (e.g., $6,000/month in California) adds pressure on employees, making the flexibility of fewer office days highly desirable.

      Hacker News Discussion

      • Capturing Productivity Gains: Many commenters note that while workers are pushed to adopt AI tools to multiply their output, they do not stand to benefit financially or receive more time off; instead, the economic gains are heavily consolidated by employers and capital owners.
      • The Reality of Salaries: A discussion emerged around how salaried employees are typically compensated. Some argue that employees are paid for their availability and time rather than direct output, making it difficult to negotiate less time for the same pay.
      • Fear and Leverage: Users highlight that instead of increased compensation, the rise of AI has brought widespread fear of layoffs and lower job security, keeping workers compliant rather than demanding a 4-day workweek.
      • Collective Action and Policy: Several participants suggest that asking an employer for a day off individually is naive due to market competition and the Prisoner's Dilemma. They argue that structural changes like historical worker protections, unions, or government-led policies like Universal Basic Income (UBI) are necessary to shift the status quo.
    1. Thedetachmen t of the pure gaze cannot be dissociated from a general disposition towards the world which is the paradoxical product of conditioning by negative economic necessities-a l ife of ease-that tends to inducean active distance from necessity

      pure gaze (artistic perspective distanced from natural world) comes from being distanced from economic necessity

    2. he pure aesthetic is rooted in an ethic,or rather, an ethos of elective distance from the necessi ties of the natural

      "pure aesthetic" or artistic lense = distance from the natural and social world

    3. the shiftfrom an art which imitates nature to an art which imi tates art, derivingfrom its own history the exclusive source of its experiments and even ofits b reaks with tradition .

      art developing it's own world of references and signals- outside of "nature"

    4. a decoding operation, which implies the implemen tation of a cognitive acqui rement, acultural code.

      why does art acc mean something to people? Not just a natural or inherent sense but an embodied knowledge

  2. social-media-ethics-automation.github.io social-media-ethics-automation.github.io
    1. Steve Jobs. December 2023. Page Version ID: 1189127326. URL: https://en.wikipedia.org/w/index.php?title=Steve_Jobs&oldid=1189127326 (visited on 2023-12-10).

      This article was about summarizes Steve Job's life and career as his role as a co founder of Apple, the biggest smart phone brand today, his departure from Apple, and his return to make and bring major products back to apple. This ultimately connects back to the chapter that in capitalism company leaders are ultimately accountable to the owners and shareholders, not just to founders, workers, or users. Job's story shows how even someone as important as a co founder can lose power when the board decides the company should move in a different direction. This connects to the chapter's point that business decisions are shaped by ownership and the need for profit, not necessarily just for creativity or vision.

    1. If your rogue has a +9 to Perception, you, the player, don’t actually have to be perceptive. You can be half-asleep, eating a slice of pizza, roll a natural 20, and find the hidden dart trap.In OSR-style play, we throw that dynamic out the window. We want you, the living, breathing human being sitting at the table, to solve the problem. When you encounter a hazard, your character sheet isn’t going to save you—your brain is.Instead of saying, “I roll a Perception check to search the room,” an OSR player is encouraged to describe their specific, physical actions in the fiction. They might say, “I pour water from my waterskin onto the floor to see if it pools naturally or drains into any hidden seams between the tiles.” Or, “I hold my torch close to the doorframe to see if the draft flickers in a way that suggests a hollow space behind the wood.”

      I think there's room for comprimise here. I rogue would know traps. Maybe the roll couldt ell them what kidn of thing was possible in this dungeon. They would then have to use their nouse. Otherwise the player knowledge of theworld would have to be very great to know what to look for.

    1. In capitalism, business decisions are accountable to the people who own the business. In a publicly traded [s10] business, that is the shareholders. The more money someone has invested in a company, the more say they have. And generally in a capitalist system, the rich have the most say in what happens (both as business owners and customers), and the poor have very little say in what happens.

      This section from the chapter stood out to me because it explains why social media companies usually prioritize profit over users wellbeing. Even though users create content and are affected by the platforms choices, the company is mainly only accountable to its owners and shareholders. This introduces ethical issues because decisions are about privacy and safety may be shaped by profit instead of and for the user. It also makes me think if social media platforms are even a safe space if the platforms company isn't looking out for it's users.

    1. hey had nearly destroyed thechurch as an institution—of the more than fty thousand Orthodox churches on the territory of the RSFSR in1917, fewer than a thousand were left in 19

      slayyyy

    2. As the instructions to the census ofcials claried, thequestion was intended to indicate belief rather than confessional belonging, and the results revealed that of the98,412 people surveyed, more than half (56.17 percent) identied as believers, and this proportion rose to two-thirds in the countryside.

      religious belief still existed, but had been decimated - and belief didn't exactly mean that they engaged in religious participation

    3. Ethnographers studying rural life, such as N. M. Matorin (1898–1936) and V. G.Bogoraz-Tan (1865–1936), produced studies of “lived religion” (zhivaia religiia) and “folk Orthodoxy” (narodnoepravoslavie) that attested to the continued religiosity of the countryside throughout the 1920s and 1930s.

      slay for twist

    4. he party accused the Orthodox Church of collaborating with the religiousunderground at home and counterrevolutionary agents abroad, and cast the 1929 law as too permissive forallowing the continued existence and even proliferation of religion. In 1937 alone, the Bolsheviks closed morethan eight thousand churches (with another six thousand in 1938), and arrested thirty-ve thousand “servantsof religious cults.” The Bolsheviks also exiled or murdered much of the Orthodox Church hierarchy. Thehistorian Mikhail Shkarovskii argues that by 1938 the Orthodox Church was “on the whole, destroyed.” Localorgans charged with managing religion were liquidated as unnecessary, thereby “eliminating even thepossibility of contact between the state and the church.

      ABSOLUTE SLAYYYY SLAYYYY ZEEE BESTTTTT

    5. Russian Soviet Federal Socialist Republic (RSFSR), which had, until then, guaranteed Soviet citizens “freedomof religious and antireligious propaganda.” It was not enough, moreover, to marginalize religion; public lifealso had to be made visibly Soviet. In effect, the only right Soviet citizens retained was the right to worshipinside the connes of specically designated religious spaces

      SLAYYYYYY religious participation was effectively made illegal! only legal in certain spaces, which had been eradicated by the government. While of course, this didn't inhalitate the orthodox face, it did lead to a far more significant religious decline than england

    6. The 1929 law was intended tobring all aspects of religious life under state control by repealing numerous provisions established in 1918: itoutlawed the religious education of children and charity work, closed monasteries, and dictated that religiouscommunities had to register with local government organs

      slayyy

    7. since it was “impermissible for icons tohang in the home of a Leninist, for a priest to baptize a Leninist’s children, and for a Leninist’s children to go tochurch.

      primary source

    8. Clubs were channels through which the party could disseminate political and culturalenlightenment, and were intended to replace the church as the centers of community life. Indeed, local activistswould often turn the local church into the village club, thereby recasting it as a secular space. In more populatedtowns, the Bolsheviks created antireligious museums

      Like England, Russia saw secular activities replace this expression of religious participation. However, this was state induced

    9. arch 19, 1922, Lenin announced that the Soviet regime wasdeclaring a “ruthless battle against the black-hundreds clergy” (besposhchadnoe srazhenie chernosotennomudukhovenstvu), and opined that “the greater number of representatives of the reactionary clergy andreactionary bourgeoisie we manage to shoot on this basis the better

      PRIMARY SOURCE FOR MILITANT PHASE OF BOLSHEVIK WAR AGAINST RELIGION

    10. The Bolsheviks were also reluctant to pursue militant antireligious policies in the borderlands, wherereligion was intimately connected with nationalism, since they were mindful of exacerbating already volatileseparatist movements.

      SLAY FOR OPPOSITE AND HOW THEY COULDN'T GO TOO FAR IN ENFORCING RELIGIOUS DECLINE!

    11. ollowing this logic, the Bolsheviks, immediately passed a series of decrees to establishthe foundations for a modern secular state. The “Decree on Land” (October 26, 1917) nationalized all monasticand church land. Another decree, “On Civil Marriage, Children, and on the Registration of Acts of Civil Status”(December 18, 1917), created a secular bureaucracy—the ofce for the registration of acts of civil status (Zapis’aktov grazhdanskogo sostoianiia, or ZAGS)—to take over the registration of births, marriages, deaths, anddivorces from religious institutions. Finally, a third decree, “On the Separation of Church from State andSchool from Church” (January 23, 1918), deprived religious organizations of their status as juridical entities andremoved religion from government and education.

      SLAY SLAYYYY literally the examples of how religious participation was forced to decline - this didn't mean, howerver, that there weren't those who still attended church - many did - but it was a struggle that lasted a long time. Unlike her British counterpart, Russia, at the start of the revolution, had a much lower level of literacy, while government controlls over Bible publishing meant that religious participation through scripture reading was heavily restricted. But, unlike her English counterpart who was predominately Anglican (and thus placed emphasis on religious participation through scripture reading), the Orthodox faith was far more ritualistic, religious participation continuing to relatively strong levels in terms of Orthodox ritual.

    12. PRIMARY SOURCE!! shows how, for the revolutionaries, the only way that socialism could fully be implemented was the banishing of religion, which was a strong element of the Tsarist Regime and, for them, a remnant of autocratic oppression. while Marxist theory implie that, upon revolution religion would become (), religious participation declining entirely as a consequence. This, however was not the case. While many were drawn to the marxist alternative of atheism, leading to a slight decline in religious participation, the orthodox had roots deeply imbedded in poltical, cultural and of course, religious sectors. As such, the new Bolshevik government sought to forcibly reduce religious participation. Give examples.

    13. had become by the twentiethcentury a means of asserting moral and political autonomy in opposition to both the church and state

      atheism was a way tp go against the state!

    14. he Orthodox Church had aprivileged place at the top of the empire’s confessional hierarchy and had historically performed an essentialpolitical role for the Russian state alongside its spiritual mission, providing transcendent legitimation for thetsar’s earthly authority.

      unlike england, the church prior to the revolution had been a significant political thingie

    Annotators

    1. How does the UST's TeachOnline office aligns (or not) with the contents of this encyclical.

      In alignment with our Catholic University's mission of goodness, knowledge and discipline; first, we've worked very hard to understand how artificial intelligence works, the best approach for artificial intelligence and, what it can and cannot do. As instructional designers we have an ethical and moral code to do no harm to our students; the creation or purveying of false information would be a moral and intellectual harm; so, to the best of our abilities, we seek to only generate accurate and factual information with artificial intelligence tools. We do this by using existing documents, meeting transcripts, and other human-generated artifacts as part of context engineering for the prompts we are creating.

      Additionally, on the topic of goodness, and in alignment with the ethical quandaries of using artificial intelligence tools that can be connected to "long chain of mediation, involving vast networks of natural resources, energy infrastructure, and above all people". That is, tools that are known to be exploitative to the environment and hurt neighboring people, –specially marginalized communities– (xAI/Grok), disregard the subsidiarity of local communities (Meta AI), and known for harming adult and children with its ability to convince them of false and violent informaton (ChatGPT); our chosen tools are Anthropic's Claude Sonnet and Opus models. That isn't to say that Anthropic is guiltless. However, it continues to stand above all other companies as being the most ethical and conscientious artificial intelligence lab – although that is not saying much, Claude has been used as a hacking tool, and it was used in Pentagon for weapon and operation planning; prior to its designation as a national security risk, ironically because they sought to enact a "red line" (that is disarm) on their AI being used on weapon systems and mass surveillance.

      As educators and instructional designers, we welcome the challenge to rethink "the organization of schools, physical spaces, evaluation methods and the role of teachers themselves... promote an authentically integral education that addresses every dimension of the person." To do this, we follow our scientific and ethical practices of our profession in the development of courses that have measurable outcomes, accurate, engaging, collaborative, applicable to real life, that hopefully lead to reflection and contemplation. Additionally, our role as educators helps "disarm" AI from its worst possible uses, and we can further assist by beating "swords into ploughshares" by helping our students understand the ethical and moral boundaries of any technological use and implement it in ways that aid humanity. We respect that our faculty engage in the work of Nehemiah, by helping to build the wall of Jerusalem; by engaging in one of the most charitable acts in humanity, that of giving away and imparting their knowledge unto the future generation.

      WIP!!!!

    2. In a particular way, we need adults to rediscover their vocation as artisans of education, prepared to work patiently each day, with the support of extensive and shared educational partnerships.

      I like that idea of a community of "artisans of education", presumably including but not limited to teachers and other educational professionals.

    1. Reviewer #1 (Public review):

      Summary:

      Wang Liao and colleagues aim to provide a comprehensive synthesis of zebrafish circadian research, with particular emphasis on the decentralized photoreceptive architecture that distinguishes teleosts from mammals, and to outline future research directions leveraging emerging technologies for translational applications. The authors frame zebrafish as occupying a "crucial evolutionary and experimental niche" and argue that the model system is uniquely suited to address open questions in chronobiology.

      Strengths:

      The review is broad in scope and up to date in its citation of recent primary literature. The coverage of physiological outputs - spanning cardiovascular rhythmicity, hepatic metabolism, immune function, reproduction, and gut homeostasis - is more comprehensive than many existing reviews in this area, and researchers seeking an entry point into any of these subfields will find a useful orientation. The figures are well-designed and effectively summarise complex regulatory relationships. The section on immune rhythmicity is a particular strength, providing mechanistic detail on how specific clock components (Clock1a, Per1b, Per2, Cry1a) differentially regulate neutrophil behaviour, bacterial killing, and cytokine expression; this level of molecular specificity distinguishes it from comparable sections in the review. The brief discussion of non-canonical clock gene functions (CLOCK in neuronal connectivity, BMAL1 in stem cell state, vascular calcification) raises genuinely interesting points that are underexplored in the field and might deserve more prominence.

      The future perspectives section makes a conceptually interesting move in suggesting that the zebrafish decentralized architecture could reframe a central question in chronobiology - from how a master clock imposes order on passive peripheral oscillators, to how semi-autonomous oscillators achieve coherence. This is the most original conceptual contribution in the manuscript, and it would benefit from much further development.

      Weaknesses:

      The core limitation of this review is that it functions primarily as an annotated bibliography rather than a critical synthesis. Section after section follows the same pattern: a physiological system is introduced, several findings from recent papers are described in sequence, and the section ends. Missing throughout is an evaluative voice - where does the field agree, where does it disagree, which findings have been replicated versus remain preliminary, and which conceptual questions are genuinely unresolved versus merely unstudied? Readers with expertise in the field will find little that reframes their understanding; readers new to the field will receive information but not the interpretive scaffolding needed to assess its significance.

      The framing of zebrafish as occupying a "crucial evolutionary and experimental niche" is asserted but not substantiated. The experimental advantages of zebrafish - optical transparency, external development, genetic tractability - are real, but they apply primarily to larval stages, typically the first two weeks of development. The review does not adequately address whether the key features it highlights, particularly peripheral photosensitivity and autonomous peripheral oscillators, have been demonstrated in adult animals, where optical transparency is lost. Many of the physiological findings described (sleep-wake cycles, cardiovascular function, reproduction, and immune function) are most relevant in adult or juvenile fish, yet the mechanistic underpinnings often come from larval studies. Whether the mechanisms generalise across developmental stages is not discussed, and this is an important gap that the review could acknowledge explicitly.

      The claim that zebrafish bridge invertebrate and mammalian models is a conventional framing that appears in most zebrafish review articles; its repetition here adds little. More interesting - and underexplored - is the comparative question of how the decentralised clock architecture of teleosts compares with that of other non-mammalian vertebrates, or indeed with invertebrate systems such as Drosophila, where peripheral tissue clocks and non-visual photoreception have also been studied. The review does not engage with this comparative dimension, which would be the natural intellectual context for the claims being made.

      The future perspectives section identifies several promising directions - optogenetic circuit mapping, whole-body longitudinal imaging, inter-organ communication, network modeling - but these are described at a high level of generality. Most are not specific to the questions raised by the zebrafish decentralized clock architecture; they would appear in any forward-looking review of circadian biology. The one conceptually distinctive idea - that zebrafish could be used to ask how distributed oscillators achieve coordinated coherence without hierarchical control - is identified but not developed into concrete experimental questions or testable predictions. The discussion of non-canonical clock gene functions in the Future Perspectives section would benefit from being more directly connected to what zebrafish specifically can offer: given that teleost genome duplication has produced additional paralogues of clock genes, there is a concrete opportunity to dissect canonical from non-canonical functions through comparative analysis of paralogues with diverged expression patterns. This point is hinted at but not made explicitly.

      Appraisal of conclusions:

      The conclusions are broadly consistent with the evidence cited, and the authors are appropriately cautious in noting that many signalling cascades and inter-tissue communication mechanisms remain incompletely characterised. The conclusion that zebrafish represents a valuable and underexploited model for circadian-disease translational research is well-supported. However, the review would be significantly strengthened if the authors distinguished more clearly between what is firmly established, what is supported by preliminary or single-study evidence, and what remains genuinely speculative.

      Likely impact and utility:

      This review will be useful as an orientation document for researchers new to zebrafish circadian biology, and the comprehensive treatment of physiological outputs across organ systems is a genuine service to the field. Its impact as an intellectual contribution is limited by the descriptive approach and the absence of original synthesis or conceptual reframing. The most interesting ideas in the manuscript - the reframing of the central/peripheral clock hierarchy question, and the potential of clock gene paralogues for probing non-canonical functions - could be further developed and, if pursued, could form the basis of a more distinctive and impactful contribution.

    2. Reviewer #2 (Public review):

      Summary:

      This review is valuable in principle because circadian rhythms in zebrafish are unexplored and therefore this degree is valuable in principle. There are a number of significant weaknesses that should be addressed for it to have an impact. First, while the review covers a broad range of topics in chronobiology, it does not put them in context. Placing zebrafish work in the context of other model organisms that are better understood and other fish species would broaden the appeal. The review could also expand to a discussion of sleep, where the understanding in zebrafish is much more advanced. Critically, providing a novel framework, identifying new areas of opportunity and limitations of the system would expand the interest to non-zebrafish research groups. In addition, there are a number of misstatements/mis-citations that are critical to correct. Therefore, I find this review potentially impactful, but its current form is likely to limit its impact.

      Strengths:

      Focusing on decentralized photo sensing is a strength because it is relatively unique to zebrafish.

      The breadth of discussion in zebrafish is a strength.

      Weaknesses:

      It might be helpful to reorganize the review with an introduction on what is known in other better studied systems to be highly conserved, then to focus in on the components of zebrafish that are discussed here.

      A weakness is the lack of integration with other model organisms and other fish systems. Therefore, the narrow focus on zebrafish is unlikely to appeal to broader audiences.

      It's surprising that there is not more discussion of sleep, which has been studied in detail, and its relationship to the clock.

      Discussions of limitations of the model, including adult vs larval analysis and challenges performing long-term behavioral analysis in fish, would be valuable.

    3. Reviewer #3 (Public review):

      Summary:

      Over the past 3 or 4 decades, our understanding of the molecular mechanism underlying the circadian clock has increased substantially. This is in large part due to successful forward and reverse genetics approaches applied to a broad range of genetic model systems, notably Drosophila, Neurospora, mouse, Arabidopsis and cyanobacteria. Although the clock components in these species are diverse, the basic operating principles are highly conserved, allowing us to build a general view of clock mechanisms. Looking forward, there are still many unanswered questions regarding how clocks are organized at the systems level and, in turn, how they are coupled to key aspects of physiology. Each model species has its own set of advantages and disadvantages for tackling particular questions. As this timely review aims to illustrate, the zebrafish has become a particularly valuable model for exploring circadian clock biology. This is in part due to its technical advantages, accessibility of early developmental stages and its directly light-entrainable peripheral clocks. This provides unparalleled opportunities for studying the circadian clock hierarchy and its links with physiology.

      Strengths:

      This review does a good job of integrating the many lines of circadian clock research where the zebrafish has been used as a model and provides an overview of many future challenges it is well-suited to tackle.

      Weaknesses:

      There are citation errors, as well as inaccurate and misleading statements that must be remedied in a revised version.

    4. Author response:

      We sincerely thank the reviewers and editors for the thorough, constructive, and insightful comments, which have greatly helped us improve the accuracy, clarity, and rigor of the manuscript. We acknowledge that the current version has several limitations, including insufficient contextualization with other model systems and lack of critical synthesis. These important weaknesses will be comprehensively addressed in a future revised version of the review.

      For the present revision, we have focused exclusively on correcting objective errors, factual inaccuracies, and citation mistakes as pointed out by the reviewers. All specific factual and reference issues raised by Reviewer 2 and Reviewer 3 have been carefully corrected in the revised manuscript, including inaccurate statements, incorrect citations, missing references, and inconsistent descriptions of zebrafish clock genes, photoreception, and physiological functions.

      We appreciate the reviewers’ thoughtful suggestions regarding the conceptual depth, comparative context, critical synthesis, and expanded discussion of sleep and model limitations. While we fully agree that these aspects would significantly strengthen the review, we plan to systematically incorporate these broader conceptual improvements in a future, more substantial revision.

    1. eLife Assessment

      This important study demonstrates that a perinuclear actomyosin network, present in some types of human cells, facilitates kinetochore-spindle attachment of chromosomes in unfavorable locations, thereby reducing their missegregation rate. This actomyosin network and its general role have been studied previously, but this study convincingly clarifies the underlying mechanism and expands the investigation to additional cell lines. The results are relevant to understanding chromosome missegregation in cancer cells.

      [Editors' note: this paper was reviewed by Review Commons.]

    2. Reviewer #1 (Public review):

      Sheidaei and colleagues report a novel and potentially important role for an early mitotic actomyosin-based mechanism, PANEM contraction, in promoting timely congression of chromosomes located at the nuclear periphery, particularly those in polar positions. The manuscript will interest researchers studying cell division, cytoskeletal dynamics, and motor proteins. Although some data overlap with the group's prior work, the authors extend those findings by optimizing key perturbations and performing more detailed analyses of chromosome movements, which together provide a clearer mechanistic explanation. The study also builds naturally on recent ideas from other groups about how chromosome positioning influences both early and later mitotic movements.

      Comments on revised version:

      In the revised manuscript, organizational issues have been largely resolved. In addition, the inclusion of new experiments in additional cell lines, along with an expanded discussion that places actomyosin contractility in the broader conceptual context of other mechanisms governing chromosome movement, has significantly strengthened the manuscript.

    3. Reviewer #3 (Public review):

      Sheidaei et al. report how chromosomes are favourably positioned to facilitate kinetochore-microtubule interactions during early mitosis. Studying kinetochore capture during early prophase is extremely difficult due to kinetochore crowding, but the team has taken up the challenge by classifying types of kinetochore movements, carefully marking kinetochore positions in early mitosis, and linking these to map their fate/next positions over time. The work is an excellent addition to the chromosome segregation field, as most of the literature has thus far focused on tracking kinetochores at slightly later stages of mitosis. The authors show that PANEM facilitates chromosome positioning toward the interior of the newly forming spindle, which in turn promotes chromosome congression. In the absence of PANEM, chromosomes end up in unfavourable locations and fail to form proper kinetochore-microtubule interactions. The work highlights the perinuclear actomyosin network in early mitosis (PANEM) as a key spatial and temporal element of chromosome congression, a step that precedes the segregation process.

      Comments on revised version:

      The authors' revisions have brought clarity to the description of movements in many of the figures. The manuscript ties a fundamental process to differences in cancer cell lines.

      The work extends their published discovery that an actomyosin network forms on the cytoplasmic side of the nuclear envelope during prophase. The current manuscript explains how this network facilitates chromosome capture and congression by tracking the motions of individual kinetochores during early mitosis. The findings are broadly useful for the cell division and cytoskeletal fields.

    4. Author response:

      The following is the authors’ response to the original reviews

      Reviewer #1 (Evidence, reproducibility and clarity):

      Summary

      Sheidaei and colleagues report a novel and potentially important role for an early mitotic actomyosinbased mechanism, PANEM contraction, in promoting timely congression of chromosomes located at the nuclear periphery, particularly those in polar positions. The manuscript will interest researchers studying cell division, cytoskeletal dynamics, and motor proteins. Although some data overlap with the group's prior work, the authors extend those findings by optimizing key perturbations and performing more detailed analyses of chromosome movements, which together provide a clearer mechanistic explanation. The study also builds naturally on recent ideas from other groups about how chromosome positioning influences both early and later mitotic movements.

      In its current form, however, the manuscript is not acceptable for publication. It suffers from major organizational problems, an overcrowded and confusing Results section and figures, and a lack of essential experimental controls and contextual discussion. These deficiencies make it difficult to evaluate the data and the authors' conclusions. A substantial structural revision is required to improve clarity and persuasiveness. In addition, several key control experiments and more conceptual context are needed to establish the specificity and relevance of PANEM relative to other microtubule- and actin-based mitotic mechanisms. Testing PANEM in additional cell lines or contexts would also strengthen the claim. I therefore recommend Major Revision, addressing the structural, conceptual, and experimental issues detailed below.

      Major Comments

      A. Structural overhaul and figure reorganization

      The Results section is overly dense, lacks clear structure, and includes descriptive content that belongs in the Methods. Many figure panels should be moved to Supplementary Materials. A substantial reorganization is required to transform the manuscript into a focused, "Reports"-type article.

      Move methodological and descriptive details (e.g., especially from the second Results subheading and Figure 2) to the Methods or Supplementary Materials.

      In these parts, we define four phases of kinetochore motion in early mitosis. Without such a description in the main text, readers would be confused about subsequent analyses. Figure 2 is also important to show examples of how the four phases develop. Although we respect this suggestion from the reviewer, we would like to keep these parts in the main text and main figure.

      Remove repetitive statements that simply restate that later phenotypes arise as consequences of delayed Phase 1 (applicable to subheadings 3 onward).

      As suggested, we have removed the statement for the delayed start of Phase 2 for peripheral kinetochores in azBB-treated cells (Page 9, second paragraph). We have also simplified the statement for the delayed start of Phase 3 and Phase 4 to avoid repetition (Page 9, third paragraph; Page 10, second paragraph).

      Figure 4I: This panel is currently unclear and should be drastically simplified.

      Following this suggestion, we simplified Figure 4I by removing the column of ‘Start’, which is easily deduced from the ‘Duration’ results and therefore does not provide much new information.

      I recommend to reorganize figures as follows:

      Figure I: Keep as single figure but simplify. Figure 1D and 1E could be combined, move unnormalized SCV to supplementary materials. Same goes for 1F.

      We have reorganized Figure 1, as suggested, and moved unnormalized data to supplemental materials.

      New Figure 2: Combine current Figures 2A, 3A, 3C, 3D, 4C, 4F, and 4H to illustrate how PANEM contraction facilitates initial interactions of peripheral chromosomes with spindle microtubules which increases speed of congression initiation.

      If we were to follow this suggestion, we would lose Figure 2B, D, Figure 3B and Figure 4A, where examples of kinetochore motions are shown in images and 3D diagrams. The new Figure would mostly consist of only graphs. Without examples of images and 3D diagrams, readers would have difficulty understanding the study. Although we respect this suggestion from the reviewer, we would like to keep Figures 2, 3 and 4, as they are (except for making Figure 4I simpler; see above).

      New Figure 3: Combine current Figures 5A, 5C, 5D, 5F, 6B, 6C, and lower panels of 4H to show how

      PANEM contraction repositions polar chromosomes and reduces chromosome volume in early mitosis to enable rapid initiation of congression.

      If we were to follow this suggestion, we would lose Figure 5B and Figure 6A, where examples of kinetochore/chromosome dynamics are shown in images and 3D diagrams. For the same reason as above, we would like to keep Figure 5 and 6 as they are, although we respect this suggestion from the reviewer.

      New Figure 4: Combine Figures 7A, 7B, 7D, 7E, 7F, expanded Supplementary Figure S7, and new data to demonstrate that PANEM actively pushes peripheral chromosomes inward which is important for efficient chromosome congression in diverse cellular contexts.

      We have conducted new experiments to demonstrate the role of PANEM in diverse cellular contexts, as detailed below. We have combined the new results with the original Figure S7 to create Figure 8 in line with this suggestion.

      On the other hand, in our view, combining Figure 7A-E and the extended Figure S7 would be confusing because the two parts address different topics. Although we respect this suggestion from the reviewer, we would like to keep Figure 7 and the extended Figure S7 (i.e. Figure 8) separate.

      B. Specificity and redundancy of actin perturbation

      To establish the specificity and relevance of PANEM, the authors should include or discuss appropriate controls:

      Apply global actin inhibitors (e.g., cytochalasin D, latrunculin A) to disrupt the entire actin cytoskeleton. These perturbations strongly affect mitotic rounding and cytokinesis but only modestly influence early chromosome movements, as reported previously (Lancaster et al., 2013; Dewey et al., 2017; Koprivec et al., 2025). The minimal effect of global inhibition must be addressed when proposing a localized actomyosin mechanism. Comment if the apparent differences in this approach and one that the authors were using arises due to different cell types.

      We did experiments along this line, using a dominant-negative LINC construct, in our previous study (Booth et al eLife 2019). LINC-DN should more specifically remove/reduce PANEM than the global actin inhibitors mentioned above. LINC-DN attenuated the reduction of CSV soon after NEBD and increased the number of polar chromosomes (Booth et al eLife 2019); i.e. in this regard, the outcome was similar to azBB treatment in the current study. One can expect that global actin inhibitors would also inhibit the PANEM formation and show effects similar to LINC-DN. By contrast, the indicated references reported that global actin inhibitors strongly affect mitotic rounding and cytokinesis but only modestly influence early chromosome movements, as the reviewer noted. One possibility is that such differences may have arisen from different cell types – this could be important, especially given that some cells form the PANEM and others do not (Figure 8A). A second possibility is that cytokinesis, mitotic rounding and PANEM formation may rely on actin polymerization to different extents. For example, the same concentration of global actin polymerization inhibitors may affect cytokinesis, but may still allow PANEM formation to proceed without observable effects on early chromosome movements. As suggested, we discussed this topic in the Discussion (page 16, third paragraph).

      Clarify why spindle-associated actin, especially near centrosomes, as reported in prior studies using human cultured cells (Kita et al., 2019; Plessner et al., 2019; Aquino-Perez et al., 2024), was not observed in this study. The Myosin-10 and actin were also observed close to centrosomes during mitosis in X.laevis mitotic spindles (Woolner et al., 2008). Possible explanations include differences in fixation, probe selection, imaging methods, or cell type. Note that some actin probes (e.g., phalloidin) poorly penetrate internal actin, and certain antibodies require harsh extraction protocols. Comment on possibility that interference with a pool of Myo10 at the centrosomes is important for effects on congression.

      As the reviewer implies, we cannot rule out that we could not detect actin associated with the spindle or centrosomes because of the difference in methods or cell lines between the current study and the literature mentioned by the reviewer. We have therefore moderated our claim in the Discussion that ‘we did not detect any actin network inside the nucleus, on the spindle or between chromosomes’ by adding ‘at least, using the method and the cell line in the current study’ to this statement (Page 14, second paragraph). We have also cited the three references mentioned by the reviewer in the Discussion (Page 14, second paragraph). Regarding Myosin10, azBB (blebbistatin variant) should have negligible effects on class-X myosin, including Myosin-10 (Limouze et al 2004 [PMID 15548862]). It is therefore unlikely that the effects of azBB that we observed in the current study are due to the inhibition of Myosin-10. We have cited Woolner et al 2008 and another paper and discussed this topic in the Discussion (Page 14, second paragraph).

      C. Expansion of PANEM functional analysis

      To strengthen the conclusions and broaden the study beyond the group's previous work, PANEM function should be tested in additional contexts (some may be considered optional but important for broader impact): [underlined by authors]

      Test PANEM function in at least one additional cell line that displays PANEM to rule out cell-line-specific effects.

      As suggested, we have studied the effect of PANEM contraction in cell lines other than U2OS. We have found that when PANEM contraction was inhibited, the reduction in chromosome scattering was diminished in RPE1 cells (new Figure 8B, C). Moreover, we have found that inhibition of PANEM contraction increased polar chromosomes during prometaphase/ metaphase in RPE1 and HCT116 cells (which form PANEM), but not in HeLa cells (which do not form PANEM) (new Figure 8D, E). These results suggest that the effects of PANEM contraction, originally observed in U2OS cells, are also present in other cell lines (RPE1 and HCT116) that form PANEM.

      Examine higher-ploidy or binucleated cells to determine whether multiple PANEM contractions are coordinated and if PANEM contraction contributes more in cells of higher ploidies or specific nuclear morphologies.

      This is an interesting suggestion, but it takes lots of time to conduct such a study, and it goes beyond the scope of this paper.

      Investigate dependency on nuclear shape or lamina stiffness; test whether PANEM force transmission requires a rigid nuclear remnant.

      This is an interesting suggestion, but it takes lots of time to conduct such a study, and it goes beyond the scope of this paper.

      Analyze PANEM's contribution under mild microtubule perturbations that are known to induce congression problems (e.g., low-dose nocodazole).

      In the current study, we found that PANEM contraction affects chromosome motions in Phase 1 and Phase 3 but not Phase 2 or Phase 4. Mild microtubule perturbation itself could affect chromosome motions in all four Phases. We do not think it would be so informative to study what additional effects the reduced PANEM contraction shows when combined with mild microtubule perturbation.

      Evaluate PANEM contraction role in unsynchronized U2OS cells, where centrosome separation can occur before NEBD in a subset of cells (Koprivec et al., 2025), and in other cell types with variable spindle elongation timing.

      Following this suggestion, we first investigated the timing of spindle elongation, relative to NEBD, in asynchronous U2OS cells (Figure 8 – figure supplement 3). We imaged cells every 5 min (it was difficult to reasonably observe enough mitotic cells using a shorter interval). Most of the cells showed no significant change in the spindle length (distance between two spindle poles) after (or around) NEBD [e.g. Cell 1 in A] or a mild reduction in it [e.g. Cell 2 in A]. Only a small number of cells (2-3 out of 26) showed a mild increase in the spindle length after (or around) NEBD [e.g. Cell 3 in A]. Because the spindle elongation after NEBD was rare and mild, it was difficult to address how the timing of spindle elongation affects the effect of PANEM on reducing chromosome scattering and on chromosome relocation from polar regions. We explained this result and discussed this topic in the Discussion section.

      Quantify not only the percentage of affected cells after azBB but also the number of chromosomes per cell with congression defects in the current and future experiments.

      It is tricky to count the number of chromosomes because they frequently overlap. Counting kinetochores is more feasible, but kinetochore signals show some non-specific background (e.g. those outside of the nucleus in prophase). We therefore quantified the chromosome volume at polar regions in azBB-treated cells (Figure 6C).

      D. Conceptual integration in Introduction and Discussion

      The manuscript should better situate its findings within the context of early mitotic chromosome movements:

      Clearly state in the Introduction and elaborate in the Discussion that initiation of congression is coupled to biorientation (Vukušić & Tolić, 2025). This provides essential context for how PANEM-mediated nuclear volume reduction supports efficient congression of polar chromosomes.

      It has been a widely accepted view in the field that chromosome congression precedes biorientation, since the publication in 2006 (Kapoor et al Science 2006). Very recently, this view has been challenged by the new publication (Vukušić & Tolić, Nat comm 2025), as indicated by this reviewer. We have mentioned this new model and discussed the new interpretation of our results based on this new model, in the Discussion (page 15; ‘It has been a widely accepted view…’).

      To explain the new interpretation of our results more clearly, we have a new diagram as a supplemental figure (Figure 9 – figure supplement 1) in the revised manuscript.

      Explain that PANEM is most critical for polar chromosomes because their peripheral positions are unfavorable for rapid biorientation (Barišić et al., 2014; Vukušić & Tolić, 2025).

      We have included such a statement in the Discussion, as a part of the new interpretation of our results based on the new model that chromosome biorientation precedes congression (see above). We have also cited the indicated two papers.

      Discuss how cell lines lacking PANEM (e.g., HeLa and others) nonetheless achieve efficient congression, and what alternative mechanisms compensate in the absence of PANEM. For example, it is well established that cells congress chromosomes after monastrol or nocodazole washout, which essentially bypasses the contribution of PANEM contraction.

      Following this suggestion, we discussed three possible mechanisms that could compensate for a lack of PANEM and facilitate kinetochore-MT interaction and chromosome congression, based on previous literature (Page 17): 1) the enhanced assembly rate of spindle MTs may facilitate kinetochore-MT interactions in N-CIN+ cancer cells, 2) chromosome biorientation may precede congression more frequently to promote the congression towards the spindle midplane, and 3) the balance between CENP-E, Dynein and chromokinesin’s activities may incline to greater chromosome-arm ejection forces towards the spindle midplane.

      Minor Comments

      These issues are more easily addressable but will significantly improve clarity and presentation.

      Introduction

      Remove the reference to Figure 1A in the Introduction. The portion of Figure 1 and related text that recapitulates the authors' previous work should be incorporated into the Introduction, not the Results.

      As suggested in the second sentence of this comment, we have moved most of the second paragraph of the first section of Results to Introduction (Page 4) and cited Figure 1A and 1B in Introduction. We would like to keep the reference to Figure 1A in the Introduction, because showing the PANEM images at the beginning of the manuscript would help readers’ understanding of our study. In addition, citing Figure 1A in the Introduction is more consistent with the suggestion in the second sentence of this comment.

      Results (by subheading)

      First subheading: When introducing the ~8-minute early mitotic interval, cite additional studies that have characterized this period: Magidson et al., 2011 (Cell); Renda et al., 2022 (Cell Reports); Koprivec et al., 2025 (bioRxiv); Vukušić & Tolić, 2025 (Nat Commun); Barišić et al., 2013 (Nat Cell Biol).

      As suggested, we cited these references at the indicated part of the first section of the Results (page 5).

      Second subheading: Cite key reviews and foundational research on kinetochore architecture and sequential chromosome movement during early mitosis: Mussachio & Desai, 2017 (Biology); Itoh et al., 2018 (Sci Rep); Magidson et al., 2011 (Cell); Vukušić & Tolić, 2025 (Nat Commun); Koprivec et al., 2025 (bioRxiv); Rieder & Alexander, 1990 (J Cell Biol); Skibbens et al., 1993 (J Cell Biol); Kapoor et al., 2006 (Science); Armond et al., 2015 (PLoS Comput Biol); Jaqaman et al., 2010 (J Cell Biol).

      Rieder & Alexander, 1990 (J Cell Biol) and Kapoor et al., 2006 (Science) have already been cited in the second section of the Results in the original manuscript. We agree that all other references should be cited in this manuscript, and they are now cited in the Introduction and/or Discussion where they fit best (e.g. Mussachio & Desai 2017 reviews the kinetochore in general and is therefore best cited in the Introduction).

      Third subheading: Clarify why some kinetochores on Figure 3A appear outside the white boundaries if these boundaries are intended to represent the nuclear envelope.

      We interpret that these are background signals in the cytoplasm, which do not come from kinetochores, because 1) before NEBD, they were outside of the nucleus, and 2) after NEBD, they did not show any characteristic kinetochore motions such as those towards a spindle pole (Phase 2) and the spindle mid-plane (Phase 4). We have commented on these background signals in the legend for Figure 3A.

      Fourth subheading: Note that congression speed is lower for centrally located kinetochores because they achieve biorientation more rapidly (Barišić et al., 2013, Nat Cell Biol; Vukušić & Tolić, 2025, Nat Commun).

      Relevant to this comment, there was an error regarding the congression speed of central kinetochores (original Figure 4H). The congression speed of peripheral kinetochores was shown correctly, but for central kinetochores it was shown incorrectly with µm per time interval (30s) shown, rather than µm per minute. We amended this error in the revised manuscript (new Figure 4H). Based on the corrected data, the speed of congression is similar between peripheral and central kinetochores. The original Figure 3G (the speed of poleward motion for central kinetochores) had a similar error, which we have also corrected in the revised manuscript. We apologize for these errors and the confusion it may have caused.

      Regarding this comment, if biorientation is achieved more rapidly for central kinetochores, Phase 3 (rather than congression speed) would be shorter for central kinetochores. Indeed, Phase 3 is slightly shorter for central kinetochores (control) than for peripheral kinetochores (control) (Figure 4C), but the difference is not statistically significant (t test; p\=0.21).

      Fifth subheading: Cite studies on polar chromosome movements: Klaasen et al., 2022 (Nature); Koprivec et al., 2025 (bioRxiv). Clarify that Figure 5F displays only those kinetochores that initiated directed congression movements.

      These two references have already been cited and discussed in this Result section of our original manuscript. However, considering this suggestion, we have discussed more about polar chromosome movements reported by Koprivec et al (page 11). Meanwhile, the reviewer is correct about Figure 5F, and we have clarified this point in the Figure 5F legend.

      Sixth subheading (currently in Discussion): Move the final paragraph of the Discussion into the Results and expand it with preliminary analyses linking PANEM contraction to congression efficiency across untreated cell types or under mild nocodazole treatment.

      As suggested, we have moved the final paragraph of the Discussion in the original manuscript to make a new final section in the Results in the revised manuscript. Moreover, as suggested, we have studied the outcome of inhibiting PANEM contraction in cell lines other than U2OS (Figure 8 B–E), and have described the new results to the new final section in the Results.

      Discussion

      1. When discussing cortical actin, cite key reviews on its presence and function during mitosis: Kunda & Baum, 2009 (Trends Cell Biol); Pollard & O'Shaughnessy, 2019 (Annu Rev Biochem); Di Pietro et al., 2016 (EMBO Rep).

      As suggested, we have cited all these review papers in the Discussion (page 17), and mentioned the role of the cortical actin on the spindle orientation and positioning (Kunda & Baum, 2009; Di Pietro et al., 2016), as well as the function of the actomyosin ring on cytokinesis (Pollard & O'Shaughnessy, 2019).

      Significance

      Advance

      This study's main strength is its novel and potentially important demonstration that contraction of PANEM, a peripheral actomyosin network that operates contracts early mitosis, contributes to the timely initiation of chromosome congression, especially for polar chromosomes. While PANEM itself was previously described by this group, this manuscript provides new mechanistic evidence, improved perturbations, and detailed chromosome tracking. To my knowledge, no prior studies have mechanistically connected this contraction to polar chromosome congression in this level of detail. The work complements dominant microtubule-centric models of chromosome congression and introduces actomyosin-based forces as a cooperating system during very early mitosis. However, the impact of the study is currently limited by major organizational issues, insufficient controls, and incomplete contextualization within existing literature. Addressing these issues will substantially improve clarity and credibility. [underlined by authors]

      We have addressed the underlined criticisms as detailed above.

      Audience

      Primary audience of this study will be researchers working in cell division, mitosis, cytoskeleton dynamics, and motor proteins. The findings may interest also the wider cell biology community, particularly those studying chromosome segregation fidelity, spindle mechanics, and cytoskeletal crosstalk. If validated and clarified, the concept of PANEM could be integrated into textbooks and models of chromosome congression and could inform studies on mitotic errors and cancer cell mechanics.

      Expertise

      My expertise lies in kinetochore-microtubule interactions, spindle mechanics, chromosome congression, and mitotic signaling pathways.

      Reviewer #2 (Evidence, reproducibility and clarity):

      In this manuscript, Sheidaei et al. reported on their study of chromosome congression during the early stages of mitotic spindle assembly. Building on their previous study (ref. #15, Booth et al., Elife, 2019), they focused on the exact role of the actin-myosin-based contraction of the nuclear envelope. First, they addressed a technical issue from their previous study, finding a way to specifically impair the actomyosin contraction of the nuclear membrane without affecting the contraction of the plasma membrane. This allowed them to study the former more specifically. They then tracked individual kinetochores to reveal which were affected by nuclear membrane contraction and at what stage of displacement towards the metaphase plate. The investigation is rigorous, with all the necessary controls performed. The images are of high quality. The analyses are accurate and supported by convincing quantifications. In summary, they found that peripheral chromosomes, which are close to the nuclear membrane, are more influenced by nuclear membrane contraction than internal chromosomes. They discovered that nuclear membrane contraction primarily contributes to the initial displacement of peripheral chromosomes by moving them towards the microtubules. The microtubules then become the sole contributors to their motion towards the pole and subsequently the midplane. This step is particularly critical for the outermost chromosomes, which are located behind the spindle pole and are most likely to be missegregated.

      Significance

      While the conclusions are somewhat intuitive and could be considered incremental with regard to previous works, they are solid and improve our understanding of mitotic fidelity. The authors had already reported the overall role of nuclear membrane contraction in reducing chromosome missegregation in their previous study, as mentioned fairly and transparently in the text. However, the reason for this is now described in more detail with solid quantification. Overall, this is good-quality work which does not drastically change our understanding of chromosome congression, but contributes to improving it. Personally, I am surprised by the impact of such a small contraction (of around one micron) on the proper capture of chromosomes and wonder whether the signalling associated with the contraction has a local impact on microtubule dynamics. However, investigating this point is clearly beyond the scope of this study, which can be published as it is. [underlined by authors]

      The suggested topic (underlined) is intriguing. However, we agree with the reviewer that it is beyond the scope of this paper. The reviewer recommends publication of our manuscript as it is.

      Reviewer #3:

      Sheidaei et al., report how chromosomes are brought to positions that facilitate kinetochore-microtubule interactions during mitosis. The study focusses on an important early step of the highly orchestrated chromosome segregation process. Studying kinetochore capture during early prophase is extremely difficult due to kinetochore crowding but the team has taken up the challenge by classifying the types of kinetochore movements, carefully marking kinetochore positions in early mitosis and linking these to map their fate/next-positions over time. The work is an excellent addition to the field as most of the literature has thus far focussed on tracking kinetochore in slightly later stages of mitosis. The authors show that the PANEM facilitates chromosome positioning towards the interior of the newly forming spindle, which in turn facilitates chromosome congression - in the absence of PANEM chromosomes end up in unfavourable locations, and they fail to form proper kinetochore-microtubule interactions. The work highlights the perinuclear actomyosin network in early mitosis (PANEM) as a key spatial and temporal element of chromosome congression which precedes the segregation process.

      Major points

      (1) The complexity of tracking has been managed by classifying kinetochore movements into 4 categories, considering motions towards or away from the spindle mid-plane. While this is a very creative solution in most cases, there may be some difficult phases that involve movement in both directions or no dominant direction (eg Phase3-like). It is unclear if all kinetochores go through phase1, 2, 3 and 4 in a sequential or a few deviate from this pattern. A comment on this would be helpful. Also, it may be interesting to compare those that deviate from the sequence, and ask how they recover in the presence and absence of azBB.

      To respond to this comment, we would like to first clarify how we selected kinetochores for our analysis. We selected kinetochores that can be individually tracked. If kinetochore tracking was difficult (before the start of Phase 4 in control and azBB-treated cells or before observing the extended Phase 3 in azBB-treated cells) because of kinetochore crowding, we did not choose such kinetochores. For example, related to the next comment of this Reviewer, we did not include kinetochores close to spindle poles (within 4 µm) at NEBD in our analysis for the following two reasons: First, these kinetochores often did not show clear and rapid movements towards a spindle pole, which we used to define Phase 2. Second, although we referred to kinetochore co-localization with a microtubule signal for the start of Phase 2, this was difficult for kinetochores close to spindle poles because of a high density of microtubules. As requested, we have added this comment to the Method section (page 25).

      With the above selection, all selected kinetochores without azBB treatment (control) showed the poleward motion (Phase 2) and congression (Phase 4) in this order, though their extents were varied among kinetochores. All selected kinetochores with azBB treatment also showed the poleward motion (Phase 2), and some of them showed congression (Phase 4) after Phase 2. Then, Phase 1 and Phase 3 were defined as intervals between NEBD and Phase 2 and between Phase 2 and Phase 4, respectively. If no Phase 4 was observed with azBB, we judged that Phase 3 continued till the end of tracking. We have added this comment to the Method section (page 25-26).

      (2) Would peripheral kinetochore close to poles behave differently compared to peripheral kinetochore close to the midplane (figure S4)? In figure 3D, are they separated? If not, would it look different?

      Since we did not include kinetochores close to spindle poles (at NEBD), for which it was difficult to define Phase 2 (see our response to the above major point 1), in our analysis, the suggested comparison is not feasible.

      (3) Uncongressed polar chromosomes (eg., CENPE inhibited cells) are known to promote tumbling of the spindle. In figure 5B with polar chromosomes, it will be helpful to indicate how the authors decouple spindle pole movements from individual kinetochore movements.

      In contrast to CENPE-inhibited cells, azBB-treated cells did not show much tumbling of the spindle, though both cells showed uncongressed polar chromosomes. The reason for this difference may be fewer uncongressed polar chromosomes in azBB-treated cells. There were still modest spindle motions in azBB-treated cells. However, because kinetochore motions were assessed relative to a spindle pole (and other reference points on the spindle) in our study (Figure 2A, C), the modest spindle motions were offset in our analyses of kinetochore motions. We have clarified the underlined part in the Method section (page 24).

      (4) The work has high quality manual tracking of objects in early mitosis- if this would be made available to the field, it can help build AI models for tracking. The authors could consider depositing the tracking data and increasing the impact of their work.

      As suggested, we have included kinetochore tracking data as supplemental data in the revised manuscript (Figure 3 – source data 1–4; Figure 5 – source data 1, 2).

      Minor points

      (1) It will be helpful for readers to see how many kinetochores/cell were considered in the tracking studies. Figure legends show kinetochore numbers but not cell numbers.

      As suggested, we have now mentioned the number of cells, where the kinetochore motions were analyzed, in the legends for Figures 3, 4, 5, and supplemental figures.

      (2) Discussion point: If cells had not separated their centrosomes before NEBD, would PANEM still be effective? Perhaps the cancer cell lines or examples as shown in Figure 6A have some clues here.

      Following this suggestion, we first investigated the timing of spindle elongation, relative to NEBD, in asynchronous U2OS cells (Figure 8 – figure supplement 3). We imaged cells every 5 min (it was difficult to reasonably observe enough mitotic cells using a shorter interval). Most of the cells showed no significant change in the spindle length (distance between two spindle poles) after (or around) NEBD [e.g. Cell 1 in A] or a mild reduction in it [e.g. Cell 2 in A]. Only a small number of cells (2-3 out of 26) showed a mild increase in the spindle length after (or around) NEBD [e.g. Cell 3 in A]. Because the spindle elongation after NEBD was rare and mild, it was difficult to address how the timing of spindle elongation affects the effect of PANEM on reducing chromosome scattering and on chromosome relocation from polar regions. We explained this result and discussed this topic in the Discussion section.

      (3) Figure 7 cartoon shows misalignment leading to missegregation. It may be useful to consider this in the context of the centrosome directed kinetochore movements via pivoting microtubules. Is this process blocked in azBB-treated cells?

      We understand that the Reviewer refers to the kinetochore pivoting mechanism around a spindle pole, which was recently reported by the Tolic group (Koprivec et al., 2026). Such a pivoting mechanism would work only when the spindle elongates (i.e. the distance between spindle poles is enlarged) after NEBD. Therefore, to address this Reviewer’s question, we tried to assess how PANEM contraction contributes to relocating polar chromosomes when the spindle elongates before or after NEBD in asynchronous U2OS cells (i.e. in the situation where the kinetochore pivoting mechanism is applied or not), as we noted above in response to Point 2. However, spindle elongation after NEBD was rare and mild, and we were unable to address this issue (see our response to Point 2). We discussed this matter in the Discussion section.

      (4) Are all the N-CIN- lines with PANEM highly sensitive to azBB? In other words, is PANEM essential for normal congression in some of these lines.

      Because blebbistatin could kill cells by inhibiting cytokinesis, the blebbistatin sensitivity of cell growth may not necessarily reflect how essential the PANEM contraction is for chromosome congression.

      Instead, we addressed more directly how essential the PANEM contraction is for chromosome congression. We analyzed chromosome congression in RPE1 and HCT116 cells (both are NCIN-) in the presence and absence of pnBB, the inhibitor of PANEM contraction (new Figure 8D, E). With pnBB, these cells showed congression defects, suggesting that the PANEM contraction is essential for chromosome congression in these N-CIN- cells.

      (5) Are congression times delayed in lines that naturally lack PANEM?

      For example, it takes 10-20 min for HeLa cells (lacking PANEM) to complete chromosome congression after the NEBD (Bancroft et al 2025: https://doi.org/10.1242/jcs.163659). This is not significantly different from the time (8-18 min) for chromosome congression we observed in U2OS cells (which form PANEM). We assume that cells lacking PANEM have developed a compensatory mechanism for efficient chromosome congression – we have discussed possible compensatory mechanisms in the last paragraph of the Discussion (page 17).

      (6) Page 23 "we first identified the end of congression" how does this relate to kinetochore oscillations that move kinetochores away from the metaphase plate?

      The start of kinetochore oscillation was defined as the end of Phase 4 if we could track the kinetochore until that point. In some cases where the kinetochore became close to the midplane (< 2.5 µm), it was not possible to track it further due to kinetochore crowding around the spindle mid-plane – in such cases, the end of Phase 4 was assigned as the end of tracking. These definitions were not necessarily clear in the original manuscript. Moreover, in the original manuscript, it was not clearly stated that the end of Phase 4 was defined in the same way for both non-polar and polar kinetochores. We have now clarified these points in the Method section (page 25).

      (7) Are spindle pole distances (spindle sizes) different in early and late mitotic cells (4min vs 6min after NEBD) in control vs azBB-treated cells? Please comment on Figure S2E (mean distance) in the context of when phase 4 is completed. Does spindle size return to normal after congression?

      In Figure S2E (Figure 1 – figure supplement 6 in the revised manuscript), we did not observe a significant difference in the spindle-pole distance (the spindle size) between control and azBBtreated cells at any individual time points. The smallest p-value was 0.094 at 6.0 min. As suggested, we have explained this in the legend for this supplementary figure. Completion of Phase 4 is highly variable across different kinetochores within the same cell; thus, a general comment on its completion timing in cells is not feasible.

      Significance:

      The current work builds upon their previous work, in which the authors demonstrated that an actomyosin network forms on the cytoplasmic side of the nuclear envelope during prophase. This work explains how the network facilitates chromosome capture and congression by tracking motions of individual kinetochores during early mitosis. The findings can be broadly useful for cell division and the cytoskeletal fields.

    1. Here, we propose a solvent-mediated molecular engineering strategy to fabricate high-performance bamboo molecular plastics (BM-plastics) with tunable H-bond networks. By employing a hydrated ZnCl₂/formic acid deep eutectic solvent (DES), we disassemble the native H-bond matrix of bamboo cellulose into a homogeneous molecular system. Subsequent ethanol stimulation triggers the rearrangement of cellulose chains, fostering dense, ordered H-bond interactions between hydroxyl and formate ester groups (Fig. 1b). This dual-step process not only overcomes the trade-off between mechanical strength and processability (Fig. 1c) but also enables scalable manufacturing under ambient conditions (Fig. 1d). In contrast to traditional processing methods that often require toxic or costly reagents and are associated with high energy consumption, our solvent-mediated molecular engineering approach delivers substantial operational advantages while significantly improving the performance characteristics of the resulting BM-plastic

      SCIENCE!

    1. If the PDF is not tagged, navigate to Convert > Recognize Text > Current File, then Accessibility > Autotag Document to add tags.

      This path is not in the Current File dialog box

    1. This annotates nothingAndrew Wooldridge ⛰️ wrote: "Record harvest sparks mass giveaway of free potatoes across Berlin | Germany | The Guardian"

    1. ex should therefore be162 modeled as a biological state when the exposure or phenotype is sex-dependent, rather than used163 only as an adjustment variable.

      yes, but what are the implications of this? Does it change it somehow in terms of the analytical strategies?

    2. Biological state is the internal context in which genetic variation and exposures affect phenotype.

      what does this mean? Isn't the biological state a phenotype? what's their difference?

    3. Lagged exposure87 definitions, exclusion of early events, and sensitivity analyses for reverse causation, such as88 removing cases diagnosed soon after exposure assessment, are needed before assigning a causal89 interpretation to the interaction

      Yes, but I do feel the authors working on GxE are aware of this issues

    4. In the red meat example, diet is84 not simply an external modifier of genetic risk, but may mark early disease, organ dysfunction,85 or illness-related dietary adaptation, as in kidney disease

      Again, this is the same problem, which is not novel, and widely recognized, of reverse causation

    5. Physical activity may reduce obesity risk through energy expenditure and76 improved insulin sensitivity, while also reflecting socioeconomic and built-environment77 conditions that determine whether regular activity is feasible, including neighborhood safety,78 walkability, work schedule, and leisure time [3; 4].

      Again, is this novel, or even novel within the literature that the authors are addressing?

    6. Yet even when the exposure is upstream, it may represent a broader behavioral pattern rather72 than a single biological factor. The exposure may also act indirectly or in parallel with other73 upstream processes.

      Is this a novel insight?

    7. An exposure may precede the biological state,66 reflect it, incompletely measure the relevant internal response, or trigger a transition into a new67 state

      There are several terms that the authors take for granted and that I think need a definition or greater context: e.g. reflect (when saying reflects biological state), exposure labels

    8. Exposure La

      This section is pointing out at a known problem, for example, in exposomics, which is that of reverse causation? What's the difference with the authors' take on this

    9. The55 issue is not only whether genotype interacts with exposure, but whether the interaction points to56 a plausible biological mechanism

      This is the key thesis

    10. the same genotype and the same exposure label do not50 necessarily produce the same clinical or biological outcome

      I'm not sure if a follow the central problem: is the problem that even in the face of same genotypes x environments we get different biological outcomes? If so, isn't that the main problem of biology? how the same genotypes can lead to different outcomes? Alternatively, why same exposures lead to different outcomes? I understand that one solution is actually looking at GxE, but I'm nor certain that the GxE model actually wants to answer that question, but rather, contribute a little more to its answer.

    11. In the conventional gene-environment interaction (G×E) model, genotype confers a47 degree of risk, while environmental exposures modify the probability of disease.

      I'd be careful with this definition, because previous works have defined GxE both as a different effect of exposure on disease risk among people with different genotypes, and as a different effect of genotypes on disease risk among people with different exposures. Therefore, never assuming a single direction of change.

    Annotators

    1. Für die Zielgruppe ab 40 ist das der entscheidende Punkt, weil viele dieser Pflegeserien speziell auf reife Haut, Anti-Aging-Wirkstoffe und nachgewiesene Formulierungen ausgerichtet sind.

      The sentence is too long: Gerade ab 40 wird dieser Punkt oft wichtiger. Viele Pflegeserien sind speziell auf reife Haut und wirksame Anti-Aging-Inhaltsstoffe abgestimmt.

    1. Expectations Your initial post is due Wednesday, at 11:59 PM ET; all discussion activities including your replies to classmates’ posts must be completed by Sunday, at 11:59 PM ET. Failure to meet the timeliness and quantity expectations will result in a point deduction.

      Delete?

      This is redundant - it's already in the assignment and when we make updates - it's in both places.

    2. To deepen the conversation, select from among the following prompts:

      Delete from template because there's custom language in each discussion?

      Want to avoid using "prompt" but also want to make it clear that students don't need to reply using ALL of the prompts/questions.

    3. Prompt Suggestions Challenge a perspective: Do you agree or disagree with their stance on mandatory ethical hardware lifecycle practices? Why? Provide evidence or examples to support your view. Expand the discussion: Did their article present a new angle on e-waste that you hadn’t considered? How does it compare to your findings? Propose a solution: How could businesses better balance cost, efficiency, and sustainability when disposing of outdated hardware? Suggest an alternative strategy or best practice. Make it personal: Have you or someone you know experienced challenges with outdated hardware disposal? How did the organization handle it, and what lessons can be learned?

      Delete callout and text.

    4. the most pressing ethical issue facing the tech industry today? How does this ethical concern impact various stakeholders, including users, companies, governments, and society as a whole? Provide examples to support your points. You may also want to consider: Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Then, lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

      Delete text

    5. When you’re ready to begin, navigate to the Post tab for detailed instructions on how to make your initial post. Check the Respond tab to review the requirements for responding to your peers. Lastly, visit the Evaluation tab for details on this discussion’s expectations and grading.

      Navigate to the Post tab for detailed instructions on how to make your initial post. Check the Respond tab to review the requirements for responding to your peers. Refer to the Evaluation tab for details on how this discussion will be graded.

      OR simplify

      Select the Post tab for detailed instructions on how to make your initial post, the Respond tab to review the requirements for responding to your peers, and the Evaluation tab for information on how this discussion will be graded.

    1. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

      Delete the lorum ispum. Keep "In this module..."

    1. It is operated by the social self:
      • distributed across participants,
      • shaped by intra-action,
      • evolving through occasions,
      • capable of interpreting cards,
      • co-creating meaning,
      • enacting movement,
      • reshaping the field.
    2. not merely comment on the situation.

      Cards placed around the GCQ do not merely comment on the situation. They help constitute the situation as a field of relation, possibility, re-orientation and next moves.

    3. intra-action:
      • participants shape the field,
      • the field shapes participants,
      • the app mediates forms of noticing,
      • the cards alter the available pathways,
      • AI reflects and extends,
      • shards preserve what might otherwise vanish.
    4. Adjacent Possible Playground

      ``` f it supports alignment but not community, it becomes a clever session tool. If it supports community but not alignment, it becomes another social platform. If it supports platform features without practice, it becomes empty infrastructure.

      The Core Play Field keeps the product whole. ```

    5. Playⁿ makes meaning shareable. The app helps shared meaning remainalive long enough to become practice

      Playⁿ makes meaning shareable. The app helps shared meaning remain alive long enough to become practice

    6. Its purpose is not to replace the card session

      Its purpose is to help the card session become more - continuous, - legible, - memorable, and - actionable.

    1. New empires would emerge from these tenuous beginnings, and by the end of the seventeenth century, Spain would lose its privileged position to its rivals. An age of colonization had begun and, with it, a great collision of cultures commenced.

      Rising globalization fueled the need for new markets, manpower for production, and raw materials—ushering in a proto-capitalist society

    1. Typically in Woodland communities, women practiced agriculture while men hunted and fished.

      Gender roles were present -> how egalitarian were these communities? Has gender stratification always existed?

    2. Corn—as well as other Mesoamerican crops—spread across North America and continues to hold an important spiritual and cultural place in many Native communities.

      ...continues to hold an important spiritual and cultural place in many Native communities.

      What effects did the production of cash crops have on a cultural scale? We mainly know it from the economic and social lens.

    3. The Salinan people of present-day California, for example, tell of a bald eagle that formed the first man out of clay and the first woman out of a feather.1 According to a Lenape tradition, the earth was made when Sky Woman fell into a watery world and, with the help of muskrat and beaver, landed safely on a turtle’s back, thus creating Turtle Island, or North America. A Choctaw tradition locates southeastern peoples’ beginnings inside the great Mother Mound earthwork, Nunih Waya, in the lower Mississippi Valley.2 Nahua people trace their beginnings to the place of the Seven Caves, from which their ancestors emerged before they migrated to what is now central Mexico.3

      Just how much did their origin stories/mythology diverge from one another? How does a civilization's topography shape their culture - the means and ways it is translated?

    4. Native Americans built settled communities and followed seasonal migration patterns, maintained peace through alliances and warred with their neighbors, and developed self-sufficient economies and maintained vast trade networks.

      What sociological aspects played a role into self-proclaimed European superiority during this century? How is it that they did not recognize the cultural diversity and advancements made by already existing communities?

    1. thus increasing student completion of reading assignments

      Using annotation in reading tasks gives students more autonomy, control, and ownership over their learning. By highlighting key ideas, asking questions, making connections, and adding personal thoughts to a text, students become more actively engaged with their reading rather than passively consuming information. This deeper interaction improves comprehension, critical thinking, and confidence in understanding complex texts.

      Annotation also supports students with different learning speeds and abilities, as each student can process and respond to the text in a way that works best for them. Furthermore, sharing annotations and discussing ideas with peers encourages collaboration and meaningful discussion, helping students learn from different perspectives while building stronger peer relationships. Overall, annotation transforms reading into a more interactive, reflective, and student-centered learning experience.

    1. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      Summary:

      In this manuscript, the authors investigate the role of Transcription Termination Factor 2 (TTF2) in the regulation of mitotic transcription. Using siRNA-mediated knockdown in two distinct human cell types combined with nascent RNA labeling (EU pulse), the authors identify an unexpected role for TTF2 in the timing of RNA Polymerase I (Pol I) reactivation following mitosis. The study suggests that this temporal misregulation may have downstream consequences for nucleolar morphology and function in interphase. The manuscript is well-written, and the figures are of high quality and clearly presented.

      Major comments:

      1. A primary limitation of the current study is that it does not deeply explore the underlying mechanism of the observed phenomenon. To strengthen the claims, the following points should be addressed:

      1a. Directionality of Phenotype: In Page 9, the authors conclude that TTF2 depletion is linked to abnormal nucleolar organization during interphase. It remains unclear if this is a direct result of mitotic misregulation or an independent interphase effect. To distinguish between these possibilities, I suggest the following experiment: perform a mitotic shake-off early in the siRNA treatment (~24h), collect mitotic cells, and allow them to re-enter G1 to image for nascent RNAs and nucleolin. This would clarify if the mitotic defect precedes and causes the interphase morphology changes. Alternatively, the authors should state that their current study cannot distinguish between these two possibilities.

      1b. Secondary Effects: The long duration of siRNA treatment (48h) raises the possibility that TTF2 knockdown misregulates the expression of other Pol I regulatory factors, leading to secondary effects. This limitation should be explicitly acknowledged in the Discussion. 2. The term "significant" is used throughout the manuscript without accompanying statistical testing.

      2a. Please provide statistical analyses (e.g., p-values) for the average plots in Figures 1-3 to substantiate the findings.

      2b. Where statistics are not performed, the language should be softened to "notable" or "observed increase" rather than "significant." 3. siRNA knockdowns are generally supported by quantification. Please provide the percentage reduction of the target protein by quantifying the blots provided in the supplemental figures. 4. To rule out the possibility that the increased nucleolin signal observed after TTF2 KD is simply due to higher protein abundance, the authors should perform a western blot to confirm that total nucleolin protein levels remain unchanged upon TTF2 depletion.

      Minor comments:

      1. The abstract and discussion refer to the role of TTF2 as a "conserved" process. As the study only tested human cell lines, "conserved" is technically inaccurate (as it implies evolutionary comparison). I recommend using "general" or "cell-type independent."
      2. While the Methods section is detailed, the Results section would benefit from brief descriptions of the treatments to improve flow.

      Example revision (Page 4): "...we treated two distinct cell lines with control and TTF2-specific siRNAs for 48 hours, followed by a 30-minute EU pulse to label nascent RNAs. Click chemistry and Hoechst labeling enabled 2-color imaging of mitotic chromosomes and nascent RNA..." 3. The data generally agree across both cell types; however, the presence of clustered signals in HeLa metaphase chromosomes is a notable divergence. It would be beneficial to include speculation in the Discussion on whether this represents a failure to silence Pol I transcription or an even earlier reactivation, and what this implies about a cancer cell line.

      Significance

      General assessment:

      The study is strong in its use of two different cell systems, providing confidence that the observed effects are not cell-line-specific. The figures are beautifully presented and the writing is clear. The primary limitation is the lack of mechanistic depth regarding how TTF2 specifically interfaces with the Pol I machinery compared to its known roles with Pol II.

      Advance:

      This work reports a previously unrecognized role for TTF2 in the temporal control of Pol I reactivation. While TTF2 is well-known for its role in terminating transcription and facilitating Pol II release during mitosis, its specific influence on the nucleolar transcription cycle provides a new perspective on how cells transition out of the mitotic state.

      Audience:

      This research will be of interest to researchers in the fields of gene regulation, the cell cycle, and nucleolar biology. Because it touches on the fundamental process of how transcriptional machinery is reset after cell division, it has implications for the broader basic research community interested in epigenetic memory and cellular identity.

    2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #1

      Evidence, reproducibility and clarity

      In this paper by the Oliveira lab a new perspective on TTF2 function during mitosis is proposed. Known for its role to terminate transcription at mitotic onset, this paper further shows an exciting involvement of TTF2 to schedule timely rDNA transcription at mitotic exit. Moreover, this role is shown to have a clear importance in the structuration of nucleoli, since TTF2 depletion is associated with premature partial assembly of nucleoli on the mitotic chromosomes and, subsequently, to fragmented nucleoli in interphase. These conclusions, which are well supported by imaging data, are original, interesting and, in fact, largely unexpected.

      The paper is very simple in its execution, based on siRNA depletion of TTF2 and monitoring of transcription by imaging using EU incorporation and rRNA-FISH, as well as nucleoli morphology and dynamics using immunostainings. Yet, it is well executed and has no major caveats. However, the authors should consider the following:

      1. The Teves lab has shown that TBP is a key factor maintaining its binding at rDNA loci during mitosis, enabling a prompt reactivation of rRNA production in interphase (Kwan et al. RNA 2024). This paper should be discussed on the light of current findings.
      2. In relation to the previous comment, I would strongly recommend the authors to analyse TBP-depleted cells, ideally using the line generated in the Teves lab, to address whether delayed rDNA transcription after mitosis leads to delayed nucleoli structuration. This assay would allow them to further confirm their model.
      3. In addition, it would be important to test if in the absence of mitotic TBP, the depletion of TTF2 does also lead to mitotic transcription.

      Significance

      Strengths: originality of the observation and simplicity of the experimental setup

      Limitations: exclusively based on imaging data

      Advance: completely unanticipated observation

      Audience: general readers interested in gene regulation

      My expertise: gene regulation through mitosis

    1. Lewis, Helen. 2026. “The Men Who Want Women to Be Quiet.” The Atlantic 336(6): 26–35. https://www.theatlantic.com/magazine/2026/06/conservative-masculinism-misogyny/686939/ (May 27, 2026; May 28, 2026).

    1. eLife Assessment

      This potentially useful paper presents an intriguing hypothesis about the evolutionary origins of the SLC25 family of mitochondrial carrier proteins common to all eukaryotic life, proposing that all members originated from the ADP/ATP carrier (AAC) and that AAC itself may have emerged from bacterial homologs such as CysZ and YihY. While the phylogenetic analyses and structural searches are reasonable methodologies to explore ancient evolutionary events, the evidence provided here is deemed to provide incomplete support for the conclusion that the mitochondrial ATP transporter is related to CysZ and Yih.

    2. Reviewer #1 (Public review):

      Summary:

      This paper tries to address an important outstanding issue, which is the evolutionary origin of the SLC25 family of mitochondrial carrier proteins, which are common to all eukaryotic life, with few exceptions. The authors have carried out phylogenetic analyses and DALI searches of AlphaFold databases of bacterial and archaeal membrane proteins. They identify two bacterial proteins, CysZ and YhiY, and they propose that they are progenitors of SLC25 family members. Whilst the paper addresses an interesting topic, the conclusions are not supported by the data and are not presented in an unbiased manner, as they highlight only features that provide some tentative support for the hypothesis. They do not address the large number sequence and structural properties that refute the hypothesis, such as the asymmetric vs three-fold pseudo-symmetric features, hexamer vs monomer, and the complete lack of any conserved motifs with similar functions. Any resemblances between CysZ/YhiY and mitochondrial carriers thus seem to be superficial and could well be coincidental, as they represent generic properties of membrane proteins rather than specific ones, indicative of an evolutionary relationship.

      Strengths:

      This paper explores the evolutionary origins of the SLC25 family of mitochondrial carrier proteins, which are found across nearly all eukaryotic organisms. They were likely to be present in the last common ancestor of all eukaryotes, around two billion years ago. The question is whether they are of bacterial, archeal or eukaryotic origin. The authors propose that two bacterial proteins, CysZ and YihY, may represent ancestral forms of these carriers, based on structural comparisons of models, a sequence motif, and phylogenetic analyses. While the research addresses an important and longstanding question, the presented evidence does not convincingly support their hypothesis.

      Weaknesses:

      A central concern is the reliance on structural similarity searches using predicted protein models, since these models are often built using known protein structures as templates, and thus these searches may produce misleading matches. The reported similarities between CysZ, YihY, and mitochondrial carriers are weak and fall within ranges expected for unrelated membrane proteins, which commonly share general structural features, such as helical bundles. Quantitative measures of similarity are low and do not support a shared evolutionary origin. The case for YhiY is extremely poor as neither structure nor sequence features support the claim. Importantly, the opening of the YihY is towards the membrane rather than the water phase, as is the case for carriers, indicating that it has a very different structure and function. The case for CysZ is somewhat better, as it is a helical bundle with two short helices somewhat resembling the matrix helices of mitochondrial carriers, and a short sequence PXDXXK that is part of one of the known sequence motifs of mitochondrial carriers, but this is where the similarities end.

      Mitochondrial carriers have a distinctive threefold pseudo-symmetrical structure and a highly complex transport mechanism involving six structural elements. This paper's hypothesis does not explain how such a high level of threefold pseudo-symmetry could have evolved from entirely asymmetric proteins. To complicate matters further, CysZ is not functional as a monomer but forms a functional hexamer, which also explains why it has two half helices rather than two transmembrane helices. Thus, the hypothesis is that CysZ, which is an asymmetric protomer of a functional hexamer, has evolved into a three-fold pseudo-symmetric protein, which is functional as a monomer. A more convincing explanation is that the threefold pseudo-symmetrical structure arose from gene triplication and fusions, with later mutations introducing asymmetry to support diverse substrate binding. In support of this notion, mitochondrial carriers transporting large molecules, such as ATP, show more asymmetry, whereas those for small molecules remain nearly symmetrical. In general, the vast majority of transport proteins arose from gene duplications and fusions of the domains.

      Although mitochondrial carriers have a similar sequence motif as found in CysZ (PXDXXK), their roles are very different. In mitochondrial carriers, this motif is located roughly in the middle of transmembrane helices H1, H3, and H5, where proline creates a pronounced kink, bringing the charged residues inward to form a salt-bridge network in the central water-filled cavity. The formation and disruption of this network is essential for the transport mechanism when switching between inward- and outward-open states. In CysZ, the motif is found at the end of a helix and in the following loop at the end of the transporter, with residues pointing outward toward the water phase. These residues are typical of membrane-water interface regions, where proline acts as a helix breaker and charged residues interact with the water phase. Thus, this motif in CysZ does not match the position or function seen in mitochondrial carriers, and its presence is likely to be coincidental, because these residues often occur in the water-membrane region. Importantly, none of the other important conserved three-fold symmetrical motifs of mitochondrial carriers is found in these bacterial proteins, such as the cytoplasmic network [YF][DE]xx[RK], cardiolipin binding sites, ER-links, and sequences of small amino acids, which are critical for its dynamic mechanism.

      The phylogenetic relationship is also overstated, as there is no sequence similarity between these proteins other than that occurring because of similar biophysical properties, such as transmembrane helices. The authors suggest that a specific mitochondrial carrier represents the ancestral member of the family, but this conclusion appears to be inferred rather than rigorously demonstrated. Key aspects, such as tree rooting and taxon sampling, are not sufficiently addressed, weakening confidence in the evolutionary claims. Further, the selection of only a few bacterial and archaeal proteomes for analysis limits the study's scope. Broader searches would be necessary to support claims about conservation and ancestry. Independent sequence searches indicate that CysZ and YihY are not widely conserved in the bacterial groups most closely related to mitochondria, undermining the argument that they are plausible ancestors.

      Overall, the presented similarities are superficial and can be explained by general features of membrane proteins rather than by specific adaptations to function. The hypothesis that CysZ and YihY are evolutionary precursors of mitochondrial carriers is not supported by the presented data.

    3. Reviewer #2 (Public review):

      Summary:

      Here, the authors performed a phylogenetic analysis of mitochondrial ATP/ADP carrier (AAC) proteins. They also performed a structure-based screen for remote homologs, seeking to reveal their evolutionary origins. The authors claim that AACs are found at the root of their family tree, and through a structure-based homolog search protocol, identify putative prokaryotic homologs.

      The proposed evolutionary history of AACs is bold and complicated, but the phylogenetic methodology and the way in which the tree is interpreted are incomplete and unconvincing. Further, the structure-based search strategy uses very relaxed cutoffs for fold similarity, which may be fine, but it does not clearly justify this decision. This is potentially very problematic, as I did not find the quantitative or qualitative assessments of fold similarity particularly compelling.

      In summary, the authors have presented a bold and extremely interesting hypothesis for the evolution of these proteins, but there is insufficient support for their claims.

      Strengths:

      (1) The authors are presenting a very interesting hypothesis about the birth of these proteins, including that they may have undergone a radical rearrangement in their sequence at some point in evolution.

      (2) The paper makes use of appropriate tools for structure-based homolog identification.

      (3) Identification of a conserved sequence motif in these twilight zone proteins would be a rare and interesting occurrence, and could be consistent with their proposed homology.

      Weaknesses:

      (1) The phylogenetic analysis and its interpretations are incomplete. The authors regularly refer to the root of the tree, and its placement is given central importance. However, the methodology by which they selected the root is unexplained. This is notable, as the proposed root is curious and quite confusing. It implies that (at least) yeast and Paramecium AACs are independently paraphyletic. While certainly not impossible, this evokes quite a complicated evolutionary history. The taxonomy of this gene family, when rooted this way, does not seem to echo the phylogeny of species, suggesting an extremely complex history of duplication/loss and horizontal gene transfer, none of which the authors discuss in detail. Perhaps more clearly and specifically: I'm very surprised by the branching order at the root, where there are three independent branches of fungal proteins, followed by the excavate proteins in a monophyletic clade, followed by several independent branches of the Paramecium proteins. I very much expect incomplete lineage sorting at this evolutionary depth, but this seems extreme to the point that I question if it is accurately placed. More directly: this very much looks like an unrooted tree, presented radially.

      (2) The Bayesian and ML trees seem quite incongruent, but this is not discussed. In fact, the text states that they "exhibit a similar tree topology." This is admittedly very difficult to assess without very carefully going over the tree, branch by branch, but there are nevertheless differences, the most obvious being paraphyly vs monophyly of taxon-specific AAC clades. Do the authors have any comments on this, and can they show some sort of consensus tree? How does this affect their interpretation?

      (3) Presenting branch support as similarly-sized points makes it nearly impossible to actually judge the strength of support.

      (4) The use of structure for remote homology detection is becoming increasingly popular, and in my opinion, is very powerful. But it is still much too early to be taken for granted. The methodology must be justified. Most importantly, the authors have not clearly described why they chose these quantitative cutoffs (I'm mostly thinking of the Dali Z-score cutoff, which here seems very low for a transmembrane protein of this size, as the Z-score is very dependent on alignment length). The authors reference categories defined by tool authors, but why a Z-score of 3, specifically? The same goes for TM scores. There are not yet any defined best practices, to my knowledge, so the authors should independently validate/justify their approach in some way and/or cite and discuss relevant literature (there have been a growing number of these screens using similar approaches in recent years).

      (5) The proposed homologs have very little quantitative structural similarity to the query structure, or to each other, as shown in Figure 3 (and hence my concerns about the methodology). Also, I did not find the structural alignments in the supplement or Figure 4 to be qualitatively compelling. They simply appear too different, and I cannot discard this qualitative assessment because the quantitative similarities are likewise very weak. It's not clear to me if this is because the folds are in fact different, or if my view of them is a presentation issue (perhaps it could be improved by visualizing more angles, or more carefully cartooning the similarities and differences).

      (6) The authors point out that the alpha-helices are ordered differently in YihY and CysZ, and that their membrane orientation is flipped. Taken at face value, I would view this as evidence against homology. This could perhaps be more reasonably explained as convergent global fold similarity resulting from different underlying structures. However, the authors imply that this may be the result of the transposition of the sequences encoding these alpha helices, yet there is no convincing description or argument concerning when and how this could have occurred. I think this would be a deeply interesting phenomenon, but there is insufficient evidence and discussion to seriously consider whether or not it is homology or convergence.

      (7) Following up on comment #5, the authors did perform a very interesting in silico experiment by transposing sequences to reorder the helices. They then note that structural similarity improved. This is very, very interesting, but without other evidence of homology between the transposed alpha helices, I do not think this disproves alternative hypotheses. Does any such evidence exist?

      (8) The authors show in Figure 5E-F that sequence transposition flips the membrane orientation, such that YihY and CysZ have extracellular termini (which you would expect from homologs, I suppose). But it is just cartooned and not discussed. Is this computationally or experimentally supported?

      (9) The putative presence of a conserved motif would be a very compelling piece of evidence consistent with homology. However, it is not clear to me in the text which proteins actually have the repeats - is it truly just CysZ? What does this mean for YihY? Further, what specifically is being proposed to be homologous? Is SLC25 repeat 2 proposed to be homologous to CysZ repeat 2 (and the same for 3 to 3)? If so, this would seem to have implications for the transposition hypothesis. The helix nomenclature (e.g., H1-6) suggests homology across the proteins (i.e, H1 is homologous to H1); however, wouldn't the presence of these conserved domains instead, for example, suggest homology between SLC H3 and CysZ H2? The authors' conclusions are not clear, and it is difficult to interpret what the implications are for assessing homology.

      (10) The sequence retrieval methods are incomplete, so it is impossible to reproduce the searches or to judge their accuracy and scope. What were the E-value cutoffs and other settings used in the searches?

      (11) The phylogenetic methods are incomplete. What substitution models were used, and how were they chosen? What branch support method was used? What were the stop conditions of the Bayesian analysis (e.g. did the authors monitor for convergence, and how)? How much of the Bayesian analysis was considered burn-in, if any? And echoing points 1 & 2 above, how were these phylogenies rooted?

      (12) Throughout, there is a distinct lack of careful, evolutionarily informative language.

      (i) In reference to the phylogeny, the authors frequently refer to "grouping," but it's not entirely clear what this means. Referring to clades and their branching order would be more informative.

      (ii) The authors refer to the excavate branch as the "most ancient." Whether or not excavates most closely resemble LECA is somewhat irrelevant, because the branch itself is not the most ancient - it is equally as ancient as its sister branch, which may be all other eukaryotes.

      (iii) Likewise, the authors refer to bacterial proteins as "the evolutionary ancestor of mitochondrial AACs," and state that "AAC emerged from the conserved sulfat transporter CysZ." But extant bacteria are not the ancestors of mitochondria - nor are extant proteins descended from other extant proteins. They are, perhaps more accurately, cousins.

      (iv) The authors refer to AACs as "evolutionarily founder member of the SLC25 carrier family," but I'm not sure that has a clear evolutionary meaning, unless the authors mean to say that the common ancestor was more AAC-like than anything-else-like. Even if the rooting is accurate, a basal branch does not necessarily reflect the ancestral state.

    4. Reviewer #3 (Public review):

      Summary:

      The most important weakness is that the authors have avoided the direct structural comparison of experimentally determined x-ray structures of AAC and CysZ. Instead, the comparisons are made through predicted membrane topologies and predicted structural models of protein homologs, which give rise to misleading results. Direct comparison of the X-ray structures of the ADP/ATP carrier and CysZ clearly shows that these proteins have very different folds. Therefore, flaws in the methods produce results that lead to the wrong conclusions, and the authors have not achieved their aims.

      Weaknesses:

      (1) Figure 2. There is something very strange about how the tree is drawn, given that S. cerevisiae AAC1, AAC2 and AAC3 share about 76-83% sequence identity but appear to be very diversified in the tree. The phylogenetic trees are only based on the sequences of three species. The authors should explain in much more detail how they made the phylogenetic trees to support their statement that all mitochondrial carriers have come from an ancient AAC.

      (2) There are at least three and seven X-ray structures of CysZ (with about 43% sequence identity to the E. coli homolog) and AAC, respectively, deposited in the Protein Data Bank. Therefore, there is no need for the approach using predicted structures as described in the manuscript. It is clear from direct comparison of the CysZ and AAC structures that they have very different folds, i.e. lengths of the transmembrane helices, their orientation and packing. CysZ has been suggested to form dimers or trimers of dimers (eLife 2018;7:e27829), with each protomer formed by two long transmembrane helices and four short helices that do not cross the membrane totally. Thus, CysZ has a different membrane topology and oligomeric state than AAC (monomer with six transmembrane helices). CysZ is therefore rightfully classified in a separate 3D domain fold from mitochondrial carriers in various protein family and domain databases.

      (3) In the 3D structures of CysZ, the conserved QYXDYPXDNHK motif is involved in a network of hydrogen bonds and salt bridges thought to hold the helical bundle together (eLife 2018;7:e27829). This motif is similar to PX[DE]XX[KR], a part of the signature motif, typical of mitochondrial carriers, which is repeated three times in the sequences and forms a three-fold pseudo-symmetrical salt bridge network of the so-called matrix gate that opens and closes during the transport cycle. Therefore, although this single motif in CysZ is similar to those of mitochondrial carriers, it is not found in a similar structural context to those in AAC structures.

      (4) It appears odd that the sulfate transporter CysZ should be more similar to nucleotide-transporting AAC than any of the other mitochondrial carriers, of which some transport sulfate.

      (5) The alphafold model of YihY is not very similar to either the crystal structures of CysZ or AAC.

      (6) The authors are relying too much on the TM-score results. The values of 0.5-0.6 between AAC and CysZ or YihY probably reflect that they contain six main helices. However, as noted in point 2, they have very different folds.

    5. Author response:

      Thank you for your decision letter with the public review and the recommendations. While we are delighted that the referees feel the work is addressing an outstanding and important issue, they have raised concerns regarding the strength of the support. We will address all the concerns in full in a revised manuscript in the due course. Please find below a couple of general points regarding the referees’ concerns and a proposal as to how we plan to address them.

      (1) The idea of the manuscript is to present a plausible solution for a long-standing question in the field of mitochondrial biology and evolution. The fact that the identified solution to the origin of AAC transporters is a remote structural homolog (as you will see in our later detailed response that it is better than any other sequence/structure available till date) is to be expected. If the actual similarities were any better than what we have identified (with a special case of circular permutation), they could have been identified by other simpler structural homology search methodologies.

      (2) A recurrent and strong disagreement of the reviewers on the findings presented in this manuscript is rooted on the fact that the structural and sequence relatedness between AAC and CysZ detected in this work are so weak that they can be co-incidental and not an actual evolutionary link. Based on the above, we now searched carefully in all available structural databases such as SCOP, CATH, ECOD etc. whether the above fold link has been noted by others independently. We notice that in the ECOD (Evolutionary Classification of Protein Domains) database only AAC and CysZ are grouped together under a single Possible homology group (X) called ‘Mitochondrial ADP/ATP carrier-like’. The ECOD database contains hierarchical classification of protein domains organized according to their evolutionary relationships and the server is maintained by Prof. Nick Grishin at The University of Texas Southwestern Medical Center.

      Link to ECOD database: http://prodata.swmed.edu/ecod/index_af2_pdb.php

      Reference: Cheng H, Schaeffer RD, Liao Y, Kinch LN, Pei J, et al. (2014) ECOD: An Evolutionary Classification of Protein Domains. PLOS Computational Biology 10(12): e1003926. https://doi.org/10.1371/journal.pcbi.1003926

      Therefore, our study and the independent findings of the ECOD database team together offers greater confidence on the proposed remote evolutionary relationship between AAC and CysZ, and that the structural and sequence similarity we report in the manuscript are not a mere co-incidence. We will also incorporate the details of possible evolutionary relationship between AAC and CysZ identified in the ECOD database in the revised version of manuscript.

      (3) One point we would like to stress is that considering all the similarities identified, it very unlikely falls into the class of ‘convergent evolution’. We will make this point explicit in the revised version.

      (4) Lastly, while we totally agree that the similarities are in the twilight zone, considering the importance of the problem, we feel that our work would induce researchers from the field of protein design to attempt possible interconversion of the two distantly related transporters thus providing an experimental rationale for the evolution of these transporters.

    1. without the extraction, exploitation, or surveillance

      found in most proprietary technology.

      They will describe what it looks like - when ordinary folks make tech that is - local first, - peer-to-peer, - decentralized, - open-source.

      LX is the curator of the Solidarity Tech track at Camp this year, and is also a co-lead of the DWeb YVR Node.

    1. eLife Assessment

      This valuable study uses a computer vision pipeline to infer the motor control of cephalopod skin, revealing that individual chromatophores exhibit anisotropic deformations and can be associated with multiple putative motor units. The evidence supporting these claims is convincing, and the authors present some limited electrophysiological validation of the findings from their computational analysis. This work will be of significant interest to biologists studying cephalopod behavior and motor control.

    2. Reviewer #1 (Public review):

      Renard, Ukrow et al. applied their recently published computational pipeline (CHROMAS) to the skin of Euprymna berryi and Sepia officinalis to track the dynamics of cephalopod chromatophore expansion. By segmenting each chromatophore into radial slices, and analyzing the co-expansion of slices across regions of the skin, they inferred the motor control underlying chromatophore groups.

      Strengths:

      - The authors demonstrate that most motor units of cephalopod skin include a subregion of multiple chromatophores, creating "virtual chromatophores" between fixed chromatophores. This is an interesting concept that challenges prevailing models of chromatophore organization, and raises interesting possibilities for how chromatophore arrays may be patterned during development.

      - This study introduces new analytical approaches of cephalopod skin that will be valuable for the quantitative study of cephalopod behavior.

      Weaknesses:

      - The authors use patch-clamp experiments in E. berryi to test their approach for inferring motor units. The stimulations indeed evoke expansions of sub-regions of each chromatophore, creating "virtual chromatophores". However, they were not able to predict these motor units from behavioral analysis before confirming them with patch-clamp, limiting the strength of this validation.

      - In S. officinalis, chromatophores are far more numerous than in E. berryi and exhibit frequent spontaneous activity, making it more challenging to distinguish shared motor drive. Patch-clamp experiments in this species would provide important validation and strengthen confidence in the method for inferring motor units.

      - Although multiple experimental conditions were tested (e.g., age, size, behavioral context, sedation, head-fixation, lighting), data is only shown from a small subset of experiments. Analyzing pooled data across conditions would allow for more generalizable conclusions.

      - Different clustering algorithms were used for the two species (HDBSCAN for E. berryi and Affinity Propagation for S. officinalis). Since Affinity Propagation appeared to better capture correlation structure in S. officinalis, it would be informative to reanalyze the E. berryi data using the same method to assess potential algorithm-dependent biases.

      Conclusion:

      The CHROMAS tool is likely to be valuable to the field, given the need for quantitative frameworks in cephalopod biology. The predictions outlined here provide a useful foundation for future experimental investigation.

    3. Reviewer #2 (Public review):

      Summary:

      Overall, this is an excellent paper, making use of a newly developed system for monitoring the behaviour of chromatophores in the skin of (mostly) free swimming bobtail squid and European cuttlefish. The manuscript is very well written, clearly presented and very well structured. The central finding, that individual chromatophores are connected to multiple motor neurones, is not new. Novelty instead comes from the ability to measure the actuation of chromatophore sections across wide areas of skin in free-swimming animals, showing the diversity of local motor units and reinforcing the notion that individual chromatophores are not necessarily the individual units of colour change, but rather local motor units that cover multiple neighbour and near neighbour chromatophore muscles. This is an excellent finding and one that will shape our understanding of the neural control of cephalopod skin colour. I have a number of minor points below that the authors will need to address before acceptance.

      Strengths:

      The methodological approach to collecting large amounts of data about local variations in the expansion of sections of chromatophores is exciting, and the analysis pipeline for clustering sections of chromatophores whose spontaneous activity correlated over time is powerful and exciting.

      Comments on revisions:

      All concerns have been addressed in the revised version of the manuscript.

    4. Reviewer #3 (Public review):

      Summary:

      This study uses high-resolution videography and a custom computer-vision pipeline to dissect the motor control of cephalopod chromatophores in Euprymna berryi and Sepia officinalis. By quantifying anisotropic chromatophore deformations and applying dimensionality reduction methods, the authors infer that individual chromatophores can be a part of multiple motor units. Clustering analyses reveal putative motor units that often span multiple chromatophores, with diverse and overlapping geometries. Chromatophore expansion dynamics are faster and more stereotyped than relaxation, consistent with active neural contraction followed by passive recoil. Together, the results show that chromatophores function not as uniform pixels but as fractionated, coordinately controlled elements that enable flexible pattern generation

      Strengths:

      The authors present compelling, direct evidence that a). chromatophore deformations are anisotropic, and indirect evidence that b). individual chromatophores can be split across multiple putative motor units. This evidence is provided through data collected over large spatial scales, but also at a sub-chromatophore resolution. This combination of scale and resolution is not possible using traditional neuroanatomical and physiological approaches alone.

      The authors also develop a new non-invasive, image analysis approach to extract information about chromatophore deformation across large spatial scales on the organism's body. In principle this approach is applicable across species and may allow for further comparative characterization of chromatophore motor control. It is therefore a promising new tool and useful resource for the community.

      Weaknesses:

      An important weakness of the work is that the methods the authors develop can only be applied during resting, spontaneous 'flickering' activity of chromatophores to yield interpretable results at the motor unit level. This is because common presynaptic input would confound the identification of individual motor units. Thus, there remains a large difficulty in linking insights about single motor unit organization to the circuit and behavioral levels.

      Another weakness of this paper is the rather limited electrophysiological validation of the computational findings. The authors present only one electrophysiology experiment in E. berryi, the species that they used only for 'methodological development' and not for detailed characterization. A complementary electrophysiological experiment in S. officinalis, or some visualization of neuron morphology confirming that motor neurons do indeed project to multiple chromatophores would strengthen the generalizability of their computational analysis. This would be particularly pertinent to validate the author's claim that some motor units contain chromatophores that are quite distant from one another on the animal.

      Overall, the authors' technical contributions and method development are an important advance. This work serves as an excellent proof of concept that their method can extract useful information about chromatophore motor control. Further validation of their method is needed to fully trust the fine-scale conclusions drawn about the distribution and composition of multi-innervated chromatophores. Furthermore, the authors raise many interesting ideas about developmental constraints on circuit wiring and potential adaptive significance of multi-innervated chromatophores for certain features of camouflage patterning. Their method may be able to help resolve some of these questions in the future if it is refined and applied across developmental stages, regions on the animal, and across species

      Comments on revisions:

      Thank you for clarifying my major point of confusion regarding how one might connect these results to behaviorally relevant camouflage. I now have a better understanding of the author's rationale in studying resting activity of motor units and believe that the clarifications added to the manuscript will help other readers who encounter similar confusion.

    5. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      Renard, Ukrow et al. applied their recently published computational pipeline (CHROMAS) to the skin of Euprymna berryi and Sepia officinalis to track the dynamics of cephalopod chromatophore expansion. By segmenting each chromatophore into radial slices and analyzing the co-expansion of slices across regions of the skin, they inferred the motor control underlying chromatophore groups.

      Strengths:

      The authors demonstrate that most motor units of cephalopod skin include a subregion of multiple chromatophores, creating "virtual chromatophores" in between the fixed chromatophores. This is an interesting concept that challenges prevailing models of chromatophore organization, and raises interesting possibilities for how chromatophore arrays may be patterned during development.

      This study introduces new analyses of cephalopod skin that will be valuable for the quantitative study of cephalopod behavior.

      Weaknesses:

      The authors chose to image spontaneous skin changes in sedated animals, rather than visually-evoked skin changes in awake, freely-moving animals. Spontaneous chromatophore changes tend to be small shimmers of expansion and contraction, rather than obvious, sizable expansions. This may make it more challenging to distinguish truly co-occurring expansions from background activity. The authors don't provide any raw data (videos) of the skin, so it is difficult to independently assess the robustness of the inferred chromatophore groupings.

      The patch-clamp experiments in E. berryi are used to test the validity of their approach for inferring motor units. The stimulations evoke expansions of sub-regions of each chromatophore, creating "virtual chromatophores" as predicted from the behavioral analysis. However, the authors were not able to predict these specific motor units from behavioral analysis before confirming them with patch-clamp, limiting the strength of the validation. It would be informative to quantify the results of the patch-clamp experiments - are the inferred motor units of similar sizes to those predicted from behavior?

      The authors report testing multiple experimental conditions (e.g., age, size, behavioral stimuli, sedation, head-fixation, and lighting), but only a small subset of these data are presented. It is difficult to determine which conditions were used for which experiments, and the manuscript would benefit from pooling data from multiple experiments to draw general conclusions about the motor control of cephalopod skin.

      The authors use a different clustering algorithm for E. berryi and S. officinalis, but do not discuss why different clustering approaches were required for the two species.

      Impact:

      The authors use their computational pipeline to generate a number of interesting predictions about chromatophore control, including motor unit size, their spatial distribution within the skin, and the independent control of subregions within individual chromatophores by putatively distinct motor neurons. While these observations are interesting, the current data do not yet fully support them.

      The CHROMAS tool is likely to be valuable to the field, given the need for quantitative frameworks in cephalopod biology. The predictions outlined here provide a useful foundation for future experimental investigation.

      We thank the reviewer for the thoughtful and detailed evaluation of our work and for recognizing the potential of the CHROMAS pipeline for studying chromatophore control.

      We agree that some aspects of the manuscript required clarification and additional explanation, and we have revised the text accordingly. We also now provide access to representative raw video recordings in the Data Availability section. In the E. berryi patch-clamp experiments, single motor neurons evoked expansions of sub-regions of chromatophores, consistent with the “virtual chromatophore” concept. We have now quantified the size of motor units across patch-clamp sessions, and the results show that the inferred motor-unit sizes broadly match those predicted from behavioral recordings, supporting the validity of our approach.

      We agree that pooling data across individuals would provide valuable insight into variability across animals. In practice, we recorded chromatophore activity from several animals (14 Euprymna berryi and 12 Sepia officinalis) under different experimental conditions during development of the experimental pipeline. However, acquiring long, stable, artifact-free recordings suitable for motor unit analysis is technically challenging. We now clarify this point in the manuscript. Specifically, we explain that multiple animals were recorded during pipeline development, while the analyses presented focus on recordings with the highest signal quality. We anticipate that the framework introduced here will enable future studies to collect larger datasets and compare motor unit organization across individuals, developmental stages, and species.

      HDBSCAN was used for E. berryi during initial exploratory analyses, and Affinity Propagation was adopted for S. officinalis because it better captured the correlation structure of those recordings. We did not re-analyze the E. berryi data with Affinity Propagation, and the implications of algorithm choice are now discussed in the Discussion.

      Reviewer #2 (Public review):

      Summary:

      Overall, this is an excellent paper, making use of a newly developed system for monitoring the behaviour of chromatophores in the skin of (mostly) free-swimming bobtail squid and European cuttlefish. The manuscript is very well-written, clearly presented and very well-structured. The central finding, that individual chromatophores are connected to multiple motor neurones, is not new. Novelty instead comes from the ability to measure the actuation of chromatophore sections across wide areas of skin in free-swimming animals, showing the diversity of local motor units and reinforcing the notion that individual chromatophores are not necessarily the individual units of colour change, but rather local motor units that cover multiple neighbour and near-neighbour chromatophore muscles. This is an excellent finding and one that will shape our understanding of the neural control of cephalopod skin colour.

      Strengths:

      The methodological approach to collecting large amounts of data about local variations in the expansion of sections of chromatophores is exciting, and the analysis pipeline for clustering sections of chromatophores whose spontaneous activity correlated over time is powerful and exciting.

      Weaknesses:

      Some minor edits and typographical errors need correcting. I also had some concerns that the preparation for the electrophysiological section of the manuscript complies with the journal's ethical requirements, so I would urge that this be carefully checked.

      We thank the reviewer for the positive evaluation of our work and for recognizing the value of the methodological approach and the clarity of the manuscript.

      We have carefully reviewed the manuscript and corrected minor typographical errors.

      Regarding the ethical considerations raised for the electrophysiological experiments, we have carefully verified that the experimental procedures comply with the journal's ethical requirements and relevant institutional guidelines.

      Reviewer #3 (Public review):

      Summary:

      This study uses high-resolution videography and a custom computer-vision pipeline to dissect the motor control of cephalopod chromatophores in Euprymna berryi and Sepia officinalis. By quantifying anisotropic chromatophore deformations and applying dimensionality reduction methods, the authors infer that individual chromatophores can be a part of multiple motor units. Clustering analyses reveal putative motor units that often span multiple chromatophores, with diverse and overlapping geometries. Chromatophore expansion dynamics are faster and more stereotyped than relaxation, consistent with active neural contraction followed by passive recoil. Together, the results show that chromatophores function not as uniform pixels but as fractionated, coordinately controlled elements that enable flexible pattern generation

      Strengths:

      The authors present compelling, direct evidence that a). chromatophore deformations are anisotropic, and indirect evidence that b) individual chromatophores can be split across multiple putative motor units. This evidence is provided through data collected over large spatial scales, but also at a sub-chromatophore resolution. This combination of scale and resolution is not possible using traditional neuroanatomical and physiological approaches alone.

      The authors also develop a new non-invasive, image analysis approach to extract information about chromatophore deformation across large spatial scales on the organism's body. In principle, this approach is applicable across species and may allow for further comparative characterization of chromatophore motor control. It is therefore a promising new tool and useful resource for the community.

      Weaknesses:

      An important weakness of the work is that the methods the authors develop can only be applied during resting, spontaneous 'flickering' activity of chromatophores. The inability to reliably apply their technique during any kind of realistic camouflage is a large limitation, as it means this method cannot be used to study the dynamics of motor control during realistic camouflage behaviors.

      Another weakness of this paper is the rather limited electrophysiological validation of the computational findings. The authors present only one electrophysiology experiment in E. berryi, the species that they used only for 'methodological development' and not for detailed characterization. A complementary electrophysiological experiment in S. officinalis, or some visualization of neuron morphology confirming that motor neurons do indeed project to multiple chromatophores, would strengthen the generalizability of their computational analysis. This would be particularly pertinent to validate the author's claim that some motor units contain chromatophores that are quite distant from one another on the animal.

      Overall, the authors' technical contributions and method development are an important advance. This work serves as an excellent proof of concept that their method can extract useful information about chromatophore motor control. Further validation of their method is needed to fully trust the fine-scale conclusions drawn about the distribution and composition of multi-innervated chromatophores. Furthermore, the authors raise many interesting ideas about developmental constraints on circuit wiring and potential adaptive significance of multi-innervated chromatophores for certain features of camouflage patterning. Their method may be able to help resolve some of these questions in the future if it is refined and applied across developmental stages, regions of the animal, and across species

      We thank the reviewer for their thoughtful evaluation and for recognizing the potential of the computational approach introduced in this study.

      Regarding the focus on spontaneous chromatophore activity, we have clarified earlier in the Results section why these events are necessary to isolate individual muscle activations. While large camouflage patterns are visually striking, they involve the coordinated activation of many groups of chromatophores by premotor circuits simultaneously, making the identification of individual motor units, our goal here, impossible. Our approach can, however, also be applied during active behavior, including camouflage; the questions addressed there would be different, focusing on how multiple motor units are coordinated to generate the resulting skin patterns, rather than resolving the structure of single motor units. This could be challenging if the patterns of premotor control are highly variable, thus making the detection of meaningful or interpretable motion correlations difficult. This remains to be tested.

      We also acknowledge that electrophysiological validation remains limited. Patch-clamp experiments were performed in Euprymna berryi to test predictions generated by the computational analysis, and these experiments confirmed that activation of single motor neurons can produce anisotropic expansion of chromatophore subregions. We now provide the associated datasets in the Data Availability section. We agree that complementary electrophysiological or anatomical experiments in Sepia officinalis would further strengthen the conclusions. Such experiments represent an important direction for future work.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      General points:

      (1) Given all the experimental conditions and animals tested, the manuscript would be much stronger if the figures represented pooled data from many animals and experiments (e.g. Figure 1C).

      We agree that pooling data from multiple animals would strengthen the manuscript. In practice, we tested these experimental conditions across several animals (14 Euprymna berryi and 12 Sepia officinalis), but we selected the segments shown in the figures for their minimal artifacts and errors. Acquiring high-quality, stable recordings of this type is extremely challenging, and the presented data represents the clearest examples suitable for analysis and visualization. We hope that in the future these methods will enable not only the collection of a larger, high-quality dataset, but also comparisons across individuals, ages, species, and different regions of the mantle.

      (2) It's very unclear what animals were used for each experiment:

      (a) E. berryi: L677 states that 14 animals were filmed, and L684 implies that non-sedated individuals were used in addition to sedated animals, but it appears all the data is from a single E. berryi with sedation?

      The original wording was unclear, so we modified the sentence for clarity. The Methods now specify that 14 animals were filmed to refine the experimental pipeline and explore different conditions, while the data presented in the Results are from a single lightly sedated individual chosen for quality and stability of chromatophore activity.

      (b) S. officinalis: L692 onwards states that lots of different conditions and animals were explored, but only minimal data from a couple of animals is described in the figures. L156 states that all (?) the data comes from one head-fixed animal and one sedated and head-fixed animal. L549: The conclusion states that the pipeline was used in freely moving animals, but it appears that all of the S. officinalis were head-fixed? This is very confusing. Rather than describing the conditions of every experiment ever performed, the manuscript would benefit from explicitly stating the experimental conditions used for each figure.

      The original text was unclear. We have clarified in the manuscript which animals and experimental conditions were used for the analyses in each figure. To clarify, E. berryi was recorded without head fixation, whereas S. officinalis data were obtained under head-fixed conditions. We did film 11 S. officinalis without head fixation, and data can in principle be extracted from these recordings. Head fixation was used both to minimize visual artifacts and to enable longer, stable recordings, which was important for capturing the highest level of apparent noise in motor unit activation—information that is critical for our analyses of motor-unit organization, though not necessary for studies of broader camouflage patterns. Our computational pipeline enables large-scale analyses that would be very difficult or impossible with traditional electrophysiology, not that all data were acquired from freely behaving animals. While fully unconstrained recordings remain technically challenging due to optical and logistical constraints, we maintain that our approach provides a valid framework for analyzing freely behaving animals.

      (c) Additionally, there is a claim that the sedated condition represents the unsedated one (e.g. L151 and L643), but no data is shown to support this. L173 references Figure 6d as evidence, but 6d doesn't exist. Only L210 provides sedation/no sedation statistics for the number of components per motor unit. However, in L643 it says "and motor unit organization remained unchanged". This data needs to be shown to include that statement.

      Reference to the inexistant 6d figure was removed. L170 provides statistics for the number of principal components per chromatophore, and L210 provides statistics for the number of components per MU. We do not think a sub-figure is necessary. We, however, agree that L643 “motor unit organisation” is potentially misleading as we only compared the number of chromatophores belonging to a single MU and not the MU shape or distribution. Changed “organization” to “size (in chromatophores)”.

      (3) The text needs considerable revision. There are many typos (including multiple instances of "refs" instead of the actual references being inserted). These issues make the manuscript much more difficult to evaluate.

      Our apologies. We have now added the missing refs.

      (4) It is not clear how convincing the chromatophore groups are. For instance, Figure 4h could alternatively be interpreted as a group of 5 chromatophores in a motor group that happen to co-vary with a sixth one at a great distance. Without seeing some of the raw data (videos), it's difficult to assess how convincing it is that these chromatophores belong to the same group. I recommend analyzing: when multiple chromatophores expand together, what is the likelihood that other chromatophores also happen to expand at the same time (given the frequency that they're all changing shape spontaneously)?

      We appreciate the reviewer’s concern. Chromatophores are assigned to the same cluster because their activity, or that of their slices, covaries consistently over time. It is, of course, possible that what appears as a single motor unit may reflect two or more motor neurons acting simultaneously during the recording. Longer video segments increase confidence in the integrity of inferred motor units, but in the absence of a ground truth for motor unit spatial organization in this species at this age, it is difficult to quantify the likelihood that two motor units are being conflated. Raw video data is provided in the Data Availability section. We note, however, that most of the time motor units cannot be readily discerned by eye, because individual chromatophores and their constituent slices fluctuate continuously, and motor-unit correlations are subtle and distributed across multiple chromatophores.

      (5) The rationale for focusing on spontaneous activity is introduced relatively late in the manuscript and would benefit from being stated earlier. Examples should be provided of what this looks like (as opposed to regular chromatophore expansion). It would be valuable to see measurements across many experiments of how expanded the chromatophores are - what is the change in surface area? And what is the frequency of expansion for each chromatophore?

      Thank you for the remark. This is true. We have added a paragraph at the beginning of the Results section to clarify the rationale for focusing on spontaneous activity.

      This section now reads:

      “Because our primary aim was to describe the composition and coordination of chromatophore motor units, it was important to examine animals in the absence of the descending commands that occur during active behavior. Spontaneous activity, typically mild and “noisy” was thus ideal to enable measurements of the motion correlations between chromatophores that reflected shared motor neuron drive, rather than shared correlations due to upstream motor neuron groupings by premotor circuits.”

      We added an example of video recording of spontaneous activity in our Data Availability section.

      While quantifying expansion magnitude and frequency across experiments would indeed be valuable, these questions fall outside the primary focus of the present study, which centers on resolving motor unit organization. In the section “Dynamics of chromatophore expansion and contraction,” we analyze the speed of expansion and contraction to demonstrate that such kinetic features can be reliably detected with the temporal resolution of our video imaging approach. By isolating single muscle activations, we establish a methodological framework that can be used in future work to quantify expansion amplitude, rate of change and frequency across preparations.

      (6) Chromatophore expansion was only measured in anesthetized E. berryi, and L679 states that chromatophore expansion was triggered by shining light on the skin. However, light-mediated chromatophore expansion may be mediated by a different mechanism, so chromatophore correlations do not necessarily reflect the underlying motor control.

      We agree that there is, in principle, a theoretical risk of direct light-mediated activation of chromatophores. Yet, the kinetics of this light mediated activation are very different, and are the object of a separate, on-going investigation by our groups. In our experiments, the illumination was applied to the whole animal rather than locally to the skin, ensuring that all chromatophores and the eyes were exposed to the same light source. By transitioning from darkness to light, we created a window in which chromatophores were partially expanded—both fully contracted and fully expanded states would show little to no decorrelation. Within this window, we observed spontaneous fluctuations in chromatophore activity, which formed the basis for our correlation analyses. To our knowledge, direct light-mediated expansion of chromatophores has not been reported in E. berryi although it may exist there. Finally, the size, shape, and orientation of the inferred motor units align with electrophysiological evidence, supporting the validity of our motor unit inferences.

      (7) Some figures might be better suited for the supplement. For instance, it's not clear what the significance of Figure 5 is (it's not currently sufficiently justified in the text).

      We have clarified the purpose of Fig. 5 in both the Results and Discussion sections. In the Results, we now explain that events are separated by amplitude to show that expansion–contraction kinetics can be reliably measured across a full range of chromatophore events, validating the precision of our videographic approach. In the Discussion, we highlight that this precision allows measurement of radial muscle speeds and opens avenues to study chromatophore biomechanics, including the contributions of intertwined forces such as radial muscles, elastic pigment sacs, and intercellular coupling.

      (8) Multiple chromatophores can belong to multiple clusters - this study reveals that this is because subsections of a chromatophore are controlled separately. But do the same sections (slices) of chromatophores ever belong to multiple clusters?

      Yes, it is possible. Dubas (1985) used videographic recordings to show that the same chromatophore muscle fibers could be activated by stimulation of different nerve bundles, supporting Florey’s (1969) electrophysiological evidence for polyneuronal excitatory innervation. From Dubas: "Usually, different muscle fibres were recruited by each nerve but sometimes a single muscle fibre responded to stimulation of each nerve. Variations of the stimulus voltage also produced gradation of the amplitude of shortening of individual muscle fibres. This supports the evidence above for multiple innervation of single muscle fibres."

      The petal-like distribution of motor-neuron influence shows overlapping territories, suggesting that some chromatophore sections may be influenced by multiple neurons. However, this overlap could arise from polyinnervation of individual muscles, the presence of gap junctions between muscles, or passive mechanical coupling due to the elastic properties of the pigment sac.

      The petal-like distribution of motor-neuron influence shows overlapping territories, suggesting that some chromatophore sections may be influenced by multiple neurons. However, this overlap could arise from polyinnervation of individual muscles, the presence of gap junctions between muscles, or passive mechanical coupling due to the elastic properties of the pigment sac.

      With the present approach, it is not possible to disentangle the relative contributions of these mechanisms, which will require targeted physiological or anatomical experiments. For this reason, we adopted a hard clustering approach for individual chromatophore slices.

      (9) All time should be labeled in seconds, not in frames, and all distances should be measured in um or mm, not in pixels.

      We chose to present figures in pixels and frames to reflect the native units of our recordings and analyses, which preserves fidelity and reproducibility of the computational pipeline. For biological interpretation, corresponding values are converted to µm in the main text, providing the relevant real-world scale. A scale for conversion is provided in the figure legend.

      Specific comments:

      (1) L36: I'm not sure the description of virtual chromatophores here is clear enough to make sense to a more general audience.

      Addressed. We retained the concept of ‘virtual chromatophores’ in the abstract and added a brief clarifying phrase to indicate that these are functional groupings of adjacent chromatophore territories that act as single units.

      (2) L50: "Rimmed by" - consider rephrasing.

      Addressed. Replaced with “surrounded”.

      (3) L64: "refs" - actual references aren't inserted. There are multiple other examples of this.

      Addressed. Added missing references.

      (4) L100: This section could use rewriting. Some of the text reads more like a figure legend.

      Addressed. We have streamlined the main text to reduce redundancy with the figure legend.

      (5) L101: Consider the opening sentence/s providing a more general introduction to the question and approach.

      Addressed.

      (6) L104: This implies that the data presented are from 14 animals of many ages. This is only relevant if the pooled data is analyzed and presented.

      We agree that the original phrasing was ambiguous. We have modified the sentence for clarity, and explain in the Methods that 14 animals were filmed to refine the pipeline and explore experimental conditions, while the analyses shown are from a single animal.

      (7) L111: HDBSCAN should be defined.

      Addressed. The acronym has been expanded.

      (8) L173: Figure 6D doesn't exist.

      Addressed. Reference to the inexistent 6d figure was removed.

      (9) L193: "excluding negative (contraction) phases" This phrase requires clarification.

      Addressed. Added “see Methods” in the legend and added clarification on the reasoning in Methods.

      (10) L204: Should explain why the switch to affinity-propagation clustering was made when a different method was used for E. berryi.

      Addressed in discussion.

      (11) Figure 3: I recommend including a diagram or image of a whole cuttlefish and showing what the corresponding imaging area was in relation to the animal so the reader gets an intuitive sense of scale.

      Thank you. We have added a supplementary figure to give the reader a sense of scale.

      (12) L221/Fig 3b: These colors are supposed to represent clusters of 3 to 5 chromatophores? The clusters look much bigger.

      The figure shows clusters of 3 to 5 chromatophores, but many adjacent clusters were assigned the same color. We have changed the colors to remove this ambiguity.

      (13) Figure 3c: This would be more powerful if it represented the combined data of many experiments to draw a general conclusion. Also, shouldn't these cluster sizes match those in 2e, e.g. they get as big as 40?

      We assume the reviewer is referring to a comparison between Figures 3c and 2e. For visualization purposes, the graph in 3c was truncated to display over 90% of the data, which explains why the largest clusters appear smaller than in 2e. We modified the legend accordingly. We agree that the results would be strengthened by pooling data from additional experiments; however, acquiring high-quality, artifact-free recordings suitable for motor unit analysis is extremely challenging. We hope that our framework will enable future studies to extend this analysis.

      (14) Figure 4: I would show some of these examples earlier, to give the reader an intuitive sense of the data and claims (though it doesn't need its own figure - provide a couple of examples, and the diagram of how much of the mantle you're sampling) then put the rest in the supplement, and include some videos too.

      We agree that providing spatial context is important for readers to develop an intuitive understanding of the dataset. However, introducing examples of motor units earlier in the manuscript would, in our view, interrupt the logical progression of the Results, where motor unit identification builds on prior analyses. To address the reviewer’s concern, we have added a new supplementary figure (Fig. S1) illustrating the size and location of the sampled mantle region. In addition, we now provide representative videos in the Data Availability section to give readers direct visual access to the underlying dynamics.

      (15) Figure 4f: Is the location of the split color in each dot accurate? It's surprising that each one is split down the middle, and the pink side is always on the right - this is unintuitive given where the motor neuron is likely to be located.

      The dots and half dots represent the membership of a chromatophore to a particular cluster.

      (16) Figure 5: I didn't find this figure sufficiently justified in the text. I would move this to the supplement.

      Addressed in General point #7.

      (17) L350: States that 12 animals were patched, but the data isn't shown. It's important to show all of this data (some of which can be in the supplement).

      Addressed. We provided the data in the Data Availability Section.

      (18) Figure 5: I would quantify how many chromatophores were in each motor group across all the recording sessions, and compare this to the equivalent behavioral analysis.

      We assume the reviewer means Fig. 6. We calculated and stated the size of motor units across patching sessions.

      (19) Figure 5c: I recommend labeling each panel with a different number so you can refer to specific data.

      We assume the reviewer means Fig. 6c. We consider the figure layout clear enough to allow readers to follow the data without additional panel numbers.

      (20) L379: Typo: repeat of "quantitative"

      Addressed.

      (21) L576: Salinity should be 33-36 ppt, not %

      Addressed.

      (22) L877: The salinity units are sg? That should be stated. Though I would use the same units for salinity throughout.

      Addressed.

      Overall, this work introduces a potentially valuable quantitative framework for studying chromatophore dynamics. Addressing the points above would substantially strengthen the manuscript and clarify the scope and support for its conclusions.

      We thank the reviewer for these many helpful comments.

      Reviewer #2 (Recommendations for the authors):

      (1) Line 64 - missing references for chromatophore colour with age.

      Addressed. Added missing refs.

      (2) Line 64-65 - would be good to have a little more detail about what is meant by 'migrating through the skin'. Is this a lateral process, or depth in the skin?

      Addressed. Changed “migrating in the thickness..” with “through the thickness..” to emphasize verticality.

      (3) Line 72 - typo, should read '...individual and groups...'

      Addressed.

      (4) Remove 'In Fig 1, ...' from line 104.

      Addressed.

      (5) Figure 1 - It's unclear why some chromatophores are uncoloured with a red dot in the centre. Are these chromatophores that do not share a cluster with neighbours? If so, wouldn't it make more sense to colour the chromatophore with a unique colour of its own? Or, at the very least, make a note in the caption to indicate that all white chromatophores are not clustered with neighbours.

      Segmented chromatophores are shown in white, with coloured slices highlighting cluster membership. Uncoloured slices represent outliers. Addressed in the figure legend.

      (6) Line 119 - the concept of a 'closed virtual chromatophore' needs a few more words of explanation. The way I interpret the text as it is, is that the motor units driving colour change are not necessarily the individual chromatophores, but a motor region containing a mixture of whole and partial chromatophores innervated by the same motor neuron. If this is the case, a few extra words of description would help here to remove any ambiguity as I think this is an important concept for the paper.

      Addressed. We added a sentence clarifying the concept.

      (7) Line 173 - Figure 6d doesn't exist in the paper. Was a different panel intended? If so, please make sure to number the figures in order of appearance in the manuscript.

      Reference to the inexistent figure 6d was removed.

      (8) Figure 3b is very difficult to see. Perhaps consider lightening the background image. Please also indicate whether the individual colours refer to individual clusters. If this is the case, then some of these clusters look much larger than the 3-5 suggested in the caption.

      This issue has been corrected.

      (9) Line 210 - remove the bold type.

      Addressed.

      (10) Line 211 - please specify which 'two groups' you are referring to here. Presumably, this is anaesthetised and non-anaesthetised.

      Addressed.

      (11) I think that the text is missing any indication of the pixel sizes involved in extracting slice metrics, particularly from the S. officinalis data. It would be great to include some data on how many pixels span the radius of an expanded chromatophore. There is some small indication of this in Figure 2a, but a panel or two with details about the pixel size of S. officinalis chromatophores and their slices would be welcome. This would help with the judgment of the robustness of the resolution of the analysis. Looking at the y-axis in Figure 5a, there is some indication that the chromatophore radius is only 1 to 8 pixels. Is this the case?

      Figure 5a doesn’t show chromatophore radius but instead the relative change in peak amplitude during an expansion event. At that point the chromatophore has likely a larger radius as you sum the baseline radius of the chromatophore + the size of the peak.

      (12) Line 246-7 - reword this sentence to avoid referring to Figure 3d in the narrative. Include it in parentheses instead.

      Addressed.

      (13) Lines 408 and 409 - missing references.

      Addressed.

      (14) Line 576 - salinity should be reported in parts per thousand, not per cent.

      Addressed.

      (15) Line 593 - how were animals <50mm fed?

      Animals smaller than 50 mm were fed Neomysis spp. or small Palaemonetes spp., as noted a few lines above the description for animals larger than 50 mm.

      (16) Line 847 - typo - '...putative motor units' ramifications...'

      Addressed.

      (17) Line 854 - better to write out the [chrom_id, label] info as narrative text rather than using the variable names.

      Addressed.

      (18) Line 876 - two typos '...were reared in an artificial...'

      Addressed.

      (19) Line 877 - please use the same salinity metric as used in the earlier part of the methods.

      Addressed.

      (20) Section 898-910 - equipment details would ideally include the location of the company. E.g. (BX51W1, Olympus, Tokyo, Japan).

      Addressed.

      Reviewer #3 (Recommendations for the authors):

      I am left with a number of questions that arise from the authors' work, some of which the authors themselves briefly mention in the technical limitations section.

      (1) In relation to the first weakness, do the authors know if the recruitment patterns they identify are likely to be the same when octopi perform visually-mediated camouflage to their environment?

      Thank you for this comment. We assume the reviewer is referring to S. officinalis. There seems to be a misunderstanding: our approach is designed to reveal the smallest independent functional units—motor units—that together generate skin patterns. The technique is fully applicable to an animal displaying camouflage, but the results would necessarily differ. Camouflage patterns are composed of relatively large shapes compared to individual motor units and arise from the coordinated activation of multiple units. Disentangling motor units requires decorrelated activity, whereas visually-evoked camouflage inherently drives correlated motor-unit activation by premotor control. To use an analogy, if our goal were to map the distribution and wiring of pixels on a screen, it would be more informative to broadcast a noise signal rather than display coherent images, as the noise produces decorrelated activity that allows the underlying structure to be resolved. We have clarified this important point in the early results section.

      (2) The authors provide indirect evidence that motor neurons innervate multiple chromatophores. Can sets of radial muscles within a chromatophore be innervated by multiple motor neurons? Is there neuroanatomical evidence or experiments that could perhaps shed light on this?

      Addressed above. Same question as #1(8).

      (3) Are multi-innervated chromatophores evenly distributed across the octopus's body? For instance, could the authors compare chromatophore recruitment over multiple patches on the animal from multiple regions?

      At present, we do not have sufficient data to quantitatively compare motor-unit structure or the distribution of multi-innervated chromatophores across different body regions of cuttlefish. However, we would not necessarily expect uniformity across the skin, as distinct body regions are associated with characteristic pattern elements (e.g., the white square on the central mantle or the thicker zebra stripes along the sides). It is therefore plausible that different motor-unit geometries and densities are differentially represented across regions to support these region-specific patterns. Future recordings spanning multiple patches and body locations will be required to test this question directly.

      (4) Relatedly, is there any idea of whether chromatophore size or age corresponds with the number of motor units within a single chromatophore?

      At present, our analyses are limited to single developmental time points, and we therefore cannot directly assess whether chromatophore size or age correlates with the number of motor neurons innervating an individual chromatophore. However, this is a question that our analysis framework is explicitly designed to address. Our custom pipeline, CHROMAS, (Ukrow, Renard et al., 2025) includes tools for longitudinal image alignment that allow chromatophores to be tracked within the same animal across development. Applying these scripts to developmental datasets enables future analyses linking chromatophore growth or age to changes in the motor innervation of single chromatophores.

      I understand that a full resolution to the issues raised above may require substantial additional experiments. At a minimum, further discussion of these points with integration of existing literature would elevate the paper.

    1. eLife Assessment

      The paper presents a valuable finding that the human brain and models that incorporate sentence structures can capture sentence-level semantics beyond word meaning, while large language models behave differently. The evidence supporting the authors' claims is solid, though the stimuli are highly controlled and some analyses could be more thorough. This work will be of interest to researchers in language neuroscience and those developing language models.

    2. Reviewer #1 (Public review):

      Summary:

      This paper investigates whether transformer-based models can represent sentence-level semantics in a human-like way. The authors designed a set of 108 sentences specifically to dissociate lexical semantics from sentence-level information and collected 7T fMRI data from 30 participants reading these sentences. They conducted representational similarity analysis (RSA) comparing brain data and model representations, as well as the human behavioral ratings. It is found that transformer-based models match brain representation better than static word embedding baseline which ignores word order but fall short of models that encode the structural relations between words. The main contributions of this paper are:

      (1) The construction of a sentence set that disentangles sentence structure from word meaning.

      (2) A comprehensive comparison of neural sentence representations (via fMRI), human behavior, and multiple computational models at the sentence level.

      Strengths:

      (1) The paper evaluates a wide variety of models, including layer-wise analysis for transformers and region-wise analysis in the human brain.

      (2) The stimulus design allows precise dissociation between lexical and sentence-level semantics. The RSA-based approach is empirically sound and intuitive.

      (3) The constructed sentences, along with the fMRI and behavioral data, represent a valuable resource for studying sentence representation.

      Weaknesses:

      (1) The rationale behind averaging sentence embeddings across multiple transformer models (with different architectures and training objectives) is unclear. These transformer-based models have different training paradigms and model architectures, which may result in misaligned semantic spaces. The averaging operation may dilute the distinct sentence representations learned by each model, potentially weakening the overall semantic encoding for sentences. Please clarify this choice or cite supporting methodology.

      (2) All structure-sensitive models discussed incorporate semantics to some extent. Including a purely syntactic baseline, such as a model based on context-free grammar, would help confirm the importance of syntactic structures.

      (3) In Figure 2, human behavioral judgments show weak correlations with neural data, and even fall below those of computational models, suggesting the behavioral judgments may not reflect the sentence structures in a brain-like way. This discrepancy between behavioral and neural data should be clarified, as it affects the interpretation of the results.

      (4) To better contextualize model and neural performance, sentence similarity should be anchored to a notion of semantic "ground truth", such as the matrix shown in Figure 1a. Comparing this reference with human judgments, brain responses, and model similarities would help establish an upper bound.

      (5) The structure of this paper is confusing. For instance, Figure 5 is cited early but appears much later. Reordering sections and figures would enhance readability.

      (6) While the analysis is broad and comprehensive, it lacks depth in some respects. For instance, it remains unclear what specific insights are gained from comparing across brain regions (e.g., whole brain, language network, and other subregions). Similarly, the results of simple-average and group-average RSA appear quite similar and may not advance the interpretation.

      (7) While explaining the grid-like pattern due to sentence length is important, this part feels somewhat disconnected from the central question of this paper (word order). It might be better placed in supplementary material.

      Comments on revised version:

      The new version of the paper has addressed my main concerns, including:

      (1) clarification about the methodology of Transformer embeddings

      (2) discussion about the purely syntactic models

      (3) discussion about the low correlation between behavioural ratings and brain activations

      (4) better structure of the paper

      (5) clarification about pre-registration

      I believe the paper has been substantially improved after revision.

    3. Reviewer #3 (Public review):

      Summary:

      Large Language Models have revolutionized Artificial Intelligence and can now match or surpass human language abilities on many tasks. This has fuelled interest in cognitive neuroscience in exposing representational similarities between Language Models and brain recordings of language comprehension. The current study breaks from this mold by: (1) Systematically identifying sentence structures for which brain and Large Language Model representations diverge. (2) Accounting for such sentence structures using a model structured by semantic roles. As such the study may now fuel interest in characterizing how Large Language Models and brain representations differ, which may prompt new more brain like language models.

      Strengths:

      * This study presents a bold challenge to a literature trend that has touted similarities between Transformer models and human cognition based on representational correlations with brain activity. This challenge is substantiated by identifying sentences for which brain and model representations of sentences diverge.

      * This study conducts a rigorous pre-registered analysis of a comprehensive selection of the state-of-the-art Large Language Models, on a controlled sentence comprehension fMRI dataset. The analysis is conducted within a Representation Similarity framework to support similarity comparisons between graph structures and brain activity without needing to vectorize graphs. Transformer models are predicted and shown to diverge from brain representations on subsets of sentences with similar word-level content but different sentence structures.

      * The study introduces a 7T fMRI sentence comprehension dataset and accompanying human sentence similarity ratings which may be a fruitful resource for developing more human-like language models. Unlike other model-based sentence datasets, the relation between grammatical structure and word-level content is controlled, and subsets of sentences for which models and brains diverge are identified.

      Weaknesses:

      * The interpretation of findings is nuanced. Although Transformers underperform as brain models on the critical subsets of controlled sentences, a Transformer outperforms all other models when evaluated on the union of all sentences when both word-level content and structure vary. Transformers also yield equivalent or better models of human behavioral data. Thus, although Transformers have demonstrable flaws as human models which are pinpointed here, in the general case (some) Transformers are more human-like than the other models considered.

      * There may be confounds between the critical sentence structure manipulations and visual processing. This is inconvenient because activation in brain regions that process semantics tends to partially correlate with low-level representations of sentence surface features encoded in visual cortex. Although the study commendably controls for confounds associated with sentence length, correlations with the key sentence structure models are most salient in visual cortex and diminish in other brain networks when V1-V4 activation is controlled for.

      * Sentence similarity computations are emphasized as the basis for unifying comparative analyses of graph structures and vector data. A strength of this approach is that correlation is not always the ideal similarity metric. However, a weakness is that similarity computations are not unified across models. This has practical consequences because different similarity metrics applied to the same model produce positive or negative correlations with brain data and repeating analyses with a different representational dissimilarity measure seems to produce some anomalous results.

    4. Author response:

      The following is the authors’ response to the original reviews.

      Reviewer #1 (Public review):

      (1) The rationale behind averaging sentence embeddings across multiple transformer models (with different architectures and training objectives) is unclear. These transformer-based models have different training paradigms and model architectures, which may result in misaligned semantic spaces. The averaging operation may dilute the distinct sentence representations learned by each model, potentially weakening the overall semantic encoding for sentences. Please clarify this choice or cite supporting methodology.

      The reviewer questions the rationale for averaging sentence embeddings across different models. However, our method involves computing correlations separately for each model, then averaging the correlations. We apologize for the confusion. We have clarified this on page 3:

      “Results for the ‘Transformers’ model are computed by computing correlations separately for five different transformer models and then taking a simple average of these correlations. Results for each individual transformer are presented in Supplementary Information Figure S2.”

      (2) All structure-sensitive models discussed incorporate semantics to some extent. Including a purely syntactic baseline, such as a model based on context-free grammar, would help confirm the importance of syntactic structures.

      Following the suggestion, we have implemented two syntactic models and discuss the results on page 10:

      “We also found that purely syntactic models based on constituency parses (see Benepar and CFG) show poor correlations with brain activity (see Supplementary Information Figure S2). Examining the corresponding RSA matrices (see Figure S1), this seems to be due to such models being overly sensitive to syntactic form, and relatively insensitive to which words are assigned to different nodes within the syntactic tree. This is most evident for the edit-distance similarity metric, and to a lesser extent also for the subtree similarity metric. This finding highlights the value of hybrid approaches designed to appropriately balance sensitivity to lexical, syntactic, and compositional information in representing semantic information at the sentence level.”

      (3) In Figure 2, human behavioral judgments show weak correlations with neural data, and even fall below those of computational models, suggesting the behavioral judgments may not reflect the sentence structures in a brain-like way. This discrepancy between behavioral and neural data should be clarified, as it affects the interpretation of the results.

      While the behavioural judgements are made by different participants and involve a different task than the neuroimaging results, nonetheless we agree the difference is surprising and warrants more detailed consideration. We have included a more detailed discussion of this issue on page 11:

      “Our study has several limitations. First, we found a surprisingly low correlation between behavioural ratings and brain activations (see Figure 2). This may be partly explained by differences in task structure. In the behavioural experiment, participants viewed many pairs of related sentences, and were explicitly asked to pay attention to differences in the words of each sentence. In contrast, in the fMRI task, participants read one sentence at a time without an explicit comparison. In addition, we suspect that presentation of so many sentence pairs with highly similar structures may have biased the way in which participants rated sentence similarity. Modifications to the behavioural task to mitigate these aspects may reduce the divergence between behavioural and brain findings.”

      (4) To better contextualize model and neural performance, sentence similarity should be anchored to a notion of semantic "ground truth", such as the matrix shown in Figure 1a. Comparing this reference with human judgments, brain responses, and model similarities would help establish an upper bound.

      While our design matrix served as the basis for constructing a set of stimuli with systematic modifications, we respectfully suggest that it should not be regarded as a ‘semantic ground truth’. Sentence pairs within each category will not have the same degrees of semantic similarity since the words and context differ across sentences in a graded manner. Furthermore, while we anticipated ‘different’ sentence pairs would be less similar than ‘swapped’ sentence pairs, and that within each of the six block diagonals the ‘modified’ or ‘substituted’ sentence pairs would be the most similar, we did not have any prediction about the magnitude of these differences. Our goal was to construct a set of sentence pairs which spanned a range of semantic similarities, and allowed for dissociation between lexical similarity and overall similarity in meaning. The design matrix is not intended to represent a ‘ground truth’ that human judgements or brain representations would be expected to conform with.

      (5) The structure of this paper is confusing. For instance, Figure 5 is cited early but appears much later. Reordering sections and figures would enhance readability.

      We agree that placement of figures was not ideal in the previous draft. We have reworked the manuscript so that all figures appear closer to their mention in the text, and the figure (now Figure 3) appears in the correct order. We have also substantially revised the discussion, and included subheadings to help guide the reader through the various different issues we include.

      (6) While the analysis is broad and comprehensive, it lacks depth in some respects. For instance, it remains unclear what specific insights are gained from comparing across brain regions (e.g., whole brain, language network, and other subregions). Similarly, the results of simple-average and group-average RSA appear quite similar and may not advance the interpretation.

      We included both analyses in line with our preregistration, and also because we believe the fact that two distinct approaches to analyzing the data yield similar results strengthens our conclusions.

      (7) While explaining the grid-like pattern due to sentence length is important, this part feels somewhat disconnected from the central question of this paper (word order). It might be better placed in supplementary material.

      We believe that the grid-like pattern in the RSA results is an important unexpected finding that warrants discussion in the main manuscript.

      Reviewer #1 (Recommendations for the authors):

      (1) Consider including a purely syntactic baseline model. For instance, parse each sentence into a constituency tree and compute tree edit distances between pairs of trees. This would allow you to construct a sentence similarity matrix based solely on syntactic structure, and may clarify the role of syntax in sentence representations.

      See our response to Public Review comment 2.

      (2) Instead of averaging embeddings across different transformer-based models, I recommend reporting RSA results for each model individually. For instance, compare one sentence-level model (e.g., SentBERT or SimCSE) and one general-purpose language model (e.g., GPT-2 or Llama).

      See our response to Public Review comment 1.

      (3) I suggest revisiting the structure of the Results section to improve the clarity and impact of your key findings. Consider which results are most central to the paper's claims and ensure they are presented in the main text. Less central analyses (e.g., the analysis on the grid-like pattern) might be better suited for the supplementary information. Presenting behavioral results prior to neuroimaging results could also improve logical flow by first validating model similarity estimates behaviorally.

      As mentioned in our response to Public Review comment 5, we have revised the ordering of the figures to improve the flow of the main manuscript. We believe that the grid-like pattern in the RSA results is an important unexpected finding that warrants discussion in the main manuscript. In addition, we believe that presenting the neuroimaging results first is appropriate as this is the primary and most important contribution of our study.

      Reviewer #2 (Public review):

      (1) The stimuli are not fully controlled for lexical content across conditions. Residual lexical differences between sentences could still influence both brain and model similarity patterns. To more cleanly isolate syntactic effects, it would be useful to systematically vary only a single structural element while keeping all other lexical content constant (e.g., the boy kicked the ball / the ball kicked the boy). It would be better to engage more with the minimal pair paradigm, which is widely used in large language model probing research.

      The reviewer rightly argues that our stimuli do not fully control for lexical content across conditions, and that a more appropriate paradigm may be to utilise minimal pairs in which only a single variable of interest (such as sentence structure) is modified. We agree that most of our sentence pairs do not constitute minimal pairs; however, this was not our objective. Our study design aimed to synthesise traditional minimal pair approaches with more recent research paradigms using naturalistic stimuli. As such, we selected stimuli which are more complex and contain more variable features than traditional minimal pair studies, but which also are tailored to highlight differences which are of particular theoretical interest.

      Because we are interested in comparing the effects of multiple sentence elements and semantic roles, a systematic pairwise comparison of minimal pairs is not necessarily optimal. Instead, we designed our stimuli to leverage the advantage of fMRI in that we can measure the brain representations corresponding to each sentence, and hence can conduct a full series of pairwise comparisons of sentence representations. We do not claim this approach to be universally superior to a minimal pair approach, but we do believe our novel approach provides additional insights and a new perspective on semantic representation relative to minimal pair studies.

      We have added the following paragraph on pages 9-10 contrasting our approach to previous minimal-pair studies:

      “Another approach that has seen widespread use is the presentation of minimal sentence pairs that differ only in one specified aspect, for example, interchanging subject and object in a sentence (Frankland 2015, Wang 2016, Frankland 2020, Giglio 2024), or altering adjective-noun phrases to influence composition (Graves 2010, Schell 2017, Fyshe 2019, Ciapparelli 2025). Our approach is an extension of these approaches utilising more naturalistic and complex sentences, designed to facilitate comparison of a wider range of structural manipulations (see Table 1). In more completely characterising the representational structure of various computational models in response to different structural contrasts, we can more comprehensively evaluate their adequacy as models of semantic processing in the brain.”

      (2) The comparisons are done across fundamentally different model types, including static embeddings, graph-based parsers, and transformers. The inherent differences in dimensionality and training objectives might make the conclusion drawn from RSA inconclusive. Transformer embeddings typically occupy much higher-dimensional, anisotropic representational spaces, and their similarity structure may reflect richer, more heterogeneous information than models explicitly encoding semantic roles. A lower RSA correlation in this study does not necessarily imply that transformers fail to encode syntactic information; rather, they may represent additional aspects of meaning or context that diverge from the narrow structural contrasts probed here.

      The reviewer notes that low RSA correlations do not necessarily imply that transformers fail to encode syntactic information. We acknowledge this in our discussion (page 10), where we also highlight that our focus is not on whether transformers encode such information, but rather what transformer representations can tell us about how sentence structure is represented in the brain. Our results indicate that transformer embeddings do not have the same geometric properties as brain representations of sentence meaning, at least for certain types of sentences where lexical information is insufficient to determine overall meaning.

      The reviewer also notes that transformer embeddings are highly anisotropic; however, we adjust for this by normalising each feature as discussed on page 14. Finally, the reviewer notes that the transformers we examine differ in architecture and training objectives. This is not critical for our study because we are not seeking to determine which architecture or training objectives are best. Our goal is simply to compare a range of approaches and see which, if any, have similar sentence representations to those formed by the brain. In fact, our results indicate that architecture and training regime make relatively little difference for our stimuli, as shown by the pattern of results for all models in Figure S2.

      (3) The interpretation of the RSA correlation largely depends on the understanding of models. The authors suggest that because hybrid models correlate better than transformers, this implies that transformers are inferior at representing syntax. However, this is not a direct test of syntactic ability. Transformers may encode syntactic information, but it may not be expressed in a way that aligns with the RSA paradigm or the chosen stimuli. RSA does not reveal what the model encodes, and the models might achieve a good correlation for non-syntactic reasons (e.g., length of sentence, orthographic similarity, lexical features).

      The reviewer argues that RSA correlations do not measure the extent to which a model encodes syntactic information. This is very similar to the previous point. We do not claim that our results show that transformers do not encode syntactic information. Rather, our claim is that sentence embeddings derived from transformers have different geometric properties to brain representations, and that brain representations are better described by models explicitly representing key semantic roles. From this we conclude that, at least for the sentences we present, the brain is highly sensitive to semantic roles in a way that transformer representations are not (at least to the same extent). We have clarified this in a modified paragraph on page 11:

      “We emphasise that our results do not show that transformers fail to represent syntactic or semantic role information. Indeed, large language models show clear capabilities of correctly interpreting sentence structure (Chang 2024), and probing studies have found that transformers represent information about syntax and word order (Clark 2019, Manning 2020). This is consistent with our finding that directly prompting GPT-4 to rate sentence similarity yields very high correlations with human judgements (see Supplementary Information Figure S3). Nonetheless, the fact that transformers can encode and utilise structural information to perform linguistic tasks does not mean that they effectively utilise this information to construct a brain-like representation of sentence meaning.”

      We also respectfully disagree with the reviewer’s suggestions that sentence length and orthographic or lexical similarities may drive model correlations with brain activity. As we discuss on page 19, we explicitly control for differences in sentence length when computing correlations. Our process for constructing our sentence set also controls for lexical similarity by generating pairs of sentences with all or mostly the same words but different orderings. We did not explicitly address orthographic similarity, but this will be strongly correlated with lexical similarity.

      Reviewer #2 (Recommendations for the authors):

      (1) Model dimensionality: the interpretability of cosine similarity diminishes as the dimensionality increases, and there are some math tricks to work around it. To make a fair comparison among models with different dimensionalities, it would be better to apply some dimensionality-insensitive distance metrics.

      We thank the reviewer for this suggestion. We repeated all vector-based similarity calculations using the Dimension Insensitive Euclidean Metric (DIEM). As shown in Figure S9, the results are broadly similar, though with overall somewhat lower brain correlations for most transformers compared to cosine similarity.

      (2) Depending on the scope of the current study, if the authors would like to establish whether transformers are inferior to graph-based models in representing syntax, a linear classifier using the model embeddings would be sufficient. I think this would be a more direct assessment of model syntax ability than correlation with brain data.

      As we discuss in our previous responses, our objective in this study was not to assess how well transformers can represent syntax. Rather, the goal was to assess whether internal transformer representations have similar geometric properties to patterns of brain activation. Our results indicate that transformers do represent sentence structure, but in a different manner to the human brain.

      Reviewer #3 (Public review):

      (1) The interpretation of findings is nuanced. Although Transformers underperform as brain models on the critical subsets of controlled sentences, a Transformer outperforms all other models when evaluated on the union of all sentences when both word-level content and structure vary. Transformers also yield equivalent or better models of human behavioral data. Thus, although Transformers have demonstrable flaws as human models, which are pinpointed here, in the general case, (some) Transformers are more human-like than the other models considered.

      The reviewer argues that we overstate some of our conclusions, as several transformers achieve higher brain correlations than the hybrid model when computed over all sentence pairs, as well as on the behavioural data. In response, we first note that our primary interest in this paper is on the block diagonal sentence pairs, as these were specifically designed to interrogate how different models represent sentence structure. The comparison with all sentence pairs is presented for comparison but is not our primary focus on this paper, as also reflected in the pre-registered prediction that our VerbNet-CN hybrid model would show higher brain correlations than transformers over this block diagonal subset.

      Second, we have included a new analysis in the revised manuscript (Figure S9) where we compute brain correlations controlling for the pattern of similarities observed in the primary visual cortex (averaged over participants), as a way to control for visual similarity. This added control substantially reduces the brain correlations of the transformers, such that they all have lower correlations than VerbNet-CN and AMR-smatch even over the set of all sentence pairs. We provide interpretation of this result in the discussion.

      Third, we would like to note one of the disadvantages of transformers as a model of mind or brain representations is that they are largely a ‘black box’ whose workings are poorly understood. One advantage of hybrid models like our simple semantic role model is that they can be much easier to interpret, thereby enabling them to be used to determine which features are most important for brain representations of sentence meaning, and what mechanisms are used to combine individual words into a full sentence. Given their relative simplicity and interpretability, we believe hybrid models have considerable value as scientific tools, even in cases where they achieve comparable correlations to transformers. We have added a short discussion of this issue in the revised manuscript (page 10).

      (2) There may be confounds between the critical sentence structure manipulations and visual representations of sentence stimuli. This is inconvenient because activation in brain regions that process semantics tends to partially correlate with visual cortex representations, and computational models tend to reflect the number of words/tokens/elements in sentences. Although the study commendably controls for confounds associated with sentence length, there could still be residual effects that remain. For instance, the Graph model correlates most strongly with the visual cortex despite these sentence length controls.

      We agree with the reviewer that this is a potential confound. As noted in the previous response, we have implemented a new control analysis in which we directly control for visual similarities as reflected in participant-averaged similarities of primary visual cortex activations in response to all stimuli. These results are shown in Figures S8-S11 in the SI. We show that transformer correlations are reduced much more than graph and hybrid models with this control. Also, we note that the AMR-smatch graph model shows high correlations with other brain regions even after removing correlations with the visual cortex (Figure S10). This indicates that the model represents a range of sentence features, including both superficial visual or length-related features, as well as semantic features that are represented in common with language and other cortical regions.

      (3) Sentence similarity computations are emphasized as the basis for unifying comparative analyses of graph structures and vector data. A strength of this approach is that correlation is not always the ideal similarity metric. However, a weakness is that similarity computations are not unified across models. This has practical consequences here because different similarity metrics applied to the same model produce positive or negative correlations with brain data.

      The reviewer notes that the method for computing similarities differs between the vector-based (mean and transformer) models, and the hybrid and syntax-based models, thereby potentially adding an additional confound to our results. We agree that this is a potential limitation, and our correlations should always be understood as applying to a model paired with a similarity metric. However, we believe that this is mostly unavoidable when comparing different formalisms. In the revised manuscript we have incorporated an entirely new similarity metric for vector-based models (DIEM similarity), as well as an extended discussion of the effect of different similarity metrics for graph and hybrid models.

      Reviewer #3 (Recommendations for the authors):

      (1) Compute separate RSAs on each sentence pair type (especially Swapped), to quantify how each sentence type manipulation contributed to the divergence between model and brain. Although the manuscript is already brimming with analyses, I think squeezing this in would be helpful because the results currently rely on qualitative inspection of group-average scatter plots to interpret how sentence pair manipulations contributed to the divergence between Transformers and humans. The Swapped condition would appear to be the centrepiece of the title and manuscript, and potentially the only condition for which confounds associated with the surface form of sentence are controlled for (because sentences should be the same words in different orders). Thus, this analysis might see to the inconvenient visual cortex correlations in Figures 3d/e.

      We respectfully disagree that computing separate RSA for each sentence pair type would be a useful additional analysis. The motivation for the construction of our stimulus set was to provide a range of variants of a given base sentence that alter the semantic meaning and lexical content (somewhat) independently. The purpose of the ‘modified’ sentences, for instance, is to construct sentences with a similar overall meaning but lower lexical similarity due to the inclusion of many modifier words. It is precisely the comparisons across the different pair types that provide information about how each model represents sentence semantics, so restricting an analysis to only a single subset would not be very informative. Another problem with this approach is that it would dramatically reduce the number of sentence pairs analysed, thereby decreasing statistical power. In the revised manuscript we have provided additional details regarding the motivation and rationale for how our stimulus set of 108 sentences was constructed, which should help to elucidate this point more clearly. The following excerpt is from page 3:

      “Within each of the six subsets, we begin with a base sentence such as `the cameraman brought the equipment to the director', which we then systematically modified in various ways to create different combinations of lexical and compositional similarity, in order to dissociate these two aspects of meaning (see Table 1 for further details).”

      (2) Explaining the motivation for the sentence stimulus types. I appreciated the careful design of the dataset, but I couldn't immediately work out the motivation for all the different sentence types, and why this selection was ideal to identify divergences with Transformers. For instance, given the goal of (approximately) controlling for lexical similarity whilst varying sentence meaning, I couldn't immediately see why stimulus blocks weren't all built from rearranging the same content words (as in the Swapped condition). The negative RSA correlation with the Mean model also made me stop and think - it seems like the more similar the words in a sentence, the more different their structure, and vice versa, but I wasn't clear that this was a design feature. Thus, a few extra words motivating the conditions could be helpful for the reader, and these might helpfully lead them to anticipate the negative RSA correlation.

      As noted in the previous response, in the revised manuscript we have expanded our explanation of the rationale for the construction of our 108 sentences. In particular, Table 1 in the methods section now includes two additional columns which summarise the intended combinations of lexical and overall sentence similarity which our sentence pairs are intended to satisfy.

      (3) Explanation for why different implementations and similarity computations between variants of ostensibly equivalent Graph / Hybrid models yielded widely divergent positive vs negative brain correlations, despite both positively capturing behavioural ratings. This might incorporate a brief intuitive explanation of how Graph model similarities were computed (e.g., what SMATCH and WWLK do). In light of the above, why do different similarity algorithms applied to the Graph model yield positive and negative correlations on the same brain (e.g., Figure S2 - Graph / Graph-WL a,b, diag-pairs). Same goes for why Hybrid and Hybrid-AMR yielded positive vs negative correlations (e.g., Figure S2 - Graph / Graph-WL a,b, diag-pairs). Acknowledge that the brain results are sensitive to similarity computations in the Discussion.

      We appreciate this suggestion. We have added an extended consideration of these issues to the discussion (pages 10-11), as well as some additional details regarding the differences between the Smatch and WWLK metrics in the methods section (page 17).

      (4) Acknowledgement and explanation of why the human similarity ratings were poor at explaining brain data in Figure 2a,b (right column diag-pairs). The poor behaviour vs brain match is indirectly implied in the Discussion as "the comparison between behavioural and fMRI data is somewhat difficult owing to the difference in task structure." However, I would suggest being upfront and explicitly mentioning and explaining the poor brain match in Figures 2a and b, because the reader will notice and wonder - especially because the models correlate strongly with the behavioural data without the models doing the human behavioral task (though this could be a possibility, see later).’

      As suggested, we have included a passing reference to this in the presentation of our main results in page 5, and a lengthier discussion on page 11:

      “Our study has several limitations. First, we found a surprisingly low correlation between behavioural ratings and brain activations (see Figure 2). This may be partly explained by differences in task structure. In the behavioural experiment, participants viewed many pairs of related sentences, and were explicitly asked to pay attention to differences in the words of each sentence. In contrast, in the fMRI task participants (who were not the same as the behavioural task participants) read one sentence at a time without an explicit comparison. In addition, we suspect that presentation of so many sentence pairs with highly similar structures may have biased the way in which participants rated sentence similarity. Modifications to the behavioural task to mitigate these aspects may reduce the divergence between behavioural and brain findings.”

      (5) Brief explanation of why model vs brain correlations tended to be strongest in the visual cortex (Figure 3d,e). Currently, this issue is only mentioned in passing, however, it seems worthy of further comment.

      We appreciate the reviewer for highlighting this issue. We have added discussion of the potential for visual confounds to several points in the revised manuscript, including the ‘Neuroscience of semantics’ subsection on page 11. As noted, we have also added a new analysis in which we compute correlations controlling for the average RSA similarities of the primary visual cortex. We find that this additional control significantly reduces correlations for most transformer models, but only has a more modest reduction on the correlations for most of the graph and hybrid models, particularly VerbNet-CN (see Figures S8-S11).

      (6) Softening/clarifying some statements that could be misconstrued as suggesting Transformers were universally inferior models. Statements made in the Abstract/Discussion initially came over to me as implying that Transformers were universally inferior models when compared to the Graph/Hybrid models - but this appears only to be true when one looks at analyses conducted within block diagonal sentence subsets. Otherwise, when analyses are conducted on all sentences (between and within blocks, Figure 5) Llama 3 L2 provides by far the strongest brain model. Transformers also appear to yield the strongest accounts of the behavioural data, whether tested on block diagonal or all sentence pairs (Figure S3). To remedy this, I would suggest softening some statements in the Abstract/Discussion that could be misconstrued as suggesting that Transformers were universally inferior. I would also suggest explicitly acknowledging that when the entire dataset was analyzed, Transformers were most accurate, and that (some) Transformers best accounted for the behavioural data.

      We agree that there was some lack of precision in certain sections of the previous draft regarding the conclusions to be drawn regarding the representational capacities of transformers. We have revised the abstract and conclusion to better reflect our intended message, which is that transformers certainly can represent sentence structure and semantic roles, but that the way in which they do this (through vector representations in their hidden layers) is significantly different to how such features are represented in the human brain. In particular, we have included this new text on page 10:

      “We emphasise that our results do not show that transformers fail to represent syntactic or semantic role information. Indeed, large language models show clear capabilities of correctly interpreting sentence structure, and probing studies have found that transformers represent information about syntax and word order. This is consistent with our finding that directly prompting GPT-4 to rate sentence similarity yields very high correlations with human judgements (see Figure S3). Nonetheless, the fact that transformers can encode and utilise structural information to perform linguistic tasks does not mean that they effectively utilise this information to construct a brain-like representation of sentence meaning.

      (7) Given that GPT-4 was already deployed to parse semantic roles for the hybrid model, and GPT-4 should be able to generate reasonable similarity ratings between sentence pairs, it struck me that an interesting addendum could be to use GPT-4 similarities derived from the human behavioral task to interpret both brain and human behavioral data. This might also help support the case for conducting analyses within a similarity-based framework.

      We appreciate this suggestion. We have added this model (GPT-4 ratings of sentence similarity) to the revised manuscript (see Figures S1-S3).

      Other changes

      As noted by reviewer 3, the full set of sentence pairs was missing from the previous draft. They have been added to the SI of the revised manuscript.

      We have renamed the Graph and Hybrid models in the manuscript to AMR-Smatch and Verbnet-CN respectively, for greater clarity as to which models these terms refer to, and also to better differentiate from the newly added constituency parse graph models.

      We have thoroughly revised the discussion section, incorporating feedback from all reviewers regarding areas needing additional depth.

      We have added subsections to the discussion to aid the reader navigating the now lengthier section.

    1. eLife Assessment

      This valuable study uses technically compelling long-term in vivo recordings and computational modeling to investigate whether hawkmoth olfactory receptor neurons show circadian modulation of spontaneous firing. The authors further propose the provocative model that post-translational mechanisms, rather than the transcriptional-translational processes, may contribute to circadian regulation of neuronal excitability. However, the evidence for circadian firing in these neurons, and for post-translational modification of Orco as the underlying mechanism, remains incomplete. In contrast, the study does provide strong evidence that the application of cyclic nucleotides can modulate Orco-dependent activity at a single time point, and reports that the temporal pattern of Orco transcript abundance is not circadian. However, the findings are incomplete to exclude a role for transcriptional-translational mechanisms and their associated multi-layered controls in circadian regulation.

    2. Joint Public Review:

      This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several assumptions that underlie their data analysis and model builds, as well as insufficient biological data including critical controls to validate and/or fully justify the model the authors are proposing.

      Strengths:

      The authors raise several intriguing model-based hypotheses regarding the mechanisms that underlie the generation of olfactory rhythms. The electrophysiological approach and the long-term recording paradigm are elegant and technically impressive. In the revised version, the authors have added additional qPCR data supporting the lack of rhythmic Orco transcript expression and included a new figure suggesting that cAMP can modulate Orco conductance.

      Major weaknesses:

      (1) The cAMP experiment was only conducted at one time-point, which is insufficient to support the central claim that "AMP and cGMP may have ZT-dependent effects on Orco conductivity".

      (2) The revised manuscript continues to rely heavily on prior publications or defers key mechanistic questions (or important manipulations) to future studies. In its current form, the evidence presented remains insufficient to support the central claim that a PTFL constitutes the primary underlying circadian clock mechanism. The proposed model is intriguing, but the data provided do not yet directly demonstrate the novel mechanism.

    3. Author response:

      The following is the authors’ response to the original reviews.

      Joint Public Review

      This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several questionable assumptions that underlie their data analysis and model builds, as well as insufficient biological data, including critical controls to validate and/or fully justify the model the authors are proposing.

      We thank the reviewers for their thorough and thoughtful comments and believe that the manuscript is much stronger now after the revision which incorporates the requested changes. We added results of new experiments and additional analyses. Although these new insights did not change the previous conclusions, we significantly reworked the Discussion and added further references to clarify the conclusions we want to make.

      Please note that we used ORN as acronym for “olfactory receptor neuron” throughout the manuscript. ORNs contain odorant receptors (ORs), and in insects these ORs associate with the olfactory receptor co-receptor (Orco) to be trafficked to the membrane of the cilium of the ORN, where they can be contacted by pheromones and odorants. In Manduca sexta, evidence is accumulating for G-protein coupled metabotropic pheromone transduction and not for OR-Orco dependent ionotropic transduction, as shown for Drosophila melanogaster. In both insect species, besides its chaperone function, Orco can form leaky cation channels, which can regulate the spontaneous spiking activity of ORNs. In this study, we explored this role of Orco.

      Strengths:

      The study is notable for its combination of long-term in vivo tip recordings with computational modeling, which is technically challenging and adds weight to the authors' claims. The link between Orco, cyclic nucleotides, and circadian regulation is potentially important for sensory neuroscience, and the modeling framework itself - a stochastic Hodgkin-Huxley formulation that explicitly incorporates channel noise - is a solid and forward-looking contribution. Together, these elements make the study conceptually bold and of clear interest to circadian and olfactory biologists.

      Major weaknesses:

      At the same time, several limitations temper the conclusions. The pharmacological evidence relies on a single antagonist and concentration, without key controls. The circadian analysis is based on relatively small numbers of neurons, with rhythms detected only in subsets, and the alignment procedure used in constant darkness raises concerns of bias. The molecular evidence is sparse, with only three qPCR timepoints, and the model, while creative, rests on assumptions that are not yet fully supported by in vivo data.

      Please see our responses to the detailed comments.

      Detailed comments are provided below:

      (1) The role for Orco proposed in the authors' model largely stems from the effects seen following the administration of (a single dose) of the Orco antagonist, OLC15. However, this hypothesis is undercut by the lack of adequate pharmacological controls, including a basic multipoint OLC15 dose-response series in addition to the administration of blockers for the other channels that are embedded in their model, but which were ruled out as being involved in the modulation of biological rhythms. In addition, these studies would (ideally) also benefit from the inclusion of the same concentration (series) of an inactive OLC15 analog to better control for off-target effects.

      The Orco agonist VUAA1 (Jones et al., 2011) binds directly to Orco and increases the channel open time probability. In M. sexta hawkmoths, we have already published that VUAA 1 increases the low spontaneous activity of ORNs in a dose-dependent fashion (Nolte et al., 2013). Chen and Luetje (2012) systematically varied the chemical structure of VUAA1 to identify new Orco ligands and discovered 22 Orco ligand candidates (OLCs) that either activated or inhibited Orco. In their heterologous expression system, Orco was most sensitive to inhibition by OLC15. Based on these results, we published a dose-response curve of OLC15 inhibition (1-100 µM) using in vivo tip recordings of pheromone-sensitive long trichoid sensilla of M. sexta (Nolte et al., 2016). There, we also demonstrated that OLC15 dose-dependently antagonizes the VUAA1-dependent activation of Orco.

      Furthermore, we tested other published Orco antagonists, which were characterized in heterologous assays, in primary cell cultures of hawkmoth ORNs, as well as in in vivo assays in intact hawkmoths. We focused on amiloride-derived antagonists, because we previously identified an amiloride-sensitive cation channel in hawkmoth ORNs. We found that, in contrast to OLC15, the amilorides HMA and MIA were not Orco-specific antagonists but instead affected different ion channel targets depending on the time of day (Nolte et al., 2016). Based on those experiments and the dose-response curves we determined that the Orco agonist VUAA1 (Jones et al., 2011) and the Orco antagonist OLC15 (Chen and Luetje, 2012) worked best in hawkmoth ORNs to target Orco pharmacologically. Due to those results and other comparative tests with other published Orco antagonists we settled since then in all further experiments on a dose of 50 µM OLC15 as most adequate to antagonize Orco functions in Manudca. In the current study, we focus on Orco without excluding the possibility that other ion channels in the ORNs contribute to the control of membrane potential rhythms.

      We have clarified the Methods section accordingly.

      (2) The expression pattern of Orco was assessed using qPCR at only three timepoints. Rhythmic transcripts can easily be missed with such sparse sampling (Hughes et al., 2017). A minimum of six evenly spaced timepoints across a 24-hour cycle would be required to confidently rule out circadian transcriptional regulation. In addition, the use of the timeless mRNA control from another study is not acceptable. Furthermore, qPCR analysis measures transcript abundance, not transcription, as the authors repeatedly state. Transcriptional studies would require nuclear run-off or, more recently, can be done with snRNAseq analysis. Taken together, these concerns undermine the authors' desire to rule out TTFL-based control that directly led them to implicate a PTTF-based model.

      We agree with the referees that more time points and a direct comparison between timeless and Orco mRNA levels should be included in this manuscript. We included these additional qPCR experiments and edited the manuscript to make clear that we measure transcript abundance, but we will not perform snRNAseq analysis due to time- and financial constraints.

      (3) The modelling presented is based on Orco as a ZT-dependent conductance tied to the cAMP oscillations that were reported by this group in the cockroach and from the presence and functionality in Manduca of homomeric Orco complexes that are devoid of tuning ORs. While these complexes have been generated in cell culture and other heterologous expression systems, as well as presumably exist in vivo in the Drosophila empty neuron and other tuning OR mutants, there is no evidence that these complexes exist in wild-type Manduca ORNs. While this doesn't necessarily undermine every aspect of their models, the authors should note the presence of Orco/OR complexes rather than Orco homomeric complexes.

      Our ELISAs found circadian oscillations in cAMP levels not only in antennae of the Madeira cockroach (Schendzielorz et al., 2014, 2012), but also in hawkmoth antennae (Schendzielorz et al., 2015). For clarification, we added the 2015 citation to the Modeling chapter in the Methods section.

      We agree with the referees that we cannot distinguish between Orco homo- and heteromers in the different compartments of our hawkmoth ORNs but we know that both are expressed in the pheromone-sensitive ORNs. Thus, as the referee suggests, we added text regarding the presence and localization of OR-Orco heteromers. Consistent data collected across different experiments (heterologous expression systems, primary cell cultures of hawkmoth ORNs, in vivo/in situ studies) support that Orco homomers are present in hawkmoth ORNs. In addition to co-expression of MsexOrco and MsexSNMP-1 with either MsexOr-1 or MsexOr-4 in a heterologous expression system, MsexOrco expression alone was already sufficient to increase intracellular Ca<sup>2+</sup> levels spontaneously as a result of its property as leaky, non-specific cation channel, and in response to VUAA1 application (Nolte et al., 2013). Both in developing hawkmoth pupae and differentiating primary cell cultures of hawkmoth ORNs, Orco expression started during a developmental time window where ORNs did not yet express pheromone receptors but where Orco affected spontaneous activity and intracellular Ca<sup>2+</sup> levels dependent on VUAA1 (Nolte et al., 2016). In vitro patch clamp studies of differentiating cultured hawkmoth ORNs during this time window of pupal development characterized ion channels/currents with properties of Orco as a leaky, non-specific cation channel/current that depends on protein kinase C and cyclic nucleotides (Dolzer et al., 2021, 2008; Krannich and Stengl, 2008; Stengl, 1993). Thus, Orco homomers are present in developing hawkmoth ORNs during a time window where ORNs already express spontaneous activity but they do not heteromerize with pheromone receptors. However, we do not know whether and in what ratio homo- and heteromers of Orco and ORs are present in the respective sensillum compartments of adult hawkmoths because all OR-specific antibodies tested did not work in immunocytochemical studies of hawkmoth antennae (Nolte et al., 2013; Stengl, 1994; Stengl and Hildebrand, 1990). Our hypothesis of differential distribution of Orco homomers in the some and dendrite compartment, and OR-Orco heteromers in the cilia is based on differential immunocytochemical localization of Drosophila ORs mainly in the cilia compartment (Benton et al., 2006).

      We clarified our manuscript accordingly.

      (4) Some aspects of the authors' models, most notably the decision to phase align/optimize their DD and OLC15 recordings, are likely to bias their interpretations.

      It is consensus that insects display daily and circadian rhythms in pheromone-dependent mating, odor-gated feeding, and egg-laying behavior that phase-locks to environmental rhythms, corresponding with daily/circadian rhythms of sensory neuron physiology (e.g., Merlin et al., 2007; Rymer et al., 2007; Schendzielorz et al., 2015, 2012). However, circadian rhythms can be easily masked by stress, like the disturbances during an experimentally very challenging long-term recording experiment over several days. In addition, we observed over the years in our animal raising facility that in 17:7 light-dark cycles the originally nocturnal hawkmoths M. sexta distribute their activity patterns over the course of the day, finding nocturnal as well as diurnal hawkmoths. Thus, light-dark cycles were not enough to ensure phase-synchronized behavioral rhythms, and it is very likely that the nocturnal hawkmoths, next to stress signals, rely heavily on pheromone/odor dependent synchronization as also found in other moth species (Ghosh et al., 2024). Because we focus on spontaneous activity and not on pheromone-dependent physiology in this study, we used isolated males that were never exposed to the female pheromones, taking phase dispersal into account. Therefore, it became necessary in free-running conditions to first determine the respective behavioral rhythm for each animal, and then to phase-align their activity patterns to allow for statistical analysis. Otherwise, circadian differences would average out in a phase-dispersed free-running population. As requested by the referees in point (7), we added RAIN to test for rhythmicity in each of our recordings and revised the manuscript accordingly.

      Furthermore, in preliminary experiments we briefly exposed hawkmoths to pheromone the night before the start of the experiment. However, we failed to obtain phase-synchronized spiking rhythms. Most likely, a circadian pattern of pheromone exposure would have been necessary as zeitgeber, which could not be used here due to long-term pheromone-dependent effects in spiking activity. These results are added as supplementary figure to Fig 3.

      (5) The tip recordings from long trichoid sensilla are critical aspects of this study. These recordings were carried out on upper sensillar tips located on the distal-most second annulus. Since there are approximately 80 annuli on the Manduca antennae, it is unclear whether the recordings are representative of the antennal response.

      We think the reviewers might have misinterpreted our description of the recording site. In the Methods, we state that we clip off the 20 most distal annuli (leaving a stump of about 60 annuli) and insert the reference electrode into the flagellum up to the second annulus from the cut end, i.e., the recording sites are located at 2/3 – 3/4 of the antenna length as seen from the head of the animal. We clarified this in the Methods section.

      In addition, our lab did show with antibody stainings against Orco that apparently all ORNs that innervate long and short trichoid sensilla along the whole flagellum express the same staining pattern (Nolte et al., 2016). Lee and Strausfeld (1990) mapped all types of antennal sensilla, and together with pheromone-dependent tip-recordings of Kaissling et al. (1989) it was shown that most of the male antennal sensilla are pheromone-sensitive long trichoid sensilla, with one of the two innervating ORNs always responding to bombykal, ensuring high sensitivity to pheromone detection. Furthermore, our patch clamp recordings of primary cell cultures of whole male antennae found largely overlapping ion channel populations across ORNs (review: (Stengl, 2010)). This would indicate that all ORNs, whether they express ORs sensitive to pheromone or general odorants, could potentially share the same Orco-dependent spontaneous activity rhythms. Furthermore, in our lab, different experimenters from different years that recorded from long trichoid sensilla on different annuli did not detect obvious differences in neither the spontaneous activity nor the pheromone responses (c.f., Dolzer et al., 2003; Gawalek and Stengl, 2018; Schneider et al., 2025). Thus, it is very likely that we are reporting a general encoding mechanism that is not locally restricted along the antennal flagellum and is very likely shared by all types of OR-Orco expressing ORNs.

      (6.1) The authors do not provide any data in support of their cAMP/cGMP-based Orco gating…

      There are publications supporting cyclic nucleotide gating of Orco in Drosophila, but only after previous phosphorylation via protein kinase C (PKC; review: (Wicher and Miazzi, 2021)). Since Orco is very conserved among insect species, it is likely that PKC- and cGMP/cAMP-dependent regulations are present for Orco in other insect species. To test this, we are currently characterizing second messenger-dependence of spontaneous spiking activity, which is the focus of a follow-up manuscript. Nevertheless, to provide more evidence for our hypothesis of the current manuscript, we added a new set of tip-recording experiments that demonstrate cAMP-dependent gating of Orco. Because of the addition of this figure, we merged figures 8-10 into Figure 8 and added the cAMP data as Figure 9.

      (6.2) … and the PTTF model proposed is somewhat disappointing.

      For a detailed introduction of our PTFL membrane clock hypothesis please see our opinion paper that we refer to in the manuscript (Stengl and Schneider, 2024). We added clarification of how Orco activation can influence cAMP levels. A more elaborate PTFL clock model including many more of the identified ion channels in hawkmoth ORNs is the focus of another manuscript to come.

      (6.3) The model seems to be influenced by their long-held proposal that insect olfactory signaling has a critical metabotropic component involving cyclic nucleotides, PKC, etc, a view that may be influenced by the use of Orco homomeric complexes generated in HEK cells.

      Indeed, we propose a metabotropic pheromone-transduction cascade, which in moths and cockroaches is based on G-protein-mediated activation of phospholipase C but not on adenylyl cyclase activation. Our hypothesis is not influenced by HEK cell heterologous expression studies of Orco but is supported by our own work comparing in vivo tip recordings of intact hawkmoths with patch clamp experiments on hawkmoth primary cell cultures of olfactory receptor neurons, which are able to respond to their species-specific pheromones in vitro (Schneider et al., 2025; Stengl, 2010; Stengl and Funk, 2013; Wicher and Miazzi, 2021). In addition, a multitude of publications by other laboratories with in vivo and in vitro studies using physiological, genetic, and immunocytochemical assays all support a metabotropic signal transduction cascade in insect olfaction (Stengl, 2010; Stengl and Funk, 2013; Takagi et al., 2025; Wicher and Miazzi, 2021). In contrast, the hypothesis suggesting a solely ionotropic pheromone- and general odor-dependent transduction cascade for all insect species is based on very sparse experimental evidence, based primarily on heterologous expression studies such as HEK cells that lack the insect’s WT molecular surroundings, and thus, cannot predict OR-Orco function in vivo. Furthermore, the ionotropic hypothesis is heavily based upon the argument that an inverse 7TM receptor cannot couple to G-proteins, which lacks careful backup via biochemical and structural studies. In addition, the ionotropic hypothesis lacks support via carefully performed physiological in vivo studies in different insect species that paid attention to analysis of the distinct kinetic components of ORN´s odor/pheromone responses and that employ physiological concentrations and durations of odor/pheromone stimuli (please see our most recent publication by Schneider et al. (2025)). We added references to the possible odor transduction mechanisms to the introduction.

      (6.4) Nevertheless, structural studies on Orco do not support a cyclic nucleotide binding site, although PKC-based phosphorylation has been implicated in the fine-tuning/adaptation of olfactory signaling.

      While structural studies did not find evidence for conserved known cyclic nucleotide binding sites on Orco, this does not exclude the presence of indirect cAMP effects via e.g., Orco subunits complexing with other molecules under direct cAMP control, such as other ion channel subunits. Furthermore, it does not exclude so far unknown binding sites, or via sites that fold out only after a specific sequence of previous phosphorylations of the many phosphorylation sites on Orco. Indeed, physiological studies in Drosophila presented evidence for cyclic nucleotide dependence of Orco after previous PKC-dependent phosphorylation (Getahun et al., 2013). Our ongoing in vivo experiments in hawkmoths further corroborate a zeitgeber time-dependent PKC- and cyclic nucleotide-dependent modulation of Orco. These detailed studies will be published in a follow-up publication. In the revised version of this manuscript, we added tip-recording experiments that indicate cAMP involvement in Orco gating (new Figure 9).

      (7) Because only 5/11 LD and 7/10 DD animals showed daily rhythms, with averages lacking clear daily modulation, the methods are not sufficiently reliable enough to reveal novel underlying mechanisms of circadian rhythm generation. The reported results are therefore not yet reliable or quantifiable. To quantify their results, the authors should apply tests for circadian rhythmicity using methods such as RAIN, JTK CYCLE, MetaCycle, or Echo. The use of FFT and Wavelet is applauded, but these methods do not have tests of significance for rhythms and can be biased when analyzing data in which there could only be 1-3 circadian cycles. Because the conclusions appear to be based on 11-12 neurons that were recorded for 2-4 days, the reader is concerned that the methods are not yet perfected to provide strong evidence for circadian regulation of spontaneous firing of ORNs. The average data (e.g., Figure 3Bii and 3Cii) highlight the apparent lack of daily rhythms. In summary, the results would be more compelling if more than 50% of the recordings had significant circadian amplitudes and with similar periods and phases.

      The long-term tip-recordings of intact hawkmoths are very challenging and take a very long time to accomplish, thus, we are very happy that we succeeded in obtaining so many of them (N=40). We are thankful to the reviewers’ suggestion to use RAIN since this analysis revealed circadian rhythms in 7 of 11 LD recordings, 8 of 12 DD recordings, and 2 of 12 OLC15 recordings. Please see also our response to (4) above, commenting the phase-dispersal of activity rhythms observed in our experiments, as well as in the behavior of hawkmoth males in the mating cage.

      (8) The statement that circadian patterns of ORN firing are lost with the Orco antagonist (OLC15) is not strongly supported. The manuscript should be revised to quantify how Orco changed circadian amplitude in the 12 recorded neurons. Measures of circadian amplitude can avoid confusing/vague statements like Line 394 “low and high frequency bands appeared to merge during the activity phase around ZT 0 in the animals that showed clear circadian rhythms (N = 5 of 11 in LD)”. The conclusion that Orco blocks circadian firing appears to be contradicted by Figure 6, which indicates that ~6 of these neurons had circadian periods detected by wavelet. The manuscript would be strengthened with details about the specificity and reproducibility of the Orco antagonist. The authors quantify the gradual decrease in firing with the slope of a linear fit to estimate how the “effectiveness [of OLC15] increased over time.” They conclude that the drug “obliterated circadian rhythms and attenuated the spontaneous activity in several, but not all experiments (N = 8 of 12).” The report would be greatly strengthened with corroborating data from additional Orco antagonists and additional doses of OLC15 (the authors use only 50 uM OLC15).

      According to the valuable suggestions of the referees, we used RAIN to detect circadian rhythms in the spiking attributes in each individual animal. Since only 2 of 12 animals displayed a circadian rhythm in OLC15, statistical comparison of circadian amplitudes is not possible. We revised the results section accordingly and added to the figure legend to make it clearer that the heat maps in Fig 5 are representative from one animal each and not averages across animals.

      As the reviewer states correctly in (7), wavelet results of circadian rhythmicity must be interpreted carefully because of the low number of circadian cycles in ~3-4 day recordings. Since the heatmaps in Figure 5 visually revealed the presence of ultradian rhythms, the main focus of the wavelet analysis in Figure 6 is in the detection and quantification of ultradian periods up to 20 h.

      We revised the Methods section to include references to previous experiments that characterized the effect of different doses of OLC15 and other Orco antagonists and agonists in M. sexta antennae (Nolte et al., 2016). Please see also our response to (1).

      (9) The manuscript includes several statements that are more speculation than conclusion. For example, there is no evidence for tuning or plasticity in this report. Statements like the following should be removed or addressed with experiments that show changes in odor response specificity or sensitivity: "ORN signalosomes are highly plastic endogenous PTFL clocks comprising receptors for circadian and ultradian Zeitgebers that allow to tune into internal physiological and external environmental rhythms as basis for active sensing." (Discussion Line 622). The paper concludes that (line 380) "mean frequency of spontaneous spiking and the frequency of bursting expressed daily modulation, and are both most likely controlled via a circadian clock that targets the leak channel Orco." This is too bold given the available results.

      We revised the manuscript accordingly and clarified which statements are supported via published evidence and which are predictions based upon our novel hypothesis published in our opinion paper (Stengl and Schneider, 2024).

      (10.1) Because Orco conductance is modulated by cyclic nucleotides, it remains highly plausible that circadian regulation occurs upstream at the level of signaling pathways (e.g., calcium, calcium-binding proteins, GPCRs, cyclases, phosphodiesterases).

      We agree with the referees that it is very likely that there are multiple layers of interconnected feedback cycles that control Orco localization and activity. Our novel hypothesis suggests interlocked TTFL and PTFL control of physiological circadian rhythms, not strictly hierarchical TTFL control, which would require a daily turnover of membrane proteins and transcriptional control via the established TTFL clock in insect ORNs. We are currently searching for TTFL control at all levels of odor/pheromone transduction using ZT-dependent transcriptomics in combination with qPCR and single-nucleus transcriptomics, involving also all the molecules suggested by the referees. These studies are ongoing, are very time- and money-consuming, and are beyond the scope of this manuscript. However, we added a set of experiments to this manuscript in which we demonstrate that the effect of increased cAMP on the spontaneous spiking activity is mediated by Orco (new Figure 9).

      (10.2) The possibility that circadian oscillations of cyclic nucleotides are generated by the canonical TTFL mechanism has not been excluded. In fact, extensive work in Drosophila has demonstrated that the TTFL-based molecular clock proteins are required for circadian rhythms in olfaction.

      Our experiments that test circadian TTFL control at different levels of the cAMP transduction cascade in hawkmoth antennae are on the way and are part of another publication. In section 6.2 we already stated that our experiments do not exclude that Orco is under indirect control of the TTFL. We revised our discussion accordingly.

      The experiments published for TTFL dependent control of Drosophila olfaction that we are aware of (Krishnan et al., 1999; Tanoue et al., 2004) do not exclude interlinked PTFL and TTFL clocks. Krishnan et al. (1999) demonstrated that the TTFL clock in antennal olfactory receptor neurons correlates with circadian rhythms in odor responses measured in electroantennogram (EAG) recordings, not in single sensillum recordings as in our experiments. EAG recordings comprise not only voltage responses of the olfactory sensory neurons but also voltage changes generated in non-neuronal antennal cells such as trichogen and tormogen cells that built the transepithelial potential gradient via vATPases that generates the high K<sup>+</sup> concentration in the sensillum lymph (Jain et al., 2024; Klein, 1992; Thurm and Küppers, 1980). In addition, EAG recordings most likely contain responses of afferent neurons originating from somata in the brain that maintain central control of the antennae. Thus, EAG recordings are difficult to interpret.

      (11) A defining feature of circadian oscillators is the feedback mechanism that generates a time delay (e.g., PERIOD/TIMELESS repressing their own transcription). While the authors describe how cyclic nucleotides can regulate Orco conductance, they do not provide a convincing explanation of how Orco activity could, in turn, feed back into the proposed PTFL to sustain oscillations. For these reasons, the authors should consider:

      (a) Providing a broader discussion of non-TTFL models of circadian rhythms (e.g., redox cycles, post-translational modifications).

      We revised the discussion accordingly.

      (b) Reassessing Orco expression using a higher-resolution temporal sampling ({greater than or equal to}6 timepoints per 24 h).

      We added those experiments to the revised version of the manuscript (see our response to (2)).

      (c) Clarifying or revising the PTFL model to explicitly address how feedback would be achieved. Alternatively, the data may be more consistent with Orco conductance rhythms being regulated by post-translational mechanisms downstream of the canonical TTFL oscillator, as suggested by the Drosophila olfactory system literature.

      We added possible negative feedback elements to the Discussion to explain how our proposed PTFL could in principle work independent of TTFL clock.

      Minor weaknesses:

      (1) The authors should compare the firing patterns of ORN neurons to the bursts, clusters, and packets of retinal efferent spikes reported in Liu JS and Passaglia CL (2011; JBR). By comparing measures in moths to measures in Limulus, the authors might be able to address the question: Is the daily firing pattern of ORN neurons likely a conserved feature of circadian control of sensory sensitivity?

      We have revised the discussion accordingly.

      (2) The methods need further details. For example, it is unclear if or how single neuron activity was discriminated and whether the results were compromised by the relatively large environmental fluctuations in temperature (21-27oC), humidity (35-60%), or other cues known to modulate spontaneous firing.

      These large fluctuations stem from doing experiments at different seasons (higher temperature and humidity in the summer months, lower temperature and humidity in winter). Throughout each individual experiment, conditions were stable. We clarified the Methods section accordingly.

      Recommendations for the authors:

      The authors should post the code for their computational model to a repository like GitHub.

      The code for the computational model is now available at https://github.com/a-c-schneider/VijayanForlinoEtAl2025_Model.git

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    1. eLife Assessment

      This study addresses an important question in aging biology by combining metabolic, genetic, and functional approaches to examine how cytosolic acetyl-CoA metabolism influences late-life fitness in replicatively aging yeast. The evidence supporting the roles of AMPK activation, mitochondrial acetyl-CoA utilization, and fatty acid synthesis in shaping distinct aging-associated phenotypes is convincing overall, with the engineered A2A strain providing a particularly elegant demonstration of coordinated metabolic regulation. However, several conclusions would benefit from clarification or moderation, particularly regarding the relationship between late-life fitness and replicative lifespan, the interpretation of "senescence," the proposed existence of distinct aging subpopulations, and the extent to which the data support mechanistic claims about lipid starvation, acetyl-CoA excess, and chromatin-based aging pathways.

    2. Reviewer #1 (Public review):

      This rigorous and creative study uses an elegant combination of metabolomics, transcriptomics, and budding yeast molecular genetics to discover that (i) activating AMPK to maintain mitochondrial respiration fueled by cytosolic Acetyl CoA and (ii) increasing fatty acid synthesis independent of respiration drive independent pathways that increase the fitness of replicatively-aged budding yeast cells, albeit without increasing their lifespan. This work will be of interest to scientists in the field of aging and metabolism. Some clarifications in the text would address the following concerns, which would increase the impact of the study:

      (1) What does activation of AMPK (via PGDP-Sak1 expression) do to the replicative lifespan? How many bud scars, in general, do the subpopulations that are older - yet have less Tom70 (increased mitochondrial fitness) - have, after the 48 hrs time point that they are examining? How many divisions occurred in this 48hr time period - i.e. is it long enough to have all cells reach the end of their replicative lifespan? This information is important to rule out that a subset of the mutant cells just divided faster and hence had more divisions within 48 hrs (growing faster and living longer are different things). Having identical growth curves doesn't indicate per se that they all divide at the same rate, as there may be a subpopulation that divides faster and a subpopulation that doesn't grow so well.

      (2) A2A cells do not have an extended replicative lifespan (RLS) but show an increase in the "low senescence" population (Figure 2). If the cells are not becoming senescent, why don't they have longer RLS? Not having a longer lifespan seems inconsistent with the statement that "bud scar counting confirmed that A2A cells reach a higher age than wild type", which comes back to how many times the cells can divide in the 48hr timepoint studied and their rate of cell division? Also, the lifespan curve shown is plotted against time, not cell division number, which does not take into account different division times of cells within the population (described above). It would be much more useful to show standard lifespan curves showing cell division numbers per lifespan per cell.

      (3) Increased "fitness" of the old cells is implied from the increased size of the colonies that the old cells can make. However, this is a measure of the fitness of the daughters per se, not the old mother cells. Are the old mothers just passing on healthier mitochondria and more lipids to the daughters, such that they can divide more times? If the aged cells have an "increased fitness", why don't they divide more times themselves (i.e. live longer?).

      (4) The statement is made that "these experiments define two classes of aging cells with distinct metabolic needs, coherent with the model of two aging trajectories previously proposed (referencing Nan Hao's work)". However, the big difference here is that in Nan Hao's work, their two aging trajectories influenced the length of lifespan, but that does not appear to be the case here. That distinction should be made clear. Perhaps the authors could also speculate as to why the A2A yeast stops dividing after presumably the same number of cell divisions, even though they have an activated AMPK and activated fatty acid synthesis pathway.

      (5) I am a bit confused by the use of the word "senescence" by this lab here and in their previous growth on galactose studies. If yeast don't senesce, which is usually defined as an irreversible arrest of the cell cycle where cells stop dividing, shouldn't the yeast that do not senesce still be dividing and hence have a longer lifespan? Should a different term be used rather than senescence? Such as "fitness late in life". The authors giving their definition of senescence may help reduce this apparent contradiction.

    3. Reviewer #2 (Public review):

      Summary:

      In this study, the authors investigate how cytosolic acetyl-CoA metabolism influences replicative aging in budding yeast. They propose that acetyl-CoA regulates aging through three major pathways: (1) mitochondrial transport to support mitochondrial function, (2) fatty acid synthesis, and (3) global protein acetylation. The data show that AMPK activation promotes mitochondrial import of acetyl-CoA and partially mitigates mitochondrial decline in a subset of aging cells.

      Furthermore, the engineered A2A strain, which enhances mitochondrial acetyl-CoA utilization while relieving inhibition of fatty acid synthesis, increases the proportion of cells exhibiting a "low senescence" phenotype.

      Overall, this is a thoughtful and potentially impactful study that advances our understanding of metabolic control of aging. Addressing the points below, particularly by refining interpretations and, where feasible, incorporating additional analyses, will further strengthen the manuscript and its conclusions.

      Strengths:

      The study has several notable strengths. It addresses an important question by shifting the focus from lifespan to preservation of late-life fitness, which is highly relevant to aging biology. The work integrates metabolic, genetic, and functional analyses to link cytosolic acetyl-CoA flux with distinct aging outcomes, and the engineering of the A2A strain provides a clear and elegant demonstration of how coordinated pathway modulation can improve cellular fitness.

      Weaknesses:

      (1) While the manuscript focuses on mitochondrial transport and fatty acid synthesis, cytosolic acetyl-CoA is also a key regulator of histone acetylation and chromatin silencing. It would strengthen the study to consider whether acetyl-CoA depletion contributes to improved fitness through enhanced rDNA silencing. Given the well-established role of rDNA instability in yeast aging, additional experiments examining rDNA silencing and stability would be valuable. For example, monitoring rDNA copy number changes (not necessarily ERCs) under AMPK activation, oleic acid supplementation, and in the A2A strain, similar to approaches used in the authors' prior work, would help clarify whether chromatin regulation contributes to the observed phenotypes.

      (2) The current data do not fully distinguish whether AMPK activation and oleic acid supplementation act on distinct subpopulations of aging cells. An alternative explanation is that oleic acid supplementation enhances mitochondrial function and acts additively with AMPK activation, thereby increasing the fraction of cells in the "low senescence" state. Since this distinction is not central to the main conclusions, I suggest softening the language around subpopulation specificity. Emphasizing instead that the A2A strain coordinately modulates multiple branches of acetyl-CoA metabolism to improve late-life fitness would maintain the strength of the central message without overinterpretation.

      (3) The manuscript proposes that lipid starvation and excess acetyl-CoA are major drivers of senescence in distinct subpopulations of wild-type aging cells. This conclusion is not yet fully supported by the presented data. Direct measurements of age-dependent divergence in acetyl-CoA and fatty acid levels at the single-cell level would be needed to substantiate this model. Based on the current evidence, a more conservative interpretation would be that aging cells exhibit differential sensitivity to perturbations in acetyl-CoA and lipid metabolism. Accordingly, I recommend revising the statement in the Abstract ("We further implicate lipid starvation and excess acetyl coenzyme A availability as major drivers of senescence...") and the corresponding discussion text to better align with the data.

    4. Reviewer #3 (Public review):

      Summary:

      These findings suggest that PGPD-SAK1 yeast show a subpopulation with lowered TOM70-GFP expression in high bud scar staining aged cells. Deletion of CAT2 or MLS1 reduces this effect. A PGPD-SAK1 acc1S1157A double mutant (called "A2A" here) shows an even larger effect of lowered tom70 expression in high bud scar staining aged cells. Utilization of various additional mutants involved in acetyl-CoA transport, carnitine shuttle, respiration, etc., leads the authors to conclude that these shifts in TOM70-GFP in aged cells are linked to the AMPK-fatty acid metabolic regulatory system.

      Strengths:

      These extensive and clearly described experiments reveal interesting changes in TOM70-GFP intensity in subsets of aged yeast in several mutants eventually identified as linked to the AMPK-fatty acid metabolic regulatory system.

      Weaknesses:

      (1) 3 biological replicates for mRNASeq is low.

      (2) While "Traditional conceptions of ageing implicate a progressive accumulation of damage leading to systemic degradation in performance until death, with evolutionary pressures acting to maximise early life fitness and fecundity at the expense of ageing health." is tangential perhaps to the data and conclusions of the study, both claims of this sentence are at best controversial, and the manuscript is no weaker for their omission.

      (3) The statement that "Here, we determine the basis of senescence and fitness loss in replicatively ageing yeast" is a bit strong as a summary of the present careful work presented here. If the authors had created yeast mutants that retained fitness indefinitely, this would be a more appropriate strength of claim to summarize the work.

    5. Author response:

      Public Reviews:

      Reviewer #1 (Public review):

      This rigorous and creative study uses an elegant combination of metabolomics, transcriptomics, and budding yeast molecular genetics to discover that (i) activating AMPK to maintain mitochondrial respiration fueled by cytosolic Acetyl CoA and (ii) increasing fatty acid synthesis independent of respiration drive independent pathways that increase the fitness of replicatively-aged budding yeast cells, albeit without increasing their lifespan. This work will be of interest to scientists in the field of aging and metabolism. Some clarifications in the text would address the following concerns, which would increase the impact of the study:

      (1) What does activation of AMPK (via PGDP-Sak1 expression) do to the replicative lifespan? How many bud scars, in general, do the subpopulations that are older - yet have less Tom70 (increased mitochondrial fitness) - have, after the 48 hrs time point that they are examining? How many divisions occurred in this 48hr time period - i.e. is it long enough to have all cells reach the end of their replicative lifespan? This information is important to rule out that a subset of the mutant cells just divided faster and hence had more divisions within 48 hrs (growing faster and living longer are different things). Having identical growth curves doesn't indicate per se that they all divide at the same rate, as there may be a subpopulation that divides faster and a subpopulation that doesn't grow so well.

      Increasing AMPK activity increases replicative lifespan [PMID: 25869125], but given our finding that AMPK activation splits the population, such replicative lifespan assays are hard to interpret. Bud scar counts have a similar issue. Hence we restricted the lifespan and bud scar analyses to wt and A2A which are more homogenous (Figures S2 B and E). A2A cells at 48h have ~25% more bud scars than wt cells. Yes, by 48h most of the cells have lost viability (Figure 2E). The reviewer is correct that you can't properly compare the lifespan curves if the cells divide at different rates, hence our follow-up test of wt at 48h vs A2A at 40h viability after we had confirmed that these timepoints captured cells at equivalent replicative ages (Figure 2D,E). This shows that viability of A2A is slightly lower than wt at matched age, indicating a slightly shorter lifespan.

      (2) A2A cells do not have an extended replicative lifespan (RLS) but show an increase in the "low senescence" population (Figure 2). If the cells are not becoming senescent, why don't they have longer RLS? Not having a longer lifespan seems inconsistent with the statement that "bud scar counting confirmed that A2A cells reach a higher age than wild type", which comes back to how many times the cells can divide in the 48hr timepoint studied and their rate of cell division? Also, the lifespan curve shown is plotted against time, not cell division number, which does not take into account different division times of cells within the population (described above). It would be much more useful to show standard lifespan curves showing cell division numbers per lifespan per cell.

      Our observation that cells can reach the end of life without senescing is consistent with other studies that have studied the life course of individual cells by microscopy [PMID: 31291577, 32675375]. These studies always highlight some proportion of the cells that reach the end of life with no or minimal senescence, though this fraction varies with the experimental system. The question of why cells lose viability without senescing is a complete unknown in the field, but reflects a wider lack of consensus as to why yeast lose viability with replicative age.

      We are wary about making strong statements on lifespan for exactly the reason the reviewer picks out. In liquid culture we can only assess viability over time, and it is clear from the comparison of liquid and solid media lifespans performed by the Gottschling lab [PMID: 19652178] that culture system has a huge effect on lifespan, with cells in classical microdissection-based lifespan assays living far longer than they do in liquid. This of course means that classical microdissection assays are not very useful for A2A so we are left with an unsatisfactory approximation. We have therefore restricted our conclusion on lifespan to simply say that lifespan of A2A cells is not extended which our data in Figures 2D,E,S2B does support (see also answer to Q1), and therefore with the majority of A2A cells showing low senescence marks and high fitness at 48h we can conclude that lifespan and fitness loss must be separable.

      We will note these limitations of lifespan measurements in the manuscript.

      (3) Increased "fitness" of the old cells is implied from the increased size of the colonies that the old cells can make. However, this is a measure of the fitness of the daughters per se, not the old mother cells. Are the old mothers just passing on healthier mitochondria and more lipids to the daughters, such that they can divide more times? If the aged cells have an "increased fitness", why don't they divide more times themselves (i.e. live longer?).

      Yes, colony growth speed is defined by daughter cell replication, and as long as the daughters and subsequent generations divide at the same rate irrespective of whether they come from a young or old mothers then the size of the colony after 24 hours varies based on the time it took the initial mother to produce a daughter. This is what the assay really measures. We note that aged wildtype mothers often do not divide at all in the first 24 hours after being put on an agar plate (hence the tiny reported colony size), even though they do eventually produce a daughter which then forms a colony, whereas A2A cells tend to produce the first daughter rapidly whether young or old. It is known that daughters of aged wildtype mothers also divide slower, which will also contribute to differences in colony size, and this may well result from a lipid and/or mitochondrial contribution, but the primary driver of colony size in 24 hours is the time the mother took to initially divide. We will add this detail to the manuscript.

      As noted above, the mechanistic basis of lifespan is unknown, but although senescence can shorten lifespan, our work and that of others shows that lifespan is still limited in the absence of senescence.

      (4) The statement is made that "these experiments define two classes of aging cells with distinct metabolic needs, coherent with the model of two aging trajectories previously proposed (referencing Nan Hao's work)". However, the big difference here is that in Nan Hao's work, their two aging trajectories influenced the length of lifespan, but that does not appear to be the case here. That distinction should be made clear. Perhaps the authors could also speculate as to why the A2A yeast stops dividing after presumably the same number of cell divisions, even though they have an activated AMPK and activated fatty acid synthesis pathway.

      We will add this distinction. As noted above, we are wary of making strong statements regarding lifespan as the assays we can do in liquid culture are limited. We are therefore similarly wary about speculating about causes for the lack of lifespan difference because in reality all we can do is rule out a big effect. We would love to speculate on why the A2A cells don't have an extended lifespan, but at this point we don't have any good ideas on this point!

      (5) I am a bit confused by the use of the word "senescence" by this lab here and in their previous growth on galactose studies. If yeast don't senesce, which is usually defined as an irreversible arrest of the cell cycle where cells stop dividing, shouldn't the yeast that do not senesce still be dividing and hence have a longer lifespan? Should a different term be used rather than senescence? Such as "fitness late in life". The authors giving their definition of senescence may help reduce this apparent contradiction.

      We completely agree, this is confusing and noted this distinction in the Introduction. Use of the term senescence to mean a loss of fitness late in life in yeast stems from the classical definition of senescence as applied to whole organisms. However, the term senescence as applied to cells has a more specific meaning in terms of the cell cycle as the reviewer notes. As an individual S. cerevisiae is both a cell and an organism, the terminology clashes. However, the marker we largely employ (Tom70-GFP) which in our hands is a very good proxy for fitness was originally defined as marking the senescence entry point (SEP), so overall we feel we can't avoid the term.

      Reviewer #2 (Public review):

      Summary:

      In this study, the authors investigate how cytosolic acetyl-CoA metabolism influences replicative aging in budding yeast. They propose that acetyl-CoA regulates aging through three major pathways: (1) mitochondrial transport to support mitochondrial function, (2) fatty acid synthesis, and (3) global protein acetylation. The data show that AMPK activation promotes mitochondrial import of acetyl-CoA and partially mitigates mitochondrial decline in a subset of aging cells.

      Furthermore, the engineered A2A strain, which enhances mitochondrial acetyl-CoA utilization while relieving inhibition of fatty acid synthesis, increases the proportion of cells exhibiting a "low senescence" phenotype.

      Overall, this is a thoughtful and potentially impactful study that advances our understanding of metab to olic control of aging. Addressing the points below, particularly by refining interpretations and, where feasible, incorporating additional analyses, will further strengthen the manuscript and its conclusions.

      Strengths:

      The study has several notable strengths. It addresses an important question by shifting the focus from lifespan to preservation of late-life fitness, which is highly relevant to aging biology. The work integrates metabolic, genetic, and functional analyses to link cytosolic acetyl-CoA flux with distinct aging outcomes, and the engineering of the A2A strain provides a clear and elegant demonstration of how coordinated pathway modulation can improve cellular fitness.

      Weaknesses:

      (1) While the manuscript focuses on mitochondrial transport and fatty acid synthesis, cytosolic acetyl-CoA is also a key regulator of histone acetylation and chromatin silencing. It would strengthen the study to consider whether acetyl-CoA depletion contributes to improved fitness through enhanced rDNA silencing. Given the well-established role of rDNA instability in yeast aging, additional experiments examining rDNA silencing and stability would be valuable. For example, monitoring rDNA copy number changes (not necessarily ERCs) under AMPK activation, oleic acid supplementation, and in the A2A strain, similar to approaches used in the authors' prior work, would help clarify whether chromatin regulation contributes to the observed phenotypes.

      We have data addressing this point that we will add to the manuscript. In short, we see no difference in gene expression from Sir2-repressed sub-telomeric regions or MAT loci, but the genome-wide gene expression dysregulation associated with age is partially suppressed in PGPD-SAK1. However, A2A does not suppress this further, so it is not critical for the suppression of senescence in A2A though we are following this up. ERC accumulation is higher in A2A at 48h, consistent with the cells being older, meaning that ERCs are unlinked to senescence onset as we have previously reported. There is a strong upregulation of transcripts from Sir2-repressed rDNA intergenic spacers with age in all genotypes, but we attribute this simply to the copy number increase of these regions on ERCs rather than a defect in silencing. We have previously looked for heritable changes in rDNA copy number arising during ageing and found (to our surprise) absolutely nothing, so we don't expect any changes under these conditions.

      (2) The current data do not fully distinguish whether AMPK activation and oleic acid supplementation act on distinct subpopulations of aging cells. An alternative explanation is that oleic acid supplementation enhances mitochondrial function and acts additively with AMPK activation, thereby increasing the fraction of cells in the "low senescence" state. Since this distinction is not central to the main conclusions, I suggest softening the language around subpopulation specificity. Emphasizing instead that the A2A strain coordinately modulates multiple branches of acetyl-CoA metabolism to improve late-life fitness would maintain the strength of the central message without overinterpretation.

      We agree that oleic acid and the lipids produced downstream of Acc1 in A2A may improve late life fitness via enhanced mitochondrial function, and in support of this Oxygen Consumption Rate is marginally (though significantly) higher in A2A than PGPD-SAK1. We will add this data to the manuscript. However, we disagree with the interpretation of an additive effect as we report throughout the study that AMPK activation and lipid biosynthesis/supplementation affect different sub-populations of cells. We do not observe populations of intermediate senescence cells, rather by flow cytometry and fitness assays we observe individual cells in binary low senescence or high senescence states.

      (3) The manuscript proposes that lipid starvation and excess acetyl-CoA are major drivers of senescence in distinct subpopulations of wild-type aging cells. This conclusion is not yet fully supported by the presented data. Direct measurements of age-dependent divergence in acetyl-CoA and fatty acid levels at the single-cell level would be needed to substantiate this model. Based on the current evidence, a more conservative interpretation would be that aging cells exhibit differential sensitivity to perturbations in acetyl-CoA and lipid metabolism. Accordingly, I recommend revising the statement in the Abstract ("We further implicate lipid starvation and excess acetyl coenzyme A availability as major drivers of senescence...") and the corresponding discussion text to better align with the data.

      We agree and will adjust the abstract to make it clearer that the lipid starvation / excess acetyl coA interpretation is a model.

      Reviewer #3 (Public review):

      Summary:

      These findings suggest that PGPD-SAK1 yeast show a subpopulation with lowered TOM70-GFP expression in high bud scar staining aged cells. Deletion of CAT2 or MLS1 reduces this effect. A PGPD-SAK1 acc1S1157A double mutant (called "A2A" here) shows an even larger effect of lowered tom70 expression in high bud scar staining aged cells. Utilization of various additional mutants involved in acetyl-CoA transport, carnitine shuttle, respiration, etc., leads the authors to conclude that these shifts in TOM70-GFP in aged cells are linked to the AMPK-fatty acid metabolic regulatory system.

      Strengths:

      These extensive and clearly described experiments reveal interesting changes in TOM70-GFP intensity in subsets of aged yeast in several mutants eventually identified as linked to the AMPK-fatty acid metabolic regulatory system.

      Weaknesses:

      (1) 3 biological replicates for mRNASeq is low.

      Thank you for pointing this out. We performed another replicate after posting the initial preprint but didn’t update the figure in the eLIFe-reviewed version. We will add this to the scatter plots and analysis in Figure 1, the findings have not changed.

      (2) While "Traditional conceptions of ageing implicate a progressive accumulation of damage leading to systemic degradation in performance until death, with evolutionary pressures acting to maximise early life fitness and fecundity at the expense of ageing health." is tangential perhaps to the data and conclusions of the study, both claims of this sentence are at best controversial, and the manuscript is no weaker for their omission.

      We actually feel that this sentence is very important to the message of the manuscript, which is that ageing does not necessarily have to involve a loss of fitness before death. Ageing is often described as the progressive wearing out of components leading to decline and death (with an old car often used as an analogy); in the ageing field this is certainly controversial, but outside the field this remains the normal understanding. We think it is important to state this widely held viewpoint with which our findings are hard to reconcile.

      Our interpretation that yeast are bet-hedging as a population growth strategy and this drives ageing in the long term is a classic antagonistic pleiotropy - we will add this term (from the citation that is already in the manuscript) and clarify in the discussion to make it obvious why we are introducing this concept in the introduction.

      (3) The statement that "Here, we determine the basis of senescence and fitness loss in replicatively ageing yeast" is a bit strong as a summary of the present careful work presented here. If the authors had created yeast mutants that retained fitness indefinitely, this would be a more appropriate strength of claim to summarize the work.

      Indeed - we will refine this sentence.

    1. eLife Assessment

      This important study provides convincing data suggesting that subcellular localization of the spatial regulator of cell division, MinD, is an intrinsic feature of the protein's ability to associate with the membrane as both a dimer and a monomer. These findings distinguish the behavior of MinD in B. subtilis from its counterpart in E. coli and suggest that there is not a need to invoke additional localization factors. The reviewers felt that the revisions, particularly the additional experiments and changes to the text to make the experimental design and conclusions clearer, improve the quality of the manuscript.

    2. Reviewer #1 (Public review):

      [Editor's note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

      Summary:

      In this work the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole to pole oscillation whereby a time average minimum of the Min proteins at mid cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites, and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.

      Strengths:

      In the current study, the authors perform a detailed biochemical characterization of the in vitro ATPase activity of MinD and demonstrate that rapid hydrolysis is elicited by adding phospholipids. They further show using a collection of substitution mutants of MinD that both monomers and dimers bind to the membrane, and ATP occupancy changes the on and off rates. Identification, quantification, and tracking of discrete Halo-MinD populations was nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.

      In the revised manuscript, the authors now demonstrate localization and tracking data for minC and minJ deletion strains, which suggest that MinJ impacts MinD membrane cycling, but MinC does not. Additional in vitro work showed that the PDZ domain of MinJ modifies MinD ATP hydrolysis rates, and the authors propose that MinJ may promote MinD dimer formation.

      Weaknesses of the revised version: No major weaknesses.

    3. Reviewer #2 (Public review):

      Summary:

      Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.

      Strengths:

      The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.

    4. Author response:

      The following is the authors’ response to the previous reviews

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      In this work the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole to pole oscillation whereby a time average minimum of the Min proteins at mid cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites, and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.

      Strengths:

      In the current study, the authors perform a detailed biochemical characterization of the in vitro ATPase activity of MinD and demonstrate that rapid hydrolysis is elicited by adding phospholipids. They further show using a collection of substitution mutants of MinD that both monomers and dimers bind to the membrane, and ATP occupancy changes the on and off rates. Identification, quantification, and tracking of discrete Halo-MinD populations was nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.

      In the revised manuscript, the authors now demonstrate localization and tracking data for minC and minJ deletion strains, which suggest that MinJ impacts MinD membrane cycling, but MinC does not. Additional in vitro work showed that the PDZ domain of MinJ modifies MinD ATP hydrolysis rates, and the authors propose that MinJ may promote MinD dimer formation.

      Weaknesses of the revised version: No major weaknesses.

      We thank this reviewer for the positive evaluation of our manuscript and the precise summary of our findings.

      Reviewer #2 (Public review):

      Summary:

      Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.

      Strengths:

      The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.

      Weaknesses:

      The authors immobilized liposomes, for which they used E. coli total lipids, to measure ATPase activity and liposome association and dissociation of B. subtilis MinD. For these experiments would be more suitable to use B. subtilis total lipids as more biologically relevant data could be gained.

      Although the work is in detail and nicely compares the function of B. subtilis Min system with E. coli Min system, it lacks the comparison of the Min system function in other rod-shaped Gram-positive bacteria. I would suggest including in the Discussion the complexity of other Min systems. Especially, this complexity is seen in other rod-shaped and spore formers such as Clostridial species in which one of these Min systems or both are present, an oscillating E. coli Min system type and more static as in B. subtilis.

      Comments on revisions:

      I'm satisfied with the authors response to my private recommendation points. However, I thought that they would also respond to my points mentioned in Public Review part, weaknesses as shown above and update the revised version accordingly.

      We are very grateful to the reviewer for the positive comments and fully agree with the points raised. Due to the overall length of the manuscript, we initially omitted a discussion of the complexity of the Min system in certain Firmicutes. However, we agree that this aspect should be considered. Accordingly, we have now added a dedicated paragraph to the Discussion section addressing this point.

      We also agree that investigating different lipid compositions, including native membranes from Bacillus subtilis, represents a logical next step to further elucidate the influence of lipids on the MinD activity cycle. However, we consider this to constitute a separate project and therefore beyond the scope of the present study.

      Recommendations for the authors:

      Reviewing Editors:

      Some minor corrections are requested-the addition of a bit more details about the complexity of Min systems in other bacteria in particular to the discussion as suggested by Reviewer 2 would be very much appreciated.

      We thank the editors for their positive assessment and the clear recommendations. We have now added a dedicated paragraph to the Discussion section addressing the complexity of the Min system in Clostridioides.

      Reviewer #1 (Recommendations for the authors):

      The following corrections are requested:

      Abstract - Line 29 - Remove the word "solely" from this statement of the abstract. It would be wise to not be so rigid for a biological system that is only partially characterized and to allow for the possibility that biological factors, including local concentrations and/or other molecules, may yet be discovered to impact MinD activation under certain conditions.

      We agree and have amended the text to avoid a to restrictive statement.

      Line 38 - Remove "do not require any unknown protein component" for the reason stated above. Currently, the experiments recapitulate activation suggesting the membrane binding and release controls dynamics without additional factors. This allows for the possibility that biological factors may yet be shown to impact MinD activation under certain conditions.

      We agree and have change the text.

      Discussion - Line 526 - Thermus thermophilus is misspelt.

      Corrected.

    1. eLife Assessment

      This important study provides novel information on multi-enzyme complexes, known as metabolons, that form between sequential enzymes in a metabolic pathway. Using an innovative NanoBiT split-luciferase system, the authors present compelling evidence that malate dehydrogenase (MDH1) and citrate synthase (CIT1) dynamically associate under different metabolic conditions in Saccharomyces cerevisiae. The findings suggest the dynamic MDH1-CIT1 interaction facilitates control of TCA pathway flux rate.

    2. Reviewer #1 (Public review):

      Summary:

      The study by the Obata group characterizes the dynamics of the canonical malate dehydrogenase-citrate synthase metabolon in yeast.

      Strengths:

      The study is well-written and appears to give clear demonstrations of this phenomenon.

      Studies of the dynamics of metabolon formation are rare; if the authors can address the concern detailed below, then they have provided such for one of the canonical metabolons in nature.

      Weaknesses:

      There is a fundamental issue with the study, which is that the authors do not provide enough support or information concerning the split luciferase system that they use. Is the binding reversible or not? How the data is interpreted is massively influenced by this fact. What are the pros and cons of this method in comparison to, for example, FLIM-FRET? The authors state that the method is semi-quantitative - can they document this? All of the conclusions are based on the quality of this method. I know that it has been used by others, but at least some preliminary documentation to address these questions is required.

      Comments on revised version:

      I feel that the authors have adequately addressed my prior concerns. I have no further critiques of their work.

    3. Reviewer #2 (Public review):

      This study explores the dynamic association between malate dehydrogenase (MDH1) and citrate synthase (CIT1) in Saccharomyces cerevisiae, with the aim of linking this interaction to respiratory metabolism. Utilizing a NanoBiT split-luciferase system, the authors monitor protein-protein interactions in vivo under various metabolic conditions.

      Major Concerns:

      (1) NanoBiT Signal May Reflect Protein Abundance Rather Than Interaction Strength<br /> In Figure 1C, the authors report increased MDH1-CIT1 interaction under respiratory (acetate) conditions and decreased interaction during fermentation (glucose), as indicated by NanoBiT luminescence. However, this signal appears to correlate strongly with the expression levels of MDH1 and CIT1, raising the possibility that the observed luminescence reflects protein abundance rather than specific interaction dynamics. To resolve this, NanoBiT signals should be normalized to the expression levels of both proteins to distinguish between abundance-driven and interaction-driven changes.

      (2) Lack of Causal Evidence<br /> The study presents a series of metabolic perturbation experiments (e.g., arsenite, AOA, antimycin A, malonate) and correlates changes in metabolite levels with NanoBiT signals. However, these data are correlative and do not establish a functional role for the MDH1-CIT1 interaction in metabolic regulation. To demonstrate causality, the authors should implement approaches to specifically disrupt the MDH1-CIT1 interaction. One strategy could involve using a 15-residue peptide (Pept1) derived from the Pro354-Pro366 region of CIT1, previously shown to mediate the interaction or introducing the cit1Δ3 (Arg362Glu) mutation, which perturbs binding. Metabolic flux analysis using ^13C-labeled glucose and mitochondrial respiration assays (e.g., Seahorse) could then assess functional consequences.

      (3) Absence of Protein Expression Controls Under Perturbation Conditions<br /> In experiments involving acetate, arsenite, AOA, antimycin A, and malonate, the authors infer changes in MDH1-CIT1 association based solely on NanoBiT signals. However, no accompanying data are provided on MDH1 and CIT1 protein levels under these conditions. This omission weakens the conclusions, as altered expression rather than interaction strength could underlie the observed luminescence changes. Immunoblotting or quantitative proteomics should be used to confirm constant protein expression across conditions.

      Conclusion:

      Although the central question is compelling and the use of NanoBiT in live cells is a strength, the manuscript requires additional experimental rigor. Specifically, normalization of interaction signals, introduction of causative perturbations, and validation of protein expression are essential to substantiate the study's claims.

      Comments on revised version:

      The manuscript is much improved.

    4. Reviewer #3 (Public review):

      Summary:

      Metabolons are multisubunit complexes that promote the physical association of sequential enzymes within a metabolic pathway. Such complexes are proposed to increase metabolic flux and efficiency by channeling reaction intermediates between enzymes. The TCA cycle enzymes malate dehydrogenase (MDH1) and citrate synthase (CIT1) have been linked to metabolon formation, yet the conditions under which these enzymes interact, and whether such interactions are dynamic in response to metabolic cues, remains unclear, particularly in the native cellular context. This study uses a nanoBIT protein-protein interaction assay to map the dynamic behavior of the MDH1-CIT1 interaction in response to multiple metabolic stimuli and challenges in yeast. Beyond mapping these interactions in real time, the authors also performed GC-MS metabolomics to map whole cell metabolite alterations across experimental conditions. Finally, the authors use microscale thermophoresis to determine components that alter the MDH1-CIT1 interaction in vitro. Collectively, the authors synthesize their collected data into a model in which the MDH1-CIT1 metabolon dissociates in conditions of low respiratory flux, and is stimulated during conditions of high respiratory flux. While their data largely support these models, some key exceptions are found that suggest this model is likely oversimplified and will require further work to understand the complexities associated with MDH1-CIT1 interaction dynamics. Nonetheless, the authors put forth an interesting and timely toolkit to begin to understand the interaction kinetics and dynamics of key metabolic enzymes that should serve as a platform to begin disentangling these important yet understudied aspects of metabolic regulation.

      Strengths:

      - The authors address an important question: how do metabolon-associated protein protein interactions change across altered metabolic conditions?

      - The development and validation of the MDH1-CIT1 nanoBIT assay provides an important tool to allow the quantification of this protein-protein interaction in vivo. Importantly, the authors demonstrate that the assay allows kinetic and real time assessment of these protein interactions, which reveal interesting and dynamic behavior across conditions.

      - The use of classic biochemical techniques to confirm that pH and various metabolites can alter the MDH1-CIT1 interaction in vitro is rigorous and supports the model put forth by the authors.

      Weaknesses:

      The authors have addressed identified weaknesses within the revision of their manuscript.

    5. Author response:

      The following is the authors’ response to the original reviews.

      eLife Assessment

      This study reports a dynamic association/dissociation between malate dehydrogenase (MDH1) and citrate synthase (CIT1) in Saccharomyces cerevisiae under different metabolic conditions that control TCA pathway flux rate. The research question is timely, the use of the NanoBiT split-luciferase system to monitor protein-protein interactions is innovative, and the significance of the findings is valuable. However, the strength of evidence needed to support the conclusions was found to be incomplete based on a lack of critical control and mechanistic experiments.

      We thank the editor for this thoughtful assessment of our work. We are encouraged that the research question, experimental approach, and overall significance were viewed positively.

      To address the concern regarding the strength of evidence, we have implemented additional controls in the revised manuscript. Specifically, we have repeated all MDH1CIT1 interaction measurements alongside strains expressing full-length NanoLUC fusion proteins to assess MDH1 and CIT1 protein abundance. The resulting data, now included as supplementary figures (Figure 2 – figure supplement 2, Figure 2 – figure supplement 3, Figure 3 – figure supplement 1, Figure 4 – figure supplement 2), demonstrate the reproducibility of the findings and indicate that the observed changes in MDH1-CIT1 interaction are not attributable to protein abundance variations.

      We agree that a detailed mechanistic dissection of how the MDH1–CIT1 complex influences metabolic pathway flux is an essential piece of evidence for establishing the functions of the metabolon. However, such analyses require extensive additional investigation beyond the scope of the present study. Accordingly, we have clarified the aims of this work in the revised manuscript to emphasize that our primary objective is to characterize the dynamic behavior of the MDH1–CIT1 interaction under different metabolic conditions and to identify key factors associated with its regulation.

      We believe these revisions strengthen the rigor of the study, better define its scope, and provide a solid foundation for future mechanistic investigations.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      The study by the Obata group characterizes the dynamics of the canonical malate dehydrogenase-citrate synthase metabolon in yeast.

      Strengths:

      The study is well-written and appears to give clear demonstrations of this phenomenon.

      Studies of the dynamics of metabolon formation are rare; if the authors can address the concern detailed below, then they have provided such for one of the canonical metabolons in nature.

      We sincerely thank the reviewer for their positive assessment and for recognizing the value of our study in characterizing the dynamics of the MDH1-CIT1 metabolon. We appreciate the recognition that studies of metabolon dynamics are rare and that our work provides a clear demonstration of this phenomenon for a canonical metabolon. We have carefully addressed the methodological concerns regarding the NanoBiT system as detailed below to further strengthen the evidence for our findings.

      Weaknesses:

      There is a fundamental issue with the study, which is that the authors do not provide enough support or information concerning the split luciferase system that they use.

      We agree that a detailed description of the NanoBiT system is essential to ensure the reliability of the methodology. As suggested, we have added a dedicated paragraph to the Introduction (Lines 90–103) to clarify these technical aspects, supported by the foundational work of Dixon et al. (2016).

      Is the binding reversible or not? How the data is interpreted is massively influenced by this fact.

      Yes, the NanoBiT system is specifically designed to be reversible. The intrinsic affinity of the subunits is low (K<sub>D</sub> = 190 μM), and the association and dissociation rate constants (k<sub>on</sub> = 500 M<sup>-1</sup>s <sup>-1</sup>, k<sub>off</sub> = 0.2 s<sup>-1</sup>) are well outside the range of typical protein-protein interactions (Dixon et al., 2016). These kinetics ensure that the assembly and disassembly of the luminescent complex are dictated solely by the interaction characteristics of the target proteins (MDH1 and CIT1) and not by the tags themselves. This allows for real-time monitoring of both the association and dissociation phases.

      What are the pros and cons of this method in comparison to, for example, FLIM-FRET?

      We have now explicitly addressed the pros and cons of our methodology compared to fluorescence-based systems:

      Pros: The NanoLUC-based reporter is 150 times brighter than conventional luciferases and has a significantly higher dynamic range (Hall et al 2016), allowing detection of weak transient interactions. Importantly for this study, fluorescence-based methods such as FLIM-FRET and BRET are difficult to implement in yeast microplate assays due to the high levels of cellular autofluorescence. NanoBiT bypasses this issue, providing a high signal-tonoise ratio.

      Cons: Unlike FRET, NanoBiT requires the application of a substrate (furimazine). We did not include this disadvantage in the manuscript because it is not critical in a yeast study. Furimazine can be applied directly to the medium and readily permeates cells.

      The authors state that the method is semi-quantitative - can they document this?

      The semi-quantitative nature of the system is supported by its high dynamic range and the linear relationship between the luminescence signal and the amount of protein complex formed, as documented in Dixon et al. (2016). By using this system in a microplate setting, we were able to monitor relative increases or decreases in interaction levels over time across multiple metabolic conditions, providing a robust comparative analysis of metabolon dynamics.

      All of the conclusions are based on the quality of this method. I know that it has been used by others, but at least some preliminary documentation to address these questions is required.

      We acknowledge the reviewer’s concern regarding the reliance on the NanoBiT system. To ensure the reliability of our conclusions, we have included several lines of evidence to validate the method and demonstrate that the observed luminescence signals accurately reflect protein-protein interaction dynamics.

      To confirm the NanoBiT results using an independent biochemical approach, we performed an in vivo pull-down assay following glucose addition (Figure 2 – figure supplement 1A). The results demonstrate a reduction in the physical association between MDH1 and CIT1. This biochemical validation directly supports the reduction in interaction observed with the NanoBiT system during the Crabtree effect.

      We have provided protein abundance data for both MDH1 and CIT1 across the experimental conditions (Figure 2 – figure supplement 1&3; Figure 3 – figure supplement 1; Figure 4 – figure supplement 2). These results show only minor changes in protein levels, confirming that the fluctuations in the NanoBiT signal are independent of protein expression and represent genuine changes in metabolon assembly.

      To ensure the findings are reproducible, we have included MDH1-CIT1 interaction results from repeated independent experiments (Figure 2 – figure supplement 1&3; Figure 3 – figure supplement 1; Figure 4 – figure supplement 1). The consistency of the results across these trials confirms the robustness of the system in monitoring the metabolic regulation of this complex.

      We hope that these additional experimental validations, alongside the detailed technical description based on the established properties of the NanoBiT system (Dixon et al., 2016; Hall et al., 2012), provide the necessary documentation to satisfy the reviewer’s concerns regarding the quality and reliability of the method.

      Reviewer #2 (Public review):

      This study explores the dynamic association between malate dehydrogenase (MDH1) and citrate synthase (CIT1) in Saccharomyces cerevisiae, with the aim of linking this interaction to respiratory metabolism. Utilizing a NanoBiT split-luciferase system, the authors monitor protein-protein interactions in vivo under various metabolic conditions.

      Major Concerns:

      (1) NanoBiT Signal May Reflect Protein Abundance Rather Than Interaction Strength

      In Figure 1C, the authors report increased MDH1-CIT1 interaction under respiratory (acetate) conditions and decreased interaction during fermentation (glucose), as indicated by NanoBiT luminescence. However, this signal appears to correlate strongly with the expression levels of MDH1 and CIT1, raising the possibility that the observed luminescence reflects protein abundance rather than specific interaction dynamics. To resolve this, NanoBiT signals should be normalized to the expression levels of both proteins to distinguish between abundance-driven and interaction-driven changes.

      We agree that distinguishing between abundance-driven and interaction-driven changes is vital. To address this, we have included new data showing the relative protein levels of MDH1 and CIT1 across all experimental conditions. The protein levels were assessed using yeast lines expressing these proteins tagged with full-length NanoLUC luciferase (Figure 2 – figure supplement 1&3, Figure 3 - figure supplement 1, Figure 4 – figure supplement 2). Using the luminescence data of these relative protein levels, we have included plots showing normalized interaction index (Figure 2 – figure supplement 1G & 3D,H,L; Figure 3 - figure supplement 1D,H,L P; Figure 4 – figure supplement 1D,H,L). This index was calculated by dividing the NanoBiT interaction signal by the product of the relative abundances of both proteins:

      In this formula, NanoBiT, MDH1, and CIT1 are the relative luminescence levels at each time point. This analysis clarified that the changes in the interaction signal significantly exceeded the fluctuations in protein levels, confirming that the dynamics are interactionspecific and not abundance-driven. To provide the most direct and transparent representation of the experimental measurements, we have chosen to keep the raw RLU data in the main figures and have moved the data related to protein abundance and normalization to figure supplements.

      (2) Lack of Causal Evidence

      The study presents a series of metabolic perturbation experiments (e.g., arsenite, AOA, antimycin A, malonate) and correlates changes in metabolite levels with NanoBiT signals. However, these data are correlative and do not establish a functional role for the MDH1CIT1 interaction in metabolic regulation. To demonstrate causality, the authors should implement approaches to specifically disrupt the MDH1-CIT1 interaction. One strategy could involve using a 15-residue peptide (Pept1) derived from the Pro354-Pro366 region of CIT1, previously shown to mediate the interaction, or introducing the cit1Δ3 (Arg362Glu) mutation, which perturbs binding. Metabolic flux analysis using ^13C-labeled glucose and mitochondrial respiration assays (e.g., Seahorse) could then assess functional consequences.

      We agree with the reviewer that the current dataset correlates metabolon assembly with metabolic states rather than establishing a direct causal proof of its functional role in regulating pathway flux.

      However, the primary objective of this manuscript was to establish the dynamic nature of the MDH1-CIT1 metabolon and to demonstrate the causal relationship between the changes in cellular conditions and metabolon dynamics through in vitro and in vivo assessments. Demonstrating that this canonical multienzyme complex undergoes reversible assembly and disassembly in vivo represents a major advance, as metabolon dynamics is a critical, yet previously unrevealed, factor involved in metabolic regulation. We aimed to define the specific environmental triggers that govern these dynamics, providing the necessary foundation for defining the functions of metabolons.

      We completely agree that establishing causality using interaction-deficient mutants coupled with metabolic flux analysis is another critical experiment to establish the functions of the TCA cycle metabolon. We have, in fact, been conducting these precise metabolic flux analyses on CIT1 mutants with disrupted interaction with MDH1. Because the functional consequences of complex disruption involve wide-reaching metabolic rerouting that requires extensive data presentation and modeling, this work forms a separate, comprehensive follow-up study that is currently in preparation for submission in the near future.

      To address this limitation in the current manuscript, we have carefully reviewed and revised the Abstract, Results, Discussion, and Conclusion sections (Lines 19-22; 205; 322-327; 341-342; 458-466). We have removed any language that may have inadvertently implied direct causality. We now explicitly state that our findings indicate the relationship between metabolon dynamics and respiratory conditions, and we have added a clear statement noting that the direct effects of this assembly on metabolic flux are the focus of our forthcoming studies.

      (3) Absence of Protein Expression Controls Under Perturbation Conditions

      In experiments involving acetate, arsenite, AOA, antimycin A, and malonate, the authors infer changes in MDH1-CIT1 association based solely on NanoBiT signals. However, no accompanying data are provided on MDH1 and CIT1 protein levels under these conditions. This omission weakens the conclusions, as altered expression rather than interaction strength could underlie the observed luminescence changes. Immunoblotting or quantitative proteomics should be used to confirm constant protein expression across conditions.

      In response to your first concern, we have now performed protein expression assessments for all experiments, including the perturbation conditions, such as acetate, arsenite, AOA (Figure 3 – figure supplement 1), antimycin A, cyanide, and malonate (Figure 4 – figure supplement 2). The results demonstrate that the protein levels of MDH1 and CIT1 remain relatively stable throughout these treatments and do not correlate with the large changes observed in the interaction signals. This is also demonstrated by the normalized interaction index, which confirms that the shifts in luminescence are driven by the dynamic assembly and disassembly of the MDH1-CIT1 metabolon rather than changes in protein concentrations.

      Conclusion:

      Although the central question is compelling and the use of NanoBiT in live cells is a strength, the manuscript requires additional experimental rigor. Specifically, normalization of interaction signals, introduction of causative perturbations, and validation of protein expression are essential to substantiate the study's claims.

      We sincerely thank the reviewer for recognizing the value of our central question and the strength of the live-cell NanoBiT system, as well as for your rigorous critique that has strengthened this manuscript. To address the concerns regarding experimental rigor, we have now provided extensive validation of MDH1 and CIT1 protein expression across all experimental conditions using yeast lines tagged with the full-length NanoLUC luciferase. These data demonstrate relatively stable protein expression, allowing us to calculate a normalized interaction index that substantiates that the observed luminescence shifts are driven by dynamic metabolon assembly rather than protein concentration. Regarding causative perturbations, we agree that introducing interaction-deficient mutants coupled with isotopic flux analysis is the critical next step to establish functional consequences. Because defining these pathway-wide rerouting events requires extensive modeling, this work will be reported in a follow-up study currently in preparation. Accordingly, we have carefully revised the manuscript to remove language implying direct causality, explicitly framing metabolon dynamics as an integral factor in metabolic regulation closely related to pathway activity and cellular metabolic states. We believe these new quantitative controls, normalizations, and textual clarifications thoroughly address the need for additional rigor and solidly substantiate our findings.

      Reviewer #3 (Public review):

      Summary:

      Metabolons are multisubunit complexes that promote the physical association of sequential enzymes within a metabolic pathway. Such complexes are proposed to increase metabolic flux and efficiency by channeling reaction intermediates between enzymes. The TCA cycle enzymes malate dehydrogenase (MDH1) and citrate synthase (CIT1) have been linked to metabolon formation, yet the conditions under which these enzymes interact, and whether such interactions are dynamic in response to metabolic cues, remain unclear, particularly in the native cellular context. This study uses a nanoBIT protein-protein interaction assay to map the dynamic behavior of the MDH1-CIT1 interaction in response to multiple metabolic stimuli and challenges in yeast. Beyond mapping these interactions in real time, the authors also performed GC-MS metabolomics to map whole-cell metabolite alterations across experimental conditions. Finally, the authors use microscale thermophoresis to determine components that alter the MDH1-CIT1 interaction in vitro. Collectively, the authors synthesize their collected data into a model in which the MDH1CIT1 metabolon dissociates in conditions of low respiratory flux, and is stimulated during conditions of high respiratory flux. While their data largely support these models, some key exceptions are found that suggest this model is likely oversimplified and will require further work to understand the complexities associated with MDH1-CIT1 interaction dynamics. Nonetheless, the authors put forth an interesting and timely toolkit to begin to understand the interaction kinetics and dynamics of key metabolic enzymes that should serve as a platform to begin disentangling these important yet understudied aspects of metabolic regulation.

      We thank the reviewer for this thoughtful and constructive summary of our work. We appreciate the recognition of the novelty and utility of our experimental approach and the integrated analysis of MDH1–CIT1 interaction dynamics.

      We agree with the reviewer that, although our data largely support a model in which MDH1– CIT1 interaction correlates with respiratory activity, there are conditions that do not fully conform to this simplified framework. In the revised manuscript, we have addressed these apparent inconsistencies by providing detailed interpretations of the counterintuitive observations (e.g., ETC inhibition) and emphasizing that the MDH1–CIT1 interaction is modulated by changes in the mitochondrial matrix microenvironment associated with respiratory activity.

      Furthermore, we have revised the Discussion to highlight that the regulation of the MDH1– CIT1 interaction is likely multifactorial, involving the combined effects of pH, metabolites, and other unknown factors, which together enable fine-tuning of metabolic flux in fluctuating environments. This expanded perspective is now more clarified.

      We agree that identifying the precise molecular determinants of MDH1–CIT1 interaction dynamics will require additional mechanistic studies, such as systematic analyses using yeast mutants. While these experiments are an important next step, they are beyond the scope of the present study. We anticipate that the toolkit and framework established here will facilitate such future investigations.

      Strengths:

      (1) The authors address an important question: how do metabolon-associated proteinprotein interactions change across altered metabolic conditions?

      (2) The development and validation of the MDH1-CIT1 nanoBIT assay provides an important tool to allow the quantification of this protein-protein interaction in vivo. Importantly, the authors demonstrate that the assay allows kinetic and real time assessment of these protein interactions, which reveal interesting and dynamic behavior across conditions.

      (3) The use of classic biochemical techniques to confirm that pH and various metabolites can alter the MDH1-CIT1 interaction in vitro is rigorous and supports the model put forth by the authors.

      We thank the reviewer for these positive and encouraging comments. We are pleased that the importance of the research question, the development of the MDH1–CIT1 NanoBiT assay, and the integration of in vivo and in vitro approaches were recognized. We especially appreciate the acknowledgment of the assay’s ability to capture dynamic and kinetic changes in protein–protein interactions, as well as the support provided by the biochemical analyses. We hope that the experimental framework established in this study will serve as a useful platform for further investigations into metabolon dynamics and metabolic regulation.

      Weaknesses:

      (1) Some of the data collected seem to be merely reported rather than synthesized and interpreted for the reader.

      We agree that explicitly synthesizing these findings is essential for clarity. To improve this, we have revised the Results section to include concise summary statements at the conclusion of each major experimental paragraph (Lines 190-191, 201, 218-219, 229-231, 241-242, 272-274, 282-283; 291-293). These additions interpret the data in relation to our main hypothesis. The discussion section was thoroughly revised to more precisely explain the logic supporting the model (Lines 381-393; 433-443, 458-466). Additionally, to bring together the entire dataset, we introduced a new summary schematic (Figure 6A). This figure visually and conceptually integrates our diverse findings, covering metabolic treatments, pH fluctuations, and complex metabolite profiles, showing how these signals work together to control multienzyme complex assembly.

      This is particularly true for data that seem to reflect more complex trends, such as the GCMS experiments that map metabolites across multiple experiments, or treatments that show somewhat counterintuitive results, such as the antimycin A treatment, which promotes rather than disrupts the MDH1-CIT1 interaction.

      We agree that our complex datasets, including the metabolomics and the seemingly counterintuitive Antimycin A results, required deeper synthesis. To clarify the broader metabolic trends, we have added Figure 6A to visually map which factors, specifically pH, malate, fumarate, and aspartate, most consistently align with complex assembly. We revised the Discussion (Lines 390-393, 439-443) to explicitly conclude that no single variable predominantly governs the interaction, but it is coordinately regulated by multiple microenvironmental cues.

      Regarding the Antimycin A (and other ETC inhibitors) discrepancy, where the interaction is enhanced despite suppressed respiration, we have expanded our interpretation (Lines 346–358) to explain this as a transient response that is not directly reflected by steadystate respiratory activity. Specifically, we propose that acute perturbations of the mitochondrial matrix microenvironment, particularly changes in pH, temporarily promote MDH1–CIT1 interaction. Thus, under these conditions, transient microenvironmental changes can dominate over steady-state respiratory output in regulating metabolon assembly.

      The discussion paragraph about the imperfect relationship between pH and interaction has been revised to highlight our conclusion that mitochondrial matrix pH can be a contributing factor rather than the primary regulator (Lines 386-393).

      (2) Some of the assertions put forth in the manuscript are not substantiated by the data presented, and the authors are at times overly reliant on previous findings from the literature to support their claims. This is particularly notable for claims about "TCA cycle flux"; the authors do not perform flux analysis anywhere in their study and should be cautious when insinuating correlations between their observations and "flux".

      We appreciate the reviewer’s careful evaluation of our terminology and fully agree that claims regarding "flux" should be reserved for studies that employ direct isotopic flux measurements. In response to this constructive feedback, we have thoroughly reviewed the manuscript to ensure that our assertions are substantiated by the presented experimental data. We have carefully evaluated the use of the term "flux" throughout the Abstract, Introduction, and Discussion, replacing it with more accurate phrases such as "pathway activity," "respiratory activity," or "mitochondrial respiration" depending on the specific context (Lines 11; 20-21; 50; 111-112; 322-327; 329; 345; 349-350; 442-443; 458466).

      We also removed a paragraph discussing the potential role of the MDH1-CIT1 metabolon in the malate-aspartate shuttle (Line 361). We realized the paragraph is highly speculative, and our data do not directly support the hypothesis. The influence of the MDH1-CIT1 on the malate-aspartate shuttle is a major finding of the upcoming manuscript reporting its effects in metabolic network flux. We apologize for mixing up the results of two separate studies.

      Furthermore, we have revised our conclusions to avoid over-reliance on prior literature in making causal claims. We now explicitly frame the dynamic assembly of the MDH1-CIT1 metabolon as an integral factor in metabolic regulation, closely related to cellular metabolic states, rather than stating that it controls pathway flux (Lines 454-462). We believe these textual revisions accurately align our claims with our current observations and remove any unsubstantiated assertions.

      (3) The manuscript presentation could be improved. For figures, at times, the axes do not have intuitive labels (example, Figure 1A), data points and details about the number of samples analyzed are missing (bar graphs and box plots), and molecular weight markers are not reported on western blots. The authors refer to the figures out of order in the text, which makes the manuscript challenging to navigate as a reader.

      We thank the reviewer for these helpful suggestions to improve the clarity and presentation of the manuscript. We have made several revisions accordingly.

      First, axis labels have been revised throughout the figures to improve clarity and make them more intuitive. Second, we have added the number of biological replicates to the figure captions and updated bar graphs and box plots to display individual data points. Third, to improve the transparency of the immunoblot data, we have included molecular weight marker position in Figure 1C and corresponding full gel images in a new Figure 1 – figure supplement 2. Other immunoblot images have been moved to Figure 2 – figure supplement 1 since they lack molecular marker images.

      In addition, we have reorganized the figure panel labeling and corresponding text to improve the flow of the Results section. Specifically, figure subpanels are now arranged according to the measured parameters rather than treatment conditions, and the relevant sections describing TCA cycle manipulation and ETC inhibition have been revised to follow this updated figure order (Lines 208–231; 251–274). These changes improve the readability and logical progression of the manuscript.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      The grammar in the abstract in the sentence which states called metabolon. This needs to be fixed.

      We thank the reviewer for pointing this out. We have revised the sentence in the Abstract to improve clarity. The revised sentence reads: “The tricarboxylic acid (TCA) cycle enzymes malate dehydrogenase (MDH1) and citrate synthase (CIT1) form a multienzyme complex, referred to as a metabolon, that channels intermediate oxaloacetate between their reaction centers.” (Lines 7-9)

      Reviewer #3 (Recommendations for the authors):

      Major points:

      (1) Much of the data reported in this manuscript reads as a summary of what was found, rather than distilling what the trends in the data mean or how they support the proposed model.

      We thank the reviewer for this comment. This concern overlaps with your previous point (Weakness 1), which we have addressed through revisions to improve synthesis and clarity. Specifically, we have added concise summary statements at the end of each major experimental section (Lines 190-191, 201, 218-219, 229-231, 241-242, 272-274, 282-283; 291-293), and we have included a new summary schematic (Figure 6A) that integrates the findings to illustrate how metabolic conditions and mitochondrial microenvironments relat to MDH1–CIT1 interaction. Together, these revisions improve the interpretation and clarify how the results support our model.

      For instance, in Figure 3, the authors use one metabolic treatment to activate the TCA cycle and two to inhibit the TCA cycle. In Figure 3M, GC-MS data are reported for select metabolites across these three conditions, as well as a control condition. However, these metabolites don't follow clean "trends" according to the predictions; as one example, malate is down in the TCA active (acetate) and one TCA inhibited condition (arsenite), whereas it is elevated in the second TCA inhibited (aminooxyacetate) condition. As an additional example, glutamate is down in the arsenite (inhibited) condition, slightly down in the acetate (activated) condition, but is unchanged in the AOA (inhibited) condition. Similar variability is seen in Figure 4M. What do these discrepancies mean? How do they support the model? As written, these data bring forth more questions than they answer.

      We appreciate the reviewer’s careful analysis of the metabolomics data in Figures 2E, 3M, and 4M. The reviewer notes that the levels of certain metabolites show complex patterns that do not simply reflect overall TCA cycle activity. We have acknowledged that our metabolomics dataset is a valuable resource for the research community and have added a brief paragraph to emphasize the complex metabolic phenotypes resulting from chemical treatments (Lines 422-431).

      As mentioned in the paragraph, this complexity is biologically expected. It is likely from the distinct primary targets of each inhibitor, such as arsenite affecting redox-sensitive enzymes and AOA disrupting the malate-aspartate shuttle, as well as off-target effects and the adaptive reorganization of intersecting metabolic networks to bypass local blockades. Rather than viewing these diverse metabolic phenotypes as discrepancies, we leveraged them to uncouple general respiratory suppression from specific metabolite pools, allowing us to independently assess their relationship with metabolon assembly.

      Furthermore, we note that our GC-MS analysis measures whole-cell metabolite levels, which represent the sum of multiple subcellular compartments and may not precisely reflect localized concentrations within the mitochondrial matrix that is directly affected by the TCA cycle. The description of this limitation of whole-cell metabolomics has been revised in Lines 417-420.

      (2) Why do the authors propose that antimycin A increases the interaction between MDH1 and CIT1 despite decreasing respiratory activity? Given the generalities proposed in Figure 6, this is important to address.

      We thank the reviewer for this comment. This point overlaps with Weakness 1, where we have addressed the apparent discrepancy associated with antimycin A (and other ETC inhibitors). Briefly, we have expanded our interpretation (Lines 349–360) to explain this effect as a transient response that is not directly aligned with steady-state respiratory activity. We propose that acute perturbations of the mitochondrial matrix microenvironment, particularly changes in pH, temporarily promote MDH1–CIT1 interaction. In addition, we have revised the Discussion (Lines 386–404) to clarify that mitochondrial matrix pH acts as a contributing factor rather than the primary regulator of the interaction. Together, these revisions reconcile the ETC inhibition by antimycin A with the overall model presented in Figure 6.

      (3) The authors use acetate to "activate" the TCA cycle; do other non-fermentable carbon sources also promote the MDH1-CIT1 interaction?

      We thank the reviewer for this insightful question. We have tested additional nonfermentable carbon sources and found that they did not significantly affect MDH1–CIT1 interaction (Figure 3—figure supplement 1). We note that raffinose present in the medium likely provides a baseline carbon source supporting oxidative metabolism, which may limit the observable effects of these treatments (Lines 149-150).

      In addition, we performed a new experiment using ethanol. While ethanol treatment enhanced the MDH1–CIT1 interaction signal, it also increased the abundance of MDH1 and CIT1, resulting in a reduced interaction index. Because ethanol induces protein accumulation under our experimental conditions, this result is not straightforward to interpret. We have included this observation and its interpretation in the revised manuscript (Lines 208–211).

      (4) The authors show that the MDH1-CIT1 interaction is sensitive to pH. Is the MDH1-CIT1 interaction affected by uncouplers in vivo?

      We thank the reviewer for suggesting a meaningful experiment. We performed a new experiment examining the effect of the uncoupler CCCP on MDH1–CIT1 interaction in vivo (Figure 4—figure supplement 4). We found that CCCP treatment increased the interaction signal, consistent with the idea that acidification of the mitochondrial matrix promotes MDH1–CIT1 association.

      However, we observe that CCCP treatment also decreased the luciferase signals from MDH1 and CIT1 fused to full-length NanoLUC in an abnormal way, making it harder to interpret the interaction index. Therefore, although these results support a possible role for pH in regulating the interaction, they should be viewed with caution and included as a figure supplement. This experiment and its interpretation have been added to the revised manuscript (Lines 276–283).

      (5) NADH is a potent suppressor of many enzymes within the TCA cycle, including MDH1 and CIT1. Can the authors modulate mitochondrial NADH through genetic manipulation of Ndi1, or through overexpression of mito-Lb-NOX (PMID: 27124460)?

      We thank the reviewer for this insightful suggestion. We agree that the mitochondrial NADH is a potential regulator of the MDH1-CIT1 interaction as it is a potent suppressor of many TCA cycle enzymes, and indeed, we have previously shown that NADH inhibit the MDH-CS interaction in vitro (Omini et al 2021 PMID: 34548590). For this reason, we investigated the mitochondrial matrix redox state that is related to the NADH levels in the current study. The reviewer’s proposed strategy of using targeted genetic tools like mito-Lb-NOX or Ndi1 manipulation to specifically influence the NADH level is an elegant approach to isolate this variable. However, implementing this system requires generating, optimizing, and validating new yeast strains that harbor the targeted NADH-modulating constructs alongside NanoBiT and full-length NanoLUC sensor systems. Because this extensive strain engineering and subsequent live-cell validation fall outside a feasible timeframe for the current manuscript revision, we must respectfully defer these experiments. We view the precise manipulation of the mitochondrial redox state via tools like mito-Lb-NOX as a complementary approach for our future work to systematically pinpoint the individual regulatory factors. We have expanded our Discussion (Lines 417-420; 462-465) to highlight the targeted genetic manipulation of the possible regulatory factors including the NADH pool, as a critical future direction for dissecting these dynamics.

      (6) The authors should correct their figures:

      (a) Axes should be easy to interpret on graphs.

      (b) Individual datapoints should be shown on bar graphs and box plots. Minimally, the number of samples evaluated should be reported.

      (c) Molecular weight markers should be reported on blots.

      We thank the reviewer for these helpful suggestions. Points (a) and (b) overlap with Weakness 3, which we have addressed through revisions to improve figure clarity and data presentation. Specifically, axis labels have been revised to be more intuitive, the number of samples is now reported in the figure captions, and bar and box plots have been updated to include individual data points. For time-course data, we retained point-line plots, as alternative formats (e.g., bar or box plots) would reduce clarity due to the density of time points.

      For point (c), we have added molecular weight markers to the immunoblot data where available (Figure 1C). In the time-course experiment in the original Figure 2, molecular weight markers were absent from the gel images. Although we are confident in the identity of the detected signals, we have moved these data to a figure supplement (Figure 2—figure supplement 1C) to reflect this limitation. Similarly, the corresponding Co-IP data are now presented as a figure supplement (Figure 2—figure supplement 1A).

      Minor points:

      (1) In the last paragraph before the results, the authors refer to "the fluorescent biosensors", but start the paragraph discussing the nanoBIT PPI. After reading the manuscript, these seem to be distinct experimental setups, but that was not evident in the first read through of the paper.

      We thank the reviewer for pointing out this source of confusion. We apologize for the lack of clarity in distinguishing between the experimental approaches. In this study, the NanoBiT system was used to measure MDH1–CIT1 interaction, whereas fluorescent biosensors were used to assess mitochondrial matrix pH, redox state, and ATP levels. We have revised the paragraph to more clearly distinguish these methodologies and their respective roles in the study (Lines 105–112).

      (2) As mentioned above, referring to multiple figures out of order within the manuscript is very jarring for the reader. The authors should consider reworking the narrative or figures to be presented in order.

      We thank the reviewer for this comment. This concern overlaps with the previous comment regarding figure organization, which we have addressed by revising both the figure labeling and the corresponding text. Specifically, figure subpanels have been reorganized to follow the measured parameters rather than treatment conditions, and the Results sections describing TCA cycle manipulation and ETC inhibition have been revised to follow the updated figure order (Lines 208–231; 251–274). These changes improve the logical flow and readability of the manuscript.

    1. When Amy says she wants to "disappear" I think this shows her insecurity about the family and insecurity at 14 years old. she cares very deeply of what people think of her. At the end she does show that her appreciation for culture has grown

    2. I think she should have embraced her culture because the traditions are different. I also think Roberts family should have known that going into this dinner.

    1. Teleshuttle ucm.teleshuttle.com › 2018 › 11 › as-we-will-think-legacy-of-ted-nelson.html Smartly Intertwingled: "As We Will Think" -- The Legacy of Ted Nelson, Original Visionary of the Web Why Nelson matters A fuller explanation of why Nelson matters is in my post from a few years ago, Digital Camelot - The Once and Future Web of Engelbart and Nelson, but here I caption its core message: If you care about modern culture and how technology is shaping it, this is worth thinking about -- A powerful eulogy for where the Web might have gone, and still may someday, and the friendship of the two people most responsible for envisioning the Web* -- Ted Nelson's eulogy for his friend Doug Engelbart, as reported by John Markoff in The Times -- with Nelson's inimitable flair.

    1. We must also make constitutional engagement a valued public ritual, model civil discourse, invest in civic educators inside and outside the classroom, and protect and celebrate civic spaces like museums, libraries, and historical sites.

      everyone has a part to play

    2. . They threw out the Articles, drafted a new constitution, and sent it to the states for ratification. In 1789, less than twelve months after the Constitution was ratified, Congress passed the Bill of Rights, adding ten amendments to the original document. The states approved several additional amendments over the coming decades to close loopholes in the original text or clarify ambiguous language.

      if this were to happen again...who would be in the room drafting it?

    1. Additional Distinguishing Information Virginia-Based Firm DAA is headquartered in Vienna, Virginia. We understand the Commonwealth’s regulatory and governance framework for public school divisions, including the Virginia Public Procurement Act and relevant sections of the Code of Virginia governing school operations and closures. Specialized Focus Unlike general-purpose consulting firms that offer redistricting as one service among many, DAA is built specifically around demographic analysis, enrollment forecasting, and school boundary planning. Our methodologies are purpose-built for K-12 applications. Our team understands the particular data quality challenges of school enrollment records. We are attuned to the community dynamics that surround redistricting decisions. And our deliverables are designed for the people who use them — school boards making policy decisions, administrators managing operations, and families trying to understand where their children will go to school. Census Bureau Expertise Two of our three principals are former Census Bureau staff who worked directly on the methods used to produce population estimates and projections for the United States. This gives us an unusually deep understanding of the federal demographic data that forms the foundation of any enrollment projection. We know not just how to use Census, ACS, and other federal data products, but how they are made — their assumptions, their error structures, and where they should and should not be trusted. This is particularly important in the post-2020 environment, where differential privacy and COVID-19 disruptions have introduced data quality challenges that many analysts do not fully appreciate. Collaborative, Not Black-Box We do not disappear for three months and return with a set of recommendations. Our phased methodology is designed so that the district is a partner at every stage — reviewing data, validating assumptions, providing local knowledge, and understanding the tradeoffs before scenarios are finalized. This approach produces better analysis, because local expertise catches things that data alone cannot, and it produces better outcomes, because the district owns the process and its results. Clients Who Come Back Several of our clients have extended their engagements beyond the original scope. Brunswick County Public Schools extended from one year to four. Others have returned for annual updates or new phases of work. We take this as the strongest possible signal that our work delivers value and that districts find us good partners to work with. Data Security and FERPA Compliance This project will require access to geocoded student enrollment records protected under the Family Educational Rights and Privacy Act (FERPA), 20 U.S.C. § 1232g. We take these obligations seriously and have established procedures that meet the requirements of every district we work with. Data Handling All student data will be transmitted via Box.com, an enterprise-grade encrypted file sharing platform that has been approved by multiple school districts for this purpose. Once received, student data is stored on encrypted, access-controlled systems accessible only to project team members. No student data is shared with third parties under any circumstances. All analysis is conducted on secured workstations with current security software and operating system patches. FERPA Compliance Our team has completed FERPA training and maintains current knowledge of student data privacy requirements. We will execute a data sharing agreement with the district that complies with FERPA’s “studies” exception (34 CFR § 99.31(a)(6)). All published reports and maps present data in aggregate form only — no individual student will be identifiable in any deliverable. Student-level records are used solely for geocoding and boundary analysis. Data Destruction Upon project completion or contract termination, we will securely destroy all student-level data in our possession and provide written certification of destruction to the district. Aggregate analytical outputs will be retained only as needed for the contracted deliverables. Staff Training All DAA personnel assigned to this project have received training on student data privacy, including FERPA obligations, data minimization principles, and incident response procedures.

      unless these sections are required, remove them.

    2. Reference 3: Norfolk, MA Public Schools Contact: Dr. Ingrid Allardi Title: Superintendent Email: allardi@norfolk.k12.ma.us Phone: (508) 528-1225 Timeline: February 2024 – May 2024 Scope: Norfolk Public Schools contracted us to develop a ten-year enrollment forecast (2024-2034) by school and grade. Our analysis considered historical enrollment patterns, school choice trends, new construction activity, and migration and birth patterns at both the local and state level.

      Change to Olathe Public Schools.

    3. Additional Deliverables (Beyond the RFP Scope) The following items are not specified as required end products in the RFP, but we typically include them at no additional cost because districts have found them useful for governance, communication, and the years of operations that follow boundary changes. Any of them can be omitted at the district’s preference. Board presentation package for October 8, 2026 — slide deck with scenario summaries, maps, data visualizations, and our analysis of recommended options. We will present in person and remain available for Board questions and discussion. Interactive web-based scenario explorer (similar to our Horseheads CSD work) — a public-facing tool that lets Board members, district staff, and community members compare scenarios side by side and look up individual addresses. Pricing for this option is in the Cost Proposal section. Methodology documentation memo capturing the forecasting and capacity methods used, so the district can re-run or update the analysis in future years. Annual update option — recurring re-projection and boundary-impact monitoring in subsequent years, priced separately.

      Don't talk about this being beyond scope. This is just what we do. No additional price involved.

    4. Project schedule. Phase windows show typical effort distribution; the dashed vertical lines mark RFP-specified milestones (initial roster, updated roster, Round 1 due, district modification requests, Round 2 due, Board presentation, Board vote).

      Make sure the dates in the above figure are readable and don't overlap the colored parts of the chart.

    5. A baseline “do nothing” forecast for BCPS shows where each school is heading by 2030-31 if no boundary changes are made. New London Academy worsens to 107% and Forest Elementary reaches 100%, while most other schools stay near current utilization. The actual Phase 5 deliverable will incorporate the housing-pipeline yield not modeled here — likely pushing eastern schools higher still. Sources: VDOE Fall Membership 2025-26 (actual); Weldon Cooper Center for Public Service, 2026-2030 K-12 Enrollment Projections; BCPS Membership Projections 2024-2029, Table 14 (capacities).

      is this correct? I thought they were declining, not increasing?

    6. The planned closure of Stewartsville Elementary — the district’s oldest operating school, built in 1912 and running at 41% of capacity in 2025-26 — adds both urgency and political sensitivity to this redistricting. The closure is projected to save approximately $1 million per year in operating costs. In March 2026, the Board voted 5-2 to close the school, prompting a community lawsuit that cited procedural requirements under VA Code § 22.1-79(8). Rather than wait for the court’s June ruling, the Board held a new public hearing and voted unanimously to delay closure to the end of 2026-27, launching this division-wide redistricting study at the same time.

      Note that we understand the sensitivity of this project, and the feelings throughout the community on how it is going.

    7. anticipate the planned closure.

      Is it officially closing? I thought part of this process for this RFP was to make sure it should close. Check this in public records -- including those we have downloaded.

    8. [Chris to confirm and add additional K-12 redistricting engagements from the past five years from the broader client list — e.g., North Penn PA, Wake NC, Orange NC. Each should include client name, dates of service, and a 2-3 sentence description of the redistricting scope.]

      Add riverview gardens school district in MO. We helped them choose an elementary school to consolidate, and built redistricting to balance their elementary and middle schools after the closure.

    9. In April 2026 we also published Enrollment Forecasting, Capacity, and Utilization Analysis: Best Practices and an Assessment of Current Models — Olathe Public Schools, a peer-benchmarked review of the exact analytical questions Bedford County faces today: how to measure functional building capacity, how to build a forecast that incorporates the housing pipeline rather than only past enrollment trends, and how to tie utilization analysis to defensible governance decisions

      Olathe is currently undergoing the same discussions as Bedford in terms of closing schools and needing to redistrict. We will bring our expertise from that engagment to this one.

    1. McBombalds is currently willing to grant the United States government only conditional access. It is willing to conduct a public demonstration for Japanese observers in international waters, or some other uninhabited area, but it is not yet ready to authorize use of the A-bomb for all lawful military uses.

      这个虚构场景展示了私营公司对政府使用其技术的限制条件。这反映了当前AI安全讨论中的核心问题:创造者是否应该有权限制政府对其技术的使用方式?这种限制是否符合国家安全利益?作者通过这个思想实验,揭示了技术创造者与政府之间复杂的权力关系。

  3. pressbooks.online.ucf.edu pressbooks.online.ucf.edu
    1. I built Hyperbook – an open-source tool for creating interactive workbooks for your CS courses (free, fast, and markdown-based) This sounds great. Do you have any demonstrations like videos or screenshots? I looked through your site and didn’t see anything. A video showing off what it can do makes it easier for me to explain when I get school IT to deploy the extension for VSCode. More on reddit.com r/CSEducation 10 14 18 February 2026

      interactive workbooks

    1. eLife Assessment

      This important study utilizes behavioral data and computational modeling to show that spatial properties of visual attention affect human planning. The methodology and statistical analyses are solid, though the way attention is conceptualized and modeled could be refined. The findings of this study will interest cognitive scientists studying attention, perception, and decision-making.

    2. Reviewer #1 (Public review):

      Summary:

      This study investigated how visuospatial attention influences the way people build simplified mental representations to support planning and decision-making. Using computational modeling and virtual maze navigation, the authors examined whether spatial proximity and the spatial arrangement of obstacles determine which elements are included in participants' internal models of a task. The study developed and tested an extension of the value-guided construal (VGC) model that incorporates features of spatial attention for selecting simpler task mental representation.

      Strengths:

      (1) Original Perspective: The study introduces an explicit attentional component to established models of planning, offering an approach that bridges perception, attention, and decision-making.

      (2) Methodological Approach: The combination of computational modeling, behavioral data, and eye-tracking provides converging measures to assess the relationship between attention and planning representations.

      (3) Cross-validated data: The study relies on the analysis of three separate datasets, two already published and an additional novel one. This allows for cross-validation of the findings and enhances the robustness of the evidence.

      (4) Focus on Individual Differences: Reports of how individual variability in attentional "spillover" correlates with the sparsity of task representations and spatial proximity add depth to the analysis.

      Appraisal of Aims and Results:

      The study sets out to determine how spatial attention shapes the construction of task representations in planning contexts. The authors provide evidence that spatial proximity and arrangement influence which environmental features are incorporated into internal models used for navigation, and that accounting for these effects improves model predictions. There is clear documentation of individual variation, with some participants showing greater attentional spillover and more sparse awareness profiles.

      Comments on revised version:

      The authors did a great job and I am very happy with the revised manuscript.

    3. Reviewer #2 (Public review):

      Summary:

      Castanheira et al. investigate the role of spatial attention for planning during three maze navigation experiments (one new experiment and two existing datasets). Effective planning in complex situations requires the construction of simplified representations of the task at hand. The authors find that these mental representations (as assessed by conscious awareness) of a given stimulus are influenced by (spatially) surrounding stimuli. Individual participants varied in the degree to which attention influenced their task representations, and this attentional effect correlated with the sparsity of representations (as measured by the range of awareness reports across all stimuli). Spatially grouping task-relevant information on either the left or right side of the maze led to mental representations more similar to optimal representations predicted by the value-guided construal (VGC) model - a normative model describing a theoretical approach to simplifying complex task information. Finally, the authors propose an update to this model, incorporating an attentional spotlight component; the revised descriptive model predicts empirical task representations better than the original (normative) VGC model.

      Strengths:

      The novelty of this study lies in the proposal and investigation of a cognitive mechanism through which a normative model like value-guided construal can enable human planning. After proposing attention as this mechanism, the authors make concrete hypotheses about mismatches between the VGC predictions and real human behavior, which are experimentally validated. Thus, not only does this study describe a possible mechanism for simplification of task information for planning, but the authors also propose a descriptive model, revising VGC to incorporate this attentional component.

      A strength of this paper is the variety of investigative approaches: analysis of existing data, novel experiment, and a computational approach to predict experimental findings from a theoretical model. Analyzing pre-existing datasets increases the size of the participant cohort and strengthens the authors' conclusions. Meanwhile, comparing the predictions of the existing normative model and the authors' own refined model is a clever approach to substantiate their claims. In addition, the authors describe several crucial controls, which are key to the interpretability of their results. In particular, the eye tracking results were critical.

      In summary, this paper constitutes an important step toward a more complete understanding of the human ability to plan.

      Comments on revised version:

      I am overall happy with the revision and agree that the authors have addressed most of the comments.

    4. Reviewer #3 (Public review):

      Summary:

      The authors build on a recent computational model of planning, the "value-guided construal" framework by Ho et al. (2022), which proposes that people plan by constructing simple models of a task, such as by attending to a subset of obstacles in a maze. They analyze both published experimental data and new experimental data from a task in which participants report attention to objects in mazes. The authors find that attention to objects is affected by spatial proximity to other objects (i.e., attentional overspill) as well as whether relevant objects are lateralized to the same hemifield. To account for these results, the authors propose a "spotlight-VGC" model, in which, after calculating attention scores based on the original VGC model, attention to objects is enhanced based on distance. They find that this model better explains participant responses when objects are lateralized to different hemifields. These results demonstrate complex interactions between filtering of task-relevant information and more classical signatures of attentional selection.

      Strengths:

      (1) The paper builds on existing modeling work in a novel manner and integrates classic results on attention into the computational framework.

      (2) The authors report new and extensive analyses of existing data that shed light on additional sources of systematic variability in responses related to attentional spillover effects

      (3) They collect new data using new stimuli in the original paradigm that directly test predictions related to the lateralization of task-relevant information, including eye tracking data that allows them to control for possible confounds.

      (4) The extended model (spotlight-VGC) provides a formal account of these new results.

      Comments on revised version:

      I also agree that the authors addressed our comments and the manuscript is much stronger now.

    5. Author response:

      The following is the authors’ response to the original reviews.

      Reviewer #1 (Public review):

      Summary:

      This study investigated how visuospatial attention influences the way people build simplified mental representations to support planning and decision-making. Using computational modeling and virtual maze navigation, the authors examined whether spatial proximity and the spatial arrangement of obstacles determine which elements are included in participants' internal models of a task. The study developed and tested an extension of the value-guided construal (VGC) model that incorporates features of spatial attention for selecting simpler task mental representation.

      Strengths:

      (1) Original Perspective:

      The study introduces an explicit attentional component to established models of planning, offering an approach that bridges perception, attention, and decisionmaking.

      (2) Methodological Approach:

      The combination of computational modeling, behavioral data, and eye-tracking provides converging measures to assess the relationship between attention and planning representations.

      (3) Cross-validated data:

      The study relies on the analysis of three separate datasets, two already published and an additional novel one. This allows for cross-validation of the findings and enhances the robustness of the evidence.

      (4) Focus on Individual Differences:

      Reports of how individual variability in attentional "spillover" correlates with the sparsity of task representations and spatial proximity add depth to the analysis.

      We thank the Reviewer for their overall positive assessment of our work and their helpful comments. We have addressed each point below.

      Weaknesses:

      (1) Clarity of the VGC model and behavioral task:

      The exposition of the VGC model lacks sufficient detail for non-expert readers. It is not clear how this model infers which maze obstacles are relevant or irrelevant for planning, nor how the maze tasks specifically operationalize "planning" versus other cognitive processes.

      The method for classifying obstacles as relevant or irrelevant to the task and connecting metacognitive awareness (i.e., participants' reports of noticing obstacles) to attentional capture is not well justified. The rationale for why awareness serves as a valid attention proxy, as opposed to behavioral or neurophysiological markers, should be clearer.

      We thank the reviewer for urging further clarity here. Our work builds closely on the previous maze navigation paradigm and VGC model developed and reported by Ho et al. Nature (2022). We directly adopted variants of their maze stimuli, computational model and obstacle awareness measures, and married these with an investigation of the role of visuospatial attention. We agree that it would be useful for the reader to have a more in-depth description of the paradigm and model, and how it operationalises planning, without needing to refer back to the original Ho et al. paper. We have now added additional explanatory sections to the Introduction and Methods as follows:

      On page 4:

      “One elegant approach to forming such a simplified representation is to adaptively select the granularity of information required to complete the task (Ho et al., 2022), known as value-guided construal (VGC). Unlike previous accounts, which model human planning as a search over all items (e.g.., tube lines), the VGC model predicts that a cognitively limited decision-maker selects a manageable subset of information over which to plan— i.e., a task representation—balancing utility and complexity (Ho et al., 2022). In our example, the VGC algorithm would plan over a few relevant tube lines rather than planning over all possible stations. To select the representation that achieves the best balance between utility and complexity, the model searches across all possible combinations of tube lines, computing the value (i.e., the plan’s utility minus its cost) of each representation for planning a specific journey. The algorithm then selects the representation with the highest value, which ensures that an ideal observer selects a representation which only includes the items (i.e., tube lines) that lead to successful planning while excluding as many items as possible to keep the plan as simple as possible. For our purposes, items included in the representation are considered taskrelevant, while items that are not represented are considered task-irrelevant. This algorithm, therefore, provides a normative standard of an efficient plan to which we can compare people’s actual plans.”

      On page 6:

      “We operationalized planning using a maze navigation paradigm, akin to our tube-related example, where participants were required to plan a route through the maze, avoiding obstacles that blocked their path. Obstacles predicted by the sVGC model to be included in the representation were considered task-relevant.”

      “At the end of every trial, participants reported their awareness of specific obstacles (see Methods for details). The level of awareness reported for different obstacles provides a read-out of what features of the environment individuals were subjectively representing while solving a particular maze. While other markers of attention and awareness (for instance, behavioural or neurophysiological variables) could also be used, here we focused on direct awareness reports in order to relate our findings both to those of Ho and colleagues and to the subjective awareness reports used in consciousness science (e.g. the Perceptual Awareness Scale (Barnett et al., 2024; Overgaard & Sandberg, 2021; Ramsøy & Overgaard, 2004; Samaha et al., 2015)). Participants were instructed to maintain central fixation while planning (see dataset dSC 1), in line with previous empirical work using this task (Ho et al., 2022).”

      To visualize our effects, we binarized the predictions of the sVGC model such that obstacles with a marginalized probability greater than 0.5 were considered taskrelevant, while other obstacles were considered task-irrelevant (e.g., Figure 2b). We now clarify this point in the caption of Figure 2.

      (2) Attention framework:

      The account of attention is largely limited to the "spotlight" model. When solving a maze, participants trace the correct trail, following it mentally with their overt or covert attention. In this perspective, relevant concepts are also rooted in attention literature pertaining to object-based attention using tasks like curve tracing (e.g., Pooresmaeili & Roelfsema, 2014) and to mental maze solving (e.g., Wong & Scholl, 2024), which may be highly relevant and add nuance to the current work. This view of attention may be more pertinent to the task than models of simultaneously tracking multiple objects cited here. Prior work (notably from the Roelfsema group) indicates that attentional engagement in curve-tracing tasks may be a continuous, bottom-up process that progressively spreads along a trajectory, in time and space, rather than a "spotlight" that simply travels along the path. The spread of attention depends on the spatial proximity to distractors - a point that could also be pertinent to the findings here.

      Moreover, the tracing of a "solution" trail in a maze may be spontaneous and not only a top-down voluntary operation (Wong & Scholl, 2024), a finding that requires a more careful framing of the link to conscious perception discussed in the manuscript.

      Conceptualizing attention as a spatial spotlight may therefore oversimplify its role in navigation and planning. Perhaps the observed attentional modulation reflects a perceptual stage of building the trail in the maze rather than a filter for a later representation for more efficient decision making and planning. A fuller discussion of whether the current model and data can distinguish between these frameworks would benefit readers.

      We thank the reviewer for highlighting relevant findings in the attention literature that were missing from our discussion. We fully agree that a complete account of the interplay of planning, navigation, and attention is likely to recruit the kind of curvetracing processes highlighted by the reviewer. However, we emphasise that our current focus is not on the process of navigation through a maze, but on the process of construing the maze itself. In other words, we are focused not on how people represent their path from A to B, but how they represent the maze itself, which they then use as a basis for planning between A and B. The VGC model predicts that a subset of obstacles will be included in this construal. We think that a spotlight model is a good starting point for this work, because attention is being deployed across the whole maze stimulus, and then becomes attached to particular objects located in particular positions. This is a distinct process from that involved in navigating the path itself. Accordingly, our stimuli were designed such that task-relevant obstacles could be presented either proximally or distally to the optimal path (e.g., Figure 1a and Supplemental Figures S1-6). An obstacle that blocks any possible path on one side of the maze is task-relevant but located a long way from the optimal path. The results of Ho and colleagues’ (2022) third experiment demonstrate how task-relevant yet distal obstacles are better remembered than task-irrelevant proximal obstacles (see Figure 4 of Ho et al., 2022). We also observed that obstacles further away from the navigation path were often represented by participants (see Figures S1-6), which cannot be explained by curve tracing alone.

      While these results cannot definitively rule out the possibility that participants automatically trace the path while also construing the maze, they suggest that the value-guided construal process is an independent predictor of participants’ representations beyond proximity to the navigated path. To make this distinction clearer, we now cite the papers alluded to by the reviewer, in the Discussion on pages 28-29, while also acknowledging the potential for investigating attention during the navigation process itself:

      “Future work may also wish to examine the relevance of visuospatial attention for the navigation process itself in this task. While our present findings speak to how individuals perceive the maze while planning, it remains unclear how attention is deployed during navigation along a path, such as how object-based attention progressively spreads along trajectories in time and space(Pooresmaeili & Roelfsema, 2014; Wong & Scholl, 2024).”

      There is also one additional nuance to the current spotlight model that we were inspired to consider by the reviewer’s comment. This is the idea that attentional effects may spread within or along the obstacles themselves. We cannot explore this in the current data because we asked for awareness of the entire obstacles, not parts of obstacles, but it may be possible to explore this in future work, for instance, with eye tracking measures.

      More generally, the growth-cone (i.e., zoom lens) model of attention for curve tracing proposed by Roelfsema and colleagues shares considerable similarities with the spotlight of attention model. Both models argue for the grouping of spatially proximal items based on attention. While the growth-cone model argues for varying sizes of zoom lenses (i.e., receptive fields of neurons) that facilitate the tracing of proximal items, both models predict that spatially proximal items are preferentially processed together because of attention. Indeed, the spotlight model could model these varying zoom lenses by altering the width of the attentional spotlight dynamically across the visual scene based on the spatial proximity of obstacles. Following related comments by Reviewer 2, we now investigate inter-individual differences in the attentional spotlight of participants and observed that these differences significantly predict participants’ mental representations (see Attentional spotlight model of task representations). We have now updated the Discussion to include consideration of these alternative model frameworks:

      On page 27:

      “Second, in the current work we were unable to distinguish whether these attentional effects are driven by a fixed spotlight of attention, or whether attention operates akin to a zoom lens, shifting the ‘width’ of the focus of attention according to the task demands (Eriksen & St. James, 1986; Müller et al., 2003; Schad & Engbert, 2012). The latter view would be consistent with growth-cone models of attention in which the focus of attention expands and contracts in accordance with task demands, mirroring the various receptive field sizes in the visual hierarchy (Pooresmaeili et al., 2014; Pooresmaeili & Roelfsema, 2014). In partial support of this idea, we found significant inter-individual differences in the width of participants’ attentional spotlight (Figure S11). It is also possible that attention is deployed within or along parts of obstacles, rather than on entire obstacles. Future work using naturalistic measures of eye movements may be able to address these questions.”

      (3) Lateralization of attention:

      The analysis considers whether relevant information is distributed bilaterally or unilaterally across the visual display, but does not sufficiently address evidence for attentional asymmetries across the left and right visual fields due to hemispheric specialization (e.g., Bartolomeo & Seidel Malkinson, 2019). Whether effects differ for left versus right hemifield arrangements is not made explicit in the presented findings.

      We thank the reviewer for this suggestion. To address this point, we fitted a three-way interaction model between VGC model prediction, lateralization index, and side (left vs right hemifield). We did not find evidence for the three-way effect (β= 0.01, SE= 0.02, 95% CI [-0.03, 0.04], p = 0.738; ΔBIC = 58.30 in favour of the null effect; see table below), suggesting that the side to which participants lateralized their attention did not influence their task representations. This result is now reported on page 12:

      “This effect did not vary significantly as a function of the specific hemifield (i.e., left vs right) in which task-relevant information was presented (β= 0.01, SE= 0.02, 95% CI [-0.03, 0.04], p = 0.738; ΔBIC = 58.30 in favour of the null effect; see table S14).”

      We also explored inter-individual differences in participants’ tendency to lateralize their attention (see also the next point). We observed that participants tended to lateralize their attention slightly more to the right-hand side for non-lateralized maze stimuli, despite the normative sVGC model predicting that participants should not lateralize their attention for these stimuli (Figure 3c). These results may speak to potential asymmetries in lateralization, but given the exploratory nature of these analyses, they should be verified and replicated in future work.

      (4) Individual differences:

      Individual differences in attentional modulation are a strength of the work, but similar analyses exploring individual variation in lateralization effects could provide further insight, and the lack of such analyses may mask important effects.

      Thank you for this suggestion. In new analyses, we explored whether i) participants exhibited differences in their tendency to lateralize their awareness reports, and ii) whether the degree to which they tended to lateralize their awareness predicted their performance on a separate set of maze stimuli. In short, we observed substantial variation in participants’ tendency to lateralize their awareness (Figure S11) and found that this tendency reflected an inter-individual difference which was stable across maze types. We report these new findings on pages 14-16.

      “Inter-individual variation in lateralization of attention

      Next, we investigated participants’ tendency to pay attention to obstacles within a single hemifield (left vs right) regardless of the sVGC model predictions. To do so, we computed an awareness lateralization index (ALI) based on participants’ self-reported awareness reports of obstacles on each trial (Figure 3a). Large positive values indicate that participants were preferentially aware of the right hemifield, whereas negative values indicate preferential awareness of the left hemifield. Values close to zero indicate that participants paid attention to both hemifields equally (see Methods for details). We observed that participants’ tendency to lateralize their awareness varied greatly across the Ho datasets 1 and 2 (Figure 3b); some participants preferentially paid attention to a single hemifield, regardless of whether the sVGC model predictions were lateralized. For the dSC1 dataset, we observed that on some trials, participants significantly lateralized their awareness (|ALI| > 0.5; Figure 3c) even though the sVGC model predictions were non-lateralized. These findings suggest that participants’ tendency to pay attention to a single hemifield may represent an observable inter-individual difference in how they allocate their awareness to form task construals.”

      “To further explore these inter-individual differences, we tested whether participants’ tendencies to lateralize their attention to a single hemifield was consistent across trials and maze stimuli. We observed that participants’ tendency to lateralize their attention to a single hemifield was similar for left and right lateralized maze stimuli (Spearman ⍴= 0.72, Figure 3d). This suggests that participants who preferentially attended to a single hemifield did so regardless of which hemifield they should attend to. More consequentially, the tendency for participants to lateralize their awareness on maze stimuli whose model predictions were also lateralized linearly correlated with participants’ tendency to lateralize their attention on non-lateralized maze stimuli (Spearman ⍴= 0.88, Figure 3d). Taken together, these findings emphasize that some individuals tend to preferentially attend to a single hemifield when planning. This tendency, importantly, represents an inter-individual difference in how participants allocate their attention across various maze types.”

      (5) Distinction between overt and covert attention:

      The current report at times equates eye movement patterns with the locus of attention. However, attention can be covertly shifted without corresponding gaze changes (see, for example, Pooresmaeili & Roelfsema, 2014).

      We fully agree, and thank the reviewer for prompting further reflection on this distinction. In the online experiments run by Ho and colleagues (i.e., datasets Ho1 and Ho2), participants’ eye movements were not tracked, and therefore, they could not disambiguate whether participants were engaging in covert or overt attention to sample maze obstacles. In our third experiment (i.e., dataset dSC1), we both recorded eye movements and explicitly instructed participants to fixate centrally while viewing the maze. This ensured that participants oriented their attention only covertly during planning (see Figure S13-14).

      We now elaborate on this important distinction in the Results section of the manuscript, page 12:

      “In addition, we monitored participants’ eye movements in dataset dSC 1 to ensure that attention shifts would be covert as opposed to overt—a distinction which could not be determined in the online samples of datasets Ho 1 and 2.”

      On page 28:

      “Importantly, while the visuospatial attention effects observed in the Ho 1 and 2 datasets are likely driven by both covert and overt shifts in attention, the findings presented in experiment 3 (i.e., dSC1 dataset) rule out the contribution of overt shifts in attention through the use of eye tracking (see Figure S13-14)(Carrasco, 2011; Pooresmaeili & Roelfsema, 2014).”

      The implications for interpreting the relationship between eye movement, memory, and attention in this setting are not fully addressed. The potential dynamics of attention along a maze trajectory and their impact on lateralization analysis would benefit from further clarification.

      We thank the reviewer for urging more clarity here. The attentional dynamics we document in our study concern how people perceive / construe the maze itself, rather than how they deploy their attention to guide active navigation. We have now sought to make this distinction clear at a number of points in the paper. The core idea is that attention acts as an early filter to select which obstacles are part of a task construal, which then affects both awareness and memory.

      We have now clarified the focus of our study in the introduction on pages 5-7:

      “Our focus in this study was to examine how participants perceive and represent their environment (the maze stimulus). This is a distinct process to how participants orient their attention during navigation itself, which is not part of our current study. To do so, we harness classical signatures of attentional selection to characterise how visuospatial attention shapes awareness of maze obstacles during planning.” … “Our focus in the present study was to examine attentional effects on participants’ perception of the maze stimulus. We did not quantify how individuals deploy their attention in the phase in which they were navigating through the maze.”

      We did not explicitly test for memory effects in our new experiments, but Ho and colleagues demonstrated that the sVGC model predicted not only awareness reports, but also participants’ memory of obstacles (see Ho et al., 2022). Indeed, task representations computed from memory or awareness reports were strikingly similar in their experiments (Spearman ⍴ = 0.86 between memory accuracy and awareness; ⍴ = 0.86 between confidence in memory and awareness). In relation to eye movements, we refer the reviewer back to our previous response, which details how eye movements were measured and controlled during maze construal.

      Figure 1 legend (b) --> (c)

      We have corrected this typo in the figure caption.

      Reviewer #2 (Public review):

      Summary:

      Castanheira et al. investigate the role of spatial attention for planning during three maze navigation experiments (one new experiment and two existing datasets). Effective planning in complex situations requires the construction of simplified representations of the task at hand. The authors find that these mental representations (as assessed by conscious awareness) of a given stimulus are influenced by (spatially) surrounding stimuli. Individual participants varied in the degree to which attention influenced their task representations, and this attentional effect correlated with the sparsity of representations (as measured by the range of awareness reports across all stimuli). Spatially grouping taskrelevant information on either the left or right side of the maze led to mental representations more similar to optimal representations predicted by the valueguided construal (VGC) model - a normative model describing a theoretical approach to simplifying complex task information. Finally, the authors propose an update to this model, incorporating an attentional spotlight component; the revised descriptive model predicts empirical task representations better than the original (normative) VGC model.

      Strengths:

      The novelty of this study lies in the proposal and investigation of a cognitive mechanism through which a normative model like value-guided construal can enable human planning. After proposing attention as this mechanism, the authors make concrete hypotheses about mismatches between the VGC predictions and real human behavior, which are experimentally validated. Thus, not only does this study describe a possible mechanism for simplification of task information for planning, but the authors also propose a descriptive model, revising VGC to incorporate this attentional component.

      A strength of this paper is the variety of investigative approaches: analysis of existing data, novel experiment, and a computational approach to predict experimental findings from a theoretical model. Analyzing pre-existing datasets increases the size of the participant cohort and strengthens the authors' conclusions. Meanwhile, comparing the predictions of the existing normative model and the authors' own refined model is a clever approach to substantiate their claims. In addition, the authors describe several crucial controls, which are key to the interpretability of their results. In particular, the eye tracking results were critical.

      In summary, this paper constitutes an important step toward a more complete understanding of the human ability to plan.

      We thank the Reviewer for their thoughtful and positive assessment of our findings. We also appreciate the constructive feedback on our methodology, which we believe has substantially improved our manuscript.

      Weaknesses:

      (1) There is a critical conceptual gap in the study and its interpretation, mainly due to the reliance on a self-report metric of awareness (rather than an objective measure of behavioral performance).

      a. Awareness is tested by a 9-point self-report scale. It is currently unclear why awareness of task-irrelevant obstacles in this task would necessarily compromise optimal planning. There is no indication of whether self-reported awareness affects performance (e.g., navigation path distance, time to complete the maze, number of errors). Such behavioral evidence of planning would be more compelling.

      We thank the reviewer for prompting further reflection on the connection between construal and navigation performance. We wish to emphasise that the primary focus of our study was on measuring and modeling participants’ task construals using perceptual awareness judgments, building on the methods developed by Ho and colleagues, rather than on navigation performance itself. However, as the reviewer points out, there is a natural relationship between construal and performance – if you represent the wrong obstacles, plans may be disrupted.

      To explore the relationship between task construals and performance on the navigation task we first regressed out the effects of the sVGC model on participants’ awareness reports and computed the mean squared residuals for each trial. We then used these values to predict participants’ navigation response times on each trial. We observed a significant negative relationship, suggesting that on trials where participants’ representations showed greater deviations from the normative model, they were in fact faster at navigating the mazes. This relationship was surprising, and at odds with the initial idea that adhering to normative VGC aids in task performance. However, we think that this direction of effect may make sense if one considers that a large part of the actual construal (rather than the normative prediction) in our data was in fact driven by effects such as lateralisation which are not accounted for by the sVGC model. If one is faster at harnessing inductive biases such as lateralisation, then one may be faster to complete the maze but also show a greater deviation from the predictions of the original model.

      To further explore these effects, we next focused on the distinction between lateralised and non-lateralised mazes. Here, we reasoned that the initial phase of lateralised attentional selection would lead to lateralised mazes being easier to navigate than nonlateralised ones. We conducted new analyses to determine whether participants navigated lateralized maze stimuli faster and with fewer moves than maze stimuli with non-lateralized model predictions. As detailed in Methods, we excluded trials in which participants significantly deviated from the optimal number of moves (9 or more moves) and took longer than 20 seconds to solve the maze. In line with our interpretation that attention operates as an inductive bias, participants were faster and deviated less from the optimal path on lateralized compared to non-lateralized mazes.

      We now report these new results on navigation performance on pages 20-21:

      “Maze navigation performance

      The previous analyses focused on participants’ task representations during planning. We next sought to explore links between participants’ task representations and maze navigation performance. Participants performed the maze navigation task near-ceiling: they solved 95% of maze stimuli in under 20 seconds, with minimal deviation from the optimal path (i.e., 9 moves or fewer). Notwithstanding this limited variance in task performance, we explored whether participants’ task construals may have impacted their navigation speed. To do so, we first regressed out the effects of the sVGC model from participants’ awareness reports and used the mean squared residuals for each trial to predict response times (see Methods for details). Surprisingly, we observed a negative relationship between mean squared residual variance and response times (β = -0.31, SE = 0.05, 95% CI [-0.41, -0.21], p < 0.001), indicating that participants were faster on trials where the sVGC model explained less variance in their awareness reports. In other words, trials in which participants deviated more from the sVGC model predictions were solved faster. We note that one reason for this may be the strong influence of the lateralisation effect on navigation performance (see paragraph below), which itself is not part of the sVGC model prediction.”

      “We then explored whether participant performance differed between lateralised and nonlateralised mazes. Here, we reasoned that the initial phase of lateralised attentional selection would lead to lateralised mazes being easier to navigate than non-lateralised ones. Consistent with this hypothesis, participants were faster (β = -0.04, SE = 5.91*10<sup>3</sup>, 95% CI [-0.06, -0.03], p< 0.001) and followed the optimal path more closely (β = -0.59, SE = 0.09, 95% CI [-0.78, -0.40], p< 0.001) when maze stimuli were more lateralized.”

      And in the Discussion section, on page 23:

      “Mental representations and task performance

      We observed that participants were faster and deviated less from the optimal path on maze stimuli that were lateralized. This effect is not predicted by the original sVGC model but dovetails with the interpretation that early visuospatial attention operates as an inductive bias to guide the formation of simplified task representations. Surprisingly, we also observed that participants were faster to navigate mazes on trials where their simplified task representation deviated from the sVGC model prediction. We interpret this seemingly contradictory finding in the following way: there are several factors beyond the sVGC model – including, for instance, maze lateralisation – that predict both construal and performance on the maze navigation task. Further work is needed to understand how inductive biases such as lateralisation shape both construal and performance, and the real-world benefits that such strategies might afford for naturalistic stimuli.”

      b. Relatedly, it would have been more convincing to have an objective measure of awareness, for instance, how the presence or absence of a "task-irrelevant" obstacle affects performance (e.g., change navigation path distance or time to complete the maze), or whether participants can accurately recall the location of obstacles.

      We thank the reviewer for prompting further reflection on the validity and robustness of our awareness measures. We emphasise however that our focus is not (primarily) on maze navigation performance, but on task construal, which as noted in our previous response may come apart from navigation performance for a variety of reasons. Our primary goal is to measure participants’ subjective awareness of the maze as a marker of their idiosyncratic (conscious) mental representation on each trial. In doing so, we build on a rich tradition of measuring subjective awareness in consciousness and perception science (for instance, work using the Perceptual Awareness Scale, or detection judgments). In this sense, we think our awareness scale (following Ho et al.) represents a valid and straightforward way of assessing our target psychological construct. However, we also agree with the Reviewer that convergent evidence from other measures is always valuable. In Ho and colleagues’ original paper, they developed a variant of the maze task where participants had to recall the location of obstacles, as well as rate their awareness (Exp 3) and a variant in which participants could hover their mouse over hidden obstacles in the maze to reveal their location – an online metric of attentional deployment (Exp 4). These data afforded us the opportunity to validate the awareness reports against an objective measure of recall, as suggested by the Reviewer. In reanalysing these data, we observed that the obstacle awareness and memory/hover measures were strikingly correlated within two independent samples of participants (Spearman ⍴ = 0.86 between memory accuracy and awareness; ⍴ = 0.86 between confidence in memory and awareness; ⍴ = 0.76 between the probability of hovering over the obstacle and awareness; ⍴ = 0.65 between the duration of the mouse hovering and awareness). These re-analyses are now reported on page 22 of our manuscript, to highlight the convergent validity of the awareness metric:

      “Finally, we examined the convergent validity of participants’ awareness reports by reanalyzing the memory recall data reported in Ho and colleagues’ experiment(Ho et al., 2022). We reasoned that participants should demonstrate similar task representations regardless of the measure used to probe the construal. In line with this prediction, we observed that the obstacle awareness reports and memory/hover measures were strikingly correlated within three independent samples of participants (Spearman ⍴ = 0.86 between memory accuracy and awareness; ⍴ = 0.86 between confidence in memory and awareness; ⍴ = 0.76 between the probability of hovering over the obstacle and awareness; ⍴ = 0.65 between the duration of the mouse hovering and awareness; see Tables S18 and S19).”

      c. Consequently, I'm not sure that we can conclude that the spatial context does impact participants' ability to plan spatial navigation or to "incorporate taskrelevant information into their construal". We know that the spatial context affects subjective (self-reported) awareness, but the authors do not present evidence that spatial context affects behavioral performance.

      Following the line of argument above, we think it’s important to separate out task construal (the simplified representation of the maze, measured by awareness reports), and the impact of this on navigation and other aspects of behaviour. The awareness reports (and other convergent measures) show that task-relevant information (as predicted by the VGC) is incorporated into the construal, a process which is modulated by spatial context. These are the key targets of our modeling. Whether this impacts performance is a distinct question, and one that we now address in our response to point a above.

      d. Another concern that may complicate interpretation is the following: Figure 3c shows improved VGC model predictions (steeper slope) for mazes with greater lateralization. However, there are notable outliers in these plots, where a high lateralization index does not correspond to good model performance. There is currently no discussion/explanation of these cases.

      The Reviewer astutely points out some outliers in our analysis. While on average lateralized maze stimuli are represented more closely to the sVGC model, there are indeed some noticeable outlier mazes. These mazes represent stimuli in which participants tended to lateralize their attention to the ‘wrong hemifield’—e.g., participants were more aware of obstacles in the right hemifield despite sVGC model predicting that obstacles on the left hemifield were task-relevant. We believe this explains the poor sVGC model fits on these trials. We note, however, that on average participants were capable of attending to the correct hemifield without explicit instructions (i.e., 9 out of 12 mazes).

      We have now included a discussion of these outliers in the results section of the paper on page 12:

      “We note that for three maze stimuli whose model predictions were lateralized there was nevertheless a poor fit to the sVGC model (see Figure 2c, right panel). These outliers correspond to maze stimuli where participants, on average, lateralized their attention to the incorrect hemifield (i.e., the opposite hemifield to that predicted by the sVGC model).”

      (2) I noticed an issue with clarity regarding task-relevance. It is currently not fully clear which obstacles are "task irrelevant". Also, the term is used inconsistently, sometimes conflating with "awareness". For example, in the "Attentional spotlight model of task representations" section, the authors state that "taskrelevant information becomes less relevant when surrounded by task-irrelevant information". But they really mean that participants become less aware of those task-relevant obstacles. I assume task-relevance is an objective characteristic related to maze organization, not to a participant's construal. Indeed, the following paragraph provides evidence of model predictions of awareness.

      We apologize for any confusion regarding the terminology of our manuscript. We indeed use the terms task-relevant and task-irrelevant to refer to obstacles that are objectively predicted by the normative sVGC model or the attentional spotlight model to be included in (>0.5) or excluded from (<0.5) task construals, respectively. This designation reflects the predictions from the computational model and does not reflect participants’ reported awareness. We then ran linear hierarchical models to predict participants’ awareness reports from these model predictions. The Reviewer is correct that the task-relevance of obstacles is indeed related to the maze’s organization, and not related to participants’ subjective reports of awareness. We have now clarified this point throughout the manuscript to better emphasize the difference between the model predictions of taskrelevance and participants’ subjective reports.

      On page 17:

      “To achieve this, we computed the predictions of the existing VGC model for each obstacle’s task relevance in a given maze, and averaged these predictions within an attentional spotlight of 3 squares (Figure 4a & S8, see Methods for details). This process yielded novel model predictions, whereby some obstacles which were once predicted as task-irrelevant by the normative sVGC are now predicted as task-relevant by the attentional spotlight model. We depict the effects of this spatial spotlight in Figure 4a: task-irrelevant stimuli (plotted in grey; see middle left obstacle) neighbouring taskrelevant obstacles (plotted in orange) become more task-relevant, whereas taskrelevant information becomes less relevant when surrounded by task-irrelevant information (see bottom right orange obstacle). This deviation in model predictions from the normative sVGC model was used to predict participants’ awareness reports. We hypothesized that this spotlight-VGC model would predict participants’ reports better than the original VGC model, which does not account for spatial attention.”

      (3) The behavioral paradigm has some distinct disadvantages, and the validity of the task is not backed up by behavioral data.

      a. I understand the need for central fixation, but it also makes the task less naturalistic.

      The fixation cross was required on every trial such that participants could maintain central fixation for our eye tracking experiment. While this design is less naturalistic, it allows us to examine the eye movements of participants. Requiring participants to fixate during the ‘planning’ phase of the experiment allowed us to isolate the effects of covert attention from changes in awareness due to overt shifts in attention. In other words, differences in participants’ awareness reports in the 3rd experiment cannot be explained by longer fixation times to specific obstacles.

      b. The task with its top-down grid view does not seem to mimic real human navigation. Though this grid may be similar to mental maps we form for navigation, the sensory stimuli corresponding to possible paths and to spatial context during real-life navigation are very different.

      We agree with the reviewer that while our task is engaging for participants and simple to follow, it does not mimic naturalistic navigation in humans. There is a natural tension in computational / experimental work in cognitive science in wanting to build closely on previous results and paradigms, while ensuring that results can generalise to real-world contexts. Here, our choice of paradigm and measures was closely built on previous papers using this task from Ho and colleagues (2022, 2023). While preparing this response, we learnt that the MIT group had also harnessed this same task to develop a novel dynamic variant of the VGC model (Chen et al., 2026) called the Just in Time model (JIT). The advantage of building on this prior work is that we are able to iteratively refine and expand the VGC approach, and (in our case) bring it into closer contact with work on modeling the deployment of spatial attention in human vision. The top-down aspect of the maze notably facilitated the study of the spatial deployment of attention. We now discuss the novel dynamic variant of the VGC model in our paper on page 27:

      “We close by reflecting on opportunities for further work in this area. First, an important next step is to explore the process by which task representations are formed, and how inductive biases might affect the process of task construal. The sVGC model is a normative model of the optimal task representation. Since it’s construction involves an exhaustive calculation over possible paths, it is not a plausible basis for a model of the psychological process by which participants actually construct task representations. More recently a process model of task construal has been proposed, the Just in Time model (JIT). The hypothesis of the JIT model is that participants’ task representations are built up over time by iteratively simulating possible paths through the maze, affording insight into the construal process (Chen et al., 2026). In future work, it would be of interest to ask whether the attentional effects we observe in our experiments could be meshed with a dynamic JIT account of construal. We speculate that visuospatial attention may operate as an early filter, limiting the space of potential construals based on coarse spatial features of the environment, constraining a dynamic selection of obstacles. Brain imaging techniques with high time resolution, such as M/EEG, may be able to shed further light on how task representations are formed as participants plan.”

      c. Behavioral performance is not reported, so it is unknown whether participants are able to properly complete the task. The task seems pretty difficult to navigate, especially when the obstacles disappear, and in combination with the central fixation.

      Behavioural performance is now reported in response to point 1a above.

      d. There is no discussion of whether/how this navigation task generalizes to other forms of planning.

      We fully agree that an important next step would be to generalise our results on construal to naturalistic forms of planning – for instance, using immersive VR mazes, and or investigating cognitive rather than perceptual construals. We have now added a line to this effect to the Discussion on page 28.

      “An important next step to further our understanding of task representations would be to extend the current paradigm to other forms of planning and more naturalistic tasks, such as navigating immersive virtual reality (VR) environments, planning over cognitive rather than perceptual representations (e.g. planning over an abstract space), or internallyguided planning based on working memory.”

      Reviewer #2 (Recommendations for the authors):

      (1) There are, of course, benefits to simple tasks like the ones described, but it would be interesting to compare the results to a possible experiment in which a top-down grid/map is used for planning, but then task execution is carried out in a simulated environment corresponding to the map. Also, perhaps beyond the scope of the questions addressed in this paper, but I am curious how unexpected obstacles affect representations. For instance, if participants plan based on a topdown map and then begin "real" navigation but encounter an unexpected obstacle that was not indicated on the map, does this modulate representations/awareness of future obstacles (near vs. far)?

      We fully agree that all of these lines of investigation would be super interesting to pursue in future studies, and we have added a line to the discussion to that effect on page 28:

      “An important next step to further our understanding of task representations would be to extend the current paradigm to other forms of planning and more naturalistic tasks, such as navigating immersive virtual reality (VR) environments, planning over cognitive rather than perceptual representations (e.g.. planning over an abstract space), or internallyguided planning based on working memory.”

      (2) Regarding self-reported awareness as a metric, an additional experiment could ask participants to recreate the maze (identify locations of obstacles after they disappear). This would be a more objective measure of awareness.

      Yes indeed, and as described above, this was a metric used by Ho and colleagues in their previous experiment. As we describe in more detail above, the task representations obtained via memory or awareness reports demonstrated striking similarity (⍴ = 0.86).

      (3) What is meant by "all possible orientations of the maze" in this Methods sentence: "For dataset dSC 1, participants solved each of these 24 mazes four times (i.e., all possible orientations of the maze)"?

      We thank the Reviewer for prompting more clarity here. We vertically and horizontally reversed mazes (i.e., left-right flipped) such that participants could not predict the location of the goal or start location. In this way, each maze stimulus had four potential orientations. This resulted in 96 trials of 24 unique mazes. We have clarified this point in the Methods section on page 30:

      Maze stimuli were vertically and horizontally reversed (i.e., left-right flipped) such that participants could not predict the location of the start or goal location. This resulted in four potential orientations of each maze across all 24 mazes, 96 trials in total.

      (4) For lateralization, it was unclear until reading the Methods that the lateralization index was calculated using the VGC-predicted level of taskrelevance. From the main text and Figure 2, I assumed you were just counting the number of task-relevant obstacles on each side, rather than also quantifying relevance. I understood after reading the Methods, but this could be clarified further.

      We agree with the Reviewer that this was not evident from the text. We have now updated the Results section of the manuscript to clarify this point on page 11:

      “To test this hypothesis, we derived a measure of task-relevant lateralization inspired by the attention literature (Ghafari et al., 2024; Keefe & Störmer, 2021; Vollebregt et al., 2015) (Figure 2a). Specifically, we separated maze stimuli across the vertical meridian and computed the ratio of task-relevant information presented on the left versus right side derived from the sVGC model. For example, the maze shown in Figure 2a has twice the amount of task-relevant information presented in the left hemifield than in the right (lat. Index= 1/3). A lateralization index of 0.0 indicates that both hemifields contain equal amounts of task-relevant information (i.e., non-lateralized). The lateralization index was computed using the continuous VGC predictions for each obstacle (see Methods).”

      (5) The explanation in the Methods of how the width of the attentional spotlight was chosen references Figure 1b and Supplementary Figure S2, but it seems that Supplementary Figure S8 explains this more in the caption. Also, I don't see how Figure S2 supports this.

      We apologize for this typo. The explanation of how we selected the width of the attentional spotlight should indeed reference supplemental Figure 15 (previously Figure S8). We have now corrected this and elaborated on this choice in the Methods section on page 35:

      “We fixed the ‘width’ of the attentional spotlight to a distance of 3 squares based on the observation that the two neighbouring obstacles positively predicted the awareness of a probe. We observed that the mean and median distance between neighbouring obstacles of the 2nd rank (i.e., second closest) was 3 squares away for all mazes (Figure S15). We therefore opted to fix the value of the attention spotlight to 3 squares based on these observations. Future work utilizing this model should consider the statistics of their maze stimuli when deciding on the ‘width’ of the attentional spotlight.”

      (6) The attentional spotlight width was assumed to be 3 squares, based on the linear regression predictions of the effect of neighboring obstacles on stimulus awareness. Given the individual differences across participants, it would be interesting to choose a different attentional spotlight size for each participant. Would a participant-specific attentional spotlight width improve the predictions of the spotlight-VGC model?

      The Reviewer highlights a very interesting question: do individuals vary in terms of their attentional spotlight? To test this hypothesis, we first estimated the size of the attentional spotlight for each individual based on lateralized maze stimuli, and then used this to generate personalized attentional spotlight model predictions for each subject based on these values (Figure S11). We restricted this analysis to the dSC1 dataset, where we had substantially more trials (96 in total).

      In brief, we observed that indeed the personalized spotlight model fit participants’ awareness reports better than both a normative sVGC model and a group-level attentional spotlight model. We interpret these findings with some caution as i) a subset of individuals had flat attentional slopes and therefore were excluded from these analyses, and ii) we believe we require additional trials to ensure a robust model fit at the individual level. While our results are encouraging, we hope future investigations into inter-individual differences will extend these findings.

      We have included these additional analyses in the main text.

      On page 18:

      “To further explore inter-individual differences in task construal, we tested whether adjusting the attentional spotlight width to each participant’s awareness reports improved the predictions of the attentional spotlight model. To do so, we first determined the width attentional spotlight of each individual in the dSC1 dataset based on lateralized maze stimuli. We then generated person-specific attentional spotlight model predictions for the non-lateralized maze stimuli to avoid overfitting the data (Figure S11). We note that 7 participants had either flat attentional slopes or negative beta coefficients, which prevented the selection of an appropriate attentional spotlight width (see Methods for details). We observed a significant improvement in model fit for the person-specific attentional spotlight model relative to both the group-level attentional spotlight model (ΔBIC= -1487.39) and the normative sVGC model (ΔBIC= -1655.29). While the limited trial numbers per participant in our current dataset warrants caution in interpreting these findings, these findings do encourage further research on inter-individual differences in attentional deployment during planning.”

      On pages 23-24:

      “Inter-individual differences in attention

      We also observed considerable inter-individual differences in attentional effects across participants (Figure 1c). While some participants were strongly influenced by the spatial context of neighbouring stimuli, others showed more limited evidence for an attentional effect (Figure 1b). Inter-individual differences in attention predicted the sparsity of participants’ simplified representations: participants with larger attention effects exhibited sparser representations. Moreover, these inter-individual differences in effects of spatial proximity could be incorporated into the attentional spotlight model by varying the width of the spotlight, resulting in better model predictions.”

      “Beyond these spatial proximity effects, we also observed that participants varied in their tendency to lateralize their attention to a single hemifield (Figure 3). This tendency was observed across all three datasets, including on maze stimuli whose value-guided model predictions were not lateralized. This suggests that although a strategy of allocating attention is sub-optimal for these maze stimuli, some individuals preferentially attend to a single hemifield in a heuristic-like fashion. This tendency to attend to a single hemifield was a robust inter-individual difference across maze stimuli (Figure 3d), and dovetails with individual-level variation in spatial proximity effects. Taken together, these findings offer novel insights into how people vary in the ways they allocate spatial attention to solve complex problems. Future research could explore how these individual differences constrain performance on other tasks that require planning and search in highdimensional spaces.”

      On page 17 of the Supplemental Materials:

      (7) The supplementary text about lateralization effects, above Supplementary Table S8, references Table S6, but it is Table S6 does not seem to display lateralization results.

      We thank the Reviewer for pointing out this typo: we now refer to the correct supplementary table (S9).

      (8) Why does it matter that "the maze stimuli were not designed to test horizontalmeridian lateralization effects"? What is the effect on power? Is it because there is not a good enough range in lateralization indices? It would be good to clarify, or just remove that explanation, since the cortical retinotopy explanation seems more convincing.

      We did not specifically design the maze stimuli such that there is an equal number of obstacles above and below the horizontal meridian. As such, the lateralization index derived along the horizontal meridian does not control for the number of obstacles in each hemifield, which may influence participants’ awareness reports. In contrast, we designed maze stimuli such that this would not be a concern for the vertical meridian. We have clarified this point in the discussion on page 27.

      “Third, while we observed clear lateralization effects along the vertical meridian (i.e., left vs right hemifield), effects along the horizontal meridian were less clear (i.e., above vs below; see Table S15-16). One potential explanation of this asymmetry is the retinotopic organization of the cortex, in which spatially adjacent stimuli can be retinotopically distant if presented on the opposite side of the vertical (but not horizontal) meridian, facilitating distractor inhibition. Importantly, while the visuospatial attention effects observed in the Ho 1 and 2 datasets are likely driven by both covert and overt shifts in attention, the findings presented in experiment 3 (i.e., dSC1 dataset) rule out the contribution of overt shifts in attention through the use of eye tracking (see Figure S13-14)(Carrasco, 2011; Pooresmaeili & Roelfsema, 2014).”

      (9) For Figure 2c, it would be helpful to directly state what each dot and line mean.

      We updated the caption of Figure 2c to clarify what we are plotting: each point represents an obstacle, and each line the linear fit for a maze stimulus.

      “Each point represents an obstacle in a maze, and each line represents the model fit for that specific maze stimulus.”

      (10) Figures and wording imply there is only a single probe obstacle per trial, but methods and model imply that participants are asked to report awareness for every obstacle. This should be clarified.

      We apologize for any confusion regarding the methodology of our study. The Reviewer is correct that participants reported their awareness of every obstacle presented on a given trial. We have clarified this in the Results section of the manuscript on page 7:

      “Note, participants reported their awareness of every obstacle presented on a given trial.”

      We have also updated the caption of Figure 1 to clarify this point:

      “Once participants finished navigating the maze, they were asked to report their awareness of every obstacle presented on a given trial in a random order.”

      (11) What is the reason for the exclusion of participants (33 for experiment 1 and 26 for experiment 2)?

      Participants were excluded from the Ho et al. datasets 1 and 2 based on their preregistered exclusion criteria, as detailed in the Methods section of their paper. In short, trials were excluded if participants took longer than 20 seconds to complete the trial, or if they spent longer than 5 seconds in the initial state. Participants were excluded if less than 80% of trials remained after reaction time exclusions or if they failed 2 out of 3 comprehension checks. We have elaborated on this point in the Methods section on page 31.

      “Participants were excluded from analyses based on pre-registered exclusion criteria as detailed in (Ho et al., 2022). In short, participants were excluded if 20% or more of their trials were removed based on reaction times, or if they failed 2 out of 3 comprehension checks.”

      (12) The supplemental figures are not referenced in order, and some are not referenced at all; this should be fixed.

      We thank the Reviewer for pointing this out and have reorganized our Supplementary materials accordingly.

      Reviewer #3 (Public review):

      Summary:

      The authors build on a recent computational model of planning, the "value-guided construal" framework by Ho et al. (2022), which proposes that people plan by constructing simple models of a task, such as by attending to a subset of obstacles in a maze. They analyze both published experimental data and new experimental data from a task in which participants report attention to objects in mazes. The authors find that attention to objects is affected by spatial proximity to other objects (i.e., attentional overspill) as well as whether relevant objects are lateralized to the same hemifield. To account for these results, the authors propose a "spotlight-VGC" model, in which, after calculating attention scores based on the original VGC model, attention to objects is enhanced based on distance. They find that this model better explains participant responses when objects are lateralized to different hemifields. These results demonstrate complex interactions between filtering of task-relevant information and more classical signatures of attentional selection.

      Strengths:

      (1) The paper builds on existing modeling work in a novel manner and integrates classic results on attention into the computational framework.

      (2) The authors report new and extensive analyses of existing data that shed light on additional sources of systematic variability in responses related to attentional spillover effects

      (3) They collect new data using new stimuli in the original paradigm that directly test predictions related to the lateralization of task-relevant information, including eye tracking data that allows them to control for possible confounds.

      (4) The extended model (spotlight-VGC) provides a formal account of these new results.

      We thank the Reviewer for their positive assessment of our manuscript and their insightful comments, which has improved the clarity of our findings.

      Weaknesses:

      (1) The spotlight-VGC model has a free parameter - the "width" of the attentional spotlight. This seems to have been fixed to be 3 squares. It would be good if the authors could describe a more principled procedure for selecting the width so that others can use the model in other contexts.

      Our choice for this parameter was informed by the spatial effects reported in Figure 1b. We observed that the two closest neighbouring obstacles to a probe had similar awareness (i.e., positive beta weights). We therefore compute the mean and median distances between obstacle pairs that were the second closest obstacle to a probe. This distance was 3 squares away, as depicted in Figure S15. We fixed the width of the attentional spotlight across all studies based on this observation. We agree that future research utilizing this model may need to tune this hyperparameter depending on the mean distance between a probe and its neighbours.

      We have clarified this point in the methods section on page 35:

      “We fixed the ‘width’ of the attentional spotlight to a distance of 3 squares based on the observation that the two neighbouring obstacles positively predicted the awareness of a probe. We observed that the mean and median distance between neighbouring obstacles of the 2nd rank (i.e., second closest) was 3 squares away for all mazes (Figure S15). We therefore opted to fix the value of the attention spotlight to 3 squares based on these observations. Future work utilizing this model should consider the statistics of their maze stimuli when deciding on the ‘width’ of the attentional spotlight.”

      Following the suggestion of Reviewer 2 point 6, we now also explored inter-individual differences in this parameter. To do so, we first used the lateralized mazes in the dSC1 dataset to determine the optimal width of the attentional spotlight for each individual.

      Then, we used this spotlight to derive model predictions for each person. We observed that these personalized attentional spotlight model predictions fit participants’ awareness reports on non-lateralized mazes better than the fixed-width spotlight model. We believe this preliminary result suggests the importance of modelling inter-individual differences in attentional deployment during planning. We report these effects on page 17.

      (2) Have the authors considered other ways in which factors such as attentional spillover and lateralization could be incorporated into the model? The spotlightVGC model, as presented, involves first computing VGC predictions and only afterwards computing spillover. This seems psychologically implausible, since it supposes that the "optimal" representation is first formed and then it gets corrupted. Is there a way to integrate these biases directly into the VGC framework, perhaps as a prior on construals? The authors gesture towards this when they talk about "inductive biases", but this is not formalized.

      We thank the reviewer for bringing up this very important point. We think that a full computational treatment of the inductive bias would be a distinct project, but now seek to expand our discussion on the mechanisms by which representations could be formed. In this context, we specifically highlight novel computational work from the MIT group that was published as a preprint in the time since we submitted our paper, and which proposes a new process account of construal, the “Just in Time” (JIT) model. We also elaborate on a possible mechanism by which visuospatial attention may aid the dynamics of the construal process. In short, we agree with the reviewer that spatial attention may bias individuals to search over a subset of potential representations based on low-level spatial characteristics of the obstacles (e.g., their spatial spread in the visual field), prior to (or in concert with) a dynamic JIT-like selection process. We now elaborate on these possibilities on pages 27-28:

      “We close by reflecting on opportunities for further work in this area. First, an important next step is to explore the process by which task representations are formed, and how inductive biases might affect the process of task construal. The sVGC model is a normative model of the optimal task representation. Since it’s construction involves an exhaustive calculation over possible paths, it is not a plausible basis for a model of the psychological process by which participants actually construct task representations. More recently a process model of task construal has been proposed, the Just in Time model (JIT). The hypothesis of the JIT model is that participants’ task representations are built up over time by iteratively simulating possible paths through the maze, affording insight into the construal process (Chen et al., 2026). In future work, it would be of interest to ask whether the attentional effects we observe in our experiments could be meshed with a dynamic JIT account of construal. We speculate that visuospatial attention may operate as an early filter, limiting the space of potential construals based on coarse spatial features of the environment, constraining a dynamic selection of obstacles. Brain imaging techniques with high time resolution, such as M/EEG, may be able to shed further light on how task representations are formed as participants plan.”

      […]

      “Fourth, it will also be necessary to elaborate on how bottom-up and top-down aspects of attentional selection are combined to guide complex task representations and plans. Foundational questions remain unanswered, for instance: can multiple spatial locations be preferentially selected at once, i.e. are there multiple spotlights (Awh & Pashler, 2000; McMains & Somers, 2004; Pylyshyn & Storm, 1988; Shaw & Shaw, 1977)? There is also discourse on how spatial attention may move from one location to another: are the intervening visual regions between attended locations similarly selected (Dubois et al., 2009; Kr & Np, 1999; McMains & Somers, 2004, 2005)? Our findings tentatively suggest that individuals are able to attend to disparate spatial regions to form sparse task representations, yet there is substantial variability in how individuals orient their attention during the task. The present paradigm and computational modelling, in conjunction with carefully designed stimuli, may help resolve these outstanding questions.”

      (3) Can the authors rule out that the lateralization effects are the result of memory biases since the main measure used is a self-report of attention?

      We thank the reviewer for bringing up this important point. In our experiments, we sought to measure participants’ subjective awareness of the maze stimuli as a readout of their conscious task representation on each trial. This approach marries an extensive literature on measures of perceptual awareness in consciousness science (e.g., using the Perceptual Awareness Scale) with computational models of planning. Participants’ memory of (their awareness of) the obstacles is inherent to this approach, but just as with similar approaches in consciousness science (e.g. measures of iconic memory in the Sperling paradigm), we think it provides a reasonably “online” measure of awareness. It’s important of course to ensure that results obtained with awareness reports are not idiosyncratic, and generalise to other approaches to quantifying task representations.

      To further bolster the convergent validity of our awareness measure, we reanalyzed the data from Ho and colleagues. In their original paper, they developed a variant of the maze-navigation task where participants were asked to recall the location of obstacles as well as report their awareness (Exp 3) and a third variant of the task where participants could hover their cursors over hidden obstacles to reveal their locations (Exp 4). These data allowed us to validate the awareness reports against objective measures of recall and mouse-tracking data. We observed that the subjective awareness reports of participants were strikingly correlated with recall/hover measures across two independent samples of participants (Spearman ⍴ = 0.86 between memory accuracy and awareness; ⍴ = 0.86 between confidence in memory and awareness; ⍴ = 0.76 between the probability of hovering over the obstacle and awareness; ⍴ = 0.65 between the duration of the mouse hovering and awareness). We believe these findings validate participants’ awareness reports. These findings are now reported on page 22 of the manuscript.

      “Finally, we examined the convergent validity of participants’ awareness reports by reanalyzing the memory recall data reported in Ho and colleagues’ experiment (Ho et al., 2022). We reasoned that participants should demonstrate similar task representations regardless of the measure used to probe the construal. In line with this prediction, we observed that the obstacle awareness reports and memory/hover measures were strikingly correlated within three independent samples of participants (Spearman ⍴ = 0.86 between memory accuracy and awareness; ⍴ = 0.86 between confidence in memory and awareness; ⍴ = 0.76 between the probability of hovering over the obstacle and awareness; ⍴ = 0.65 between the duration of the mouse hovering and awareness; see Tables S18 and S19).”

    6. eLife Assessment

      This important study utilizes behavioral data and computational modeling to show that spatial properties of visual attention affect human planning. The methodology and statistical analyses are solid, though the way attention is conceptualized and modeled could be refined. The findings of this study will interest cognitive scientists studying attention, perception, and decision-making.

    7. Reviewer #1 (Public review):

      Summary: This study investigated how visuospatial attention influences the way people build simplified mental representations to support planning and decision-making. Using computational modeling and virtual maze navigation, the authors examined whether spatial proximity and the spatial arrangement of obstacles determine which elements are included in participants' internal models of a task. The study developed and tested an extension of the value-guided construal (VGC) model that incorporates features of spatial attention for selecting simpler task mental representation.

      Strengths:

      (1) Original Perspective: The study introduces an explicit attentional component to established models of planning, offering an approach that bridges perception, attention, and decision-making.

      (2) Methodological Approach: The combination of computational modeling, behavioral data, and eye-tracking provides converging measures to assess the relationship between attention and planning representations.

      (3) Cross-validated data: The study relies on the analysis of three separate datasets, two already published and an additional novel one. This allows for cross-validation of the findings and enhances the robustness of the evidence.

      (4) Focus on Individual Differences: Reports of how individual variability in attentional "spillover" correlates with the sparsity of task representations and spatial proximity add depth to the analysis.

      Weaknesses:

      (1) Clarity of the VGC model and behavioral task: The exposition of the VGC model lacks sufficient detail for non-expert readers. It is not clear how this model infers which maze obstacles are relevant or irrelevant for planning, nor how the maze tasks specifically operationalize "planning" versus other cognitive processes.

      The method for classifying obstacles as relevant or irrelevant to the task and connecting metacognitive awareness (i.e., participants' reports of noticing obstacles) to attentional capture is not well justified. The rationale for why awareness serves as a valid attention proxy, as opposed to behavioral or neurophysiological markers, should be clearer.

      (2) Attention framework: The account of attention is largely limited to the "spotlight" model. When solving a maze, participants trace the correct trail, following it mentally with their overt or covert attention. In this perspective, relevant concepts are also rooted in attention literature pertaining to object-based attention using tasks like curve tracing (e.g., Pooresmaeili & Roelfsema, 2014) and to mental maze solving (e.g., Wong & Scholl, 2024), which may be highly relevant and add nuance to the current work. This view of attention may be more pertinent to the task than models of simultaneously tracking multiple objects cited here. Prior work (notably from the Roelfsema group) indicates that attentional engagement in curve-tracing tasks may be a continuous, bottom-up process that progressively spreads along a trajectory, in time and space, rather than a "spotlight" that simply travels along the path. The spread of attention depends on the spatial proximity to distractors - a point that could also be pertinent to the findings here.

      Moreover, the tracing of a "solution" trail in a maze may be spontaneous and not only a top-down voluntary operation (Wong & Scholl, 2024), a finding that requires a more careful framing of the link to conscious perception discussed in the manuscript.

      Conceptualizing attention as a spatial spotlight may therefore oversimplify its role in navigation and planning. Perhaps the observed attentional modulation reflects a perceptual stage of building the trail in the maze rather than a filter for a later representation for more efficient decision making and planning. A fuller discussion of whether the current model and data can distinguish between these frameworks would benefit readers.

      (3) Lateralization of attention: The analysis considers whether relevant information is distributed bilaterally or unilaterally across the visual display, but does not sufficiently address evidence for attentional asymmetries across the left and right visual fields due to hemispheric specialization (e.g., Bartolomeo & Seidel Malkinson, 2019). Whether effects differ for left versus right hemifield arrangements is not made explicit in the presented findings.

      (4) Individual differences: Individual differences in attentional modulation are a strength of the work, but similar analyses exploring individual variation in lateralization effects could provide further insight, and the lack of such analyses may mask important effects.

      (5) Distinction between overt and covert attention: The current report at times equates eye movement patterns with the locus of attention. However, attention can be covertly shifted without corresponding gaze changes (see, for example, Pooresmaeili & Roelfsema, 2014).

      The implications for interpreting the relationship between eye movement, memory, and attention in this setting are not fully addressed. The potential dynamics of attention along a maze trajectory and their impact on lateralization analysis would benefit from further clarification.

      Appraisal of Aims and Results:

      The study sets out to determine how spatial attention shapes the construction of task representations in planning contexts. The authors provide evidence that spatial proximity and arrangement influence which environmental features are incorporated into internal models used for navigation, and that accounting for these effects improves model predictions. There is clear documentation of individual variation, with some participants showing greater attentional spillover and more sparse awareness profiles.

      However, some conceptual and methodological aspects would be clearer with greater engagement with the broader literature on attention dynamics, a more explicit justification of operational choices, and more targeted lateralization analyses.

    8. Reviewer #2 (Public review):

      Summary:

      Castanheira et al. investigate the role of spatial attention for planning during three maze navigation experiments (one new experiment and two existing datasets). Effective planning in complex situations requires the construction of simplified representations of the task at hand. The authors find that these mental representations (as assessed by conscious awareness) of a given stimulus are influenced by (spatially) surrounding stimuli. Individual participants varied in the degree to which attention influenced their task representations, and this attentional effect correlated with the sparsity of representations (as measured by the range of awareness reports across all stimuli). Spatially grouping task-relevant information on either the left or right side of the maze led to mental representations more similar to optimal representations predicted by the value-guided construal (VGC) model - a normative model describing a theoretical approach to simplifying complex task information. Finally, the authors propose an update to this model, incorporating an attentional spotlight component; the revised descriptive model predicts empirical task representations better than the original (normative) VGC model.

      Strengths:

      The novelty of this study lies in the proposal and investigation of a cognitive mechanism through which a normative model like value-guided construal can enable human planning. After proposing attention as this mechanism, the authors make concrete hypotheses about mismatches between the VGC predictions and real human behavior, which are experimentally validated. Thus, not only does this study describe a possible mechanism for simplification of task information for planning, but the authors also propose a descriptive model, revising VGC to incorporate this attentional component.

      A strength of this paper is the variety of investigative approaches: analysis of existing data, novel experiment, and a computational approach to predict experimental findings from a theoretical model. Analyzing pre-existing datasets increases the size of the participant cohort and strengthens the authors' conclusions. Meanwhile, comparing the predictions of the existing normative model and the authors' own refined model is a clever approach to substantiate their claims. In addition, the authors describe several crucial controls, which are key to the interpretability of their results. In particular, the eye tracking results were critical.

      In summary, this paper constitutes an important step toward a more complete understanding of the human ability to plan.

      Weaknesses:

      (1) There is a critical conceptual gap in the study and its interpretation, mainly due to the reliance on a self-report metric of awareness (rather than an objective measure of behavioral performance).

      a. Awareness is tested by a 9-point self-report scale. It is currently unclear why awareness of task-irrelevant obstacles in this task would necessarily compromise optimal planning. There is no indication of whether self-reported awareness affects performance (e.g., navigation path distance, time to complete the maze, number of errors). Such behavioral evidence of planning would be more compelling.

      b. Relatedly, it would have been more convincing to have an objective measure of awareness, for instance, how the presence or absence of a "task-irrelevant" obstacle affects performance (e.g., change navigation path distance or time to complete the maze), or whether participants can accurately recall the location of obstacles.

      c. Consequently, I'm not sure that we can conclude that the spatial context does impact participants' ability to plan spatial navigation or to "incorporate task-relevant information into their construal". We know that the spatial context affects subjective (self-reported) awareness, but the authors do not present evidence that spatial context affects behavioral performance.

      d. Another concern that may complicate interpretation is the following: Figure 3c shows improved VGC model predictions (steeper slope) for mazes with greater lateralization. However, there are notable outliers in these plots, where a high lateralization index does not correspond to good model performance. There is currently no discussion/explanation of these cases.

      (2) I noticed an issue with clarity regarding task-relevance. It is currently not fully clear which obstacles are "task irrelevant". Also, the term is used inconsistently, sometimes conflating with "awareness". For example, in the "Attentional spotlight model of task representations" section, the authors state that "task-relevant information becomes less relevant when surrounded by task-irrelevant information". But they really mean that participants become less aware of those task-relevant obstacles. I assume task-relevance is an objective characteristic related to maze organization, not to a participant's construal. Indeed, the following paragraph provides evidence of model predictions of awareness.

      (3) The behavioral paradigm has some distinct disadvantages, and the validity of the task is not backed up by behavioral data.

      a. I understand the need for central fixation, but it also makes the task less naturalistic.

      b. The task with its top-down grid view does not seem to mimic real human navigation. Though this grid may be similar to mental maps we form for navigation, the sensory stimuli corresponding to possible paths and to spatial context during real-life navigation are very different.

      c. Behavioral performance is not reported, so it is unknown whether participants are able to properly complete the task. The task seems pretty difficult to navigate, especially when the obstacles disappear, and in combination with the central fixation.

      d. There is no discussion of whether/how this navigation task generalizes to other forms of planning.

    9. Reviewer #3 (Public review):

      Summary:

      The authors build on a recent computational model of planning, the "value-guided construal" framework by Ho et al. (2022), which proposes that people plan by constructing simple models of a task, such as by attending to a subset of obstacles in a maze. They analyze both published experimental data and new experimental data from a task in which participants report attention to objects in mazes. The authors find that attention to objects is affected by spatial proximity to other objects (i.e., attentional overspill) as well as whether relevant objects are lateralized to the same hemifield. To account for these results, the authors propose a "spotlight-VGC" model, in which, after calculating attention scores based on the original VGC model, attention to objects is enhanced based on distance. They find that this model better explains participant responses when objects are lateralized to different hemifields. These results demonstrate complex interactions between filtering of task-relevant information and more classical signatures of attentional selection.

      Strengths:

      (1) The paper builds on existing modeling work in a novel manner and integrates classic results on attention into the computational framework.

      (2) The authors report new and extensive analyses of existing data that shed light on additional sources of systematic variability in responses related to attentional spillover effects

      (3) They collect new data using new stimuli in the original paradigm that directly test predictions related to the lateralization of task-relevant information, including eye tracking data that allows them to control for possible confounds.

      (4) The extended model (spotlight-VGC) provides a formal account of these new results.

      Weaknesses:

      (1) The spotlight-VGC model has a free parameter - the "width" of the attentional spotlight. This seems to have been fixed to be 3 squares. It would be good if the authors could describe a more principled procedure for selecting the width so that others can use the model in other contexts.

      (2) Have the authors considered other ways in which factors such as attentional spillover and lateralization could be incorporated into the model? The spotlight-VGC model, as presented, involves first computing VGC predictions and only afterwards computing spillover. This seems psychologically implausible, since it supposes that the "optimal" representation is first formed and then it gets corrupted. Is there a way to integrate these biases directly into the VGC framework, perhaps as a prior on construals? The authors gesture towards this when they talk about "inductive biases", but this is not formalized.

      (3) Can the authors rule out that the lateralization effects are the result of memory biases since the main measure used is a self-report of attention?

    1. But there’s another reason the PSL exploded so much over the past decade. Culinary food trend analyst Suzy Badaracco told Vox in 2014, “Pumpkin became recognized as part of the comfort food trend during the recession in 2008,” due to its association with Thanksgiving and the holidays. In tough times, we’re more likely to crave foods that bring back happy memories.

      This shows the seasonal novelty impact of the drink, and associations with interesting and comforting times.

    1. We thank Prof. Wood for his interest in our preprint and for noting relevant prior work. We will cite the indicated references — the 2020 amino acid resuscitation screen (iScience), the PRDP model (BBRC 2020), and the 2013 work on arrested protein synthesis and persistence (AAC)— in our revised manuscript.

      We respectfully disagree, however, that our work overlaps conceptually. Using integrative metabolomics, proteomics, and genome-scale in silico flux modeling in Salmonella and other pathogens, we show that L-serine and arginine act as antagonistic signals that respectively accelerate and delay resuscitation, including within macrophages and in a murine infection model. The findings that a metabolite can actively delay awakening, and that these signals operate in pathogens during host infection, are to our knowledge new and complementary to prior nutrient-sensing work in laboratory E. coli.

      We will also ensure our terminology regarding resuscitation dynamics is precise and that differing observations in the literature are appropriately discussed. We appreciate the engagement.

    1. A thread is

      A - thread - comment box - a reply chain - ~ is not neutral

      The design of the space shapes

      the quality - - of the thought. - -

      //

      Spaces like that cannot be designed top down

      they need to be grown, cultivated from the ground up

      through intentional meta-reflective dedicated practice

      • imagine
      • experiment
      • explore
      • experience working with co-evolving instance first exemplars
      • extend
      • expe

      taking a universal seed that actually works

      and capable of autopoiesis

    2. communities are shaped by their affordances

      communities shaped by affordances -

      we shape affordances

      and the affordances shape what we do

    3. shaped by the architecture of platforms

      shaped by architecture of the platforms

      • chat window
      • comment threads
      • post
      • replies
      • turn-taking
      • linear sequences

      need new spaces / game n fields

      shaped by human centric interpersonal architecture for over-web interplaynforms

    4. seven dynamics

      Instead of asking only, “What seven dynamics are at play?” we ask:

      • What dynamics may be at play?
      • Which are well-supported?
      • Which are plausible interpretations?
      • Which are speculative?
      • How did the prompt shape the answer?
      • What would a critic say?
      • What is being left out?
    1. No-show rate in 1–2h is 2–4× the 2–6h rate (e.g. MY: 2.5% vs 0.7%) — passengers who book close to their ride time are far more likely to cancel.

      Do pax pay for these?

    1. I love how the post highlights Delicious as a powerful tool for collaborative learning! It’s amazing to see how bookmarking resources can foster creativity among students. Imagine integrating that into geometry projects—endless possibilities! top game

    1. Takeaways:

      1) Diffie-Hellman job is it allows protocols to send messages without the ability to remove private keys.

      2) Elliptic curve formula is y^2 = x^3 +ax +b, a mathematical curves used to create secure public and private keys.

      3) The Elliptic Curve Cryptography is very secure because it is harder to solve than Diffie-Hellman algorithm

    1. Is to

      One important sentence in this source is: “I think the new American dream is to leave.” This sentence shows that many people no longer believes that. the traditional American Dream can be achieved in America. Instead of becoming rich or successful, people now wants peace, stability, and less pressure in life. This idea connects to our radio play because Mr. Zhou also only wants safety and survival for his family during the war. (because the Payment interface, I can not select the correct text in the source)

    1. Takeaways:

      1) AES takes 128-bit of message and encrypts it into 128 bit ciphertext.

      2) AES organizes data in 4x4 grid for 128 bits (16 bytes)

      3) AES works by transforming data through substitution, shifting, mixing and adding keys to make the original messages encrypted.

    Annotators

    1. Takeway:

      1) GPU can perform a large number of computations in parallel very quickly

      2) 256-bit security is very strong because possible number of combination is about 2^256 possibilities

      3) 256 bit encryption is secure because number of possible keys is so large that it could longer than the age of universe to guess the correct key.

    1. Takeways:

      1) The main difference with Bitcoin is that the system does not rely on a bank to verify transactions. But instead it uses a decentralized verification system that relies on mathematics and cryptography.<br /> 2) Producing a digital signature involves functions that depend on both the message itself and the user's private/secret key.<br /> 3) A blockchain is like a digital ledger that records transactions securely and tracks assets across networks

    1. I’m tired of talking to AI
      • The author expresses profound frustration with the pervasive infiltration of AI-generated answers into daily and professional communications.
      • Encountering malware-spreading repositories on GitHub, the author sought a resolution via an open discussion, only to repeatedly receive copy-pasted AI answers that offered no practical utility.
      • In a workplace scenario, a business owner repeatedly forwarded unread ChatGPT screenshots rather than engaging with or directly answering the author's specific business questions.
      • Online interpersonal interactions have also been compromised, illustrated by an instance where the author discovered they were conversing with an AI agent after exchanging multiple messages on Reddit.
      • The core grievance highlights a growing societal loss of genuine human connection, as individuals increasingly forward raw AI text instead of thinking for themselves or conversing sincerely.

      Hacker News Discussion

      • Erosion of Workplace Culture: Many commenters emphasized that relying on AI to respond to colleagues destroys organic trust-building opportunities. Reaching out to teammates is often less about extracting text and more about establishing communication, context, and validation.
      • Lazy Delegation and Management Failures: Participants noted that heavy corporate pushes for AI productivity have caused a misunderstanding of boundaries. Instead of using it to handle grunt work, some employees lazily offload all cognitive overhead to chatbots without reviewing or fact-checking the output.
      • Analogy to "Let Me Google That For You": Sending a raw, unverified AI response to a direct question is widely viewed as passive-aggressive and insulting. It conveys a strong signal that the sender did not respect the asker's time enough to even read the answer they forwarded.
      • Existential Risk to Job Security: Several users pointed out that individuals who mindlessly pass along unedited AI screenshots are strongly signaling that their entire job function can be replaced by an LLM, making them prime candidates for corporate layoffs.
      • The Effort to Remain Human: Some users shared that they have intentionally begun introducing written idiosyncrasies into their messages to prove they are human, though others countered that future AI models will inevitably mimic these individual quirks anyway.
    1. I tracked 430 hours of Claude Code usage. 73% was wasted on these 9 patterns.
      • Data Logged via Proxy: Over a 90-day period, a developer tracked all Claude Code activity using an HTTP proxy to capture full payloads, token counts, and costs directly interfacing with the Anthropic API.
      • The Scale: The dataset spanning this study consists of 430 hours of actual work, 6 million input tokens, and a total spend of $1,340 on API costs.
      • The Waste Discovery: Analysis revealed that only 27% of the total tokens processed did actual "productive work." The remaining 73% were consumed by nine hidden, automated inefficiency patterns.
      • The Solution: By identifying and resolving these nine patterns—each requiring roughly a 30-second fix—productive token efficiency can be increased from 27% to approximately 65% without changing the underlying model or losing functionality.
      • The 9 Major Cost Culprits:
        1. CLAUDE.md Bloat (~14% waste): Large, overly dense, or un-optimized systemic instructions files consume massive, unnecessary overhead tokens on every single interaction. Fix: Compress, aggressively prune rules, or split instructions into context-specific modular files.
        2. Conversation History Re-read (~13% waste): Long chat sessions exponentially multiply costs, as message #30 costs 30 times more than message #1 due to processing the entire accumulated history. Fix: Use a structured context-refresh cadence to summarize and discard older, unnecessary messages without losing the current task state.
        3. Hook Injection (~11% waste): Context injected via automated UserPromptSubmit hooks unnecessarily loads extra code and data into the prompt context for tasks that don't require them. Fix: Replace indiscriminate global hooks with conditional triggers that only attach context when explicit keywords or file types are targeted.
        4. Cache Misses (~10% waste): Expired prompt caches (which have a short 5-minute lifespan) force expensive, full-price re-tokenization of the codebase context when work pauses briefly. Fix: Set up an automated low-cost "keep-alive" ping task every 4 minutes to maintain the prompt cache active during active development blocks.
        5. Skill Loading (~7% waste): Inactive or irrelevant scripts (such as loading complex front-end UI design skills during a pure backend task) create up to 13,500 token overheads per command. Fix: Explicitly disable global skill auto-loading and isolate advanced capabilities to dedicated subdirectories or specific active profiles.
        6. Extended Thinking (~5% waste): Leaving the reasoning engine globally enabled forces Claude to burn 3,000+ reasoning tokens on simple commands (like basic camelCase naming changes) where deep logic is completely unnecessary. Fix: Disable extended thinking globally by default and explicitly toggle it on only for complex architectural or bug-hunting queries.
        7. Git Diff Inflation (~5% waste): Unfiltered or massive git diff outputs being fed into the context window when reviewing changes, rather than targeting specific file modifications. Fix: Configure the workflow to stream only targeted file diffs or summary statistics rather than pulling full repository diff text into active prompts.
        8. Directory Map Re-indexing (~4% waste): Redundant and frequent re-scanning of the entire project directory tree structure instead of utilizing cached file maps. Fix: Adjust system configuration to enforce a strict file-map caching policy that limits full directory re-indexing to manual project structural changes.
        9. File Read Overlap (~4% waste): Repeatedly reading the exact same source files multiple times within a short interaction window because the system lacks a localized, short-term memory of recent file states. Fix: Implement a session-level temporary cache structure that prevents the agent from re-fetching un-mutated target files in consecutive turns.
      • Debunked Optimization Myths: Lowering costs by switching to a smaller model (like Claude Haiku) for simple tasks only yields a negligible ~3% cost reduction, while aggressively running the /clear command between every minor task proves to be completely counterproductive.
      • Actionable Optimization Script: To automatically detect and patch these specific inefficiencies within a local workspace, the text recommends running a dedicated optimization script shared by the author.
    1. The labs understand how valuable these problems are: that's why they're building their own outsourced configuration shops, and why an entire upmarket class of reinforcement learning businesses exist.

      大多数人认为大模型实验室会直接解决所有复杂问题,不需要外部帮助。但作者认为实验室明白这些复杂问题的价值,这就是他们为什么建立自己的外部配置服务,以及为什么存在整个高端强化学习企业类别。这承认了实验室在某些领域需要专业合作伙伴,挑战了实验室可以独立解决所有问题的主流观点。

    2. The critical insight in the Oz analogy is that roughly half of any real workflow that is non-agentic carries no lab advantage. They are no better than you are at writing the deterministic software underneath the model layer.

      大多数人认为AI将取代所有软件工程工作,人类只需构建AI代理层。但作者认为真实工作流程中约有一半是非代理性的,这部分工作大模型实验室没有任何优势。大模型公司在编写模型层下方的确定性软件方面并不比专业应用公司更好。这为专注于构建复杂工作流程中非AI部分的企业提供了重要机会。

    3. The model is fungible underneath; the system of work is not. The next generation of enterprise software is going to be built off the road.

      大多数人认为底层AI模型是企业的核心竞争力,模型越好产品越强。但作者认为模型是可替代的,而'工作系统'才是真正的护城河。下一代企业软件将建立在'黄砖路'之外,专注于特定行业的工作流程、数据捕获和治理。这些系统拥有端到端的工作流程所有权,这是大模型实验室无法轻易复制的优势。

    4. Running every query through Opus 4.7 is the fastest path to negative gross margins. The best Rest of Oz companies route across tiers of models — frontier models for the hardest tasks, mid-tier for the bulk, smaller custom or fine-tuned models where they've earned the right to use them.

      大多数人认为使用最先进的大模型总是最佳选择,能提供最佳结果。但作者认为这是通往负毛利的最快路径。相反,'Oz的其他部分'公司会根据任务难度分层使用不同级别的模型,只为最困难的任务使用前沿模型,为批量任务使用中等模型,为特定工作使用小型定制或微调模型。这种成本优化策略使它们能够提供更具竞争力的价格。

    5. The labs are already routing internally — different model classes for different requests, ensembles under the hood. What they can't do is route across vendors, or evaluate a competitor's model for a specific sub-task, or use an open-source fine-tune for the narrow piece where it's actually best.

      大多数人认为大模型实验室拥有绝对优势,可以解决所有AI问题。但作者认为实验室在模型选择上存在结构性限制,无法跨供应商评估模型或为特定子任务使用开源微调模型。这为专注于特定领域的企业提供了机会,它们可以选择最适合每个子任务的模型,而不仅限于自家实验室的模型。

    6. The labs really are coming for a huge swath of the application surface. But 'the application layer' isn't just one homogenous opportunity.

      大多数人认为AI将完全吞噬应用层,所有软件都会被大模型取代。但作者认为应用层并非同质化机会,存在不同类型的机遇。作者将应用分为'黄砖路'和'Oz的其他部分',认为垂直领域的复杂应用不会被大模型完全替代,因为价值不仅来自底层模型能力,还来自特定行业的可信赖、合规和运营化的支撑架构。