- Oct 2024
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www.ebhsoc.org www.ebhsoc.org
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the dynamic development of intangible cultural features such as symbols, myths, languages, beliefs, values, norms, rituals, and attitudes
View 1: everyone engages in culture!
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The quadruple bottom line of cultural entrepreneurship
I like this lingo. T'is true - the artistic innovation sphere might hardly ever be highlighted in "conventional" entrepreneurship...
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the cultural entrepreneur as an agent for “1) changing people's understanding of what is possible (changing frame of reference),
I like this!
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the automotive industry guru Henry Ford. Businessmen like Swift or Ford, because they undertook creative action that went beyond what was known or accepted at their time, had no choice but to draw their ideas and strategies from the deep sources of culture and value in which they were immersed.
culture (and business) being revolutionized alongside each other. But seriously, Henry Ford changed culture even if that's not what we predominantly associate with him
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It is also culturally specific, an example of "local knowledge," and "common sense," which differ, sometimes radically, across societies.
the expertise of the cultural entrepreneur goes beyond possessing cultural capital - but affecting how we perceive cultural capital, how we negotiate its contours ... interesting...
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they may not be employed directly or indirectly in the cultural industries per se, but they continue to produce cultural goods with or without pay... As aspiring creative workers often combine jobs, this feature leads to a slash-mark bisecting their working identity: café worker/ songwriter, courier/drummer, color consultant/ painter, administrator/jewelry designer, and so on
I like this distinction, though, as it clarifies that everyone can be a cultural entrepreneur, as long as you're working "creatively"
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for a small number of individuals, the beliefs of others are not given but can be changed. I shall refer to those people as cultural entrepreneur
interesting - cultural entrepreneurs as those who can change the beliefs of others...
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changing people's motivation for taking part in economic development
the commercial aspect is never (entirely) absent...
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t means being multi-skilled in hand work, design work, publicity and promotions, management and business and having some idea of manufacturing, as well as being in possession of creative vision, imagination
this is an interesting definition (also circular in nature) -- the cultural entrepreneur will exhibit all the best qualities of someone undertaking a career in cultural sectors...
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Occasionally entrepreneurs really open up supposedly settled matters, calling into question interpretations that define products or technologies
so we've come full circle - a cultural entrepreneur CAN be someone working in "industry" who has a profound impact on the wider culture (not just someone working in the "cultural industry/ies")
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The alternative is to use the word “cultural” as an adjective attached to the good or service that the entrepreneur provides.
much more basic... a cultural entrepreneur produces a cultural good...
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they have found very little in the literature when it comes to definitions of the term “arts entrepreneurship.
hah
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Cultural entrepreneurs are cultural because they are about the cultural.
not a circular argument at all!
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The economics has to be an instrument for them in order to realize cultural values... To be clear, we are working on a moral picture here and try to figure out what makes a good cultural entrepreneur. Someone who sees in cultural trade a way of adding profit becomes suspect as culture is his instrument and not his mission. He is rather a businessman. That does not make him a bad character but he is miscast as a cultural entrepreneur.
interesting - a values (morality) based argument for entrepreneurship!
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ttempt to define a cultural entrepreneur: “An individual who identifies an opportunity and acts upon it in order to create social, cultural, or economic value."
interesting - this might also serve as an inclusive definition of entrepreneur (without the "cultural" modifier). But people in the arts definitely highlight the cultural (and maybe the social) value of their creations, while also recognizing the economic nature of their practices...
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here is not one totalitarian culture industry—there are many “cultural industries.” Each culture, defined anthropologically, has its cultural industries. The same goes for each art form and each way of media communication.
yep!
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Cultural entrepreneurs may, of course, have a focus on financial revenues. The economic value is always one part of their calculation. For some, maximum profit is the only objective. However, for most artists, earning money is most likely only one of several goals.
seems like something of a theme in a lot of the weeks we've looked at so far!
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Those who wrote in English before the late nineteenth century mostly used the word “civilization” and its derivatives to describe the cultivated society.
interesting ... what makes us "cultured" or "cultivated"?
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we are dealing with a word that depicts the transformation of an object or a subject from one state to another. Cicero (146-43 BC) turned to agriculture to clarify how the human soul must be cultivated to a mature state, just like the seed grows to become adulta fructus.
creativity --> creation --> culture --> cultivation --> GROWTH
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Many entrepreneurs are identified by others as more “industrious” than the common person.
ok ... an industrious person doesn't necessarily work in an industry, but labours to create ... and cultural industries create all manner of material (that may "elevate" their citizens...)
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the anthropologist’s definition of culture as part of: Institutions [that] are the humanly devised constraints that structure political, economic and social interaction. They consist of both informal constraints (sanctions, taboos, customs, traditions, and codes of conduct), and formal rules (constitutions, laws, property rights). “Culture” is not static—it changes over time.
ok - so let's explore these different directions in greater depth...
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Horkheimer and Adorno's claim was that the Kulturindustrie produces the opposite of what they labeled “authentic culture." By this they meant culture that is a means in itself and culture that fosters human imagination in a different way than the culture industry does. The authentic culture, according to Horkheimer and Adorno, leaves room for independent thought. The clash results in a “sell-out of culture," in which the true meaning of culture is replaced by “well-calculated stupidities of amusement”
classic Frankfurt School thinking...
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we now identify a wide range of objects for which the leverage from a lower state to a higher one can be the concern of an entrepreneur. Now we also see the entrepreneur as someone who could be equally or more interested in some kind of social change.
art and culture can create social change, yes?
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cultural entrepreneurship is now radically different in the new long-tail economy, which is increasingly shifting away from a focus on a relatively small number of mainstream bestsellers at the head of the demand curve and toward a huge number of niches in the tail.
so, just as culture is always in flux, so too are the cultural industries. As capitalism develops, so too does the innovation sectors...
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This chapter was eliminated by Schumpeter in the second edition 20 years later. Swedberg (2006) claims “that when Schumpeter wrote the first edition he was a very young and enormously ambitious economist in Europe who wanted to take the academic world by storm; when he prepared the [English] translation in the 1930s he was a tenured professor at Harvard and had developed a much better sense for what mainstream economics was about—and also what it demanded of its practitioners if they wanted to remain in the mainstream.
ouch!
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Whereas the passive artist works statically and non-entrepreneurially with artistic adaptations according to the cultural zeitgeist, the dynamic cultural entrepreneur creates more radical artistic development.
interesting - "regular" folk typically are passive, but the artist, the true original, they CREATE!
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ultural entrepreneurs can be either “social” or “artistic” or a combination of the two. It all depends on what we accept as included in the “culture” concept.
yep - so let's look a little closer at culture then...
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Entrepreneur
Before the course started, some of you expressed a desire to learn about the history of entrepreneurship (who the first one was, etc). This is about as close as we can get (describing how the term came to be...)
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“take things from one state to another” meaning of the word entrepreneurship
a wider scope than traditional commercial entrepreneurship...
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The term “cultural entrepreneurship” has been increasingly used during the new millennium, mirroring the rapidly growing importance of the “quaternary sector of the economy,” i.e. knowledge-based industries, including culture. It seems to be used mostly in connection with “the cultural industries,” a term that is used for production and services related to both commercial mass culture and the fine arts
so, cultural entrepreneurship tends to be specific to entrepreneurship concerning cultural fields/industries ... NOT the cultural impact of entrepreneurship efforts...
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The true artist, in other words, should presumably be conceptualized as an entre-preneur; and as the economic entrepreneur has his imitators and followers, so does the artist. Both the artist and the entrepreneur are dynamic, active, and energetic and show leadership qualities, while their followers are passive and static and accept the way things are.
aha! Now we're getting somewhere!
What do you think of this analogy? Artists don't typically think of themselves as entrepreneurs (too much ideological baggage), but successful artists have to be entrepreneurial!
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'cultural entrepreneur' is used here as a synecdoche for the (mostly) young neo-bohemian person operating in freelance mode at the interstices of the flexible labor market (within and without the creative industries) and self-driven cultural production.
ah, neoliberalism (and liquid times) strike again...
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ccording to Schumpeter the innovative entrepreneur creates a new good or service. Others learn from it,
good ole Schumpeter...
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undertakers as persons who by “the natural selection of the fittest [were] to undertake, to organize, and to manage,”
love it! Let's all be "undertakers"
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Looking ahead, we aim to expand BFVD by predicting viral multimer structures, taking advantage of their compact genome size, and making them searchable using Foldseek Multimer (30).
Would you consider expanding it to all viral proteins in UniProt, or to a higher clustering resolution than UniRef30 (such as UniRef50 or UniRef90)?
In general, it would be interesting to know more about this trade off. Some questions I would love to know the answers to:
- How much computational cost and database size did you save by using UniRef30 over all UniProt viral proteins? It looks like you did 351K that represent 3M sequences. Is 3M too many to do?
- How taxonomically mixed are the UniRef30 clusters? For example, I looked at the representative sequence for a protein PB1 in influenza A and it's sequence PB1 in influenza B, so they're very closely related. Is this usually the case because viruses are so diverse?
- Do PDB and ViralZone provide more resolution for the virsues they do cover? For example, if I'm more interested in Eukaryotic viruses, will I get more exact results using one of those databases because they don't reduce down so much (e.g. UniRef30)?
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To demonstrate BFVD’s utility, we repeated and extended a part of a recent study by Say et al. (14) that annotated putative bacteriophages within metagenomically assembled contigs from wastewater. Say et al. developed a pipeline for enhanced annotations by integrating structural information from the AFDB with sequence data. Here, we applied the steps of their pipeline to one of the metagenomic samples from their study: the Granulated Activated Carbon sample 6 (GAC6). In addition to using the AFDB like they did, we included BFVD and ViralZone as reference databases for structural similarity search (Fig. 1h). Like Say et al., we found that the sequence-similarity based tool Bakta (28) could annotate on average 8% of the putative bacteriophage proteins on each contig, while Foldseek with the AFDB as reference annotated on average 51% of them. By using BFVD, we could annotate a comparable fraction of 46% of the putative bacteriophage proteins, despite the tremendous size difference between the AFDB and BFVD. However, when we searched the sample structures against the combined structure set of the AFDB and BFVD, we observed only a marginal increase in annotation performance. This suggests that the AFDB likely includes some BFVD bacte-riophage structures indirectly, through prophages embedded in bacterial genomes covered by the AFDB. While ViralZone improved Bakta’s annotations, its contribution was limited compared to the AFDB and BFVD, likely due to its focus on eukaryotic viruses.
I think it could be interesting to repeat this experiment but with a metagenome where the viruses of interest are not bacteriophages. As written, this doesn't really highlight the benefit of BFVD.
It may also be interesting to report the additional metadata you receive from annotating with BFVD instead of AFDB. If the phage structures come from hits to prophages, AFDB would presumably provide "host" information while BFVD would provide viral taxonomy (or at least taxonomy of sequences in the cluster that have a hit).
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Indeed, among the low-confidence structure predictions (pLDDT < 50), the majority (78%) had fewer than 30 homologs.
Maybe I'm misunderstanding, but I thought in the previous paragraph you stated that most of these sequences had high pLDDT, so were high confidence? That makes this sentence confusing, as well as the following one.
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Focusing on the shortest proteins (≤ 70 residues), we found that 99% of them were singletons. Unlike longer proteins, only 4% of the shortest proteins exhibited low confidence scores (pLDDT < 50). This is consistent with a previous report of high pLDDTs in sequences shorter than 100 residues (26).
Would you be willing to add a summary sentence here? I take this to mean that the structures are highly confident but that they are very unique?
There is some evidence in humans that short ORFs (<100 amino acids) are evolutionarily young and not shared between closely related species, leading to the hypothesis that they may be a reservoir of functional innovation. I'm curious if there might be anything similar posited about the evolution of these things, or if the tools aren't accurate enough in this case to put forth these types of ideas
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To limit the computational demand of structure prediction, we split 3,002 sequences longer than 1,500 residues (< 1% of all) into 6,730 sequence fragments.
Can you state more clearly what you did here? Did you split them into 1,500 residue chunks or divide them in half/thirds etc, or something more clever like relying on domain annotations
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a database of 67,715 predicted protein structures from 4,463 species of eukaryotic viruses.
It would be helpful to know here whether they subsampled to representative genomes or clustered sequences and picked representatives to better compare against the approach taken here.
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doc-10-1g-prod-01-apps-viewer.googleusercontent.com doc-10-1g-prod-01-apps-viewer.googleusercontent.com
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or does it partake of the contempla-tion of sages and philosophers about the meaning of the universe.
science is an occupation or method, cannot interpret meaning of the universe
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Weber was fundamentally at odds with those who argued for a moralitybased on science.
Weber didn't believe morality should be based on science as that was not the nature of science
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Hence, the relativity ofvalue orientations leading to different cognitive choices has nothing to do withquestions of scientific validity
subjectivity of choices researcher makes does not take away from scientific validity.
Annotators
URL
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static1.squarespace.com static1.squarespace.comSkellig1
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ay and there was just a square black gap behindthe hearth. The floor was soaking wet and it stank of disinfectant. Dadwas filthy and wet and grinning. He took me into the backyard. Thetoilet was standing there in the middle of the thistles and weeds.“Thought it’d make a nice garden seat for us,” he said.The gas fire and the plywood were down by the garage door, but theyhadn’t been taken inside.He looked at me and winked. “Come and see what I found.”He led me down to the garage door.“Hold your nose,” he said. He bent down and started to open anewspaper parcel. “Ready?”It was a parcel of birds. Four of them.“Found them behind the fire,” he said. “Must have got stuck in thechimney and couldn’t get out again.”You could make out that three of them were pigeons because of theirgray and white feathers. The last one was pigeon-shaped, but it was allblack.“This was the last one I found,” he said. “It was under a heap of sootand dust that had fallen down the chimney.”“Is it a pigeon as well?”“Yes. Been there a long, long time, that’s all.”He took my hand.“Touch it,” he said. “Feel it. Go on, it’s okay.”
they found dead birds that fell down the chimney. It is also sad and painful.
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indigenousfisheriestrainingframework.wordpress.com indigenousfisheriestrainingframework.wordpress.com
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Paradoxically, perhaps, a final lesson of these storias for us is that it is high time we turned the lens round on ourselves. For the underlying truth is that we have asked questions here of our own understanding of entrepreneurship, and have been found wanting.
powerful implications
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Our method of microstoria attempts to undermine the hegemonic postcolonial conception of the term ‘entrepreneur’. Through approaching the less obvious characters in the prevailing entrepreneur tale, we are bringing forward alternative narratives upon which to discuss entrepre-neurship
wanting to tell a different story, with different characters that weave a different plot
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it is precisely our loud approaches to entrepreneurship, made in the name of progress, which are killing so much of life around us.
a grand narrative minimizes or marginalizes alternative stories
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recognizing the idea of the barefoot entrepreneur as the other, as a subaltern voice
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We want to emphasize instead the vulnerability that exists among these barefoot entrepre-neurs—but also a deep sense of strength. It is their lives that are at stake everyday, where the lack of protection, care and freedom, and minimum subsistence, push them to act and enact their lives on a survival basis
its a variegated reality...
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mere caricature (see Pralahad, 2005) in which our barefoot entrepreneurs are depicted as superbly creative entrepreneurs, resilient and value-conscious consumers
also not the point...
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The storias we have shared here do not seek to provide a romanticized view of poor entrepre-neurs
not the point...
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barefoot entrepreneurs reflect stories and practices that contain a plethora of meaning, values and relationships which question the prominent view of the entre-preneur.
!!
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the embroidering of garments or the creation of small sculptures made from anything disposable
curiously, my kids were involved in a "venture" at their school where they collected old milk bags and "knit" them into sleeping mats. Not barefoot, but social entrepreneurship that was inherently viatorized (?!)
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There is a flux, a flow (Csikszentmihalyi, 1992) to things, which the barefoot entrepreneur has to ride, just as any artist or creative in their work.
!
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we see venture creation around crafting or other art forms
turning the disposable into something valuable. Upcycling...
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barefoot entrepreneurs because they have no other option but to create something for themselves out of the void that they exist in. Each person has different reasons for doing so; how-ever, their reasons are often linked to the community, and the will to help both themselves, and also others around them to alleviate some of their problems.
the barefoot entrepreneur doesn't exist in a vacuum but in a community instead!
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Children who are regarded without knowledge and the skills to generate ‘wealth’ are paradoxi-cally able in the urban hostile context of their existence to develop the necessary skills to create new business opportunities that sustain their existence when protection by family and state does not exist. Clearly Critical Entrepreneurship Studies (CES) needs to include taking account of such ‘other’ forms of entrepreneurship, especially the role of children in the construction of subsistence entrepreneurship
yep
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we see a very collective form of social creativity in practice. The ‘boundaries’ being crossed are those of an organizational, hierarchical, industrial nature. The workers worked as mosquitoes, as a single joint cloud, contributing to their survival like the shoal of sardines under attack from shark and catapulting the business ahead
! collective rather than individual effort
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Their work is also collective. The cartonero co-create the ‘opportunities’ that are invisible to others, in and through their social creativity. In doing so, their activities go beyond the category of ‘necessity’ entrepreneurship (Acs, 2006), all too easily seen as the poor relation of ‘opportunity’ led entrepreneurship.
learning stories allows us to finesse a definition of barefoot entrepreneuring that is distinct from "necessity" entrepreneurship (understood as the shadowy side of conventional opportunity-seeking entrepreneurship)
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driven out, looked down upon and yet valued; the scarcity of resources, the necessity for ‘bootstrap’ or ‘parsimonious’ approaches (McGrath, 1997) to entrepreneurship and the need for bricolage—all are characteristic of entrepreneurship as widely discussed, and yet they speak to us in new ways about the possibility of an ‘altermodern’ (Bourriaud, 2009b) society characterized by fragmentation and hybridization.
interesting
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The everydayness of their creative practice, the sense in which they trade in original and innovative ways to make a living—this is to act entrepreneurially. They embody the ‘entrepreneuring’ spirit
!!
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begging appears as the antithesis of the mercurial business entrepreneur that is supposed to bring economic modernization, capital and progress to the city. Stigmatized and labelled as a dan-ger to progress, beggars negotiate their own ways of interpreting the economic, their space and their business activities
...
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the discourse of entrepreneurship is not just about ‘massive successes’, but about ‘struggle, stress, debt and failure’
!!
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microstorias do not form a unitary discourse, a unique and grand history, but instead unfold the unconventional, the forgot-ten and the improper
aha! Clear link to the other reading (and hence, the title of the week as an all-inclusive "unconventional" umbrella -- broader in scope than intended by the other reading...)
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we did not plan specific questions upon which to interrogate their ‘entrepreneurship’ or capacity for creativity. Theirs was a language of deprivation and exclusion; the one our microstorias wanted to raise. That is, before we intervene with our translations and analytical frameworks, we wanted to hear in their own language what they went through as ‘barefoot entrepreneurs’ and in this way avoid the obses-sive attempt to impose (colonialist) representations that define who they are and what they do.
good approach... inductive vs. deductive research
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There is also an enduring belief in the entrepreneur’s power to have a positive effect on others (see Arndt, 1983 for more on the so-called ‘trickle down myth’). This view of entrepreneurship puts emphasis on an economic system that values success as part of the ‘creation’ of business, at the expense of individuals and communities who are marginalized or excluded from the discourse
connecting the mainstream and marginal contexts...
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We met these people in the streets. The streets of these cities are where most of the excluded members of society make their trade in informal and disorganized ways (Burrell, 1997). There they beg, collect rubbish, dance or play music, sell in small kiosks, whatever they have, in order to make a living. We approached them in the streets and engaged with them in the movement and moment of our street encounter that brought a fluid conversation about their and our existence
field-research -- opportunistic, and "real" -- these stories aren't processed like the published accounts of successful (commercial) entrepreneurs...
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our main criteria for constructing our microstorias of barefoot entrepreneurs, accept-ing the limitations and moral paradoxes (Geertz, 1968) when researching and analysing the other, was to approach only people who have been disfranchised and who operate at the mar-gins of society. That is, people who are ignored or overlooked by mainstream institutions and therefore do not receive any state or (inter)national support for their minimum survival and existence; those who have to carve their own living out of poverty; those who do not have the freedom to be the person that ‘they are able to be’
privileging the "nobodies" (rather than ignoring them).
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we attempted to de-colonize ‘the entrepreneur’
ethnographic connection with the research subject (empathy)
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though our focus on the marginal, the excluded and the ‘barefoot entrepreneur’ inevita-bly calls for analytical separation between us and the ‘other’, we write this article in the firm belief that we are part of something that we need to transform together. Our universal commonality is an axiological principle of being ‘human’.
politics clarified
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abandon this economic utopia that exploits nature and the poor for a more creative and organic integration and interdependence (of communities and peo-ples). This reflects, for him, a matter of bringing the ‘invisible’ (organizational) sectors into the forefront of life and of letting them, finally, have their say and ‘do their thing’
there is a definite link to communities and "tribes" here (though not consumer based!), networks of support...
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‘to speak of labour is to speak of the already enfranchised’ (p. 79). Hence to speak of the entrepreneur, is to speak of individuals who have already been given, or born into a state of pos-sibility and enfranchisement
Privilege!
The same thing is true when we speak of "capital" (economic, human, social, cultural...)
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the conventional knowledge we apply to understanding the economy of places and spaces in poverty are entirely meaningless.
the languages and experiences of those living "barefoot" require different ways of thinking ...
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moving to an economic system that serves the people rather than people serving the economy
a political and social agenda, not just economic!
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Currently the discourse of the entrepreneur is driven with values that promote a great [white] man narrative of progression and economic achievement via ‘creative destruction’ or business innovation in the omnipresent market
just another reminder...
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the entrepreneur is defined as an individual who takes on certain tasks based solely on a perception of market oppor-tunities and how to exploit them
the (conventional) enterprising individual...
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the Canadian aboriginal approach to economic development is predominantly collective, centred on the community or ‘nation’ for the purposes of ending dependency through economic self-sufficiency, controlling activities on tradi-tional lands, improving socio-economic circumstances, strengthening traditional culture, values and languages (and reflecting the same in development activities). These studies also question the postcoloniality of the ‘entrepreneur’ discourse and the economy of dependency that it creates (Khan et al., 2007). For example, Frederick and Foley (2006) maintain that colonial and postcolo-nial [entrepreneur] practices deprive indigenous communities of their land, culture and their basic human rights.
link to indigenous entrepreneurship...
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lives, like Imin’s, do speak of experiences and narratives that present a sig-nificantly different, and arguably no less legitimate story (Boje, 2008) to the one generally employed to define, describe or analyse ‘the entrepreneur’ (e.g. Palomino, 2003). Imin’s life does not provide a grand narrative of successful business, growth or innovation (e.g. Peredo, 2003). Imin’s life does not contribute to what Weiskopf and Steyaert (2009) describe as the holy trinity of the entrepreneur—the strong entrepreneurial figure, a neo-positivist tradition of research and the belief of optimistic policy-making that is grounded in the discourse of neolib-eral economic success. Imin’s life does not embed the myth of noble armoured knight entrepre-neurs (Sørensen, 2008). But his life does reflect distinctively different and no less valuable experiences and stories that have a bearing on our understanding of entrepreneurship
!!
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The mainstream discourse of entrepreneurship imposes an ideological and hegemonic reading that defines entrepreneurial behaviour and the enterprise economy
mainstream context, again
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In microstoria we found a method that breaks with the hegemonic power of grand narratives (theories), such as those economic theories that have come to ‘represent’ entrepreneurship
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the sublime
there's that term again!
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we approach our study of the barefoot entrepreneurs by employing ‘microstoria’ as a method (Ginzburg, 1993). Microstoria precisely addresses Max-Neef’s concerns about the representation of the poor by engaging with stories of little people, i.e. indigenous, peasants, minorities, poor, marginal and so forth
a narrative based approach - people's stories about their lives and their struggles (either marginal or mainstream) tell you who they are, their values, etc...
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two great attributes that the mosquito cloud has: first, it sticks together and second, it has no chief mosquito, therefore no one can behead the cloud
also interesting ! The tribe, but more nomadic than monadic!
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we concentrate on those everyday entrepreneur[ial] stories that populate the mar-gins of our societies; those that belong to the disenfranchised and dislocated voices of the ‘barefoot entrepreneurs’.
!! barefoot entrepreneurs are obviously unconventional...
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dominant economic theory assigns no value to tasks carried out at subsistence and domestic levels. In other words, such (economic) theory is unable to embrace the poorer sectors of the world
poorer populations aren't concerned with efficiency or maximizing growth...
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the entrepreneur is defined within an economic system that legitimizes values, actions and identities that do not reflect life among poor or neglected communi-ties in the developing world or the poor south
context
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We are emboldened to think again about ‘who is the entrepreneur?
their mission statement - to re-frame "marginal" actors and actions as entrepreneurial ... so "unconventional" can become the norm?
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collecting stories of people who otherwise will be left out
refuting the great-man or hero-centric entrepreneurial ideology
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survival tactics, self-reliance and creative practices (see De Certeau, 1988) reflect the more mundane (Rehn and Taalas, 2004) everydayness of the entrepreneur
those who live on the "margins of the neoliberal economic world" are still part of it!
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the ‘everydayness’ (Steyaert and Katz, 2004) of entrepreneuring that takes place at the margins of our societies
mundane acts of living (often necessary for survival...)
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‘the word entrepreneur has no meaning’ (p. 85). The entrepreneur emerges as a fairy tale character, a mythological role.
reminder of the mythic and discursive function of the entrepreneur ...
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www.washingtonpost.com www.washingtonpost.com
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The vast inequalities in income and opportunity that automation has given us so far could be significantly reduced
AI has an equal potential to displace people in the workplace or flatten the wealth gap.
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Incentives could be provided to change the research agenda, which today is acutely focused on automation, to explore complementary uses of AI instead.
What could inventivize companies to focus on a complementary use of AI?
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the end game includes a working class of no economic or political power, unable to improve its lot, left to live off whatever universal basic income the Silicon Valley plutocrats are willing to provide.
Policy and political action are determinent in whether or not future AI integration will be utlized to support people or maximize profits for investors.
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www.sciencedirect.com www.sciencedirect.com
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The other side of the coin, however, is the riskthat people who have become the entrepreneurs of their own lives willsuffer alienation rather than self-fulfilment
bad stuff
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the rise of impassioned entrepreneurship becomes a vehicleto imbue professional life with meaning. Connecting passions that linkprivate and professional lives help people gain emotional and culturalcompetencie
good stuff
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The inducement to manage and transform one's self and/or tribeinto an entrepreneurial business producing and selling emotional la-bour, such as intimate videos, has a substantial effect on entrepreneurs'psyche
a fancy way of saying that the constant need to update and maintain an attractive self-image in order to cultivate followers is damaging to one's mental health.
Are autopreneurs "unconventional entrepreneurs"? Are they part of a tribe?
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Whether this involves a person's work, play, or love life, they mustact like a superhero. The quest is driven by the unfettered desire to existand develop one's own identity (
more... the constant emphasis on performance, leading to an illogical and unhealthy end...
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The danger to which the autopreneur is exposed is an element thatcomplements the others presented in this article and helps define aframework within which the unconventional entrepreneurship is placed(see Fig. 1).
curious! I missed this reference to Ashman's piece in my first read...
Are autopreneurs "unconventional entrepreneurs"? Are they part of a tribe?
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Neoliberalism is the dominant ideology of liquid times (Scharff,2016), a form of governmentality in people's lives where they become“entrepreneurs of themselves
!!
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today it is the entrepreneurwho represents neoliberal alienation
yowza
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For most people today, unconventional entrepreneurship representsabsolute emancipation, allowing the perfect combination of privatepassion and professional success. Emancipation is usually defined as theprocess of being liberated from constraints that can be physical, in-tellectual, moral, or spiritual
ok
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passion can provide the stability, meaning, and social recognition
can't get my highlighter to cover the text that I want! Liquid times call out for anchors - for we need stability. Passions can become that anchor (for the self, and a sense of community).
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ach individualbecomes an entrepreneurial subject, living their lives like a companyand behaving entrepreneurially at every level (Scharff, 2016). Peopleare asked to use “technologies of the self”(Foucault, 1988) to shapetheir identity corporeally and cognitively so that they may be re-cognised both off- and online
social media = social networking = networked entrepreneurship, promoting our selves (via our interests & passions).
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The fig-urehead of work today is the entrepreneur, not in the Schumpeteriansense of the term but the unconventional entrepreneur driven by theprimacy of consumption in contemporary life.
cool -- so the unconventional figure has now become the archetype of current (liquid) times.
Do you buy what they're selling?
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nconventional entrepreneurs are supposed toabsorb and express liquidity by eliminating the barriers between private(consumption) and professional (production) lives.
reinforcing Szeman...
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n-trepreneurs are the embodiment of liquid times where planning be-comes impossible
kind of a cool statement with big time implications...
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tribes areconstrued here as sets of individuals who are not homogeneous (interms of their objective social characteristics) but linked by one and thesame passion
tribes brief definition -- if you really want to get into it, consult Maffesoli, Michel (1996). The Time of the Tribes: The Decline of Individualism in Mass Society.-- in this book Maffesoli claims that neotribalism will replace individualism in a postmodern society that follows modernity (Zygmunt Bauman's notion of liquid society charts some of the same ground ... since modern times are solid times). Neo-tribes are typically consumer tribes (groupings), but also ephemeral and lacking stability (i.e. liquid...)
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With the ad-vent of liquid times, society went from being work-centred to a situa-tion where people's lives increasingly centred on consumption
ripe for tribal entrepreneurship ...
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Unconventional entrepreneurs want to please the commu-nity but some adjustments and choices have to be made to fit their ownagenda. Even if their endeavour is conducted with passion, it is also aproject that, at some point, is expected to deliver a return on invest-ment. The balance seems hard to maintain but remains essential forunconventional entrepreneurs who do not want to disappoint theirtribe
unconventional entrepreneurs likely face a trickier balancing act than conventional entrepreneurs.
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To exploit cultural opportunity, they accept thecultural mission (Pedeliento, Bettinelli, Andreini, & Bergamaschi, 2018)of bridging the inside community with outside society.
the role, further explained...
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Beyond cultural competency, unconventional entrepreneurs manageand commercialise emotional bonds. This type of entrepreneurship re-quires significant and novel forms of emotional labour from both theentrepreneur and the broader tribe (
clear link to emotional intelligence (3 weeks hence) (but also really useful for the concept of emotional labour -- often the most difficult to monetize!)
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veryday lifemoved from being stable and secure to greater uncertainty and rapidchange. He also applies the adjective in a very precise context. Societyis to be considered liquid if the situation people find themselves in andwherein they act changes before they have the time to consolidate theirbehaviour into procedures and habits.
sketching the contours of liquid times...
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Unconventional entrepreneurs are also able to transition beyondtheir status as fan consumers to become entrepreneurs operating be-yond the scope of the tribe. Such entrepreneurs have their feet in twocamps, interacting with tribes at a professional level while maintainingtribal roots at an individual level.
so, transcendence, but connection at the same time... bit of a paradox (but that resonates with material from Richard Branson's narrative...)
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There is a connective passion (Ranfagni & Runfola,2018) between the personal and professional spheres of the peopleinvolved
see fig.1 again!
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Tribes have a considerableimpact on the entrepreneurs' start-up process, exercising this throughon- or offline exchanges of the experiences, ideas, and productionsgenerated by the activity in question.
yep
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act as cultural intermediaries combining the needs of thetribe and the features of the mass-market into a “cultural package”thatcan be shared and instrumentalised.
the role of unconventional entrepreneurs -- a kind of cultural translator...
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nconventional en-trepreneurs are not subject to the kind of transformation where in-dividuals transition from their status as consumers (or users) to fully-fledged entrepreneurs
interesting!
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The main distinction between unconventional entrepreneurs andtheir conventional counterparts thus pertains to the role that the tribeplays, and the activities that the entrepreneur develops - and continuesto develop in conjunction with the tribe - long after the company hasbeen founded
according to this definition, unconventional entrepreneurship is tribal in nature.
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launching innovative new venturessometimes involves relatively unconventional processes and compe-tencies. The person driving the process will be someone who in his/herprivate life is a big fan of a given sporting, cultural, or other activity. Totake full advantage of the passion (shared with other fans, i.e., thetribe), the person imagines (and often self-produces) a system that willbe adopted first by fellow fans and then unerringly by other consumers
the unconventional entrepreneur, fuelled by passion, connects with a tribe of like-minded others, and is able to monetize that passion by selling it to others in the tribe...
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hat trip is what fired me up to come home and finallystart GoPro to create ‘the invisible camera,’a wearable camera so convenientthat you forget you've got it on”
a vacation spurred a vocation, fuelled by avocation (passion)
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entrepreneurial passion as an intense positive emotion to-wards entrepreneurial tasks and activities important to the en-trepreneur's self-identity. Having passion for value creation andinfluencing the world tends to be the main trait associated with en-trepreneurship
entrepreneurial passion is central to all entrepreneurial activities -- without it one wouldn't carry on in the face of adversity, one wouldn't have the strength to "swim against the current" etc. BUT, domain passion is different...
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“con-necting passions”help people reconcile the quest for individual growthwith their need to engage in a business activity (
I found the term "need" to be potentially interesting here. How does this read differently if the word "desire" replaced "need"?
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the sys-tematic pursuit of a sufficiently substantial, interesting, and fulfillingamateur activity or hobby can help develop new skills, derive meaning,and positively transform live
Do you have any experience with this? It doesn't have to be an "extreme sport" like bodybuilding or surfing...
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unrelated to professional experience but linked to personal aptitudesand leisure preferences
passions related to the personal!
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In li-quid times, society requires people to take an entrepreneurial stance,not primarily due to the need to incorporate technological innovationsor maximize profit, but because entrepreneurship offers a way to copewith precariousness and uncertainty.
and as a rejoinder, the (also familiar) Szeman-esque response that uncertainty is everywhere, and we all have to be entrepreneurs to cope with the rampant riskiness of everything.
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in the first section, we focus on the role of passion as astabilising force.
laying out the structure of the subsequent analysis - part 1
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Within a relativelyshort period of time, they succumb to a sense of disappointment thatweighs on them day after day. They expected more meaningfulness intheir daily lives, hoping for less routine and boredom. This is com-pounded by a lack of recognition, a feeling that who they are and whatthey do is undervalued. For many, passion becomes a way out of thisimpasse
Ouch - the authors are talking about you (in a couple of months)!
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most passions foster the development of competencies,skills, and knowledge. In turn, this sparks innovation
love it - the things you care most about are what you spend most of your time developing, in turn cultivating (shared) interests, creating new innovation, new products, new opportunities for developing that passion...
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Conventionally, and corresponding to the stereotypical entrepreneur,opportunity recognition consists in recognising the opportunity firstand then developing an organisational development path as describedin the traditional literature.
more background
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an entrepreneur's commitment may be fuelled by motives su-perseding the rational search for profit;
does this make inherent sense to you? Because it kind-of flies in the face of neoliberal instrumentalism...
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By definition, “conventional”entrepreneurs operate in an un-certain environment (Knight, 1921) where they are able to createSchumpeterian opportunities in view of the opportunity-seeking activ-ities or alertness and readiness to recognize them (Casson, 1982;Kirzner, 1979). This means they are prepared to take responsibility forovercoming the challenges inherent in ever-present uncertainty. By sodoing, they make things easier for other actors (wage-earners, banks,etc.), but in exchange expect to be rewarded.
more (familiar) background
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The unconventional entrepreneur distances himself from the my-thical figure of the Schumpeterian entrepreneur.
cool phrasing
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domain passion cannot beassimilated with the kind of entrepreneurial passion that was so central
crucial point...
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entrepreneurs try to stay in tune withliquid times, in much the same way as the “autopreneurs”that Ashman,Patterson, and Brown (2018) describe
unconventional entrepreneurs try to stay afloat in unconventional (liquid) times.
Note the link to an earlier article!
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Liquid times have not created the unconventional entrepreneur
interesting... link to next highlight...
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We are witnessing the proliferation of individuals who have becometheir own Pygmalion and are on a never-ending identity quest to givemeaning to their lives. Unlike past eras when identity and communityrelations were fixed institutionally, in liquid times, self-identity must beroutinely created and sustained through a multiplicity of experiences.
What's your take on this?
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when theindividual more intensively feels the liquidity of life
entrepreneurship fills a void that's especially felt at certain times in life...
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What liquid times have changed is themagnitude of the phenomenon
!!
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Life in a liquid society became precarious, unfolding under condi-tions of uncertainty, instability, and insecurity (Bauman, 2007; Beck,2009), with the only constant being accelerated change
yep...
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great popularity in his own community made it much easier for him totry and conquer the rest of the world
the importance of the network (and successful intelligence...)
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Unconventionally, we argue that opportu-nity recognition is not a necessary first step, and that the desperatesearch for opportunities could end in failure.
starting to deviate from the norm...
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n the second section,we look at the entrepreneur's passion, and particularly the role that fantribes play in this variant.
structure - part 2
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In the en-trepreneurship discipline, an established convention corresponds to thestandard –if not canonical –profile of the Schumpeterian entrepreneur.
background...
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www.biorxiv.org www.biorxiv.org
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
This study explores the sequence characteristics and features of high-occupancy target (HOT) loci across the human genome. The computational analyses presented in this paper provide information into the correlation of TF binding and regulatory networks at HOT loci that were regarded as lacking sequence specificity.
By leveraging hundreds of ChIP-seq datasets from the ENCODE Project to delineate HOT loci in HepG2, K562, and H1-hESC cells, the investigators identified the regulatory significance and participation in 3D chromatin interactions of HOT loci. Subsequent exploration focused on the interaction of DNA-associated proteins (DAPs) with HOT loci using computational models. The models established that the potential formation of HOT loci is likely embedded in their DNA sequences and is significantly influenced by GC contents. Further inquiry exposed contrasting roles of HOT loci in housekeeping and tissue-specific functions spanning various cell types, with distinctions between embryonic and differentiated states, including instances of polymorphic variability. The authors conclude with a speculative model that HOT loci serve as anchors where phase-separated transcriptional condensates form. The findings presented here open avenues for future research, encouraging more exploration of the functional implications of HOT loci.
Strengths:
The concept of using computational models to define characteristics of HOT loci is refreshing and allows researchers to take a different approach to identifying potential targets. The major strengths of the study lies in the very large number of datasets analyzed, with hundreds of ChIP-seq data sets for both HepG2 and K562 cells as part of the ENCODE project. Such quantitative power allowed the authors to delve deeply into HOT loci, which were previously thought to be artifacts.
Weaknesses:
While this study contributes to our knowledge of HOT loci, there are critical weaknesses that need to be addressed. There are questions on the validity of the assumptions made for certain analyses. The speculative nature of the proposed model involving transcriptional condensates needs either further validation or be toned down. Furthermore, some apparent contradictions exist among the main conclusions, and these either need to be better explained or corrected. Lastly, several figure panels could be better explained or described in the figure legends.
We thank the reviewer for their valuable comments.
- We have extended the study and included a new chapter focusing on the condensate hypothesis, added more supporting evidence (including the ones suggested by the reviewer), and made explicit statements on the speculative nature of this model.
- We have restructured the text to remove the sentences which might be construed as contradictory.
Reviewer #2 (Public Review):
Summary:
The paper 'Sequence characteristic and an accurate model of abundant hyperactive loci in human genome' by Hydaiberdiev and Ovcharenko offers comprehensive analyses and insights about the 'high-occupancy target' (HOT) loci in the human genome. These are considered genomic regions that overlap with transcription factor binding sites. The authors provided very comprehensive analyses of the TF composition characteristics of these HOT loci. They showed that these HOT loci tend to overlap with annotated promoters and enhancers, GC-rich regions, open chromatin signals, and highly conserved regions, and that these loci are also enriched with potentially causal variants with different traits.
Strengths:
Overall, the HOT loci' definition is clear and the data of HOT regions across the genome can be a useful dataset for studies that use HepG2 or K562 as a model. I appreciate the authors' efforts in presenting many analyses and plots backing up each statement.
Weaknesses:
It is noteworthy that the HOT concept and their signature characteristics as being highly functional regions of the genome are not presented for the first time here. Additionally, I find the main manuscript, though very comprehensive, long-winded and can be put in a shorter, more digestible format without sacrificing scientific content.
The introduction's mention of the blacklisted region can be rather misleading because when I read it, I was anticipating that we are uncovering new regulatory regions within the blacklisted region. However, the paper does not seem to address the question of whether the HOT regions overlap, if any, with the ENCODE blacklisted regions afterward. This plays into the central assessment that this manuscript is long-winded.
The introduction also mentioned that HOT regions correspond to 'genomic regions that seemingly get bound by a large number of TFs with no apparent DNA sequence specificity' (this point of 'no sequence specificity' is reiterated in the discussion lines 485-486). However, later on in the paper, the authors also presented models such as convolutional neural networks that take in one-hot-encoded DNA sequence to predict HOT performed really well. It means that the sequence contexts with potential motifs can still play a role in forming the HOT loci. At the same time, lines 59-60 also cited studies that "detected putative drive motifs at the core segments of the HOT loci". The authors should edit the manuscript to clarify (or eradicate) contradictory statements.
We thank the reviewer for their valuable comments. Below are our responses to each paragraph in the given order:
We added a statement in the commenting and summarizing other publications that studied the functional aspects of HOT loci with the following sentence in the introduction part:
“Other studies have concluded that these regions are highly functionally consequential regions enriched in epigenetic signals of active regulatory elements such as histone modification regions and high chromatin accessibility”.
We significantly shortened the manuscript by a) moving the detailed analyses of the computational model to the supplemental materials, and b) shortening the discussions by around half, focusing on core analyses that would be most beneficial to the field.
Given that the ENCODE blacklisted regions are the regions that are recommended by the ENCODE guidelines to be avoided in mapping the ChIP-seq (and other NGS), we excluded them from our analyzed regions before mapping to the genome. Instead, we relied on the conclusions of other publications on HOT loci that the initial assessments of a fraction of HOT loci were the result of factoring in these loci which later were included in blacklisted regions.
We addressed the potential confusion by using the expression of “no sequence specificity” by a) changing the sentence in the introduction by adding a clarification as “... with no apparent DNA sequence specificity in terms of detectible binding motifs of corresponding motifs” and b) removing that part from the sentence in the discussions.
Reviewer #3 (Public Review):
Summary:
Hudaiberdiev and Ovcharenko investigate regions within the genome where a high abundance of DNA-associated proteins are located and identify DNA sequence features enriched in these regions, their conservation in evolution, and variation in disease. Using ChIP-seq binding profiles of over 1,000 proteins in three human cell lines (HepG2, K562, and H1) as a data source they're able to identify nearly 44,000 high-occupancy target loci (HOT) that form at promoter and enhancer regions, thus suggesting these HOT loci regulate housekeeping and cell identity genes. Their primary investigative tool is HepG2 cells, but they employ K562 and H1 cells as tools to validate these assertions in other human cell types. Their analyses use RNA pol II signal, super-enhancer, regular-enhancer, and epigenetic marks to support the identification of these regions. The work is notable, in that it identifies a set of proteins that are invariantly associated with high-occupancy enhancers and promoters and argues for the integration of these molecules at different genomic loci. These observations are leveraged by the authors to argue HOT loci as potential sites of transcriptional condensates, a claim that they are well poised to provide information in support of. This work would benefit from refinement and some additional work to support the claims.
Comments:
(1) Condensates are thought to be scaffolded by one or more proteins or RNA molecules that are associated together to induce phase separation. The authors can readily provide from their analysis a check of whether HOT loci exist within different condensate compartments (or a marker for them). Generally, ChIPSeq signal from MED1 and Ronin (THAP11) would be anticipated to correspond with transcriptional condensates of different flavors, other coactivator proteins (e.g., BRD4), would be useful to include as well. Similarly, condensate scaffolding proteins of facultative and constitutive heterochromatin (HP1a and EZH2/1) would augment the authors' model by providing further evidence that HOT Loci occur at transcriptional condensates and not heterochromatin condensates. Sites of splicing might be informative as well, splicing condensates (or nuclear speckles) are scaffolded by SRRM/SON, which is probably not in their data set, but members of the serine arginine-rich splicing factor family of proteins can serve as a proxy-SRSF2 is the best studied of this set. This would provide a significant improvement to their proposed model and be expected since the authors note that these proteins occur at the enhancers and promoter regions of highly expressed genes.
(2) It is curious that MAX is found to be highly enriched without its binding partner Myc, is Myc's signal simply lower in abundance, or is it absent from HOT loci? How could it be possible that a pair of proteins, which bind DNA as a heterodimer are found in HOT loci without invoking a condensate model to interpret the results?
(3) Numerous studies have linked the physical properties of transcription factor proteins to their role in the genome. The authors here provide a limited analysis of the proteins found at different HOT-loci by employing go terms. Is there evidence for specific types of structural motifs, disordered motifs, or related properties of these proteins present in specific loci?
(4) Condensates themselves possess different emergent properties, but it is a product of the proteins and RNAs that concentrate in them and not a result of any one specific function (condensates can have multiple functions!)
(5) Transcriptional condensates serve as functional bodies. The notion the authors present in their discussion is not held by practitioners of condensate science, in that condensates exist to perform biochemical functions and are dissolved in response to satisfying that need, not that they serve simply as reservoirs of active molecules. For example, transcriptional condensates form at enhancers or promoters that concentrate factors involved in the activation and expression of that gene and are subsequently dissolved in response to a regulatory signal (in transcription this can be the nascently synthesized RNA itself or other factors). The association reactions driving the formation of active biochemical machinery within condensates are materially changed, as are the kinetics of assembly. It is unnecessary and inaccurate to qualify transcriptional condensates as depots for transcriptional machinery.
6) This work has the potential to advance the field forward by providing a detailed perspective on what proteins are located in what regions of the genome. Publication of this information alongside the manuscript would advance the field materially.
We thank the reviewer for constructive comments and suggestions. Below are our point-by-point responses:
(1) We added a new short section “Transcriptional condensates as a model for explaining the HOT regions” with additional support for the condensate hypothesis, wherein some of the points raised here were addressed. Specifically, we used a curated LLPS proteins (CD-CODE) database and provided statistics of those annotation condensate-related DAPs.
Regarding the DAPs mentioned in this question, we observed that the distributions corresponding ChIP-seq peaks confirm the patterns expected by the reviewer (Author response image 1). Namely:
- MED1 and Ronin (THAP11) are abundant in the HOT loci, being present 67% and 64% of HOT loci respectively.
- While the BRD4 is present in 28% of the HOT loci, we observed that the DAPs with annotated LLPS activity ranged from 3% to 73%, providing further support for the condensate hypothesis.
- ENCODE database does not contain ChIP-seq dataset for HP1A. EZH2 peaks were absent in the HOT loci (0.4% overlap), suggesting the lack of heterochromatin condensate involvement.
- Serine-rich splicing factor family proteins were present only in 7.7% of the HOT loci, suggesting the absence or limited overlap with splicing condensates or nuclear speckles.
Author response image 1.
(2) In this study we selected the TF ChIP-seq datasets with stringent quality metrics, excluding those which had attached audit warning and errors. As a result, the set of DAPs analyzed in HepG2 did not include MYC, since the corresponding ChIP-seq dataset had the audit warning tags of "borderline replicate concordance, insufficient read length, insufficient read depth, extremely low read depth". Analyses in K562 and H1 did include MYC (alongside MAX) ChIP-seq dataset.
To address this question, we added the mentioned ChIP-seq dataset (ENCODE ID: ENCFF800JFG) and analyzed the colocalization patterns of MYC and MAX. We observed that the MYC ChIP-seq peaks in HepG2 display spurious results, overlapping with only 5% of HOT loci. Meanwhile in K562 and H1, MYC and MAX are jointly present in 54% and 44% of the HOT loci, respectively (Author response image 2).
Author response image 2.
These observations were also supported by Jaccard indices between the MYC and MAX ChIP-seq peaks. To do this analysis, we calculated the pairwise Jaccard indices between MYC and MAX and divided them by the average Jaccard indices of 2000 randomly selected DAP pairs. In K562 and H1, the Jaccard indices between MYC and MAX are 5.72x and 2.53x greater than the random background, respectively. For HepG2, the ratio was 0.21x, clearly indicating that HepG2 MYC ChIP-seq dataset is likely erroneous.
Author response image 3.
(3) Despite numerous publications focusing on different structural domains in transcription factors, we could not find an extensive database or a survey study focusing on annotations of structural motifs in human TFs. Therefore, surveying such a scale would be outside of this study’s scope. We added only the analysis of intrinsically disordered regions, as it pertains to the condensate hypothesis. To emphasize this shortcoming, we added the following sentence to the end of the discussions section.
“Further, one of the hallmarks of LLPS proteins that have been associated with their abilities to phase-separate is the overrepresentation of certain structural motifs, which we did not pursue due to size limitations.”
(4, 5) We agree with these statements and thank the reviewer for pointing out this faulty statement. We modified the sections in the discussions related to the condensates and removed the part where we implied that the condensate model could be because of mostly a single function of TF reservoir.
(6) We added a table to the supplemental materials (Zenodo repository) with detailed annotation of HOT and non-HOT DAP-bound loci in the genome.
Recommendations for the authors:
Reviewing Editor (Recommendations For The Authors):
The clause with "inadequate" would be dropped if the authors sufficiently address reviewer concerns about clarity of writing, including:
(1) Editing the title to better reflect the findings of the paper.
(2) Making clear that the condensate model is speculative and not explicitly tested in this study (and may be better described as a hypothesis).
(3) Resolving apparent contradictions regarding DNA sequence specificity and the interpretation of ChIP-seq signal intensity.
(4) Better specifying and justifying model parameters, thresholds, and assumptions.
(5) Shortening the manuscript to emphasize the main, well-supported claims and to enhance readability (especially the discussion section).
We thank the Editor for their work. We followed their advice and implemented changes and additions to address all 5 points.
Reviewer #1 (Recommendations For The Authors):
(1) The title "Sequence characteristics and an accurate model of abundant hyperactive loci in the human genome" does not accurately reflect the findings of the paper. We are unclear as to what the 'accurate model' refers to. Is it the proposed model 'based on the existence of large transcriptional condensates' (abstract)? If so, there are concerns below regarding this statement (see comment 2). If the authors are referring to the computational modeling presented in Figure 5, it is unclear that any one of them performed that much better than the others and the best single model was not identified. Furthermore, the models being developed in the study constitute only a portion of the paper and lacked validation through additional datasets. Additionally, sequence characteristics were not a primary focus of the study. Only figure 5 talks about the model and sequence characteristics, the rest of the figures are left out of the equation.
We agree with and thank the reviewer for this idea of clarifying the intended meaning.
(1) We changed the title and clarified that the computational model is meant:
“Functional characteristics and a computational model of abundant hyperactive loci in the human genome”.
(2) Shortened the part of the manuscript discussing the computational models and pointed out the CNNs as “the best single model”.
(2) The abstract and discussion (and perhaps the title) propose a model of transcriptional condensates in relation to HOT loci. However, there is no data provided in the manuscript that relates to condensates. Therefore, anything relating to condensates is primarily speculative. This distinction needs to be properly made, especially in the abstract (and cannot be included in the title). Otherwise, these statements are misleading. Although the field of transcriptional condensates is relatively new, there have been several factors studied. The authors could include in Figure 2d which factors have been shown to form transcriptional condensates. This might provide some support for the model, though it would still largely remain speculative unless further testing is done.
We added a new short chapter “Transcriptional condensates as a model for explaining the HOT regions”, with additional analyses testing the condensates hypothesis. We provided supportive evidence by analyzing the metrics used as hallmarks of condensates including the distributions of annotated condensate-related proteins, nascent transcription, and protein-RNA interaction levels in HOT loci. Still, we acknowledge that this is a speculative hypothesis and we clarified that with the following statement in the discussions:
“It is important to note here that our proposed condensate model is a speculative hypothesis. Further experimental studies in the field are needed to confirm or reject it.”
(3) Several apparent contradictions exist throughout the manuscript. For example, "HOT locus formation are likely encoded in their DNA sequences" (lines 329-330) vs the proposed model of formation through condensates (abstract). These two statements do not seem compatible, or at the very least, the authors can explain how they are consistent with each other. Another example: "ChIP-seq signal intensity as a proxy for... binding affinity" (line 229) vs. "ChIP-seq signal intensities do not seem to be a function of the DNA-binding properties of the DAPs" (lines 259-260). The first statement is the assumption for subsequent analyses, which has its own concerns (see comment 4). But the conclusion from that analysis seems to contradict the assumption, at least as it is stated.
In this study, we argue that the two statements may not necessarily contradict each other. We aimed to a) demonstrate that the observed intensity of DAP-DNA interactions as measured by ChIP-seq experiments at HOT loci cannot be explained with direct DNA-binding events of the DAPs alone and b) propose a hypothesis that this observation can be at least partially explained if the HOT loci have the propensity to either facilitate or take part in the formation of transcriptional condensates.
One of the conditions for condensates to form at enhancers was shown to be the presence of strong binding sites of key TFs (Shrinivas et al. 2019 “Enhancer features that drive the formation of transcriptional condensates”), where the study was conducted using only one TF (OCT4) and one coactivator (MED1). To the best of our knowledge, no such study has been conducted involving many TFs and cofactors simultaneously. We also know that the factors that lead to liquid-to-liquid phase separation include weak multivalent IDR-IDR, IDR-DNA, and IDR-RNA interactions. As a result, the observed total sum of ChIP-seq peaks in HOT loci is the direct DNA-binding events combined with the indirect DAP-DNA interactions, some of which may be facilitated by condensates. And, the fact that CNNs can recognize the HOT loci with high accuracy suggests that there must be an underlying motif grammar specific to HOT loci.
We emphasized this conclusion in the discussions.
The comment on using the ChIP-seq signal as a proxy for DNA-binding affinity is addressed under comment 4.
(4) In lines 229-230, the authors used "the ChIP-seq signal intensity as a proxy for the DAP binding affinity." What is the basis for this assumption? If there is a study that can be referenced, it should be added. However, ChIP-seq signal intensity is generally regarded as a combination of abundance, frequency, or percentage of cells with binding. RNA Pol2 is a good example of this as it has no specific binding affinity but the peak heights indicate level of expression. Therefore, the analyses and conclusions in Figure 4, particularly panel A, are problematic. In addition, clarification from lines 258-260 is needed as it contradicts the earlier premise of the section (see comment 3).
We thank the reviewer for pointing out this error. The main conclusion of the paragraph is that the average ChIP-seq signal values at HOT loci do not correlate well with the sequence-specificity of TFs. We reworded the paragraph stating that we are analyzing the patterns of ChIP-seq signals across the HOT loci, removing the part that we use them as a proxy for sequence-specific binding affinity.
(5) In Figure 1A, the authors show that "the distribution of the number of loci is not multimodal, but rather follows a uniform spectrum, and thus, this definition of HOT loci is ad-hoc" (lines 92-95). The threshold to determine how a locus is considered to be HOT is unclear. How did the authors decide to use the current threshold given the uniform spectrum observed? How does this method of calling HOT loci compare to previous studies? How much overlap is there in the HOT loci in this study versus previous ones?
We moved the corresponding explanation from the supplemental methods to the main methods section of the manuscript.
Briefly, our reasoning was as follows: assuming that an average TFBS is 8bp long and given that we analyze the loci of length 400bp, we can set the theoretical maximum number of simultaneous binding events to be 50. Hence, if there are >50 TF ChIP-seq peaks in a given 400bp locus, it is highly unlikely that the majority of ChIP-seq peaks can be explained by direct TF-DNA interactions. The condition of >50 TFs corresponded to the last four bins of our binning scale, which was used as an operational definition for HOT loci.
We have compared our definition of HOT loci to those reported in previous studies by Remaker et al. and Boyle et al. The results of our analyses are in lines 147-154.
(6) In Figure 3B, the authors state that of "the loop anchor regions with >3 overlapping loops, 51% contained at least one HOT locus, suggesting an interplay between chromatin loops and HOT loci." However, it is unclear how "51%" is calculated from the figure. Similarly, in the following sentence, "94% of HOT loci are located in regions with at least one chromatin interaction". It is unclear as to how the number was obtained based on the referenced figure.
Initially, the x-axis on the Figure 3B was missing, making it hard to understand what we meant. We added the x-axis numbers and changed the “51%” to “more than half”. We intend to say that, of the loci with 4 and 5 overlapping loops, exactly 50% contain at least one HOT locus. However, since for x=6 the percentage is 100% (since there’s only one such locus), the percentage is technically “more than half”.
The percentage of HOT loci engaging in chromatin interaction regions (91%) was calculated by simply overlapping the HOT regions with Hi-C long-range contact anchors. The details of extracting these regions using FitHiChip are described in Supplemental Methods 1.3.
(7) While we have a limited basis to evaluate computational models, we would like to see a clearer explanation of the model set-up in terms of the number of trained vs. test datasets. In addition, it would be interesting to see if the models can be applied to data from different cell lines.
We added the table with the sizes of the datasets used for classification in Supplemental Methods 1.6.1.
Evaluating the models trained on the HOT loci of HepG2 and K562 on other cell lines would pose challenges since the number of available ENCODE TF ChIP-seq datasets is significantly less compared to the mentioned cell lines. Therefore, we conducted the proposed analysis between the studied cell lines. Specifically, we used the CNN models trained on HOT and regular enhancers of HepG2 and K562. Then, we evaluated each model on the test sets of each classification experiment (Author response image 4). We observed that the classification results of the HOT loci demonstrated a higher level of tissue-specificity compared to the same classification results of the regular enhancers.
Author response image 4.
(8) Lines 349-351. The significance of highly expressed genes being more prone to having multiple HOT loci, and vice versa, appears conventional and remains unclear. Intuitively, it makes sense for higher expressed genes to have more of the transcriptional machinery bound, and would bias the analysis. One way to circumvent this is to only analyze sequence-specific TFs and remove ones that are directly related to transcription machinery.
We thank the reviewer for this suggestion. Our attempt to re-annotate the HOT loci with only sequence-specific TFs led to a significantly different set of loci, which would not be strictly comparable to the HOT loci defined by this study. Analyzing these new sets of loci would create a noticeable departure from the flow of the manuscript and further extend the already long scope of the study.
Moreover, numerous studies have shown that super-enhancers recruit large numbers of TFs via transcriptional condensates (Boija et al., 2018; Cho et al., 2018; Sabari et al., 2018). We hope that our results can serve as data-driven supportive evidence for those studies.
(9) Lines 393-396. We would like to see a reference to the models shown in the figures, if these models have been published previously.
We could not understand the question. The lines 393-396 contains the following sentence:
“However, many of the features of the loci that we’ve analyzed so far demonstrated similar patterns (GC contents, target gene expressions, ChIP-seq signal values etc.) when compared to the DAP-bound loci in HepG2 and K562, suggesting that albeit limited, the distribution of the DAPs in H1 likely reflects the true distribution of HOT loci.”
In case the question was about the models that we trained to classify the HOT loci, we included the models and codebase to Zenodo and GitHub repository.
(10) Values in Figure 7D are not reflected in the text. Specifically, the text states "Average ... phastCons of the developmental HOT loci are 1.3x higher than K562 and HepG2 HOT loci (Figure 7D)" (lines 408-409). Figure 7D shows conservation scores between HOT enhancers vs promoters for each cell line, and does not seem to reflect the text.
We modified the figure to reflect the statement appropriately.
(11) Methodology should include a justification for the use of the Mann-Whitney U-test (non-parametric) over other statistical tests.
We added the following description to the methods section:
“For calculating the statistical significance, we used the non-parametric Mann-Whitney U-test when the compared data points are non-linearly correlated and multi-modal. When the data distributions are bell-curve shaped, the Student’s t-test was used.“
Minor:
(1) Figure 2b was never mentioned in the paper. This can be added alongside Figure S6C, line 148.
Indeed, Figure 2B was supposed to be listed together with Figure S6C, which was omitted by mistake. It was corrected.
(2) Supplementary Figure 8 has two Cs. Needs to be corrected to D.
Fixed.
(3) Figure 3B is missing labels on the x-axis.
Fixed.
(4) The horizontal bar graph on the bottom left of Figure 1E needs to be described in the figure legend.
Description added to the figure caption.
(5) Line 345, Fig 15A should be Fig S15A.
Corrected.
Reviewer #2 (Recommendations For The Authors):
I listed all my concerns about the paper in the public comments. I think the manuscript is very comprehensive and it is valuable, but it should be cut short and presented in a more digestible way.
We thank the reviewer for their valuable comments and suggestions. We addressed all the concerns listed in the public comments. We shortened the manuscript by reducing the paragraph that focuses on computational classification models and reduced the discussions by about half in length.
Line 55: What are chromatin-associated proteins, i.e. are they histone modifications?
To clarify the definition used from the citation we changed the sentence to the following:
“For instance, Partridge et al. studied the HOT loci in the context of 208 proteins including TFs, cofactors, and chromatin regulators which they called chromatin-associated proteins.”
Though most of the paper can be cut short to avoid analysis paralysis for readers, there are details that still need filling in. For example, how did the authors perform PCA analysis, i.e. what are the features of each data point in the PCA analysis? Lines 214-215: How do we calculate the number of multi-way contacts in Hi-C data?
We added clarifying descriptions and changed the mentioned sentences to the following:
PCA:
“To analyze the signatures of unique DAPs in HOT loci, we performed a PCA analysis where each HOT locus is represented by a binary (presence/absence) vector of length equal to the total number of DAPs analyzed.”
Multi-way contacts on loop anchors:
“To investigate further, we analyzed the loop anchor regions harboring HOT loci and observed that the number of multi-way contacts on loop anchors (i.e. loci which serve as anchors to multiple loops) correlates with the number of bound DAPs (rho=0.84 p-value<10E-4; Pearson correlation). “
- Lines 251-252: How did the referenced study categorize DAPs? It is important for any manuscript to be self-contained.
We added the explanation and changed the sentence to the following:
“To test this hypothesis, we classified the DAPs into those two categories using the definitions provided in the study (Lambert et al. 2018) 28, where the TFs are classified by manual curation through extensive literature review and supported by annotations such as the presence of DNA-binding domains and validated binding motifs. Based on this classification, we categorized the ChIP-seq signal values into these two groups.“
- Lines 181-185, sentences starting with 'To test' can be moved to the methods, leaving only brief mentions of the statistic tests if needed.
We removed the mentioned sentence and moved to the supplemental methods (1.4).
- Lines 217-220: I find this sentence extremely redundant unless it can offer more specific insights about a particular set of DAPs or if the DAPs are closer/or a proven distal enhancer to a confirmed causal gene.
We removed the mentioned sentence from the text.
- Lines 243-246: How did the authors determine the set DAPs that have stabilizing effects, and how exactly are the 'stabilizing effects' observed/measured?
We added explanations to Supplemental Methods 3.1 and Fig S18, S19.
While addressing this comment we realized that the reported value of the ratio is 1.91x, not 1.7x. We corrected that value in the main text and added the p-value.
- When discussing the phastCons scores analyses, such as in lines 268-271, how did the authors calculate the relationship between phastCons scores and HOT loci, i.e. was the score averaged across the 400-bp locus to obtain a locus-specific conservation score?
Yes, per-locus conservation scores were averaged over the bps of loci. We added this clarification to the methods.
- Line 311: What is the role of the 'control sets' in the analyses of the sequence's relationship with HOT?
In this specific case, the control sets are used as background or negative sets to set up the classification tasks. In other words, we are asking, whether the HOT loci can be distinguished when compared to random chromatin-accessible regions, promoters, or regular enhancers. We clarified this in the text.
- I also find the discussion about different machine learning methods that classify HOT loci based on sequence contexts quite redundant UNLESS the authors decide to go further into the features' importance (such as motifs) in the models that predict/ are associated with HOT loci, which in itself can constitute another study.
We agree with the reviewer, and shortened the part with the discussions of models by limiting it to only 3 main models and moved the rest to the supplemental materials.
- Can the authors clarify where they obtain data on super-enhancers?
We obtained the super-enhancer definitions from the original study (Hnisz et al. 2013, PMID: 24119843) where the super-enhancers were defined for multiple cell lines. We clarified this in the methods.
- Figure 1B, the x and y axis should be clarified.
We clarified it by using MAX as an example case in the figure caption as follows:
“Prevalence of DAPs in HOT loci. Each dot represents a DAP. X-axis: percentage of HOT loci in which DAP is present (e.g. MAX is present in 80% of HOT loci). Y-axis: percentage of total peaks of DAPs that are located in HOT loci (e.g. 45% of all the ChIP-seq peaks of MAX is located in the HOT loci). Dot color and size are proportional to the total number of ChIP-seq peaks of DAP.”
Reviewer #3 (Recommendations For The Authors):
The list of proteins associated with different types of genomic loci at a meta level (enhancers, promoters, and gene body etc.), and an annotation of the genome at the specific loci level.
The authors use a wide range of acronyms throughout the text and figure legends, they do a reasonably good job, but the main text section "HOT-loci are enriched in causal variants" and Figure 8 would be materially improved if they held it to the same standard.
Size is a physical property and not a physicochemical property.
We thank the reviewer for their comments and suggestions. We added a table to supplemental files with detailed annotations of analyzed loci.
We reviewed the section “HOT loci are enriched in causal variants” and corrected a few mismatches in the acronyms.
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eLife Assessment
This valuable study explores the sequence characteristics and conservation of high-occupancy target loci, regions in the human genome such as promoters and enhancers that are bound by a multitude of transcription factors. The computational analyses presented in this study are solid. This study would be a helpful resource for researchers performing ChIP-seq based analyses of transcription factor binding.
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Reviewer #1 (Public review):
Summary:
This study explores the sequence characteristics and features of high-occupancy target (HOT) loci across the human genome. The computational analyses presented in this paper provide information into the correlation of TF binding and regulatory networks at HOT loci that were regarded as lacking sequence specificity.
By leveraging hundreds of ChIP-seq datasets from the ENCODE Project to delineate HOT loci in HepG2, K562, and H1-hESC cells, the investigators identified the regulatory significance and participation in 3D chromatin interactions of HOT loci. Subsequent exploration focused on the interaction of DNA-associated proteins (DAPs) with HOT loci using computational models. The models established that the potential formation of HOT loci is likely embedded in their DNA sequences and is significantly influenced by GC contents. Further inquiry exposed contrasting roles of HOT loci in housekeeping and tissue-specific functions spanning various cell types, with distinctions between embryonic and differentiated states, including instances of polymorphic variability. The authors conclude with a speculative model that HOT loci serve as anchors where phase-separated transcriptional condensates form. The findings presented here open avenues for future research, encouraging more exploration of the functional implications of HOT loci.
Strengths:
The concept of using computational models to define characteristics of HOT loci is refreshing and allows researchers to take a different approach in identifying potential targets. The major strengths of the study lie in the very large number of datasets analyzed, with hundreds of ChIP-seq data sets for both HepG2 and K562 cells as part of the ENCODE project. Such quantitative power allowed the authors to delve deeply into HOT loci, which were previously thought to be artifacts.
Weaknesses:
While this study contributes to our knowledge of HOT loci, there are critical weaknesses that need to be addressed. There are questions on the validity of the assumptions made for certain analyses. The speculative nature of the proposed model involving transcriptional condensates needs either further validation or be toned down. Furthermore, some apparent contradictions exist among the main conclusions, and these either need to be better explained or corrected. Lastly, several figure panels could be better explained or described in the figure legends.
Update After Revisions:
The authors have addressed the above comments and concerns appropriately. The addition of the new Figure 9 is particularly compelling and strengthens the authors' conclusions. This reviewer has no further concerns.
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Reviewer #2 (Public review):
Summary:
The paper by Hydaiberdiev and Ovcharenko offers comprehensive analyses and insights about the 'high-occupancy target' (HOT) loci in the human genome. These are considered genomic regions that overlap with transcription factor binding sites. The authors provided very comprehensive analyses of the TF composition characteristics of these HOT loci. They showed that these HOT loci tend to overlap with annotated promoters and enhancers, GC-rich regions, open chromatin signals, and highly conserved regions and that these loci are also enriched with potentially causal variants with different traits.
Strengths:
Overall, the HOT loci' definition is clear and the data of HOT regions across the genome can be a useful dataset for studies that use HepG2 or K562 as a model. I appreciate the authors' efforts in presenting many analyses and plots backing up each statement.
Comments on revised version:
In the second round of review, I think the authors have sufficiently addressed all of my previous comments. The study itself is very comprehensive, tackling all aspects of the HOT loci, though I still find the paper to be unnecessarily long and long-winded. That said, being consistent with the long and detailed paper, the provided Github repository and Zenodo archive is well-documented. I appreciate that the authors include detailed readme about the different datafiles available for readers. The list of HOT loci is probably the most useful asset in this manuscript and the authors did a good job documenting data availability in both Github and Zenodo.
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Reviewer #3 (Public review):
Summary:
Hudaiberdiev and Ovcharenko investigate regions within the genome where a high abundance of DNA associated proteins are located and identify DNA sequence feature enriched in these regions, their conservation in evolution, and variation in disease. Using ChIP-seq binding profiles of over 1,000 proteins in three human cell lines (HepG2, K562, and H1) as a data source they're able to identify nearly 44,000 high-occupancy target loci (HOT) that form at promoter and enhancer regions, thus suggesting these HOT loci regulate housekeeping and cell identity genes. Their primary investigative tool is HepG2 cells, but they employ K562 and H1 cells as tools to validate these assertions in other human cell types. Their analyses use RNA pol II signal, super enhancer, regular enhancer and epigentic marks to support the identification of these regions. The work is notable, in that it identifies a set of proteins that are invariantly associated with high-occupancy enhancers and promoters and argues for the integration of these molecules at different genomic loci. These observations are leveraged by the authors to argue HOT loci as potential sites of transcriptional condensates, a claim that they provide information in support of. Transcriptional condensates are an important "family" of condensates, regulating different types of genes and this work supports the hypothesis that they possess similar protein partner molecules as those thought to define such bodies.
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www.nature.com www.nature.com
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This equality, formalized by Gustav Kirchhoff in 1860, is known as Kirchhoff’s law of thermal radiation and has long guided designs to control the emitted radiation. Removing the constraint of Kirchhoff’s law unlocks a multitude of applications and designs for thermal emitters. Decoupling the absorptivity and emissivity relationship can be leveraged to achieve novel functions, ranging from reducing re-emission losses to the Sun in the context of solar energy harvesting systems to radiative camouflage.
"Thermal emission has generally been thought to obey reciprocity, where the absorbed and emitted radiation from a body are equal for a given wavelength and angular channel."
This hypothesis is wrong. It is a serious problem that this law is maintained within the physics curriculum. Even if one does not care about verification (as seems to be an almost indredible notion but convictions of some well regarded serious physicists, nonetheless) it has also stifled innnovation as has now been shown by developing a solar energy based device that would have been precluded from consideration by this law. This law makes it to the CSwP (Citizen Scientists with Parity) top 10 list of consequential problems maintained simply by the dynamics of truth by consensus and authroity.
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sourcebooks.fordham.edu sourcebooks.fordham.edu
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A poor man he was, and on a lean farm, yet he was not content to send me to a local school [at Venusia, his home town] under the pedant Flavius, though boys of pretensions, sons of prominent centurions, went there with their school bags and writing tablets slung over their left arms, and carrying their teacher the fee in their hands on the Ides of eight months in the year. On the contrary, he had the spirit to bring me even as a child to Rome, to be taught those liberal arts which a senator or eques requires for his children.
While it is stated that the father was a poor man, to own land in Venusia was a sign of wealth. It is possible that the term "poor" is used in comparison to his peers from his hometown.
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Sir Schoolmaster---show pity upon your simple scholars, at least if you wish to have many a long-haired boy attendant upon your lectures, and the class seated around your critical table love you. Then would no teacher of arithmetic or swift writing have a greater ring of pupils around him. Hot and bright are the days now under the flaming constellation of the lion; and fervid July is ripening the bursting harvest. So let your Scythian scourge with its dreadful thongs, such as flogged Marsyas of Celaenae, and your formidable cane---the schoolmaster's scepter---be laid aside, and sleep until the Ides of October. Surely in summer time, if the boys keep their health, they do enough.
This entire passage can be shortened to this: " Sir schoolmaster- leave the students be during the summer, they need a break from being hit with a ruler for mistakes."
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I would even promise the whole sum if I did not fear that if I did so, my generosity might be corrupted to serve private interests, as I see is the case in many places where teachers are employed at the public charge. There is only one way of preventing the evil, and that is by leaving the right of employing the teachers to the parents alone, who will be careful to make a right choice if they are obliged to find part of the money. You cannot make your children a better present than this, nor can you do your place a better turn."
What Pliny is suggesting, as a generalization, is similar to the modern day concept of a public school board.
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" "How is that?" I asked. "It is a matter of urgent importance to you who are fathers," and it so chanced that luckily quite a number of fathers were listening to me, "that your children should get their education here at home."
Pliny the Younger had such a following due to his being an elected official in ancient Rome. Not to be confused his his uncle Pliny the Elder.
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The following letter by Pliny to the famous historian Tacitus is witness to the interest taken in education under the Empire.
This passage would make far more sense to those not well versed in Roman history if it included the information that Pliny the Younger was a notable scholar of his day.
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Even the lower classes were not usually illiterate (witness the numerous wall scribblings at Pompeii), although there was no system of free public schools.
This is quite an interesting point. The Roman Republic was one of the few places in the world(outside of the modern era) that had writing literacy as a common skill.
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rws511.pbworks.com rws511.pbworks.com
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we should have been singing love songs to them allalong
I agree that society as a whole needs to be kinder to addicts. But as someone who has struggled with addiction, I do think there should be a balance. Addicts need love and compassion, but they also need to understand how their behaviors affect others (and themselves) so they are motivated to change. If it wasn't for the people who told me I needed to do better, I might not have changed. And I tried my whole childhood to love the addiction out of my parents but it didn't work, they remained just as drunk and violent.
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The good cage saved them.
Interesting. My parents were abusive alcoholics and I started drinking around 12 to numb the mental anguish of living in a violent household. Once I moved away for college I definitely drank less, but I still needed treatment because of the triggers in daily life. Veterans with PTSD struggle with addiction more than the general population, so I'm not sure about this claim. After going through life-altering trauma, sometimes a "good cage" doesn't solve everything (at least for humans). Is this applicable to humans? Our brains process memory different from rats
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legacy.cs.indiana.edu legacy.cs.indiana.edu
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Logic is a branch of mathematics that can deal with functions that are not continuous. Many reljearchers believe that it can play the role in software engineering that continuous mathematics plays in mechanical and electrical engineering. Unfortunately, this has not yet been verified in practice. The large number of states ant1 lack of regularity in the software result in ex- tremely complex mathematical expressions. Disciplined use of these expressions is beyond the computational capacity of both the human programmer and current computer systems. There is progress in this area, but it is very slow, and we are far from being able to handle even small software systems. With current techniques
When Parnas say it isn't verified that logic does play a role in software engineering it isn't true anymore. For example Drracket beginner lang uses logic a lot. Although Conditionals is the only one we learned so far we can use a cond inside a cond and can make a pretty complex system.
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www.biorxiv.org www.biorxiv.org
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eLife Assessment
This important study identifies biallelic variants of DNAH3 in unrelated infertile men and reports infertility in DNAH3 knockout mice. The authors demonstrate that compromised DNAH3 activity decreases the expression of IDA-associated proteins in the spermatozoa of human patients and knockout mice, providing convincing evidence that DNAH3 is a novel pathogenic gene for asthenoteratozoospermia and male infertility. The study will be of substantial interest to clinicians, reproductive counselors, embryologists, and basic researchers working on infertility and assisted reproductive technology.
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Reviewer #1 (Public Review):
Summary:
Wang and colleagues identify biallelic variants of DNAH3 in four unrelated Han Chinese infertile men through whole-exome sequencing, which contributes to abnormal sperm flagellar morphology and ultrastructure. To investigate the importance of DNAH3 in male infertility, the authors generated crispant Dnah3 knockout (KO) male mice. They observed that KO mice are also infertile, showing a severe reduction in sperm movement with abnormal IDA (inner dynein arms) and mitochondrion structure. Moreover, nonfunctional DNAH3 expression decreased the expression of IDA-associated proteins in the spermatozoa of patients and KO mice, which are involved in the disruption of sperm motility. Interestingly, the infertility of patients and KO mice is rescued by intracytoplasmic sperm injection (ICSI). Taken together, the authors propose that DNAH3 is a novel pathogenic gene for asthenoterozoospermia and male infertility.
Strengths:
This work investigates the role of DNAH3 in sperm mobility and male infertility. By using gold-standard molecular biology techniques, the authors demonstrate with exquisite resolution the importance of DNAH3 in sperm morphology, showing strong evidence of its role in male infertility. Overall, this is a very interesting, well-written, and appealing article. All aspects of the study design and methods are well described and appropriate to address the main question of the manuscript. The conclusions drawn are consistent with the analyses conducted and supported by the data.
Weaknesses:
The paper is solid, and in its current form, I have not detected relevant weaknesses.
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Reviewer #2 (Public Review):
Wang et al. investigated the role of dynein axonemal heavy chain 3 (DNAH3) in male infertility. They found that variants of DNAH3 were present in four infertile men, and the deficiency of DNAH3 in sperm affects sperm mobility. Additionally, they showed that Dnah3 knockout male mice are infertile. Furthermore, they demonstrated that DNAH3 influences inner dynein arms by regulating several DNAH proteins. Importantly, they showed that intracytoplasmic sperm injection (ICSI) can rescue the infertility in Dnah3 knockout mice and two patients with DNAH3 variants.
Strengths:
The conclusions of this paper are well-supported by data.
Weaknesses:
The sample/patient size is small; however, the findings are consistent with those of a recent study on DNAH3 in male infertility with 432 patients.
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Reviewer #3 (Public Review):
Summary:
(1) To further explore the genetic basis of asthenoteratozoospermia, the authors performed whole-exome sequencing analyses among infertile males affected by asthenoteratozoospermia. Four unrelated Han Chinese patients were found to carry biallelic variations of DNAH3, a gene encoding IDA-associated protein.<br /> (2) To verify the function of IDA associated protein DNAH3, the authors generated a Dnah3-KO mouse model and revealed that the loss of DNAH3 leads to severe male infertility as a result of the severe reduction in sperm movement with the abnormal IDA and mitochondrion structures.<br /> (3) Mechanically, they confirmed decreased expression of IDA-associated proteins (including DNAH1, DNAH6 and DNALI1) in the spermatozoa from patients with DNAH3 mutations and Dnah3-KO male mice.<br /> (4) Then, they also found that male infertility caused by DNAH3 deficiency could be rescued by intracytoplasmic sperm injection (ICSI) treatment in humans and mice.
Strengths:
(1) In addition to existing research, the authors provided novel variants of DNAH3 as important factors leading to asthenoteratozoospermia. This further expands the spectrum of pathogenic variants in asthenoteratozoospermia.<br /> (2) By mechanistic studies, they found that DNAH3 deficiency led to decreased expression of IDA-associated proteins, which may be used to explain the disruption of sperm motility and reduced fertility caused by DNAH3 deficiency.<br /> (3) Then, successful ICSI outcomes were observed in patients with DNAH3 mutations and Dnah3 KO mice, which will provide an important reference for genetic counselling and clinical treatment of male infertility.
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Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
Wang and colleagues identify biallelic variants of DNAH3 in four unrelated Han Chinese infertile men through whole-exome sequencing, which contributes to abnormal sperm flagellar morphology and ultrastructure. To investigate the importance of DNAH3 in male infertility, the authors generated crispant Dnah3 knockout (KO) male mice. They observed that KO mice are also infertile, showing a severe reduction in sperm movement with abnormal IDA (inner dynein arms) and mitochondrion structure. Moreover, nonfunctional DNAH3 expression decreased the expression of IDA-associated proteins in the spermatozoa of patients and KO mice, which are involved in the disruption of sperm motility. Interestingly, the infertility of patients and KO mice is rescued by intracytoplasmic sperm injection (ICSI). Taken together, the authors propose that DNAH3 is a novel pathogenic gene for asthenoterozoospermia and male infertility.
Strengths:
This work investigates the role of DNAH3 in sperm mobility and male infertility. By using gold-standard molecular biology techniques, the authors demonstrate with exquisite resolution the importance of DNAH3 in sperm morphology, showing strong evidence of its role in male infertility. Overall, this is a very interesting, well-written, and appealing article. All aspects of the study design and methods are well described and appropriate to address the main question of the manuscript. The conclusions drawn are consistent with the analyses conducted and supported by the data.
Weaknesses:
The paper is solid, and in its current form, I have not detected relevant weaknesses.
We thank the comments from the reviewer very much.
Reviewer #2 (Public Review):
Wang et al. investigated the role of dynein axonemal heavy chain 3 (DNAH3) in male infertility. They found that variants of DNAH3 were present in four infertile men, and the deficiency of DNAH3 in sperm affects sperm mobility. Additionally, they showed that Dnah3 knockout male mice are infertile. Furthermore, they demonstrated that DNAH3 influences inner dynein arms by regulating several DNAH proteins. Importantly, they showed that intracytoplasmic sperm injection (ICSI) can rescue the infertility in Dnah3 knockout mice and two patients with DNAH3 variants.
Strengths:
The conclusions of this paper are well-supported by data.
Weaknesses:
The sample/patient size is small; however, the findings are consistent with those of a recent study on DNAH3 in male infertility involving 432 patients.
We extend our sincere gratitude to the expert reviewers for their valuable comments and insightful suggestions.
A cohort of 587 unrelated infertile men with asthenoteratozoospermia was recruited to investigate the potential genetic etiology using WES. In addition to mutations in DNAH3 identified in four patients, mutations in serval other genes previous reported by our group, including CFAP65 (Zhang et al., 2019. PMID: 31571197), DNAH8 (Yang et al., 2020. PMID: 32681648), DNAH12 (Li et al., 2022. PMID: 34791246), FISIP2 (Zheng et al., 2023. PMID: 35654582), CEP128 (Zhang et al., 2022. PMID: 35296684), CEP78 (Zhang et al., 2022. PMID: 36206347), CT55 (Zhang et al., 2023. PMID: 36481789), SPATA20 (Wang et al., 2023. PMID: 36415156), TENT5D (Zhang et al., 2024. PMID: 38228861), CFAP52 (Jin et al., 2023. PMID: 38126872), CEP70 (Ruan et al., 2023. PMID: 36967801), PRSS55 (Liu et al., 2022. PMID: 35821214), as well as other unreported variants were also identified.
Reviewer #3 (Public Review):
Summary:
(1) To further explore the genetic basis of asthenoteratozoospermia, the authors performed whole-exome sequencing analyses among infertile males affected by asthenoteratozoospermia. Four unrelated Han Chinese patients were found to carry biallelic variations of DNAH3, a gene encoding IDA-associated protein.
(2) To verify the function of IDA associated protein DNAH3, the authors generated a Dnah3-KO mouse model and revealed that the loss of DNAH3 leads to severe male infertility as a result of the severe reduction in sperm movement with the abnormal IDA and mitochondrion structures.
(3) Mechanically, they confirmed decreased expression of IDA-associated proteins (including DNAH1, DNAH6 and DNALI1) in the spermatozoa from patients with DNAH3 mutations and Dnah3-KO male mice.
(4) Then, they also found that male infertility caused by DNAH3 deficiency could be rescued by intracytoplasmic sperm injection (ICSI) treatment in humans and mice.
Strengths:
(1) In addition to existing research, the authors provided novel variants of DNAH3 as important factors leading to asthenoteratozoospermia. This further expands the spectrum of pathogenic variants in asthenoteratozoospermia.
(2) By mechanistic studies, they found that DNAH3 deficiency led to decreased expression of IDA-associated proteins, which may be used to explain the disruption of sperm motility and reduced fertility caused by DNAH3 deficiency.
(3) Then, successful ICSI outcomes were observed in patients with DNAH3 mutations and Dnah3 KO mice, which will provide an important reference for genetic counselling and clinical treatment of male infertility.
We are very grateful for the reviewer's careful comments.
Recommendations for the authors:
Reviewer #1 (Recommendations for The Authors):
I have carefully read the revised versions of this manuscript, and I would like to thank the authors for addressing all my previous concerns.
I have no additional comments or suggestions.
We thank the reviewer for reviewing our revised manuscript.
Reviewer #2 (Recommendations for The Authors):
(1) Statistical analyses should be provided alongside the quantification (Fig S1B, S7C).
According to the suggestions of the reviewer, we have added statistical analyses of the corresponding quantification in the legends of Figure S1 and Figure S7.
(2) The numbers of sperms counted in Fig S1A should be listed.
In response to reviewer's valuable suggestions. We have listed the corresponding ratio of different morphological defects in sperm tail of the patients in Figure S1A.
(3) Due to the high similarities in experimental design, data and conclusions between the current study and previously published work by Meng et al. (2024), as well as the very similar titles of the two studies, it is crucial to emphasize the differences in the Discussion section.
Many thanks for reviewer's kind suggestions for our revised manuscript.
Employing whole-exome sequencing (WES) on infertile men to identify candidate variants, followed by in-silico and functional analysis of these variants, and generating mouse models using CRISPR-Cas9 technology, has proven to be an efficient and widely used approach for uncovering the causative genes of male infertility associated with sperm defects. Both our study and the recent work by Meng et al. utilized this approach to verify whether DNAH3 mutations are a cause of asthenoteratozoospermia. Additionally, we have also updated the title of our study to: 'DNAH3 deficiency causes flagellar inner dynein arm loss and male infertility in humans and mice'.
Meng et al. reported DNAH3 mutations in asthenoteratozoospermia affected patients, revealing multiple morphological defects in sperm tail. Moreover, ultrastructural abnormalities of the flagellar axoneme in the patients were evident in these patients, characterized by a disrupted '9+2' arrangement and the notable absence of IDAs. Additionally, they generated Dnah3 KO mice, which were infertile and exhibited moderate morphological abnormalities. While the '9+2' microtubule arrangement in the flagella of their Dnah3 KO mice remained intact, the IDAs on the microtubules were partially absent. In our study, we observed similar phenotypic differences between DNAH3-deficient patients and Dnah3 KO mice. Both studies suggest that DNAH3 plays a crucial role in human and mouse male reproduction.
However, there are notable differences between the two studies. Firstly, the phenotypes of Dnah3 KO mice showed slight differences. Meng et al. generated two Dnah3 KO mouse models (KO1 and KO2), and both of which exhibited significantly higher sperm motility and progressive motility than in our study, where nearly all sperm were completely immobile. Furthermore, their Dnah3 KO2 mice even displayed motility comparable to WT mice and retained partial fertility. We speculate that these differences may be attributed to variations in mouse genetic background or the presence of a truncated DNAH3 protein resulting from specific knockout strategies. Secondly, we conducted additional research and uncovered novel findings. We revealed that male infertility caused by DNAH3 mutations follows an autosomal recessive inheritance pattern, as confirmed through Sanger sequencing of the patients' parents. We also discovered the dynamic expression and localization of DNAH3 during spermatogenesis in humans and mice through immunofluorescent staining. We further found that DNAH3 deficiency had no impact on ciliary development in the oviduct or on oogenesis in mice, resulting in normal female fertility. Moreover, in the absence of DNAH3 in both humans and mice, the expression of IDA-associated proteins, including DNAH1, DNAH6 and DNALI1, was decreased, while the expression of ODA-associated proteins remained unaffected, indicating that DNAH3 is involved in sperm axonemal development, specifically through its role in the assembly of IDAs. Collectively, our study corroborates the findings of Meng et al., and provides additional unique insights, comprehensively elucidating the critical role of DNAH3 in human and mouse spermatogenesis.
We have added these discussions in line 275 to line 306.
Reviewer #3 (Recommendations for The Authors):
I have no more recommendations for the authors.
We thank the reviewer for reviewing our revised manuscript.
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eLife Assessment
This study, which proposes a new role of ATG6 in plant immune response, makes a valuable contribution to our understanding of plant immunity. The results suggest a direct interaction between ATG6 and NPR1, a salicylic acid receptor protein, and they will be of interest to scientists studying the regulation of plant immunity. The data presented are convincing, although the discrepancies between data from fluorescence microscopy and protein blots, particularly in the interpretation of ATG6-mCherry fusion proteins. Addressing these inconsistencies would enhance the study's overall impact.
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Reviewer #1 (Public Review):
The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.
The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.
Comments on latest version:
The authors have already addressed all my comments. I have no further issues with the manuscript.
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Reviewer #2 (Public Review):
The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.
Comments on latest version:
The term "invasion" has to be replaced with infection, as it doesn't have much meaning to this particular study. I already explained this point in the first review, but authors did not address it throughout the manuscript.
In fig. 1e there's no statistical analysis. How can one show measurements from multiple samples without statistical analysis? All the data points have to be shown in the graph and statistics performed. In the arg6-npr1 and snrk-npr1 pairs no nuclear marker is included. How can one know where the nucleus is, particularly in such poor quality low res. images? The nucleus marker has to be included in this analysis and shown. This is an important aspect of the study as nuclear localization of ATG6 is proposed to be essential for its new function. Co-localization provided in the fig. S2 cannot complement this analysis, particularly since no cytoplasmic fraction is present for NPR1-GFP in fig. S2.
In the alignment in fig 2c, it is not explained what are the species the atg6 is taken from. The predicted NLS has to be shown in the context of either the entire protein sequence alignment or at least individual domain alignment with the indication of conserved residues (consensus). They have to include more species in the analysis, instead of including 3 proteins from a single species. Also, the predicted NLS in atg6 doesn't really have the classical type architecture, which might be an indication that it is a weak NLS, consistent with the fact that the protein has significant cytoplasmic accumulation. They also need to provide the NLS prediction cut-off score, as this parameter is a measure of NLS strength.
Line 150: the NLS sequence "FLKEKKKKK" is a wrong sequence.
In fig. 3d no explanation for the error bars is included, and what type of statistical analysis is performed is not explained.
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Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
Reviewer #1 (Public Review):
The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.<br /> The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.
The authors have addressed most of my previous concerns.
Thank you so much for acknowledging our research. It is incredibly rewarding to see our work recognized. We hope that our findings will inspire new perspectives and foster further exploration in this area.
Reviewer #2 (Public Review):
The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.
Comments on revised version:
The authors demonstrate the correlation between overexertion of atg6 and higher stability and activity of npr1. They claim a novel activity of atg6 in the nucleus.
Overall, the experimental scope of the study is solid, however, the over-interpretation of the results substantially reduces the significance and value of this study for the target plant immunity readership.
Thank you very much for you constructive and insightful comments, as well as for acknowledging the experimental scope of this study. In addition, we have made every effort to address the over-interpretation of the results, as per your comments, ensuring they are more accurate and concise. In the revised version, the modified content has been highlighted in blue to clearly indicate the changes made.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
The authors have addressed most of my concerns. I have no further comments.
Thank you so much for acknowledging our research. It is incredibly rewarding to see our work recognized. We hope that our findings will inspire new perspectives and foster further exploration in this area.
Reviewer #2 (Recommendations For The Authors):
As I previously commented, in fig. 2a and c, the discrepancy between levels of atg6-mcherry in microscope image vs WB has to be explained. The explanation provided by the authors is incomplete and may mislead. The most likely reason for the difference is that the fluorescence signal in fig. 2a is predominantly from free mCherry, rather than the atg6-mcherry fusion. This has to be included in the main text to avoid misleading the reader.
Thank you very much for you constructive and insightful comments, in response to your comments, we have incorporated the necessary explanations into the revised manuscript (lines 160-164).
In fig. 1B, the PD fraction has to show the size range of free GST. Also, please use "anti" to indicate that these are immunoblots,.
Thank you for pointing this out. In the revised manuscript, we identified the range of free GST and used "anti:GST and anti:His" to indicate that these are immunoblots.
In fig 1C, the WB has to show the free GFP band in the input and IP fractions together with NPR1, rather than in separate blots.
Thank you for bringing this to our attention. Fig. 1c has been replaced, and the updated image now shows the free GFP band in the input and IP fractions together with NPR1-GFP.
In fig. 1d, the bifc signal has to be quantified from multiple images across the biological repeats. Also, there's no significance in showing the chlorophyll autofluorescence. What is the purpose of this? They need to use a nuclear marker instead.
Thank you for your suggestion. Based on your input, we utilized ImageJ software to quantify the YFP fluorescence signal. A total of n = 15 independent images were analyzed, and the corresponding results have been added to Figure 1e. Monitoring chlorophyll autofluorescence serves as a useful background signal, aiding in the distinction between the fluorescence signal of the target protein and background noise. This approach helps reduce potential signal overlap or interference during the experiment, thereby enhancing the reliability of the results.
Please provide a sequence alignment with multiple ATGs to show the conservation of the presumed bipartite NLS. This information has to be included in the main data.
Thank you very much for your constructive and insightful comments. We analyzed the putative nuclear localization signal (NLS) in the ATG6 protein sequence using the online INSP (Identification of Nuclear Signal Peptide) prediction software (http://www.csbio.sjtu.edu.cn/bioinf/INSP/). The prediction results indicated the presence of a potential nuclear localization sequence "FLKEKKKKK" within the ATG6 protein, spanning from the 217th to the 223rd amino acid. Additionally, we utilized INSP to investigate the nuclear localization sequences of various ATG proteins (TaATG6a [1], TaATG6b [1], TaATG6c [1], SlATG8h [2]) that have been previously reported to localize in the nucleus. This analysis revealed a relatively conserved NLS sequence motif: "E/K-K/E-K-K-L/K-K" in these ATG proteins. In line with your suggestion, the results of this sequence comparison have been incorporated into the revised manuscript as Figure 2c. The revised manuscript includes a description of the corresponding results. (lines 146-156).
Fig. 3d and f, how many blots are used for this quantification? Please include all the individual analyzed blots in the supplementary data. In addition, if you present such quantification with error bars, then statistical analysis is required.
Thank you for pointing this out. In Figure 3d, three independent blots were utilized for this quantification. In Figure 3f, two independent blots were used. The individual analyzed blots have been included in the supplementary Figure 7. We also conducted a statistical analysis as shown in Fig 3d and f, with a detailed description included in the legend section (lines 858 and 861).
In fig. 4, please indicate what is the normalizing gene. Also, what are the error bars?
Thank you for pointing this out. In Fig.4, values are means ± SD (n = 3 biological replicates). The AtActin gene was used as the internal control. We have included a detailed description in the figure notes
In fig. 4b the labeling is missing.
Thank you for bringing this to our attention. We have included the labeling for Fig. 4 in the revised manuscript.
Lines 236-239: this statement contradicts the data in fig. 5b: the levels of NPR1-GFP are actually reduced in the presence of atg6 at 24h. So, this result has to be described more accurately by stating that the increase is transient, and it is evident more at 8h, but not at 20-24h.
Thank you very much for you constructive and insightful comments. We have revised the description of this section to provide a more accurate account of the results (lines 253-258).
Reference
(1) Yue J, Sun H, Zhang W, et al. Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity. BMC Plant Biol. 2015;15:95.
(2) Li F, Zhang M, Zhang C, et al. Nuclear autophagy degrades a geminivirus nuclear protein to restrict viral infection in solanaceous plants. New Phytol. 2020;225:1746-1761.
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Our object is to study the ways in which peoplehave tried to develop a scientific approach tothe investigation of human social behaviour. Butwe cannot begin by definitively stating what thismeans. As we shall see, the history of socialscience shows a great variety of approaches,and we shall have to note that there are manydifficult philosophical problems here that are asyet unresolved
social science and philosophical
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not only in the School of Law, but also indepartments of Criminology, or ForensicStudies, Sociology, Economics, Philosophy,Political Science, and Psychology, some of whichare classified as social sciences and some not.He would find that in some universities Historyis classified as a social science and in others it isin another division, usually called ‘Humanities’.
social sciences and humantities have something in common
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eLife Assessment
This important work substantially advances our understanding of nocturnal animal navigation and the ways that animals use polarized light. The evidence supporting the conclusions is convincing, with elegant behavioural experiments in actively navigating ants. The work will be of interest to biologists working on animal navigation or sensory ecology.
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Reviewer #1 (Public review):
Freas et al. investigated if the exceedingly dim polarization pattern produced by the moon can be used by animal to guide a genuine navigational task. The sun and moon are celestial beacons for directional information, but they can be obscured by clouds, canopy, or the horizon. However, even when hidden from view, these celestial bodies provide directional information through the polarized light patterns in the sky. While the sun's polarization pattern is famously used by many animals for compass orientation, until now it has never been shown that the extremely dim polarization pattern of the moon can be used for navigation. To test this, Freas et al. studied nocturnal bull ants, by placing a linear polarizer in the homing path on a freely navigating ant 45 degrees shifted to the moon's natural polarization pattern. They recorded the homing direction of an ant before entering the polarizer, under the polarizer, and again after leaving the area covered by the polarizer. The results very clearly show, that ants walking under the linear polarizer change their homing direction by about 45 degrees in comparison to the homing direction under the natural polarization pattern and change it back after leaving the area covered by the polarizer again. These results can be repeated throughout the lunar month, showing that bull ants can use the moon's polarization pattern even under crescent moon conditions. Finally, the authors show, that the degree in which the ants change their homing direction is dependent on the length of their home vector, just as it is for the solar polarization pattern.
The behavioral experiments are very well designed, and the statistical analyses are appropriate for the data presented. The authors' conclusions are nicely supported by the data and clearly show nocturnal bull ants use the dim polarization pattern of the moon for homing, in the same way many animals use the sun's polarization pattern during the day. This is the first proof of the use of the lunar polarization pattern in any animal.
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Reviewer #2 (Public review):
Summary:
The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).
Strengths:
The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.
Weaknesses:
The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be re-worked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.
Impact:
The authors have discovered that nocturnal bull ants, while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.
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Reviewer #3 (Public review):
Summary:
This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.
Strengths:
The study was conducted carefully and is clearly explained here.
Weaknesses:
The revised manuscript is much improved.
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Freas et al. investigated if the exceedingly dim polarization pattern produced by the moon can be used by animals to guide a genuine navigational task. The sun and moon have long been celestial beacons for directional information, but they can be obscured by clouds, canopy, or the horizon. However, even when hidden from view, these celestial bodies provide directional information through the polarized light patterns in the sky. While the sun's polarization pattern is famously used by many animals for compass orientation, until now it has never been shown that the extremely dim polarization pattern of the moon can be used for navigation. To test this, Freas et al. studied nocturnal bull ants, by placing a linear polarizer in the homing path on freely navigating ants 45 degrees shifted to the moon's natural polarization pattern. They recorded the homing direction of an ant before entering the polarizer, under the polarizer, and again after leaving the area covered by the polarizer. The results very clearly show, that ants walking under the linear polarizer change their homing direction by about 45 degrees in comparison to the homing direction under the natural polarization pattern and change it back after leaving the area covered by the polarizer again. These results can be repeated throughout the lunar month, showing that bull ants can use the moon's polarization pattern even under crescent moon conditions. Finally, the authors show, that the degree in which the ants change their homing direction is dependent on the length of their home vector, just as it is for the solar polarization pattern.
The behavioral experiments are very well designed, and the statistical analyses are appropriate for the data presented. The authors' conclusions are nicely supported by the data and clearly show that nocturnal bull ants use the dim polarization pattern of the moon for homing, in the same way many animals use the sun's polarization pattern during the day. This is the first proof of the use of the lunar polarization pattern in any animal.
Reviewer #2 (Public Review):
Summary:
The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).
Strengths:
The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.
Weaknesses:
The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be reworked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.
Rewrote these sections and added figure panel to Figure 6.
Impact:
The authors have discovered that nocturnal bull ants while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this is the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.
Reviewer #3 (Public Review):
Summary:
This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.
Strengths:
The study was conducted carefully and is clearly explained here.
Weaknesses:
I have only a few comments and suggestions, that I hope will make the manuscript clearer and easier to understand.
Time compensation or periodic snapshots
In the introduction, the authors compare their discovery with that in dung beetles, which have only been observed to use lunar skylight to hold their course, not to travel to a specific location as the ants must. It is not entirely clear from the discussion whether the authors are suggesting that the ants navigate home by using a time-compensated lunar compass, or that they update their polarization compass with reference to other cues as the pattern of lunar skylight gradually shifts over the course of the night - though in the discussion they appear to lean towards the latter without addressing the former. Any clues in this direction might help us understand how ants adapted to navigate using solar skylight polarization might adapt use to lunar skylight polarization and account for its different schedule. I would guess that the waxing and waning moon data can be interpreted to this effect.
Added a paragraph discussing this distinction in mechanisms and the limits of the current data set in untangling them. An interesting topic for a follow up to be sure.
Effects of moon fullness and phase on precision
As well as the noted effect on shift magnitudes, the distributions of exit headings and reorientations also appear to differ in their precision (i.e., mean vector length) across moon phases, with somewhat shorter vectors for smaller fractions of the moon illuminated. Although these distributions are a composite of the two distributions of angles subtracted from one another to obtain these turn angles, the precision of the resulting distribution should be proportional to the original distributions. It would be interesting to know whether these differences result from poorer overall orientation precision, or more variability in reorientation, on quarter moon and crescent moon nights, and to what extent this might be attributed to sky brightness or degree of polarization.
See below for response to this and the next reviewer comment
N.B. The Watson-Williams tests for difference in mean angle are also sensitive to differences in sample variance. This can be ruled out with another variety of the test, also proposed by Watson and Williams, to check for unequal variances, for which the F statistic is = (n2-1)*(n1-R1) / (n1-1)*(n2-R2) or its inverse, whichever is >1.
We have looked at the amount of variance from the mean heading direction in terms of both the shifts and the reorientations and found no significant difference in variance between all relevant conditions. It is possible (and probably likely) that with a higher n we might find these differences but with the current data set we cannot make statistical statements regarding degradations in navigational precision.
As an additional analysis to address the Watson-Williams test‘s sensitivity to changes in variance, we have added var test comparisons for each of the comparisons, which is a well-established test to compare variance changes. None of these were significantly different, suggesting the observed differences in the WW tests are due to changes in the mean vector and not the distribution. We have added this test to the text.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
I have only very few minor suggestions to improve the manuscript:
(1) While I fully agree with the authors that their study, to the best of my knowledge, provides the first proof (in any animal) of the use of the moon's polarization pattern, the many repetitions of this fact disturb the flow of the text and could be cut at several instances.
Yes, it is indeed repeated to an annoying degree.
We have removed these beyond bookending mentions (Abstract and Discussion).
(2) In my opinion, the authors did not change the "ambient polarization pattern" when using the linear polarization filter (e.g., l. 55, 170, 177 ...). The linear polarizer presents an artificial polarization pattern with a much higher degree of polarization in comparison to the ambient polarization pattern. I would suggest re-phrasing this, to emphasize the artificial nature of the polarization pattern under the polarizer.
We have made these suggested changes throughout the text to clarify. We no longer say the ambient pattern was
(3) Line 377: I do not see the link between the sentence and Figure 7
Changed where in the discussion we refer to Figure 7.
(4) Figure 7 upper part: In my opinion, the upper part of Figure 7 does not add any additional value to the illustration of the data as compared to Figure 5 and could be cut.
We thought it might be easier for some reader to see the shifts as a dial representation with the shift magnitude converted to 0-100% rather than the shifts in Figure 5. This makes it somewhat like a graphical abstract summarising the whole study.
I agree that Figure 5 tells the same story but a reader that has little background in directional stats might find figure 7 more intuitive. This was the intent at least.
If it becomes a sticking point, then we can remove the upper portion.
Reviewer #2 (Recommendations For The Authors):
Minor corrections and queries
Line 117: THE majority
Corrected
Lines 129-130: Do you have a reference to support this statement? I am unaware of experiments that show that homing ants count their steps, but I could have missed it.
We have added the references that unpack the ant pedometer.
Line 140: remove "the" in this line.
Removed
Line 170: We need more details here about the spectral transmission properties of the polariser (and indeed which brand of filter, etc.). For instance, does it allow the transmission of UV light?
Added
Line 239: "...tested identicALLY to ...."
Corrected
Lines 242-258 (Vector testing): I must admit I found the description of these experiments very difficult to follow. I read this section several times and felt no wiser as a result. I think some thought needs to be given to better introduce the reader to the rationale behind the experiment (e.g., start by expanding lines 243-246, and maybe add a methods figure that shows the different experimental procedures).
I have rewritten this section of the methods to clearly state the experiment rational and to be clearer as to the methodology.
Also added a methods panel to Figure 6.
Line 247: "reoriented only halfway". What does this mean? Do you mean with half the expected angle?
Yes, this is a bit unclear. We have altered for clarity:
‘only altered their headings by about half of the 45° e-vector shift (25.2°± 3.7°), despite being tested on near-full-moon nights.’
Results section (in general): In Figure 1 (which is a very nice figure!) you go to all the trouble of defining b degrees (exit headings) and c degrees (reorientation headings), which are very intuitive for interpreting the results, and then you totally abandon these convenient angles in favour of an amorphous Greek symbol Phi (Figs. 2-6) to describe BOTH exit and reorientation headings. Why?? It becomes even more confusing when headings described by Phi can be typically greater than 300 degrees in the figures, but they are never even close to this in the text (where you seem to have gone back to using the b degrees and c degrees angles, without explicitly saying so). Personally, I think the b degrees and c degrees angles are more intuitive (and should be used in both the text and the figures), but if you do insist on using Phi then you should use it consistently in both the text and the figures.
Replaced Phi with b° and c° for both figures and in the text.
Finally, for reorientation angles in Figure 4A, you say that the angle is 16.5 degrees. This angle should have been 143.5 degrees to be consistent with other figures.
Yes, the reorientation was erroneously copied from the shift data (it is identical in both the +45 shift and reorientation for Figure 4A). This has now been corrected
Line 280, and many other lines: Wherever you refer to two panels of the same figure, they should be written as (say) Figure 2A, B not Figure 2AB.
Changed as requested throughout the text.
Line 295 (Waxing lunar phases): For these experiments, which nest are you using? 1 or 2?
We have added that this is nest 1.
Figure 3B: The title of this panel should be "Waxing Crescent Moon" I think.
Ah yes, this is incorrect in the original submission. I have fixed this.
Lines 312-313: Here it sounds as though the ants went right back to the full +/- 45 degrees orientations when they clearly didn't (it was -26.6 degrees and 189.9 degrees). Maybe tone the language down a bit here.
Changed this to make clear the orientation shift is only ‘towards’ the ambient lunar e-vector.
Line 327: Insert "see" before "Figure 5"
Added
Line 329: See comment for Line 295.
We have added that this is nest 1.
Lines 357-373 (Vector testing): Again, because of the somewhat confusing methods section describing these experiments, these results were hard to follow, both here and in the Discussion. I don't really understand what you have shown here. Re-think how you present this (and maybe re-working the Methods will be half the battle won).
I have rewritten these sections to try to make clear these are ant tested with differences in vector length 6m vs. 2m, tested at the same location. Hopefully this is much clearer, but I think if these portions remain a bit confusing that a full rename of the conditions is in order. Something like long vector and short vector would help but comes with the problem of not truly describing what the purpose of the test is which is to control for location, thus the current condition names. As it stands, I hope the new clarifications adequately describe the reasoning while keeping the condition names. Of course, I am happy to make more changes here as making this clear to readers is important for driving home that the path integrator is in play.
See current change to results as an example: ‘Both forgers with a long ~6m remaining vector (Halfway Release), or a short ~2m remaining vector (Halfway Collection & Release), tested at the same location_,_ exhibited significant shifts to the right of initial headings when the e-vector was rotated clockwise +45°.’
Line 361: I think this should be 16.8 not 6.8
Yes, you are correct. Fixed in text (16.8).
Line 365: I think this should be -12.7 not 12.7
Yes, you are correct. Fixed in text (–12.7).
Line 408: "morning twilight". Should this be "morning solar twilight"? Plus "M midas" should be "M. midas"
Added and fixed respectively.
Line 440. "location" is spelt wrong.
Fixed spelling.
Line 444: "...WITH longer accumulated vectors, ..."
Added ‘with’ to sentence.
Line 447: Remove "that just as"
Removed.
Line 448: "Moonlight polarised light" should be "Polarised moonlight"
Corrected.
Lines 450-453: This sentence makes little sense scientifically or grammatically. A "limiting factor" can't be "accomplished". Please rephrase and explain in more detail.
This sentence has been rephrased:
‘The limiting factors to lunar cue use for navigation would instead be the ant’s detection threshold to either absolute light intensity, polarization sensitivity and spectral sensitivity. Moonlight is less UV rich compared to direct sunlight and the spectrum changes across the lunar cycle (Palmer and Johnsen 2015).’
Line 474: Re-write as "... due to the incorporation of the celestial compass into the path integrator..."
Added.
Reviewer #3 (Recommendations For The Authors):
Minor comments
Line 84 I am not sure that we can infer attentional processes in orientation to lunar skylight, at least it has not yet been investigated.
Yes, this is a good point. We have changed ‘attend’ to ‘use’.
Line 90 This description of polarized light is a little vague; what is meant by the phrase "waves which occur along a single plane"? (What about the magnetic component? These waves can be redirected, are they then still polarized? Circular polarization?). I would recommend looking at how polarized light is described in textbooks on optics.
We have rewritten the polarised light section to be clearer using optics and light physics for background.
Line 92 The phrase "e-vector" has not been described or introduced up to this point.
We now introduce e-vector and define it.
‘Polarised light comprises light waves which occur along a single plane and are produced as a by-product of light passing through the upper atmosphere (Horváth & Varjú 2004; Horváth et al., 2014). The scattering of this light creates an e-vector pattern in the sky, which is arranged in concentric circles around the sun or moon's position with the maximum degree of polarisation located 90° from the source. Hence when the sun/moon is near the horizon, the pattern of polarised skylight is particularly simple with uniform direction of polarisation approximately parallel to the north-south axes (Dacke et al., 1999, 2003; Reid et al. 2011; Zeil et al., 2014).’
Happy to make further changes as well.
Line 107 Diurnal dung beetles can also orient to lunar skylight if roused at night (Smolka et al., 2016), provided the sky is bright enough. Perhaps diurnal ants might do the same?
Added the diurnal dung beetles mention as well as the reference.
Also, a very good suggestion using diurnal bull ants.
Line 146 Instead of lunar calendar the authors appear to mean "lunar cycle".
Changed
Line 165 In Figure 1B, it looks like visual access to the sky was only partly "unobstructed". Indeed foliage covers as least part of the sky right up to the zenith.
We have added that the sky is partially obstructed.
Line 179 This could also presumably be checked with a camera?
For this testing we tried to keep equipment to a minimum for a single researcher walking to and from the field site given the lack of public transport between 1 and 4am. But yes, for future work a camera based confirmation system would be easier.
Line 243 The abbreviation "PI" has not been described or introduced up to this point.
Changes to ‘path integration derived vector lengths….’
Line 267 The method for comparing the leftwards and rightwards shifts should be described in full here (presumably one set of shifts was mirrored onto the other?).
We have added the below description to indicate the full description of the mirroring done to counterclockwise shifts.
‘To assess shift magnitude between −45° and +45° foragers within conditions, we calculated the mirror of shift in each −45° condition, allowing shift magnitude comparisons within each condition. Mirroring the −45° conditions was calculated by mirroring each shift across the 0° to 180° plane and was then compared to the corresponding unaltered +45 condition.’
Discussion Might the brightness and spectrum of lunar skylight also play a role here?
We have added a section to the discussion to mention the aspects of moonlight which may be important to these animals, including the spectrum, brightness and polarisation intensity.
Line 451 The sensitivity threshold to absolute light intensity would not be the only limiting factor here. Polarization sensitivity and spectral sensitivity may also play a role (moonlight is less UV rich than sunlight and the spectrum of twilight changes across the lunar cycle: Palmer & Johnsen, 2015).
Added this clarification.
Line 478 Instead of the "masculine ordinal" symbol used (U+006F) here a degree symbol (U+00B0) should be used.
Ah thank you, we have replaced this everywhere in the text.
Line 485 It should be possible to calculate the misalignment between polarization pattern before and after this interruption of celestial cues. Does the magnitude of this misalignment help predict the size of the reorientation?
Reorientations are highly correlated with the shift size under the filter, which makes sense as larger shifts mean that foragers need to turn back more to reorient to both the ambient pattern and to return to their visual route. Reorientation sizes do not show a consistent reduction compared to under-the-filter shifts when the lunar phase is low and is potentially harder to detect.
I have reworked this line in the text as I do not think there is much evidence for misalignment and it might be more precise to say that overnight periods where the moon is not visible may adversely impact the path integrator estimate, though it is currently unknown the full impact of this celestial cue gap of if other cues might also play a role.
Line 642 "from their" should be "relative to"
Changed as requested
Figure 1B Some mention should be made of the differences in vegetation density.
Added a sentence to the figure caption discussing the differences in both vegetation along the horizon and canopy cover.
Figures 2-6 A reference line at 0 degrees change might help the reader to assess the size of orientation changes visually. Confidence intervals around the mean orientation change would also help here.
We have now added circular grid lines and confidence intervals to the circular plots. These should help make the heading changes clear to readers.
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When postcards of cross-dressed people are included in a sexuality collection, isthere a presumption that cross-dressing can or should be treated as a sexual identity?Does the HSC imbue these postcards with specific evidentiary value, as objects thatare “document[ing] historical shifts in the social construction of sexuality,” whenthey may have more relevance for other historical phenomena?
definitional power of archives
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eLife Assessment
In this compelling study, the authors examine the interactions between stellate cells and PV+ interneurons in the medial entorhinal cortex. Huang et al. focus on the spatial distribution of synaptic inputs and demonstrate that closely located neuron pairs receive common inputs, suggesting a structured functional organization in the entorhinal cortex. Advanced dual whole-cell patch recordings further reveal patterns of postsynaptic activation, indicating intensive interactions within clusters of these neurons, with weaker interactions between clusters. These findings offer significant insights into the functional dynamics of the entorhinal cortex and the circuit mechanisms that shape grid cell activity. This study is important not only for the field of MEC and grid cells, but also for broader fields of continuous attractor networks and neural circuits.
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thewasteland.info thewasteland.info
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Elizabeth and Leicester Beating oars
The external forces that Eliot alludes to throughout the Fire Sermon take on a vivid image as he references Elizabeth l and Leicester and describes them “beating the oars.” This phrase appears to be an unconventional way of describing an act of rowing. On the surface, the use of “beating” emphasizes the exertion and physical degradation as a result of an intense physical activity and further hints at the external forces that shape and, thus, can be blamed for degrading individual relationships. At the end of this section, however, Eliot appears to adopt a different central notion, where external factors are not the ones to blame for the degrading relationships in a modern society; instead it is the internal lack of commitment that breaks them down from within.
Interestingly, a related phrase, “to put an oar in someone else’s boat,” emerged during the Tudor period (of which Elizabeth was the last monarch), meaning “to involve yourself in a situation when other people do not want you to” (Cambridge Dictionary). This phrase once again seemingly points to external interference in personal matters and further reinforces the idea that external forces play a role in guiding individuals’ lives. This phrase, however, is a descendant of an innuendo related to Henry Vll, which references his wife’s affair. In this case, directly the affair itslef and a lack of emotional commitment becomes this unwelcome cause that interferes within their relationship. With this context, the affair itself—and the lack of emotional commitment within the relationship—becomes the unwelcome force that disrupts and degrades the relationship from within, rather than being an external force imposed upon it.
Elizabeth and Leicester “beating the oars” seems to be a play on this phrase. Eliot’s invocation of a relationship between Elizabeth and Leicester, who were never married but maintained an intimate connection, and a reference to a Tudor idiom deepen his critique of modern relationships. The act of “beating the oars” implies a sense of physical and emotional exertion, leaving the individuals involved devastated and symbolizing how uncommitted intimate relationships often lead to emotional depletion rather than fulfillment.
Eliot exemplified similar notions in the previous accounts of intimate relationships marked by neglect and abuse. The line we previously discussed “Why do you never speak? Speak to me” describes the emotional disconnect that accompanies relationships devoid of commitment. This discontent parallels the passive endurance of sexual advances described in the previous scene, signifying that relationships centered solely on physical intimacy fail to provide any true connection. In such relationships, even physical connection becomes a degrading and, in a sense, disconnecting experience.
This degradation is further exemplified through the notion of rowing. The “southwest” wind, which blows into a northern direction, and their travel “down [the] stream” directly signify a cold and deteriorating nature of these purely physical relationships.
The Fire Sermon itself suggests an idea where human internal passions burn them from within. Unlike Buddha’s teachings, however, which propose the possibility of liberation from these primitive desires, Eliot once again presents a more pessimistic view. Humanity, in his portrayal, is incapable of freeing itself from these desires. Each repetition of “burning, burning, burning, burning” could potentially symbolize the loss of one of the senses—sight, smell, sound, and taste. This loss reflects humanity’s growing disconnection from the world, as people focus increasingly on physical touch as their only means of connection. The plea “Oh Lord thou pluckest me out” signifies the desire for liberation. This unsuccessful plea, however, shortens to “Oh Lord thou pluckest,” interrupted by the final “burning,” which represents the ultimate degradation: the loss of the fifth sense, touch - the last, even though hollow, chance of connection.
In these references, Eliot abandons the notion of external forces controlling individual relationships and proposes that it is an internal emptiness and a lack of genuine connection, suppressed by hollow physical intimacy, that degrades them from within.
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Author response:
The following is the authors’ response to the previous reviews.
eLife Assessment
This is a valuable study in the Jurkat T cell line that calls attention to phosphorylation of formin-like 1 β role and its role in polarization of CD63 positive extracellular vesicles (referred to as exosomes). The evidence presented in the Jurkat model is solid, but concerns have been raised about the statistical analysis and more details would be required to fully assess the significance of the results. For example, ANOVA is the method described, but it requires large amounts of normally distributed data in multiple groups and cannot be used to make pairwise comparisons within groups, which would require a post-hoc method (which is not discussed). In addition, the data showing forming-like 1 β in primary human T cells without and with a CAR are provided without quantification and don't investigate any of the novel claims, so doesn't address the relevance of Formin-like 1 β beyond the Jurkat model. Nonetheless, the consistent trends in the body of the study provide solid support for the claims.
We acknowledge this general statement on statistics. Thus, we have now discussed and provided more details on the post-hoc method (Tukey), as a new Supplementary data S13 (p-values after applying tukey's method -post hoc- to the one-way anova for all the pairwise comparisons). Additionally, we have now provided quantitative data on the percentage of primary cells with and without CAR that show FMNL1 accumulations at the immune synapse (Suppl. Fig. S7). Regarding the data in primary human T cells, we have already changed the title of the manuscript to strictly adjust it to the main body of the data and our conclusions in the well-established Jurkat synapse model. We also want to emphasize that we have not pretended to extrapolate the relevance of our data regarding FMNL1 and exosomes beyond the Jurkat model. Thus, we have included some additional sentences and/or nuances in the Discussion to somewhat soften our statements in this regard (i.e. “…..provided that the FMNL1 effect on exosome secretion in Jurkat cells can be extended to primary T lymphocytes”) and to clarify this important point.
Reviewer 1:
(1) The main findings have been obtained in clones of Jurkat cells. They have not been confirmed in primary T cells. The only experiment performed in primary cells is shown in Figure S7 (primary human T lymphoblasts) for which only the distribution of FMNL1 is shown without quantification. No results presenting the effect of FMNL1 KO and expression of mutants in primary T cells are shown.
Referee is right regarding the extension of exosome secretion studies to primary human T lymphocytes. Unfortunately, it is well known that primary T lymphocytes are extremely difficult to transfect. Moreover, the expression of our large bi-cistronic large plasmids (>15 Kb) is very inefficient, coupled with the challenge of expressing large proteins, such as the 180 kDa YFP-FMNL1 chimeric variants. The convergence of all these undesirable factors synergistically hampers these studies and we have been unable to consistently achieve enough transfection efficiency to perform these experiments. However, the role of FMNL1 on MTOC/MVB polarization in Jurkat cells, confirmed in this manuscript, has been already extended to primary CD8+ T cell clones (DOI10.1016/j.immuni.2007.01.008). Given that exosome secretion requires
MTOC/MVB polarization both in Jurkat and primary T lymphoblasts (10.1038/cdd.2010.184, 10.3389/fimmu.2019.00851), this suggests FMNL1 may also control exosome secretion in primary T cells, although the formal demonstration will require further research.
A new sentence has been included in the Discussion to address this important point. Regarding the second request, we have quantified the images mentioned in Suppl. Fig. S7, and the percentages of fixed T cells showing FMNL1 accumulations at the immune synapse are included in the figure legend.
(2) Analysis in- depth of the defect in actin remodeling (quantification of the images, analysis of some key actors of actin remodeling) is still lacking. Only Factin is shown, no attempt to look more precisely at actors of actin remodeling has been done.
The referee is right. Since we have obtained new results on the role of FMNL1 on actin remodeling, we have focused on this formin, which is already a key actor in this process. In this context, we have previously shown that the formin Dia1, another major actor of actin remodeling in T lymphocytes along with FMNL1 (DOI10.1016/j.immuni.2007.01.008), does not undergo phosphorylation upon PKC activation (Suppl. Fig. 5 in https://doi.org/10.1080/20013078.2020.1759926). Since our aim was to unravel the PKC-mediated pathway controlling actin remodeling, we have ruled out more studies on Dia1. Therefore, we have included a new sentence to emphasize the specific role of FMNL1 phosphorylation, but not Dia1, in this regard. Nonetheless, future studies aimed to identifying new important players in this or related pathways could offer significant insights.
(3) The defect in the secretion of extracellular vesicles is still very preliminary. Examples of STED images given by the authors are nice, yet no quantification is performed.
The referee is right regarding this point and we acknowledge this comment. Accordingly, we have now quantified the STED images and provided numerical data on the percentages of cells exhibiting the observed phenotypes (see the figure legend for Fig. 10).
(4) Results shown in Figure S12 on the colocalization of proteins phosphorylated on Ser/Thr are still not convincing. It seems indeed that "phospho-PKC" is labeling more preferentially the CMAC positive cells (Raji) than the Jurkat T cells. It is thus particularly difficult to conclude on the colocalization and even more on the recruitment of phosphorylated-FMNL1 at the IS. Thus, these experiments are not conclusive and cannot be the basis even for their cautious conclusion: "Although all these data did not allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limit of confocal and STED microscopes, the results are compatible with the idea that both endogenous FMNL1 and YFP-FMNL1bWT are specifically phosphorylated at the cIS".
The referee may be correct regarding the detail of the "phospho-PKC" labeling. However, it cannot be overlooked that Raji cells also contain proteins that are or may be potential PKC substrates. As a matter of fact, Raji cells also express FMNL1. In addition, MHCII triggering in B cells induces PKC activation (https://doi.org/10.1002/eji.200323351). Regarding which cell type is preferentially labeled, this is a variable topic depending on the analyzed synapse.
It is true that there are likely several PKC substrates, both in Jurkat in Raji cells, but our point is that one of these substrates either colocalizes with FMNL1 or is FMNL1 itself. We do not claim at any point that FMNL1 is the only PKC substrate, neither in Jurkat or in Raji cells.
Apparently, the referee has either overlooked our results or we did not emphasize them sufficiently. Our results effectively validated the PKC substrate antibody, both on endogenous phospho-FMNL1 and phospho-YFPFMNL1β by WB (Fig. 3). Moreover, the phospho-PKC does not recognize
YFP-FMNL1β S1086A or S1086D variants (Fig. 3). Last, but not least, when FMNL1 is interfered in the Jurkat cell, the phospho-PKC does not colocalize with FMNL1, but it strongly colocalizes at the synapse with expressed YFPFMNL1βWT in the Jurkat cell (Fig. S11). Indeed YFP-FMNL1β belonged to the Jurkat cell. Taken together these results demonstrate: 1. the specificity of phospho-PKC antibody, 2. the phospho-PKC antibody certainly recognizes phosphorylated YFP-FMNL1β but not its non-phosphorylatable mutant variants, 3. the colocalization of phospho-PKC with anti-FMNL1 is specific. We have included some sentences to clarify these points and to avoid possible misunderstandings by potential readers. We acknowledge the referee for his/her clarifying point, and we firmly believe our mentioned cautious conclusion is strictly correct, although we have tuned it to consider the possibility that a different PKC substrate could be closely associated to FMNL1, producing the observed colocalization: “Although all these data do not yet allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limits of super resolution microscopy and the possibility that another PKC substrate may be associated to FMNL1 or very close to FMNL1, in a strictly S1086-dependent manner”.
To clear any doubt regarding which cell is labelled with phospho-PKC, we have changed the lower panels in Suppl. Fig. S12, and now is more evident that FMNL1 and phospho-PKC belong to the Jurkat cell.
The study would benefit from a more careful statistical analysis. The dot plots showing polarity are presented for one experiment. Yet, the distribution of the polarity is broad. Results of the 3 independent experiments should be shown and a statistical analysis performed on the independent experiments.
The referee is right and we have now included further post-hoc analyses data (Tukey) at Suppl. Fig S13. Tukey’s test values were included for all the dot plot figures. We have not included all the plots from 3 different experiments since the manuscript already contains 10+12 multi panel figures and is too large. However, we have stated in the figure legend that these independent experiments are representative of the data obtained from 3 independent experiments. Referee’s consideration regarding the broad distribution of polarity data is correct. We included in the first version of the manuscript a sentence in this regard, that it may have been overlooked: “Remarkably, one important feature of the IS consists of both the onset of the initial cell-cell contacts and the establishment of a mature, fully productive IS, are intrinsically stochastic, rapid and asynchronous processes (87, 88) (43). Thus, the score of the PI corresponding to the distance of MTOC/MVB with respect the IS (42) may be contaminated by background MTOC/MVB polarization, in great part due to the stochastic nature of IS formation (87)”.
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: They could easily outcompete otherreptiles, which were lethargic ectotherms.And by getting big quickly, they occupiedthe large-animal niches, and prevented thesmall, energy-hungry endothermic mammals from getting bigger thems
how the dino's outcompeted other animals
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: mesotherm
what the inbetween state is called
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He and his colleagues tackled the problemby examining the relationship between ananimal's growth rate—how fast it becomesa full-sized adult—and its resting metabolicrate (RMR), a measure of energy expenditure
How they went about solving the problem
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Thus, we concluded that the regulatory, immature phenotype of the CD103+ DC2 found in vivo during Hp infection might be result of TGFβ signalling and likely a result of direct immunomodulatory effect of the TGFβ mimic molecules secreted by Hp.
How similar are naïve DCs generated via in vitro stimulation by human TGF-beta compared to the CD103+ DCs generated by proximity to Hpb granulomas? Do you know whether the helminths are producing other factors, such as additional cytokine mimics, that further bias the phenotype of these DCs?
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Here, we identified that DCs derived in presence of TGFβ had a reduced ability to stimulate T cell proliferation, as measured by CTV
Did you test the effect of TGM on the ability of DC2 cells to stimulate T cell proliferation?
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TGFβ or purified TGM
Do you have any data describing how the specific activities of endogenous TGF-beta and Hpb TGM differ?
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To do this, we took advantage of the fact that at 7DPI, the granulomas approximately half of the size of the Peyer’s Patch and are visible by naked eye
It is certainly fortunate that Hpb infection produces these granulomas. In the case of infections that do not produce this phenotype, how might you have assessed the location-specific effect of helminth infection?
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RA production by both subsets of DC2 was significantly increased during Hpb infection, with this effect being most marked for the CD103- subset
I find it really interesting that Hpb is modulating the specific metabolite production of different subsets of DCs during infection. Do you have any data or hypotheses regarding how Hpb affects the downstream capabilities of specific subsets of DCs once they have differentiated?
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eLife Assessment
The research has the potential to be a valuable addition to the field, and the conclusions are solid, but there is a need for more reproducible data to address existing discrepancies and enhance its impact.
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Reviewer #1 (Public review):
Summary:
The authors aim to investigate the role of ORMDL3 in regulating Type 1 interferon (IFN) responses and its effect on tumor growth inhibition. The study focuses on the mechanisms involving the RIG-I pathway and USP10-mediated degradation and attempts to establish a link between ORMDL3 expression and the effectiveness of cancer therapy. The authors also explore the broader implications of ORMDL3 in immune signaling, particularly within the context of Type 1 IFN signaling and its therapeutic potential.
Strengths:
• The manuscript explores a novel aspect of cancer immunology by examining the relationship between ORMDL3 and Type 1 IFN signaling, potentially offering new therapeutic avenues.<br /> • A variety of experimental approaches are employed, including knockdown models, overexpression assays, and protein interaction analyses, to elucidate the role of ORMDL3 in modulating immune responses.<br /> • The findings suggest a potential mechanism by which ORMDL3 affects the tumor microenvironment and immune responses, which could have significant implications for understanding cancer progression and therapy.
Weaknesses:
• The study does not clearly establish the relationship between Type 1 IFN and cancer therapy, and more robust data are needed to support the claim that tumor growth inhibition occurs via Type 1 IFN upregulation following ORMDL3 knockdown.<br /> • There is ambiguity regarding whether ORMDL3 has a positive or negative role in the Type 1 IFN pathway, especially given conflicting findings in the literature that link higher ORMDL3 levels to increased Type 1 IFN expression.<br /> • The use of certain experimental models, such as HEK293T cells (which are not typical Type 1 IFN producers), raises concerns about the validity and generalizability of the results. Further clarity is needed regarding the rationale for using the same tag in overexpression experiments.<br /> • The manuscript contains several inconsistencies and lacks detailed explanations of critical areas, such as the mechanism by which ORMDL3 facilitates USP10 transfer to RIG-I despite no direct interaction between ORMDL3 and RIG-I.
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Reviewer #2 (Public review):
Summary:
The authors identified ORMDL3 as a negative regulator of the RLR pathway and anti-tumor immunity. Mechanistically, ORMDL3 interacts with MAVS and further promotes RIG-I for proteasome degradation. In addition, the deubiquitinating enzyme USP10 stabilizes RIG-I and ORMDL3 disturbs this process. Moreover, in subcutaneous syngeneic tumor models in C57BL/6 mice, they showed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8-positive T cells and IFN production in the tumor microenvironment (TME).
Strengths:
The paper has a clearly arranged structure and the English is easy to understand. It is well written. The results are clearly supporting the conclusion.
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Author response:
• The study does not clearly establish the relationship between Type 1 IFN and cancer therapy, and more robust data are needed to support the claim that tumor growth inhibition occurs via Type 1 IFN upregulation following ORMDL3 knockdown.
We thank the reviewer’s concern. In Figure 6 we detected the expression of IFNB1 and ISGs in MC38 and LLC tumor upon ORMDL3 knockdown. At the mean time, we also used IHC to explore the abundance of RIG-I and ORMDL3 in these tumors. In addition, in figure S5 we performed western blots to detect the expression of RIG-I with or without ORMDL3 knockdown. All these results support our hypothesis that that ORMDL3 is a negative regulator of interferon via modulating RIG-I abundance.
• There is ambiguity regarding whether ORMDL3 has a positive or negative role in the Type 1 IFN pathway, especially given conflicting findings in the literature that link higher ORMDL3 levels to increased Type 1 IFN expression.
We appreciate the reviewer’s concern. In our system and experiments, we validated that ORMDL3 is a negative regulator of interferon, although there is also literature that links higher ORMDL3 levels to increased type-I IFN response. ORMDL3 has been reported associated with rhinovirus-induced childhood asthma (Nature. 2007;448(7152):470-473; N Engl J Med. 2013 Apr 11;368(15):1398-407), and ORMDL3 level is positively associated with rhinovirus abundance (N Engl J Med. 2013 Apr 11;368(15):1398-407). There are reports indicating that ORMDL3 supports the replication of rhinovirus (for example, Am J Respir Cell Mol Biol. 2020 Jun;62(6):783-792). This phenomenon is consistent with our findings that higher ORMDL3 expression leads to lower interferon production, which facilitates viral replication. We believe that the different experimental conclusions obtained in these experiments are due to different experiment condition and different stimulation. In our research, we provided comprehensive studies at the molecular, cellular, and animal levels to support the conclusion that ORMDL3 is a negative regulator of type-I interferon.
• The use of certain experimental models, such as HEK293T cells (which are not typical Type 1 IFN producers), raises concerns about the validity and generalizability of the results. Further clarity is needed regarding the rationale for using the same tag in overexpression experiments.
We thank the reviewer’s suggestion. Besides HEK293T, in Figure 1C and 1D we also used A549 and BMDM to overexpress ORMDL3 and stimulate them with polyI:C or polyG:C, Our results showed that ORMDL3 especially inhibits RLR signaling. Additionally, in Figure 3H we found that the endogenous RIG-I expression decreased when we overexpressed ORMDL3 in BMDM. Regarding the issue of using different protein tags, we plan to use different tags to validate our results.
• The manuscript contains several inconsistencies and lacks detailed explanations of critical areas, such as the mechanism by which ORMDL3 facilitates USP10 transfer to RIG-I despite no direct interaction between ORMDL3 and RIG-I.
There are some ERMC (ER-mitochondria contact) proteins that mediate the interaction between ER and mitochondria. ORMDL3 locates in ER, and it has been reported to be associated with calcium transportation. At the meantime, the calcium transfer between ER and mitochondria plays an important role in protein synthesis. It is possible that some ERMC proteins mediate the interaction between ORMDL3 and MAVS. In addition, we also validated that ORMDL3 interacts with USP10 (Figure 5B). Although ORMDL3 and RIG-I do not interact directly, we generated a mechanistic model that ORMDL3 and MAVS recruit USP10 and RIG-I to ERMCS respectively, thus USP10 could form a complex with RIG-I (Figure 5C) and regulate the stability of RIG-I upon RNA sensing.
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eLife Assessment
The authors tested the hypothesis that age-dependent factors in human sera affect the core circadian clock or its outputs in cultured fibroblasts, and they provide compelling evidence that genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions, genes associated with oxidative phosphorylation and Alzheimer's Disease lose rhythmicity in the aged condition, while the expression of cycling genes associated with cholesterol biosynthesis increase in the cells entrained with old serum. Together, the findings suggest that yet to be identified age-dependent blood-borne factors affect circadian rhythms in the periphery. The paper provides fundamental insights and a possible explanation for previous observations showing that circadian gene expression in peripheral tissues tends to dampen or phase-shift with age.
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Reviewer #1 (Public review):
Aging is associated with a number of physiologic changes including perturbed circadian rhythms. However, mechanisms by which rhythms are altered remain unknown. Here authors tested the hypothesis that age-dependent factors in the sera affect the core clock or outputs of the core clock in cultured fibroblasts. They find that both sera from young and old donors are equally potent at driving robust ~24h oscillations in gene expression, and report the surprising finding that the cyclic transcriptome after stimulation by young or old sera differs markedly. In particular, genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions, while genes associated with oxidative phosphorylation and Alzheimer's Disease lose rhythmicity in the aged condition. Also, the expression of cycling genes associated with cholesterol biosynthesis increases in the cells entrained with old serum. Together, the findings suggest that age-dependent blood-borne factors, yet to be identified, affect circadian rhythms in the periphery. The most interesting aspect of the paper is that the data suggest that the same system (BJ-5TA), may significantly change its rhythmic transcriptome depending on how the cells are synchronized. While there is a succinct discussion point on this, it should be expanded and described whether there are parallels with previous works, as well as what would be possible mechanisms for such an effect.
Comments on revised version:
The authors have done a thorough revision of their manuscripts and provided convincing answers to all of my points. In particular, I applaud the authors for having added raw luminescence traces, and for providing Figure S5 on the amplitudes. Perhaps the authors could add a comment in the final text that the amplitudes are fairly low, 10^0.1 = 1.25 which means that the bulk of those genes has rhythms of at most 25%, which could reflect that the synchronization of the cells is partial.
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“My friend,” said Dupin, in a kind tone, “you are alarming yourself unnecessarily — you are indeed. We mean you no harm whatever. I pledge you the honor of a gentleman, and of a Frenchman, that we intend you no injury. I perfectly well know that you are innocent of the atrocities in the Rue Morgue.{z} It will not do, however, to deny that you are in some measure implicated in them. [page 564:] From what I have already said, you must know that I have had means of information about this matter — means of which you could never have dreamed. Now the thing stands thus. You have done nothing which you could have avoided — nothing, certainly, which renders you culpable.
it must be really odd to point out that, the sailor is kind of innocent. nnocent of any intentional wrongdoing. His only crime, if it can be called that, was failing to control his pet. The orangutan's actions were driven by its animal instincts, not human malice, which adds to the peculiarity of the case.
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“How was it possible,”
well, i got the same question. i think there was chances that the guessing might still go off. Dupin is kind of too certained to make the judgement. leading him to conclusions that may seem like leaps of intuition but are meant to appear logically sound.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
(1) The main hypothesis/conclusion is summarized in the abstract: "Our study presents an intriguing model of cilia length regulation via controlling IFT speed through the modulation of the size of the IFT complex." The data clearly document the remarkable correlation between IFT velocity and ciliary length in the different cells/tissues/organs analyzed. The experimental test of this idea, i.e., the knock-down of GFP-IFT88, further supports the conclusion but needs to be interpreted more carefully. While IFT particle size and train velocity were reduced in the IFT88 morphants, the number of IFT particles is even more decreased. Thus, the contributions of the reduction in train size and velocity to ciliary length are, in my opinion, not unambiguous. Also, the concept that larger trains move faster, likely because they dock more motors and/or better coordinating kinesin-2 and that faster IFT causes cilia to be longer, is to my knowledge, not further supported by observations in other systems (see below).
Thank you for your comments. We agree with the reviewer that the final section on IFT train size, velocity, and ciliary length regulation requires additional evidence. The purpose of the knockdown experiments was to investigate the potential relationship between IFT speed and IFT train size. We hypothesize that a deficiency in IFT88 proteins may disrupt the regular assembly of IFT particles, leading to the formation of shorter IFT trains. Indeed, we observed a shorter IFT particles and slight reduction in the transport speed of IFT particles in the morphants. Certainly, it would be more convincing to distinguish these IFT trains through ultrastructural analysis. However, with current techniques, performing such analysis on the zebrafish model will be very difficult due to the limited sample size. In the revised version, we have tempered the conclusions in these sections, as suggested by other reviewers as well.
(2) I think the manuscript would be strengthened if the IFT frequency would also be analyzed in the five types of cilia. This could be done based on the existing kymographs from the spinning disk videos. As mentioned above, transport frequency in addition to train size and velocity is an important part of estimating the total number of IFT particles, which bind the actual cargoes, entering/moving in cilia.
Thank you. We have analyzed the entry frequency of IFT in five types of cilia, both anterior and posterior. The analysis indicates that longer cilia also exhibit a higher frequency of fluorescent particles entering the cilia. These results are presented in Figure 3J.
(3) Here, the variation in IFT velocity in cilia of different lengths within one species is documented - the results document a remarkable correlation between IFT velocity and ciliary length. These data need to be compared to observations from the literature. For example, the velocity of IFT in the quite long (~ 100 um) olfactory cilia of mice is similar to that observed in the rather short cilia of fibroblasts (~0.6 um/s). In Chlamydomonas, IFT velocity is not different in long flagella mutants compared to controls. Probably data are also available for C. elegans or other systems. Discussing these data would provide a broader perspective on the applicability of the model outside of zebrafish.
Thank you for your suggestions. We believe the most significant novelty of our manuscript is the discovery that IFT velocities are closely related to cilia length in an in vivo model system. Our data suggest that longer cilia may require faster IFT transport to maintain their stable length, powered by larger IFT trains. We did observe substantial variability in IFT velocities across different studies. For example, anterograde IFT transport ranges from 0.2 µm/s in mouse olfactory neurons (Williams et al, 2014) to 0.8 µm/s in 293T cells (See et al, 2016) and 0.4 µm/s in IMCD-3 cells (Broekhuis et al, 2014). Even in NIH-3T3 cells, two studies report significant differences, despite using the same IFT reporters: 0.3 µm/s versus 0.9 µm/s (Kunova Bosakova et al, 2018; Luo et al, 2017). These findings suggest that cell types and culture conditions can influence IFT velocities in vitro, which may not accurately represent in vivo conditions. Interestingly, research on mouse olfactory neurons showed a strong correlation between anterograde and retrograde IFT velocities. Additionally, IFT velocity is closely related to the cell types within the olfactory neuron population, consistent with our results (Williams et al., 2014).
Reviewer #2 (Public Review):
Summary:
In this study, the authors study intraflagellar transport (IFT) in cilia of diverse organs in zebrafish. They elucidate that IFT88-GFP (an IFT-B core complex protein) can substitute for endogenous IFT88 in promoting ciliogenesis and use it as a reporter to visualize IFT dynamics in living zebrafish embryos. They observe striking differences in cilia lengths and velocity of IFT trains in different cilia types, with smaller cilia lengths correlating with lower IFT speed. They generate several mutants and show that disrupting the function of different kinesin-2 motors and BBSome or altering post-translational modifications of tubulin does not have a significant impact on IFT velocity. They however observe that when the amount of IFT88 is reduced it impacts the cilia length, IFT velocity as well as the number and size of IFT trains. They also show that the IFT train size is slightly smaller in one of the organs with shorter cilia (spinal cord). Based on their observations they propose that IFT velocity determines cilia length and go one step further to propose that IFT velocity is regulated by the size of IFT trains.
Strengths:
The main highlight of this study is the direct visualization of IFT dynamics in multiple organs of a living complex multi-cellular organism, zebrafish. The quality of the imaging is really good. Further, the authors have developed phenomenal resources to study IFT in zebrafish which would allow us to explore several mechanisms involved in IFT regulation in future studies. They make some interesting findings in mutants with disrupted function of kinesin-2, BBSome, and tubulin modifying enzymes which are interesting to compare with cilia studies in other model organisms. Also, their observation of a possible link between cilia length and IFT speed is potentially fascinating.
Weaknesses:
The manuscript as it stands, has several issues.
(1) The study does not provide a qualitative description of cilia organization in different cell types, the cilia length variation within the same organ, and IFT dynamics. The methodology is also described minimally and must be detailed with more care such that similar studies can be done in other laboratories.
Thank you for your comments. We found that cilia length is generally consistent within the same cell types we examined, including those in the pronephric duct, spinal cord, and epidermal cells. However, we observed variability in cilia length within ear crista cilia. Upon comparing IFT velocities, we found no differences among these cilia, further confirming our conclusion that IFT velocity is directly related to cell type rather than cilia length. These new results are presented in Figure S4 of the revised version.
We apologize for the lack of methodological details in the original manuscript. Following the reviewer's suggestion, we have added a detailed description of the methods used to generate the transgenic line and to perform IFT velocity analysis. These details are included in Figure S2 and are thoroughly described in the methods section of the revised manuscript.
(2) They provide remarkable new observations for all the mutants. However, discussion regarding what the findings imply and how these observations align (or contradict) with what has been observed in cilia studies in other organisms is incomprehensive.
Thank you for this suggestion. We initially submitted this paper as a report, which have word limits. We believe the main finding of our work is that IFT velocity is directly associated with cell type, with longer cilia requiring higher velocities to maintain their length. This association of IFT velocity with cell type has also been observed in mouse olfactory neurons(Williams et al., 2014). We have included a discussion of our findings, along with related data published in other organisms, in the revised version.
(3) The analysis of IFT velocities, the main parameter they compare between experiments, is not described at all. The IFT velocities appear variable in several kymographs (and movies) and are visually difficult to see in shorter cilia. It is unclear how they make sure that the velocity readout is robust. Perhaps, a more automated approach is necessary to obtain more precise velocity estimates.
Thank you for these comments. To measure the IFT velocities, we first used ImageJ software to generate a kymograph, where moving particles appear as oblique lines. The velocity of these particles can be calculated based on the slope of the lines (Zhou et al, 2001). In the initial version, most of the lines were drawn manually. To eliminate potential artifacts, we also used KymographDirect software to automatically trace the particle paths. The velocities obtained with this method were similar to those calculated manually. These new data are now shown in Figure S2 B-D. For shorter cilia, we only used particles with clear moving paths for our calculations. In the revised version, we have included a detailed description of the velocity analysis methods.
(4) They claim that IFT speeds are determined by the size of IFT trains, based on their observations in samples with a reduced amount of IFT88. If this was indeed the case, the velocity of a brighter IFT train (larger train) would be higher than the velocity of a dimmer IFT train (smaller train) within the same cilia. This is not apparent from the movies and such a correlation should be verified to make their claim stronger.
Thank you for these excellent suggestions. We measured the particle size and fluorescence intensity of 3 dpf crista cilia using high-resolution images acquired with Abberior STEDYCON. The results showed a positive correlation between the two. These data have been added to the revised version in Figure 5I, which includes both control and ift88 morphant data.
(5) They make an even larger claim that the cilia length (and IFT velocity) in different organs is different due to differences in the sizes of IFT trains. This is based on a marginal difference they observe between the cilia of crista and the spinal cord in immunofluorescence experiments (Figure 5C). Inferring that this minor difference is key to the striking difference in cilia length and IFT velocity is incorrect in my opinion.
Impact:
Overall, I think this work develops an exciting new multicellular model organism to study IFT mechanisms. Zebrafish is a vertebrate where we can perform genetic modifications with relative ease. This could be an ideal model to study not just the role of IFT in connection with ciliary function but also ciliopathies. Further, from an evolutionary perspective, it is fascinating to compare IFT mechanisms in zebrafish with unicellular protists like Chlamydomonas, simple multicellular organisms like C elegans, and primary mammalian cell cultures. Having said that, the underlying storyline of this study is flawed in my opinion and I would recommend the authors to report the striking findings and methodology in more detail while significantly toning down their proposed hypothesis on ciliary length regulation. Given the technological advancements made in this study, I think it is fine if it is a descriptive manuscript and doesn't necessarily need a breakthrough hypothesis based on preliminary evidence.
Thanks for with these comments. We agree with this reviewer that more evidences are required to explain why IFT is transported faster in longer cilia. In the revised version, we have modified and softened this section, focusing primarily on the novel findings of IFT velocity differences between cilia of varying lengths.
Reviewer #3 (Public Review):
Summary:
A known feature of cilia in vertebrates and many, if not all, invertebrates is the striking heterogeneity of their lengths among different cell types. The underlying mechanisms, however, remain largely elusive. In the manuscript, the authors addressed this question from the angle of intraflagellar transport (IFT), a cilia-specific bidirectional transportation machinery essential to biogenesis, homeostasis, and functions of cilia, by using zebrafish as a model organism. They conducted a series of experiments and proposed an interesting mechanism. Furthermore, they achieved in situ live imaging of IFT in zebrafish larvae, which is a technical advance in the field.
Strengths:
The authors initially demonstrated that ectopically expressed Ift88-GFP through a certain heatshock induction protocol fully sustained the normal development of mutant zebrafish that would otherwise be dead by 7 dpf due to the lack of this critical component of IFT-B complex.
Accordingly, cilia formations were also fully restored in the tissues examined. By imaging the IFT using Ift88-GFP in the mutant fish as a marker, they unexpectedly found that both anterograde and retrograde velocities of IFT trains varied among cilia of different cell types and appeared to be positively correlated with the length of the cilia.
For insights into the possible cause(s) of the heterogeneity in IFT velocities, the authors assessed the effects of IFT kinesin Kif3b and Kif17, BBSome, and glycylation or glutamylation of axonemal tubulin on IFT and excluded their contributions. They also used a cilia-localized ATP reporter to exclude the possibility of different ciliary ATP concentrations. When they compared the size of Ift88-GFP puncta in crista cilia, which are long, and spinal cord cilia, which are relatively short, by imaging with a cutting-edge super-resolution microscope, they noticed a positive correlation between the puncta size, which presumably reflected the size of IFT trains, and the length of the cilia.
Finally, they investigated whether it is the size of IFT trains that dictates the ciliary length. They injected a low dose (0.5 ng/embryo) of ift88 MO and showed that, although such a dosage did not induce the body curvature of the zebrafish larvae, crista cilia were shorter and contained less Ift88-GFP puncta. The particle size was also reduced. These data collectively suggested mildly downregulated expression levels of Ift88-GFP. Surprisingly, they observed significant reductions in both retrograde and anterograde IFT velocities. Therefore, they proposed that longer IFT trains would facilitate faster IFT and result in longer cilia.
Weaknesses:
The current manuscript, however, contains serious flaws that markedly limit the credibility of major results and findings. Firstly, important experimental information is frequently missing, including (but not limited to) developmental stages of zebrafish larvae assayed (Figures 1, 3, and 5), how the embryos or larvae were treated to express Ift88-GFP (Figures 3-5), and descriptions on sample sizes and the number of independent experiments or larvae examined in statistical results (Figures 3-5, S3, S6). For instance, although Figure 1B appears to be the standard experimental scheme, the authors provided results from 30-hpf larvae (Figure 3) that, according to Figure 1B, are supposed to neither express Ift88-GFP nor be genotyped because both the first round of heat shock treatment and the genotyping were arranged at 48 hpf. Similarly, the results that ovl larvae containing Tg(hsp70l:ift88 GFP) (again, because the genotype is not disclosed in the manuscript, one can only deduce) display normal body curvature at 2 dpf after the injection of 0.5 ng of ift88 MO (Fig 5D) is quite confusing because the larvae should also have been negative for Ift88-GFP and thus displayed body curvature. Secondly, some inferences are more or less logically flawed. The authors tend to use negative results on specific assays to exclude all possibilities. For instance, the negative results in Figures 4A-B are not sufficient to "suggest that the variability in IFT speeds among different cilia cannot be attributed to the use of different motor proteins" because the authors have not checked dynein-2 and other IFT kinesins. In fact, in their previous publication (Zhao et al., 2012), the authors actually demonstrated that different IFT kinesins have different effects on ciliogenesis and ciliary length in different tissues. Furthermore, instead of also examining cilia affected by Kif3b or Kif17 mutation, they only examined crista cilia, which are not sensitive to the mutations. Similarly, their results in Figures 4C-G only excluded the importance of tubulin glycylation or glutamylation in IFT. Thirdly, the conclusive model is based on certain assumptions, e.g., constant IFT velocities in a given cell type. The authors, however, do not discuss other possibilities.
Thank you for pointing out the flaws in our experiments. We apologize for any confusion caused by the lack of detail in our descriptions. Regarding Figure 2B, we want to clarify that it depicts the procedure for heat shock experiments conducted for the ovl mutants' rescue assay, not the experimental procedure for IFT imaging. In the revised version, we have included detailed methods on how to induce the expression of Ift88-GFP via heat shock and the subsequent image processing. The procedure for heat induction is also shown in Figure S2A. We have also added the sample sizes for each experiment and descriptions of the statistical tests used in the appropriate sections of the revised version.
Regarding the comments on the relationship between IFT speed variability and motor proteins, we completely agree with the reviewer. We have revised our description of this part accordingly.
Lastly, the results shown in Figure 5D are from a wild-type background, not ovl mutants. We aimed to demonstrate that a lower dose of ift88 morpholino (0.5 ng) can partially knock down Ift88, allowing embryos to maintain a generally normal body axis, while the cilia in the ear crista became significantly shorter.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Minor
(I recommend adding page numbers and probably line numbers. This makes commenting easier)
We have added page numbers and line numbers in the revised manuscript.
Intro: Furthermore, ultra-high-resolution microscopy showed a close association between cilia length in different organs and the size of IFT fluorescent particles, indicating the presence of larger IFT trains in longer cilia.
This correlation is not that strong and data are only available for 2 types of cilia.
Thanks. We have modified this part.
P5) cilia (Fig. 1D) -> (Fig. S1)
Thanks. We have corrected this.
P5) "These movies provide a great opportunity to compare IFT across different cilia." Rewrite: "This approach allows one to determine the velocity and frequency based of IFT based on kymographs" or similar.
Thank you for your correction, we have changed it in the revised manuscript.
This observation suggests that cargo and motor proteins are more effectively coordinated in transporting materials, resulting in increased IFT velocity-a novel regulatory mechanism governing IFT speed in vertebrate cilia.
This is a somewhat cryptic phrase, rewrite?
We have modified this sentence.
P6 and elsewhere: "IFT in the absence of Kif17 or Bbs proteins" I wonder if it would be better to provide subheadings summarizing the main observation instead of descriptive titles. This includes the title of the manuscript.
Thanks for this suggestion. We have changed the title of subheadings in the revised manuscript. We prefer to keep the current title of this manuscript, as we think this paper is mainly to describe IFT in different types of cilia.
Is it known whether IFT protein and motors are alternatively spliced in the various ciliated cells of zebrafish? In this context, is it known whether the cells express IFT proteins at different levels?
We analyzed the transcript isoforms of several ciliary genes, including ift88, ift52, ift70, ift172, and kif3a. Most of these IFT genes possess only a single transcript isoform. The Kif3a motor proteins have two isoforms (long and short isoforms), however, the shorter isoform contains only the motor domain and is presumed to be nonfunctional for IFT. While we cannot completely rule out this possibility, we consider it unlikely that the variation in IFT speed is due to alternative splicing in ciliary tissues.
P6) The relation between osm-3 and Kif17 needs to be introduced briefly.
Thank you for pointing this out. We have added it in the proper place of the revised manuscript.
P6) "IFT was driven by kinesin or dynein motor proteins along the ciliary axoneme." "is driven"?
Delete phrase and IFT to the next sentence?
We have deleted this sentence.
P7) "Moreover, the mutants were able to survive to adulthood and there is no difference in the fertility or sperm motility between mutants and control siblings, which is slightly different from those observed in mouse mutants(Gadadhar et al., 2021)." Could some of these data be shown?
Thanks for this suggestion. When crossed with wild-type females, all homozygous mutants showed no difference in fertility compared to controls. The percentage of fertilization rates in mutants was 90.5% (n = 7), which was similar to wild-type (87.2%, n = 7). We determined the trajectories of free-swimming sperm by high-speed video microscopy. The vast majority of sperm in ttll3 mutant, similar to wild-type sperm, swim almost entirely along a straight path, which is different from what was observed in the mouse mutant (where 86% of TTLL3-/-TTLL8-/- sperm rotate in situ). We assessed cilia motility in the pronephric ducts of 5dpf embryos using high-speed video microscopy. The ttll3 mutant exhibited a rhythmic sinusoidal wave pattern similar to the control, and there was no significant difference in ciliary beating frequency. These new data are now included in Figure S7C-H.
P7) "which has been shown early to reduce" earlier
We have changed it. Thanks.
Maybe the authors could speculate how the cells ensure the assembly of larger/faster trains in certain cells. Are the relative expression levels known or worth exploring?
Thank you for these suggestions. We believe that longer cilia may maintain larger IFT particle pools in the basal body region, facilitating the assembly of large IFT trains. The higher frequency of IFT injection in longer cilia further supports this hypothesis. It is likely that cells with longer cilia have higher expression levels of IFT proteins. However, due to the lack of proper antibodies for IFT proteins in zebrafish, it is currently unfeasible to compare this. This experiment is certainly worth investigating in the future. We have added this discussion in the revised manuscript.
Reviewer #2 (Recommendations for The Authors):
Here are detailed comments for the authors:
(1) The authors need to describe their methodology of imaging and what they observe in much greater detail. How were the different cilia types organized? Approximately how many were observed in every organ? How were they oriented? Were there length variations between cilia in the same organ? While imaging, were individual cilium mostly lying in a single focal plane of imaging or the authors often performed z-scans over multiple planes. Velocity measurement is highly variable if individual cilia are spanning over a large volume, with only part of it in focus in single plane acquisition.
Thank you for your comments. We apologize for the lack of details in the methodology. We have added a detailed description in the 'Materials and Methods' section and illustrated the experimental paradigm in Figure S2A of the revised manuscript. In most tissues we examined, the length of cilia was relatively uniform, except in the crista. The cilia in the crista were significantly longer, with lengths varying between 5 and 30 μm, compared to those in other tissues. We categorized the cilia lengths in the crista into three groups at intervals of 10 μm and measured the anterograde and retrograde velocities of IFT in each group. The results, shown in Figure S4, revealed no significant difference in IFT velocity among the different cilia lengths within the same tissue. Regarding the imaging, all IFT movies were captured in a single focal plane. In most cases, we did not observe significant velocity variability within the same cilium.
(2) It is very difficult to directly observe the large differences in IFT velocity from the kymographs, especially in the case of shorter cilia and retrograde motion in them. The quality of the example kymographs could be improved and more zoomed in several cases.
Thank you for this suggestion. We have modified this.
(3) The authors do not describe at all, how velocity analysis was done on the kymographs? Were lines drawn manually on the kymographs? From the movies and the kymographs it is visible that the IFT motion is often variable and sometimes gets stuck. How did the authors determine the velocities of such trains? A single slope through the entire train or part of the train? Were they consistent with this? Such variable motion is not so easy to discern in the case of really short cilia. The authors could use a more automatic way of extracting velocities from kymographs using tools such as kymodirect or kymobutler. Keeping in mind that IFT velocity is the main parameter studied in this work, it is important that the analysis is robust.
We apologize for the previous lack of detailed description. We utilized ImageJ software to generate kymographs, where particles appear as lines. For a moving particle, this line appears oblique. We manually drew lines on the kymographs, and the velocity of particles was calculated based on the slope (Zhou et al., 2001). We only analyzed particles that tracked the full length of the cilia. Following the reviewer's suggestions, we also used the automatic software KymographDirect to calculate the velocity of IFT particles. The results were similar to those calculated using the previous method. These new data are now shown in Figure S2B-D. For shorter cilia, we only used particles with clear moving paths for our calculations. In the revised version, we have included a detailed description of the velocity analysis methods.
(4) In line with the previous point, as visible from the kymographs the velocity is significantly slower near the transition zone. Did the authors make sure they are not including the region around the transition zone while measuring the IFT velocity, especially in the case of shorter cilia?
Thank you for the comment. In the revised manuscript, we automatically extracted the path of particle using KymographDirect software. Quantification of each particle's velocity versus position in crista reveals that anterograde IFT proceeds from the base to the tip at a relatively constant speed, whereas retrograde IFT undergoes a slightly acceleration process when returning to the base (Fig. S2E). This finding differs from observations in C. elegans, which dynein-2 first accelerating and then decelerating back to 1.2 μm/s adjacent to the ciliary base (Yi et al, 2017). We believe it is very unlikely that the slow IFT velocity is due to the calculation of IFT only in the transition zone of shorter cilia.
(5) There are several fascinating findings in this work that the authors do not discuss properly. Firstly, do the authors have a hypothesis as to why IFT speeds are so radically different in different cilia types, given that they are driven by the same motor proteins and have the same ATP levels? They make a big claim in this paper that IFT train sizes correlate with train velocities. IFT trains have a highly ordered structure with regular binding sites for motor proteins. So, a smaller train would have a proportional number of motors attached to them. Why (and how) are the motors moving trains so slowly in some cilia and not in others? If there is no clear answer, the authors must put forward the open question with greater clarity.
Thank you for the comment. We hypothesize that if multiple motors drive the movement of cargoes synergistically, it could increase the speed of IFT transport. An example supporting this hypothesis is the principle of multiple-unit high-speed trains, which use multiple motors in each individual car to achieve high speeds. Of course, this is just one hypothesis, and we cannot exclude other possibilities, such as the use of different adaptors in different cell types. We have revised our conclusions accordingly in the updated manuscript.
(6) They find that IFT speeds do not change in kif17 mutants. Are the cilia length also similar (does not appear to be the case in Figure 4 and Figure S3)? Cilia length needs to be quantified. Further, they mention that in C elegans, heterotrimeric kinesin-2 and homodimeric kinesin-2 coordinate IFT. However, from several previous studies, we know that in Chlamydomonas and in mammalian cilia IFT is driven primarily by heterotrimeric kinesin-2 with no evidence that homodimeric kinesin-2 is linked with driving IFT. It appears to be the same in zebrafish. This is an interesting finding and needs to be discussed far more comprehensively.
Thank you for your comments. We have previously shown that the number and length of crista cilia were grossly normal in kif17 mutants (Zhao et al, 2012). The length of crista cilia displayed slight variability even in wild-type larvae. We quantified the length of cilia in both the crista and neuromast within different mutants, and our analysis revealed no significant difference (see Author response image 1). We agree with the reviewer that Kif17 may play a minor role in driving IFT in cilia. However, previous studies have shown that KIF17 exhibits robust, processive particle movement in both the anterograde and retrograde directions along the entire olfactory sensory neuron cilia in mice. This suggests that, although not essential, KIF17 may also be involved in IFT (Williams et al., 2014). We have added more discussion about Kif17 and heterotrimeric kinesin in the appropriate section of the revised manuscript.
Author response image 1.
Statistical significance is based on Kruskal-Wallis statistic, Dunn's multiple comparisons test. n.s., not significant, p＞0.05.
(7) Again, they find that IFT speeds do not change in BBS-4 mutants. I have the same comment about the cilia length as for kif17 mutants. Further, the discussion for this finding is lacking. The authors mention that IFT is disrupted in BBSome mutants of C elegans. Is this the case in other organisms as well? Structural studies on IFT trains reveal that BBSomes are not part of the core structure, while other studies reveal that BBSomes are not essential for IFT. So perhaps the results here are not too surprising.
We agree with the reviewer that BBSome is possibly not essential for IFT in most cilia. However, in the cilia of olfactory sensory neurons, BBSome is involved in IFT in both mice and nematodes (Ou et al, 2005; Williams et al., 2014). We have added more discussion about BBSome in the appropriate section of the revised manuscript.
(8) No change in IFT velocities in kif3b mutants is rather surprising. The authors suggest that Kif3C homodimerizes to carry out IFT in the absence of Kif3B. Even if that is the case, the individual homodimer constituents of heterotrimeric kinesin-2 have been shown in previous studies to have different motor properties when homodimerized artificially. Why is IFT not affected in these mutants? This should be discussed. Also, the cilia lengths should be quantified.
We think the presence of the Kif3A/Kif3C/KAP3 trimeric kinesin may substitute for the Kif3A/Kif3B/KAP3 motors in kif3b mutants, which show normal length of cristae cilia. The Kif3A/Kif3C/KAP3 trimeric kinesin may have similar transport speeds as the Kif3A/Kif3B/KAP3 motors. We did not propose that the Kif3C homodimer can drive the cargoes alone. We apologize for this misunderstanding. Additionally, we have reevaluated the IFT velocities among different lengths of cristae cilia and found no difference between longer and shorter cilia within the same cell types.
(9) The findings with tubulin modifications should also be discussed in comparison to what has been observed in other organisms.
We have added further discussion about this result in the revised manuscript.
(10) The authors find that IFT velocity is lower in ift88 morphants. They also find that the cilia length is shorter (in which cilia type?). Immunofluorescence experiments show that the IFT particle number and size are lower in the ift88 morphants. How many organisms did they look at for this data? What is the experimental variability in intensity measurements in immunofluorescence experiments? Wouldn't the authors expect much higher variability in ift88 morphants (between individual organisms) due to different amounts of IFT88 than for wildtype?
Thank you for your comments. We apologize for the lack of information regarding the number of organisms observed in Figure 5. These numbers have been added to the figure legends in the revised manuscript. When a low dose of ift88 morpholino was injected, we observed significant shortening of cilia in the ear crista, along with reduced IFT speed. We measured the fluorescence intensity of different IFT particles and found a positive correlation between IFT particle size and fluorescence intensity (Fig 5I). Moreover, the variability of cilia length in cristae is slightly higher in ift88 morphants. These new data have been included in the revised version.
(11) From their observations they make the claim that IFT velocity is directly proportional to IFT train size. Now within every cilium, IFT trains have large size variations, given the variable intensities for different IFT trains. The authors themselves show that they resolve far more trains when imaging with STED (possibly because they are able to visualize the smaller trains). Is the IFT velocity within the same cilium directly correlated with the intensity of the train, both for wildtype and ift88 morphants? That is the most direct way the authors can test that their hypothesis is true. Higher intensity (larger train size) results in faster velocity. From a qualitative look at their movies, I do not see any strong evidence for that.
Thank you for your comments. We have measured the particle size and fluorescence intensity of 3dpf crista cilia using high-resolution images acquired with Abberior STEDYCON. The results, shown in Figure 5I, demonstrate a positive correlation between particle size and fluorescence intensity.
(12) Are the sizes of both anterograde and retrograde trains lower in ift88 morphants? It's not clear from the data. It should be clearly stated that the authors speculate this and this is not directly evident from the data.
Because the size of IFT fluorescence particles is based on immunostaining results, not live imaging, we cannot determine whether they are anterograde or retrograde IFT particles.
Therefore, we can only speculate that possibly both anterograde and retrograde trains are reduced in ift88 morphants.
(13) The biggest claim in this paper is that the cilia lengths in different organs are different due to differences in IFT train sizes. This is based on highly preliminary data shown in Figure 5C (how many organisms did they measure?). The difference is marginal and the dataset for spinal cord cilia is really small. The internal variability within the same cilia type is larger than the difference. How is this tiny difference resulting in such a large difference in IFT speeds? I believe their conclusions based on this data are incorrect.
From our results, we believe that IFT velocity is related to cell types rather than the length of cilia (Fig. S4), which has also been mentioned in previous studies (Williams et al., 2014). We agree with the reviewer that the evidence for faster IFT speed due to larger train size is not very solid. We have accordingly softened our conclusion and mentioned other possibilities in the revised version.
Minor comments:
(1) The authors only mention the number of IFT particles for their data. They should provide the number of cilia and the number of organisms as well.
Thank you for your suggestion. We added the number of cilia and organisms next to the number of particles in Figure 3, Figure S2-S5 and Table S1 of the revised manuscript.
(2) Cilia and flagella are similar structurally but not the same. The authors should change the following sentence: In contrast to the localization of most organelles within cells, cilia (also known as flagellar) are microtubule-based structures that extend from the cell surface, facilitating a more straightforward quantification of their size.
Thank you for the detailed review. We have changed it in our revised manuscript.
(3) The authors should provide references here. For example, Chlamydomonas has two flagella with lengths ranging from 10 to 14 μm, while sensory cilia in C. elegans vary from approximately 1.5 μm to 7.5 μm. In most mammalian cells, the primary cilium typically measures between 3 and 10 μm.
We have added it in our revised manuscript.
(4) They should mention ovl mutants are IFT88 mutants when they introduce it in the main text.
We have added it in our revised manuscript.
(5) Correct the grammar here: The velocity of IFT within different cilia also seems unchanged (Figure 4F, Movie S9, Table S1).
We have changed it.
(6) Correct the grammar here: Similarly, the IFT speeds also exhibited only slight changes in ccp5 morphants, which decreased the deglutamylase activities of Ccp5 and resulted in a hyperglutamylated tubulin
We have changed it.
Reviewer #3 (Recommendations For The Authors):
Introduction:
1st paragraph, "flagellar" should be "flagella"; 2nd paragraph, "result a wide range of" should be "result in a...".
We have changed it.
Results and discussion:
"...certain specialized cell types, including olfactory epithelia and pronephric duct, ...": olfactory epithelia and pronephric duct are tissues, not cells.
"...the GFP fluorescence of the transgene was prominently enriched in the cilia (Fig 1D)" : Fig 2D?
"The velocity of IFT within different cilia was also seems unchanged (Fig. 4 F, Movie S9, Table S1)": "was" and "seems" cannot be used together.
"...driven by b-actin2 promotor": -actin2?
"...each dynein motor protein might propel multiple IFT complexes": The "protein" should be deleted.
Thanks. We have corrected all of these mistakes.
Figures:
Figure 1: Dyes and antibodies used other than the anti-acetylated tubulin antibody should mentioned. The developmental stages of zebrafish used for the imaging are mostly missing.
Thanks. In the revised version, we have updated the figure legends to include descriptions of the antibodies, developmental stages, as well as N numbers.
Figure 2B: What "hphs" means should be explained somewhere.
Thanks. We have added full name for these abbreviations.
Figures 3A-E: For clarity, the cilia whose IFT kymographs are shown should be marked. "Representative particle traces are marked with white lines in panels D and E" (legend): they are actually black lines. The authors should also clearly disclose the developmental stages of zebrafish used for the imaging.
Thank you for your comments. In the revised manuscript, the cilia used to generate the kymograph are marked by yellow arrows. We have updated the legend to change "white" to "black." Additionally, we have included the developmental stages of zebrafish used for imaging in Figure 3A.
Figures 3G-K: The authors used quantification results from 4-dpf larvae and 30-hpf embryos for comparisons. Nevertheless, according to their experimental scheme in Figure 2B, 30-hpf embryos were not subjected to heat-shock treatment and genotyping. How could they express Ift88-GFP for the imaging? How could the authors choose larvae of the right genotypes? In addition, even if the authors heat-shocked them in time but forgot to mention, there are issues that need to be clarified experimentally and/or through citations, at least through discussions. Firstly, at 30 hpf, those motile cilia are probably still elongating. If this is the case, their final lengths would be longer than those presented (H; the authors need to disclose whether the lengths were measured from ciliary Ift88-GFP or another marker). In other words, the correlation with IFT velocities (H and I) might no longer exist when mature cilia were measured. Similarly, cilia undergo gradual disassembly during the cell cycle. Epidermal cells at 30-hpf are likely proliferating actively, and the average length of their cilia (H) would be shorter than that measured from quiescent epidermal cells in later stages.
Thank you for these comments. First, we want to clarify that Figure 2B depicts the procedure for heat shock experiments conducted for the ovl mutants' rescue assay, not the experimental procedure for IFT imaging. We visualized IFT in five types of cilia using Tg (hsp70l: ift88-GFP) embryos without the ovl mutant background. In the revised manuscript, we have provided a detailed description of embryo treatment in the 'Materials and Methods' section and illustrated the experimental paradigm in Figure S2A.
Regarding the ciliary length differences between different developmental stages, we quantified cilia length in epidermal cells at 30 hpf versus 4 dpf, and in pronephric duct cilia at 30 hpf versus 48 hpf. Our analysis found no significant difference in length between earlier and later stages. Additionally, IFT velocities were comparable between these stages. These findings suggest that slower IFT velocities may not be attributed to the selection of different embryonic stages. Furthermore, we demonstrated that longer and shorter cilia maintain similar IFT velocities in crista cilia, indicating that elongated cilia within the same cell type exhibit comparable IFT velocities. These new results are presented in Figures S4 and S5 in the revised version.
Secondly, do IFT velocities differ between elongating and mature cilia or remain relatively constant for a given cell type? The authors apparently take the latter for granted without even discussing the possibility of the former. In addition, whether the quantification results were from cilia of one or multiple fish, an important parameter to reflect the reproducibility, and sample sizes for the length data are not disclosed. The lack of descriptions on sample sizes and the number of independent experiments or larvae examined are actually common for statistical results in this manuscript.
Thank you for your comments. We apologize for omitting the basic description of sample sizes and the number of cilia analyzed. We have addressed these issues in the revised manuscript. The length of 4dpf Crista cilia is variable, with longer cilia reaching up to 30 µm and shorter cilia measuring only around 5 µm within the same crista. We categorized the cilia length of Crista into three groups at intervals of 10 µm and measured anterograde and retrograde velocities of IFT in each group. The results revealed no significant difference in IFT velocity among elongating and mature cilia within crista. These supplementary data are now included in Figure S4.
Figures 4A-B: When mutating neither Kif17 nor Kif3b affected the IFT of crista cilia, the data unlikely "suggest that the variability in IFT speeds among different cilia cannot be attributed to the use of different motor proteins". In fact, in the cited publication (Zhao et al., 2012), the authors used the same and additional mutants (Kif3c and Kif3cl) to demonstrate that different IFT-related kinesin motors have different effects on ciliogenesis and ciliary length in different tissues, results actually implying tissue-specific contributions of different kinesin motors to IFT. Furthermore, although likely only cytoplasmic dynein-2 is involved in the retrograde IFT, the authors cannot exclude the possibility that different combinations or isoforms of its many subunits and regulators contribute to the velocity regulation. Therefore, the authors need to reconsider their wording. This reviewer would suggest that the authors examine the IFT status of cilia that were previously reported to be shortened in the Kif3b mutant to see whether the correlation between ciliary length and IFT velocities still stands. This would actually be a critical assay to assess whether the proposed correlation is only a coincidence or indeed has a certain causality.
Thank you for your comments. The shortened cilia observed in Kif3b mutants may be attributed to the presence of maternal Kif3b proteins, making it challenging to exclude the involvement of Kif3b motor. Regarding the relationship between IFT speed variability and motor proteins, we agree with the reviewer that we cannot entirely dismiss the possibility of different motors or adaptors being involved. We have revised our description of this aspect accordingly.
Figures 4C-G: Similarly, when the authors found that tubulin glycylation or glutamylation has little effect on IFT, they cannot use these observations to exclude possible influences of other types of tubulin modifications on IFT. They should only stick to their observations.
Yes, we agree. We have changed the description in the revised manuscript.
Figure 5:
A-C: When the authors only compared immotile cilia of crista with motile cilia of the spinal cord, it is hard to say whether the difference in particle size is correlated with ciliary length or motility. Cilia from more tissues should be included to strengthen their point, especially when the authors want to make this point the central one.
D: The authors showed that ovl larvae containing Tg(hsp70l:ift88 GFP) (as they do not indicate the genotype, this reviewer can only deduce) display normal body curvature at 2 dpf after the injection of 0.5 ng of ift88 MO. Such a result, however, is quite confusing. According to their experimental scheme in Figure 2B, these larvae were not subjected to heat shock induction for Ift88-GFP. Do ovl larvae containing Tg(hsp70l:ift88 GFP) naturally display normal body curvature at 2 dpf?
Thank you for your comments. Due to technical limitations, comparing IFT particle size across different cilia using STED is challenging. We agree with this reviewer that the evidence supporting this aspect is relatively weak. Accordingly, we have modified and softened our conclusion in the revised version.
Regarding the injection of ift88 morpholino, we want to clarify that we are injecting it into wildtype embryos, not oval mutants. The lower dose of ift88 morpholino (0.5ng) partially knocked down Ift88, allowing embryos to maintain a grossly normal body axis while resulting in shorter cilia in the ear crista.
E: The authors need to indicate the developmental stage of the larvae examined. One piece of missing data is global expression levels of both endogenous (maternal) Ift88 and exogenous
Ift88-GFP in zebrafish larvae that are either uninjected, 8-ng-ift88 MO-injected, or 0.5-ng-ift88 MO-injected, preferably at multiple time points up to 3 dpf. The results will clarify (1) the total levels of Ift88 following time; (2) the extent of downregulation the MO injections achieved at different developmental stages; and importantly (3) whether the low MO dosage (0. 5 ng) indeed allowed a persistent downregulation to affect IFT trains at 3 dpf, a time the authors made the assays for Figures 5F-J to reach the model (K). It will be great to include wild-type larvae for comparison.
Thank you for these valuable suggestions. The ift88 morpholino (MO) was designed to block the splicing of ift88 transcripts and has been used in multiple studies. This morpholino specifically blocks the expression of endogenous ift88, while the expression of the Ift88-GFP transgene remains unaffected. It would be beneficial to titrate the expression level of Ift88 in the morphants at different stages. Unfortunately, we do not have access to a zebrafish Ift88 antibody. We assessed the effects of a lower amount of MO based on our observation that the fish maintained a normal body axis while exhibiting shorter cilia. Ideally, the amount of Ift88 should be lower in the morphants, considering the presence of ciliogenesis defects. We have included additional comments regarding this limitation in the revised version.
Movies:
Movies 1-5: Elapsed time is not provided. Furthermore, cilia in the pronephric duct and spinal cord are known to beat rapidly. Their motilities, however, appear to be largely compromised in Movies 3 and 4. Although the quantification results in Fig 3G imply that the authors imaged 30hpf embryos for such cilia, there is no statement on real conditions.
Thank you for your comments. We apologize for missing elapsed time in our movies. We have addressed this issue in the revised manuscript. Motile cilia are difficult to image due to their fast beating. To immobilize the moving cilia and enable the capture of IFT movement within the cilia, we gently press the embryo with a round cover glass to inhibit the beating of cilia. Data from each embryo were collected within 5 minutes to avoid the impact of embryo death on the results. We have added detail description in the 'Materials and Methods' section.
Materials:
The sequence of morpholino oligonucleotide against ift88 is missing.
We have added the sequence of ift88 morpholino in the revised manuscript.
References:
Important references are missing, including (1) the paper by Leventea et al., 2016 (PMID: 27263414), which shows cilia morphologies in various zebrafish tissues with more detailed descriptions of tissue anatomies and experimental techniques; (2) papers documenting that dynein motors "move faster than Kinesin motors" in IFT of C. reinhardtii and C. elegans cilia; and (3) the paper by Li et al., 2020 (PMID: 33112235), in which the authors constructed a hybrid IFT kinesin to markedly reduced anterograde IFT velocity (~ 2.8 fold) and IFT injection rate in C. reinhardtii cilia and found only a mild reduction (~15%) in ciliary length. This paper is important because it is a pioneer one that elegantly investigated the relationship between IFT velocity and ciliary length. The findings, however, do not necessarily contradict the current manuscript due to differences in, e.g., model organisms and methodology.
Thank you for the detailed review, we have cited these literatures in the proper place of the revised manuscript.
Reference
Broekhuis JR, Verhey KJ, Jansen G (2014) Regulation of cilium length and intraflagellar transport by the RCK-kinases ICK and MOK in renal epithelial cells. PLoS One 9: e108470
Kunova Bosakova M, Varecha M, Hampl M, Duran I, Nita A, Buchtova M, Dosedelova H, Machat R, Xie Y, Ni Z et al (2018) Regulation of ciliary function by fibroblast growth factor signaling identifies FGFR3-related disorders achondroplasia and thanatophoric dysplasia as ciliopathies. Hum Mol Genet 27: 1093-1105
Luo W, Ruba A, Takao D, Zweifel LP, Lim RYH, Verhey KJ, Yang W (2017) Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium. Sci Rep 7: 15793 Ou G, Blacque OE, Snow JJ, Leroux MR, Scholey JM (2005) Functional coordination of intraflagellar transport motors. Nature 436: 583-587
See SK, Hoogendoorn S, Chung AH, Ye F, Steinman JB, Sakata-Kato T, Miller RM, Cupido T, Zalyte R, Carter AP et al (2016) Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity. ACS Chem Biol 11: 53-60
Williams CL, McIntyre JC, Norris SR, Jenkins PM, Zhang L, Pei Q, Verhey K, Martens JR (2014) Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia. Nat Commun 5: 5813
Yi P, Li WJ, Dong MQ, Ou G (2017) Dynein-Driven Retrograde Intraflagellar Transport Is Triphasic in C. elegans Sensory Cilia. Curr Biol 27: 1448-1461 e1447
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eLife Assessment
The manuscript represents a valuable conceptual and technical contribution to our understanding of ciliogenesis and intraflagellar transport in vertebrates. Through a series of solid and technically superb live imaging experiments to directly visualize intraflagellar transport in various zebrafish ciliated tissues, the authors unveil the surprising breadth of intraflagellar transport speed among differing organs and link this to cell type-specific differences in cilia length and intraflagellar transport train size. This work will be of broad interest to researchers in numerous fields, including development, cell biology, and imaging.
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en.wikipedia.org en.wikipedia.org
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The Farewell Sermon is also delivered.
The last speech by the prophet in the movie
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Khalid ibn Walid, a Meccan general who has killed many Muslims, converts to Islam.
Oww, so the first convert from the Meccan side was Khalid ibn Walid in the movie.
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The film begins with Muhammad sending an invitation to accept Islam to the surrounding rulers: Heraclius, the Byzantine Emperor; Muqawqis, the Patriarch of Alexandria; Kisra, the Sasanian Emperor.
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