① Broad saucer shaped or irregular depressions in the cervical area usually on the facial tooth surface ① Geniş, tabak şeklinde ya da düzensiz çöküntüler, genellikle dişin servikal bölgesinde ve yüzeysel olarak bulunur.

② The surface appears smooth, hard and polished ② Yüzey, pürüzsüz, sert ve parlak görünür.

③ Commonly seen at gingival third of the labial surface of anterior teeth ③ Ön dişlerin dudak yüzeyinin gingival üçte birlik bölümünde yaygın olarak görülür.

④ Erosive lesion is generally glazed and has no demarcation from adjacent surface ④ Erozyon lezyonu genellikle cam gibi parlaktır ve bitişik yüzeylerden belirgin bir sınırı yoktur.

⑤ The tooth is sensitive to chemical, physical and mechanical stimuli ⑤ Diş kimyasal, fiziksel ve mekanik uyaranlara karşı hassastır.

① These are wedge type defects usually occur in cervical areas of tooth due to excessive occlusal stresses or parafunctional habits such as bruxism. ① Bunlar genellikle aşırı oklüzal stresler veya bruksizm gibi parafonksiyonel alışkanlıklar nedeniyle dişlerin servikal bölgelerinde oluşan kama tipi defektlerdir.

② Abfractions are the microfractures which appear in the enamel on cervical area of tooth, and flexe under heavy loads. ② Abfraksiyonlar, dişin servikal bölgesinde mine üzerinde görülen mikroçatlaklardır ve ağır yükler altında esnerler.

③ Abfraction lesion appears as a wedge shaped defect with sharp line angles. ③ Abfraksiyon lezyonu, keskin hat açılarla kama şeklinde bir defekt olarak görünür.

④ In the early stages they may appear as minor irregular cracks or fracture lines or wedge shaped defects in the cervical region of the tooth. ④ Erken evrelerde, dişin servikal bölgesinde küçük düzensiz çatlaklar, kırık çizgileri veya kama şeklinde defektler olarak ortaya çıkabilirler.

⑤ But in later stages, they appear as grooves extending into dentin. ⑤ Ancak ilerleyen evrelerde dentine uzanan oluklar şeklinde görünürler.

⑥ Etiology (In 1984, Lee and Eakle described lateral forces as the cause of the breakdown of the tooth structure. ⑥ Etiyoloji (1984 yılında Lee ve Eakle, diş yapısının bozulmasının nedeni olarak yan kuvvetleri tanımlamıştır.

⑦ Grippo showed that the forces could be static, such as those produced by swallowing and clenching, or cyclic, those generated during chewing action. ⑦ Grippo ise bu kuvvetlerin yutkunma ve sıkma gibi statik ya da çiğneme sırasında oluşan döngüsel kuvvetler olabileceğini göstermiştir.

⑧ The abfractive lesions are caused by flexure and ultimate material fatigue of susceptible teeth at locations away from the point of loading. ⑧ Abfraktif lezyonlar, yükleme noktasından uzak bölgelerde hassas dişlerin bükülmesi ve nihai malzeme yorgunluğu nedeniyle oluşur.

⑨ The breakdown is dependent on the magnitude, duration, frequency and location of the forces. ⑨ Bozulma, kuvvetlerin büyüklüğüne, süresine, sıklığına ve konumuna bağlıdır.

⑩ If a tooth has an abfraction, the occlusal loading on the tooth can be tested in centric occlusion and in excursive movements with occlusal marking paper. ⑩ Eğer bir dişte abfraksiyon varsa, diş üzerindeki oklüzal yükleme, merkezi oklüzyon ve hareketli oklüzyon hareketlerinde oklüzal işaretleme kağıdıyla test edilebilir.

⑪ The tooth with abfraction will show a heavy marking on one of the inclines of a cusp. ⑪ Abfraksiyonlu dişte, bir tüberün eğimlerinden birinde yoğun işaretleme görülecektir.

⑫ This damaging lateral force produces stress lines in the tooth and results in tooth breakdown. ⑫ Bu zararlı yan kuvvet, dişte stres çizgileri oluşturur ve dişin bozulmasına yol açar.

⑬ If during lateral excursions, there is cuspid rise, the loading forces of the excursive movement will be directed into the canines. ⑬ Yan hareketler sırasında, kanin yükselmesi varsa, hareketli oklüzyon kuvvetleri kaninlere yönlendirilir.

⑭ Abfractions are commonly found in cases where malaligned canines cause initial lateral guidance forces to be exerted on the lingual incline of the buccal cusp of the maxillary premolar. ⑭ Abfraksiyonlar, maloklüzyonlu kaninlerin maksiller premoların bukkal tüberünün lingual eğimine ilk yan rehberlik kuvvetlerini uyguladığı durumlarda sıkça görülür.

① Faulty oral hygiene practice ① Hatalı ağız hijyeni uygulamaları

② – Horizontal brushing technique or improper brushing technique ② – Yatay fırçalama tekniği ya da yanlış fırçalama tekniği

③ – Overzealous brushing ③ – Aşırı istekli (şiddetli) fırçalama

④ – Use of toothbrush with hard bristles ④ – Sert kıllı diş fırçası kullanımı

⑤ – Use of abrasive toothpaste/toothpowder ⑤ – Aşındırıcı diş macunu ya da diş tozu kullanımı

⑥ – Excessive time, force and frequency of brushing ⑥ – Fırçalama süresinin, kuvvetinin ve sıklığının aşırı olması

⑦ – Excessive use of interproximal brushes ⑦ – Ara yüz fırçalarının aşırı kullanımı

① It is defined as a loss of tooth substance caused by a chemical process that does not involve known bacterial action. ① Bilinen bakteriyel etkiyi içermeyen kimyasal bir süreçle oluşan diş dokusu kaybı olarak tanımlanır.

② In general, erosion appears as sharply defined wedge shaped or irregular depression caused by chemicals in the cervical area usually on the facial tooth surface. ② Genellikle servikal bölgede, özellikle dişin yüzeyel kısmında, kimyasalların neden olduğu keskin sınırlı kama şeklinde veya düzensiz çöküntüler şeklinde görülür.

③ It is classified according to the source of the acid, i.e. either intrinsic or extrinsic. ③ Asit kaynağına göre sınıflandırılır; yani içsel (intrinsic) ya da dışsal (extrinsic) olabilir.

④ Intrinsic sources of acids originate in the stomach and are associated with eating disorders, such as anorexia and bulimia nervosa or with acid reflux and regurgitation. ④ İçsel asit kaynakları mide kökenlidir ve anoreksiya, bulimia nervoza gibi yeme bozuklukları ya da asit reflüsü ve regürjitasyon ile ilişkilidir.

⑤ Extrinsic sources are acids contained in dietary components, such as carbonated soft drinks and fruit and fruit juices. ⑤ Dışsal kaynaklar ise gazlı içecekler, meyveler ve meyve suları gibi besin bileşenlerinde bulunan asitlerdir.

① Noncarious cervical lesions are defined as any gradual loss of tooth structure characterized by the formation of smooth, polished surfaces, irrespective of their etiology. ① Etkene bakılmaksızın, düzgün ve cilalı yüzeylerin oluşumuyla karakterize edilen, diş dokusunun yavaş yavaş kaybı olarak tanımlanırlar.

② In these, loss of tooth structure occurs due to other lesions rather than caries. ② Bu lezyonlarda diş dokusu kaybı çürük dışı nedenlere bağlı olarak meydana gelir.

③ Noncarious cervical lesions can also be described as ‘wasting disease of the teeth’. ③ Çürüksüz servikal lezyonlar, dişlerin "aşındırıcı hastalığı" olarak da tanımlanabilir.

④ These lesions include abrasion, erosion and abfraction. ④ Bu lezyonlar aşınma (abrasion), erozyon (erosion) ve abfraksiyonu (abfraction) içerir.

⑤ These can occur pathologically, though they are seen in general associated with the aging process. ⑤ Bu lezyonlar patolojik olarak meydana gelebilir ancak genellikle yaşlanma süreciyle ilişkilidir.

⑥ These may show independently or simultaneously. ⑥ Bu lezyonlar tek başına ya da aynı anda bir arada görülebilir.

⑦ These are usually seen in more than 50 percent of the population. ⑦ Bu lezyonlar genellikle toplumun %50’sinden fazlasında görülür.

⑧ Noncarious cervical lesions cannot be referred as inflammatory lesions or developmental abnormalities but considered as regressive alteration of the teeth. ⑧ Çürüksüz servikal lezyonlar, inflamatuar lezyonlar veya gelişimsel anormallikler olarak değil; dişlerin gerileyici (regresif) değişimi olarak kabul edilir.

⑨ The clinical picture of these lesions can vary from shallow grooves to broad scooped out lesions, to large notched or wedge shaped defects. ⑨ Bu lezyonların klinik görünümü, yüzeyel oluklardan geniş oyuklara, büyük çentikli veya kama şeklindeki defektlere kadar değişebilir.

⑩ Improper diagnosis and thus treatment can cause continuous loss of tooth structure, dentin hypersensitivity, pulpal involvement or even the loss of tooth. ⑩ Yanlış tanı ve buna bağlı yanlış tedavi, sürekli diş dokusu kaybı, dentin hassasiyeti, pulpa tutulumu hatta diş kaybına yol açabilir.

⑪ The recent studies have shown improvements in the field of understanding etiology, diagnosis, treatment and prevention of these lesions. ⑪ Son çalışmalar bu lezyonların etiyolojisinin anlaşılması, tanısı, tedavisi ve önlenmesi konusunda ilerlemeler göstermiştir.

Diagnosis is best carried out with an explorer after cleaning the tooth surface. ① Tanı, diş yüzeyi temizlendikten sonra bir sond yardımıyla en iyi şekilde yapılır.

② Radiographs can be used as adjuncts but they should be free from overlapping or burnout. ② Radyografiler yardımcı olarak kullanılabilir ancak üst üste binme (overlapping) ya da yanma (burnout) olmamalıdır.

① Commonly seen when there is periodontal attachment loss exposing the root surface. ① Kök yüzeyinin açığa çıktığı periodontal bağlantı kaybı durumlarında yaygın olarak görülür.

② Usual site of occurrence is at or apical to the CEJ ② Genellikle görüldüğü yer, semento-mine birleşiminde (CEJ) veya onun apikalindedir.

③ A cervical carious lesion can be shallow saucer shaped or deep notch shaped ③ Servikal çürük lezyonu, sığ tabak şeklinde ya da derin çentik şeklinde olabilir.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Jiao D et al reported the induction of synthetic lethality by combined inhibition of anti-apoptotic BCL-2 family proteins and WSB2, a substrate receptor in CRL5 ubiquitin ligase complex. Mechanistically, WSB2 interacts with NOXA to promote its ubiquitylation and degradation. Cancer cells deficient in WSB2, as well as heart and liver tissues from Wsb2-/- mice exhibit high susceptibility to apoptosis induced by inhibitors of BCL-2 family proteins. The anti-apoptotic activity of WSB2 is partially dependent on NOXA.

Overall, the finding that WSB2 disruption triggers synthetic lethality to BCL-2 family protein inhibitors by destabilizing NOXA is rather novel. The manuscript is largely hypothesis-driven, with experiments that are adequately designed and executed. However, there are quite a few issues for the authors to address, including those listed below.

Specific comments from the previous round of review:

(1) At the beginning of the Results section, a clear statement is needed as to why the authors are interested in WSB2 and what brought them to analyze "the genetic co-dependency between WSB2 and other proteins".

(2) In general, the biochemical evidence supporting the role of WSB2 as a SOCS box-containing substrate-binding receptor of CRL5 E3 in promoting NOXA ubiquitylation and degradation is relatively weak. First, since NOXA2 binds to WSB2 on its SOCS box, which consists of a BC box for Elongin B/C binding and a CUL5 box for CUL5 binding, it is crucial to determine whether the binding of NOXA on the SOCS box affects the formation of CRL5WSB2 complex. The authors should demonstrate the endogenous binding between NOXA and the CRL5WSB2 complex. Additionally, the authors may also consider manipulating CUL5, SAG, or ElonginB/C to assess if it would affect NOXA protein turnover in two independent cell lines. Second, in all the experiments designed to detect NOXA ubiquitylation in cells, the authors utilized immunoprecipitation (IP) with FLAG-NOXA/NOXA, followed by immunoblotting (IB) with HA-Ub. However, it is possible that the observed poly-Ub bands could be partly attributed to the ubiquitylation of other NOXA binding proteins. Therefore, the authors need to consider performing IP with HA-Ub and subsequently IB with NOXA. Alternatively, they could use Ni-beads to pull down all His-Ub-tagged proteins under denaturing conditions, followed by the detection of FLAG-tagged NOXA using anti-FLAG Ab. The authors are encouraged to perform one of these suggested experiments to exclude the possibility of this concern. Furthermore, an in vitro ubiquitylation assay is crucial to conclusively demonstrate that the polyubiquitylation of NOXA is indeed mediated by the CRL5WSB2 complex.

(3) In their attempt to map the binding regions between NOXA and WSB2, the authors utilized exogenous proteins of both WSB2 and NOXA. To strengthen their findings, it would be more convincing to perform IP with exogenous wt/mutant WSB2 or NOXA and subsequently perform IB to detect endogenous NOXA or WSB2, respectively. Additionally, an in vitro binding assay using purified proteins would provide further evidence of a direct binding between NOXA and WSB2.

Comments on latest version:

The authors have adequately addressed my previous comments.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

I In this manuscript, Jiao D et al reported the induction of synthetic lethal by combined inhibition of anti-apoptotic BCL-2 family proteins and WSB2, a substrate receptor in CRL5 ubiquitin ligase complex. Mechanistically, WSB2 interacts with NOXA to promote its ubiquitylation and degradation. Cancer cells deficient in WSB2, as well as heart and liver tissues from Wsb2-/- mice exhibit high susceptibility to apoptosis induced by inhibitors of BCL-2 family proteins. The anti-apoptotic activity of WSB2 is partially dependent on NOXA.

Overall, the finding, that WSB2 disruption triggers synthetic lethality to BCL-2 family protein inhibitors by destabilizing NOXA, is rather novel. The manuscript is largely hypothesis-driven, with experiments that are adequately designed and executed. However, there are quite a few issues for the authors to address, including those listed below.

Specific comments:

(1) At the beginning of the Results section, a clear statement is needed as to why the authors are interested in WSB2 and what brought them to analyze "the genetic co-dependency between WSB2 and other proteins".

We thank the reviewer for raising this important point. We agree that a clear rationale should be provided at the beginning of the Results section. As reported in previous studies [Ref: 1, 2, 3], strong synthetic interactions have been observed between WSB2 and several mitochondrial apoptosis-related factors, including MCL-1, BCL-xL, and MARCH5. We have referenced these findings in the Discussion section. Motivated by these studies, we became interested in the role of WSB2 and aimed to investigate the specific mechanisms underlying its synthetic lethality with anti-apoptotic BCL-2 family members. We will revise the beginning of the Results section to clearly state this rationale.

(1) McDonald, E.R., 3rd et al. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577-592 e510 (2017).

(2) DeWeirdt, P.C. et al. Genetic screens in isogenic mammalian cell lines without single cell cloning. Nat Commun 11, 752 (2020).

(3) DeWeirdt, P.C. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat Biotechnol 39, 94-104 (2021).

(2) In general, the biochemical evidence supporting the role of WSB2 as a SOCS box-containing substrate-binding receptor of CRL5 E3 in promoting NOXA ubiquitylation and degradation is relatively weak. First, since NOXA binds to WSB2 on its SOCS box, which consists of a BC box for Elongin B/C binding and a CUL5 box for CUL5 binding, it is crucial to determine whether the binding of NOXA on the SOCS box affects the formation of CRL5WSB2 complex. The authors should demonstrate the endogenous binding between NOXA and the CRL5WSB2 complex. Additionally, the authors may also consider manipulating CUL5, SAG, or ElonginB/C to assess if it would affect NOXA protein turnover in two independent cell lines.

We thank the reviewer for raising this important point. To determine whether endogenous NOXA binds to the intact CRL5^WSB2 complex, we performed co-immunoprecipitation assays using an antibody against NOXA. Indeed, NOXA co-immunoprecipitated with all subunits of the CRL5^WSB2 complex (Figure 2—figure supplement 1D), suggesting that NOXA binding to WSB2 does not disrupt interactions between WSB2 and the other CRL5 subunits. Moreover, depletion of CRL5 complex components (RBX2/SAG, CUL5, ELOB, or ELOC) through siRNAs in C4-2B or Huh-7 cells also resulted in a marked increase in NOXA protein levels.

Second, in all the experiments designed to detect NOXA ubiquitylation in cells, the authors utilized immunoprecipitation (IP) with FLAG-NOXA/NOXA, followed by immunoblotting (IB) with HA-Ub. However, it is possible that the observed poly-Ub bands could be partly attributed to the ubiquitylation of other NOXA binding proteins. Therefore, the authors need to consider performing IP with HA-Ub and subsequently IB with NOXA. Alternatively, they could use Ni-beads to pull down all His-Ub-tagged proteins under denaturing conditions, followed by the detection of FLAG-tagged NOXA using anti-FLAG Ab. The authors are encouraged to perform one of these suggested experiments to exclude the possibility of this concern. Furthermore, an in vitro ubiquitylation assay is crucial to conclusively demonstrate that the polyubiquitylation of NOXA is indeed mediated by the CRL5WSB2 complex.

We appreciate the reviewer for raising these important considerations regarding our ubiquitylation assays. We fully acknowledge the reviewer's concern that classical ubiquitination assays could potentially detect ubiquitination of proteins interacting with NOXA. However, we would like to clarify that our experimental conditions effectively mitigate this issue. Specifically, cells were lysed using buffer containing 1% SDS followed by boiling at 105°C for 5 minutes. These rigorous denaturing conditions ensure disruption of non-covalent protein interactions, thereby effectively eliminating the possibility of detecting ubiquitination signals from NOXA-associated proteins.

Regarding the suggestion to perform an in vitro ubiquitination assay, we agree this experiment would indeed provide additional evidence. However, due to significant technical complexities associated with reconstituting CRL5-based E3 ubiquitin ligase activity in vitro—which would require the expression and purification of at least six recombinant proteins—such experiments are rarely performed in this context. Furthermore, NOXA is uniquely localized as a membrane protein on the mitochondrial outer membrane, posing additional significant challenges for protein expression and purification. Given the robustness of our current in vivo ubiquitylation assay under stringent denaturing conditions, we believe our existing data sufficiently and conclusively demonstrate NOXA ubiquitination mediated by the CRL5^WSB2 complex.

(3) In their attempt to map the binding regions between NOXA and WSB2, the authors utilized exogenous proteins of both WSB2 and NOXA. To strengthen their findings, it would be more convincing to perform IP with exogenous wt/mutant WSB2 or NOXA and subsequently perform IB to detect endogenous NOXA or WSB2, respectively. Additionally, an in vitro binding assay using purified proteins would provide further evidence of a direct binding between NOXA and WSB2.

We thank the reviewer for raising these important issues. In response to the reviewer’s suggestion to map the binding regions between NOXA and WSB2 more convincingly, we have indeed performed semi-endogenous Co-IP assays, which yielded results consistent with our exogenous protein experiments (Figure 3—figure supplement 1A, B). Concerning the recommendation to further validate direct interaction using purified recombinant proteins, we encountered substantial technical difficulties in obtaining pure and soluble recombinant WSB2 protein. Additionally, given that NOXA is an outer mitochondrial membrane protein and the interaction occurs on mitochondria, we believe that an in vitro binding assay may have limited physiological relevance. We hope the reviewer can appreciate these practical challenges and our current evidence supporting the strong interaction between NOXA and WSB2.

Reviewer #2 (Public Review):

Summary:

Exploring the DEP-MAP database and two drug-screen databases, the authors identify WSB2 as an interactor of several BCL2 proteins. In follow-up experiments, they show that CRL5/WSB2 controls NOXA protein levels via K48 ubiquitination following direct protein-protein interaction, and cell death sensitivity in the context of BH3 mimetic treatment, where WSB2 depletion synergizes with drug treatment.

Strengths:

The authors use a set of orthogonal methods across different model cell lines and a new WSB2 KO mouse model to confirm their findings. They also manage to correlate WSB2 expression with poor prognosis in prostate and liver cancer, supporting the idea that targeting WSB2 may sensitize cancers for treatment with BH3 mimetics.

Weaknesses:

The conclusions drawn based on the findings in cancer patients are very speculative, as regulation of NOXA cannot be the sole function of CRL5/WSB2 and it is hence unclear what causes correlation with patient survival. Moreover, the authors do not provide a clear mechanistic explanation of how exactly higher levels of NOXA promote apoptosis in the absence of WSB2. This would be important knowledge, as usually high NOXA levels correlate with high MCL1, as they are turned over together, but in situations like this, or loss of other E3 ligases, such as MARCH, the buffering capacity of MCL1 is outrun, allowing excess NOXA to kill (likely by neutralizing other BCL2 proteins it usually does not bind to, such as BCLX). Moreover, a necroptosis-inducing role of NOXA has been postulated. Neither of these options is interrogated here.

Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors):

(1) Figure 2J. The authors showed that "the mRNA levels of NOXA were even reduced in WSB2-KO cells compared to parental cells". What is the possible mechanism? This point should at least be discussed.

We thank the reviewer for raising these important issues. The underlying mechanisms for the significantly lower mRNA levels of NOXA following the KO of WSB2 are not fully understood at present. However, we propose that this could represent a form of negative feedback regulation at the level of gene expression. Specifically, when the protein levels of BNIP3/3L rise sharply, it may activate mechanisms that suppress their own mRNA synthesis or stability, serving as a buffering system to prevent further protein accumulation. Such negative feedback loops may be critical for maintaining cellular homeostasis and avoiding excessive protein production. Moreover, this phenomenon is frequently observed in other studies investigating substrates targeted by E3 ubiquitin ligases for degradation. We have elaborated on this point in the Discussion section.

(2) Figure 2M. A previous study has clearly demonstrated that NOXA is subjected to ubiquitylation and degradation by CRL5 E3 ligase (PMID: 27591266). This paper should be cited. Also, in that publication, NOXA ubiquitylation is via the K11 linkage, not the K48 linkage. The authors should include K11R mutant in their assay.

We thank the reviewer for raising this important issue. We thank the reviewer for suggesting the relevant reference (PMID: 27591266), which we have now cited accordingly. Additionally, we would like to clarify that our new in vivo ubiquitination assays included the K11R and K11-only ubiquitin mutants, and our data demonstrate that WSB2-mediated NOXA ubiquitination indeed involves the K11 linkage ubiquitination（Figure 2—figure supplement 1E).

(3) Figure 3H, J. The authors stated, "By mutating these lysine residues to arginine, we found that WSB2-mediated NOXA ubiquitination was completely abolished". Which one of the three lysine residues is playing the dominant role?

We thank the reviewer for raising this important issue. To address this, we generated FLAG-NOXA mutants individually substituting lysine residues K35, K41, and K48 with arginine. In vivo ubiquitination assays demonstrated that lysine 48 (K48) is the predominant residue responsible for WSB2-mediated NOXA ubiquitination (Figure 3—figure supplement 1C).

(4) Figure 3N. The authors need to show that the fusion peptide containing C-terminal NOXA peptide competitively inhibits the interaction between endogenous WSB2 and NOXA and extends the protein half-life of NOXA, leading to NOXA accumulation.

We sincerely thank the reviewer for raising these important issues. As suggested, we investigated whether the fusion peptide containing the C-terminal NOXA sequence competitively disrupts the interaction between endogenous WSB2 and NOXA, subsequently influencing NOXA stability. Our results demonstrated that treatment with this fusion peptide indeed significantly reduced the endogenous interaction between WSB2 and NOXA (Figure 3—figure supplement 1D). Furthermore, we observed that the peptide dose-dependently increased endogenous NOXA protein levels and prolonged its protein half-life, thereby resulting in the accumulation of NOXA (Figure 3N; Figure 3—figure supplement 1E, F). These findings collectively indicate that the fusion peptide competitively inhibits the WSB2-NOXA interaction, stabilizes NOXA protein, and enhances its accumulation.

(5) Figure 4. a) It would be better to investigate whether WSB2 knockdown can sensitize cancer cells to the treatment with ABT-737 or AZD5991, evidenced by a decrease in both IC50 values and clonogenic survival rates and whether such sensitization is dependent on NOXA. b) The authors need to show the levels of cleaved caspase-3/7/9 and the percentages of apoptotic cells in shNC cells upon silencing of WSB2 in Figure 4A-F. c) It will be more convincing to repeat the experiment to show synthetic lethality by WSB2 disruption and MCL-1 inhibitor AZD5991 treatment using another cell line, such as WSB2-deficient Huh-7 cells in Figure 4 I&J.

We sincerely thank the reviewer for these valuable and constructive suggestions. Regarding point (a): We believe that our current Western blot and flow cytometry data (Figure 4G–L) have already provided strong evidence that WSB2 depletion enhances apoptosis in response to ABT-737 and AZD5991. Therefore, we consider that additional IC50 and clonogenic survival assays, while informative, may not be essential for supporting our conclusion. Furthermore, as shown in Figure 5A–F, we found that silencing NOXA largely, though not completely, reversed the enhanced apoptosis triggered by these inhibitors in WSB2-deficient cells, suggesting that the sensitization effect is at least partially dependent on NOXA.

Regarding point (b): We have shown that WSB2 knockout alone had no impact on the levels of cleaved caspase-3/7/9 or the percentages of apoptotic cells in Huh-7 and C4-2B cells (Figure 4G-L and Figure 4—figure supplement 1A-D), indicating that WSB2 loss does not induce apoptosis on its own under basal conditions.

Regarding point (c): We appreciate the reviewer’s suggestion and have now repeated the experiment in WSB2 knockout Huh-7 cells. The new results further support the synthetic lethality between WSB2 loss and AZD5991 treatment (Figure 4—figure supplement 1C, D).

(6) Figure 5A/C/E. The effect of siNOXA is minor, if any, for cleavage of caspases. The same thing for Figure 6F/H.

We appreciate the reviewer’s insightful observation regarding the relatively modest effect of shNOXA on caspase cleavage in Figures 5A/C/E and Figures 6F/H. Indeed, we acknowledge that the reduction in caspase cleavage following NOXA knockdown is moderate. However, consistent with our discussions in the manuscript, NOXA knockdown significantly—but not completely—rescued the increased apoptosis observed in WSB2-deficient cells treated with BCL-2 family inhibitors. This suggests that while NOXA plays a notable role, additional mechanisms or unidentified targets may also be involved in WSB2-mediated regulation of apoptosis.

(7) Figure 5 I&J. The authors may consider performing IHC staining, immunofluorescence, or WB analysis to show the levels of NOXA and cleaved caspases or PARP in xenograft tumors. This would provide in vivo evidence of significant apoptosis induction resulting from the co-administration of ABT-737 and R8-C-terminal NOXA peptide.

We appreciate the reviewer's thoughtful suggestion regarding additional immunohistochemical or immunofluorescence analyses in xenograft tumors. However, due to current limitations in available antibodies suitable for reliable detection of NOXA by IHC and IF, we are unable to perform these experiments. We greatly appreciate the reviewer's understanding of this technical constraint. Nevertheless, our existing data collectively supports the conclusion that the combination of ABT-737 and R8-C-terminal NOXA peptide significantly enhances apoptosis in vivo.

(8) Figure 7. Does an inverse correlation exist between the protein levels of WSB2 and NOXA in RPAD or LIHC tissue microarrays? On page 12, in the first paragraph, Figure 7M-P was cited incorrectly.

We sincerely thank the reviewer for raising this important issue. As mentioned above, due to current limitations regarding the availability of suitable antibodies that can reliably detect NOXA by IHC, we regret that it is not feasible to experimentally address this question at this time.

Additionally, we have carefully corrected the citation error involving Figure 7M-P on page 12, as pointed out by the reviewer.

(9) Figure S1D. BCL-W levels were reduced upon WSB2 overexpression, which should be acknowledged.

We sincerely thank the reviewer for raising this important issue. We acknowledge that BCL-W protein levels were slightly reduced upon WSB2 overexpression in Figure S1D. However, this effect is distinct from the pronounced reduction observed in NOXA protein levels. We have revised the manuscript to clarify this point. Additionally, we recognize that transient overexpression systems may occasionally lead to non-specific or artifactual changes. Our exogenous expression and co-immunoprecipitation experiments did not support an interaction between BCL-W and WSB2. Therefore, the observed reduction of BCL-W under these conditions may not reflect a physiologically relevant regulation.

(10) Figure S4. Given WSB2 KO mice are viable; the authors may consider determining whether these mice are more sensitive to radiation-induced tissue damage or but more resistant to radiation-induced tumorigenesis?

We sincerely thank the reviewer for this insightful and biologically meaningful suggestion. We agree that investigating the potential role of WSB2 in radiation-induced tissue damage and tumorigenesis would be of great interest. However, conducting such experiments requires access to specialized irradiation facilities, which are currently unavailable to us. Nevertheless, we recognize the value of this line of investigation and plan to explore it in our future studies.

(11) All data were displayed as mean{plus minus}SD. However, for data from three independent experiments, it is more appropriate to present the results as mean{plus minus}SEM, not mean{plus minus}SD.

We sincerely thank the reviewer for highlighting this important issue. In line with the reviewer's suggestion, we have revised the manuscript accordingly and now present data from three independent experiments as mean ± SEM.

(12) The figure legends require careful review: i) The low dose of ABT-199 (Figure 6H) and the dose of ABT-199 used in Figure 6I are missing. ii) The legends for Figure S1D-E are incorrect. iii) The name of the antibody in the legend of Figure S3C is incorrect.

We sincerely thank the reviewer for raising these important issues. We have carefully corrected all the errors mentioned. In addition, we have thoroughly reviewed the manuscript to prevent similar errors.

Reviewer #2 (Recommendations For The Authors):

The authors focus on NOXA, after initially identifying WSB2 to interact with several BCL2 proteins. The rationale behind this is that WSB2 depletion or overexpression affects NOXA levels, but none of the other BCL2 proteins tested, as stated in the text. Yet, BCLW is also depleted upon overexpression of WSB2 (Supplementary Figure 1). How does this phenomenon relate to the sensitization noted, is BCL-W higher in WSB2 KO cells? It does not seem so though. This warrants discussion.

We appreciate the reviewer for raising this important issue. Our results showed that overexpression of WSB2 markedly reduced NOXA levels, while the levels of other BCL-2 family proteins remained unaffected or minimally affected, such as BCL-W (Figure 2—figure supplement 1A). Furthermore, depletion of WSB2 through shRNA-mediated KD or CRISPR/Cas9-mediated KO in C4-2B cells or Huh-7 cells led to a marked increase in the steady-state levels of endogenous NOXA, without affecting other BCL-2 family proteins examined, included BCL-W (Figure 2A-C, Figure 2—figure supplement 2A, B).

If WSB2 depletion does not affect MCL1 levels, how does excess NOXA actually kill? Does it bind to any (other) prosurvival proteins under conditions of WSB2 depletion? Is the MCL1 half-life changed?

We appreciate the reviewer for raising this important point. NOXA is a BH3-only protein known to promote apoptosis primarily by binding to and neutralizing anti-apoptotic BCL-2 family members, especially MCL-1, via its BH3 domain. It can inhibit MCL-1 either through competitive binding or by facilitating its ubiquitination and subsequent proteasomal degradation. In our system, the total protein levels of MCL-1 remained unchanged in WSB2 knockout cells, suggesting that NOXA may not be promoting apoptosis through enhanced MCL-1 degradation. Instead, we speculate that the accumulation of NOXA in WSB2-deficient cells enhances apoptosis by sequestering MCL-1 through direct binding, thereby freeing pro-apoptotic effectors such as BAK and BAX. In line with our observations, Nakao et al. reported that deletion of the mitochondrial E3 ligase MARCH5 led to a pronounced increase in NOXA expression, while leaving MCL-1 protein levels unchanged in leukemia cell lines (Leukemia. 2023 ;37:1028-1038., PMID: 36973350).

Additionally, NOXA has been reported to interact with other anti-apoptotic proteins, including BCL-XL. It is therefore possible that under conditions of WSB2 depletion, excess NOXA may also bind to BCL-XL and relieve its inhibition of BAX/BAK, further contributing to apoptosis. Future experiments assessing NOXA binding partners in WSB2-deficient cells would help clarify this mechanism.

I think some initial insights into the mechanism underlying the sensitization would add a lot to this study. Is there a role of BFL1/A1 in any of these cell lines, as it can also rather selectively bind to NOXA and is sometimes deregulated in cancer?

We appreciate the reviewer for raising this important issue. While BFL1/A1 is indeed another anti-apoptotic BCL-2 family member that can selectively bind to NOXA and has been implicated in cancer, our study primarily focuses on the WSB2-NOXA axis. However, given its potential involvement in apoptosis regulation, it would be an interesting direction for future studies to explore whether BFL1/A1 contributes to NOXA-mediated sensitization in specific cellular contexts.

Otherwise, this is a very nice and convincing study.

❶ Patients treated with radiotherapy suffer substantial changes to the oral mucosa and often require new complete or partial dentures. ❶ Radyoterapi gören hastalar ağız mukozasında önemli değişiklikler yaşar ve sıklıkla yeni tam veya parsiyel protezlere ihtiyaç duyarlar.

❷ Ideally, the oral soft tissue must be adequately healed before necessary prosthodontic procedures can be initiated. ❷ İdeal olarak, gerekli protez uygulamalarına başlanmadan önce ağız yumuşak dokusunun yeterince iyileşmiş olması gerekir.

❸ There are suggestions that a latent period of at least 6 months to 1 year should be provided. ❸ En az 6 ay ile 1 yıl arasında bir latent dönem bırakılması önerilmektedir.

❶ Limited mouth opening can interfere with proper oral hygiene and dental treatment. ❶ Sınırlı ağız açıklığı, uygun ağız hijyeni ve diş tedavisini engelleyebilir.

❷ Tongue blades can be used to gradually increase the mandibular opening. ❷ Alt çenenin açılmasını kademeli olarak artırmak için dil spatulaları kullanılabilir.

❸ Dynamic bite opening appliances have also been used. ❸ Dinamik ısırık açma aparatı da kullanılmaktadır.

❹ Primary treatment is essentially to exercise the involved muscles. ❹ Temel tedavi, ilgili kasların egzersiz yapılmasıdır.

❺ For patients who experience reduced mouth opening the intensity and frequency of the exercises should be increased. ❺ Ağız açıklığı azalan hastalarda egzersizlerin yoğunluğu ve sıklığı artırılmalıdır.

❻ Trismus and fibrosis will continue following RT, which will increase in severity with time. ❻ Trismus ve fibrozis radyoterapiden sonra devam edecek ve zamanla şiddeti artacaktır.

❼ This condition will only improve with constant exercise regimen. ❼ Bu durum ancak sürekli bir egzersiz programı ile iyileşir.

❽ Exercise should be performed deliberately at regular intervals followed by a period of rest. ❽ Egzersizler düzenli aralıklarla bilinçli şekilde yapılmalı ve ardından dinlenme dönemi gelmelidir.

❾ More frequently and diligently the exercise regimen, the more beneficial the result. ❾ Egzersiz programı ne kadar sık ve titizlikle uygulanırsa sonuç o kadar faydalı olur.

❿ Chronic trismus gradually converts into fibrosis of the muscles and at this late stage stretching of muscle is not favored as a solution. ❿ Kronik trismus zamanla kasların fibrozisine dönüşür ve bu ileri aşamada kas germe çözüm olarak tercih edilmez.

⓫ Exercise must begin early in treatment regimen. ⓫ Egzersiz, tedavi programının erken döneminde başlamalıdır.

❶ Trismus may begin shortly after radiation begins. ❶ Trismus, radyoterapi başladıktan kısa bir süre sonra başlayabilir.

❷ Patients suffering from tumors of the palate, nasopharynx, and maxillary sinus are most likely to develop the trismus. ❷ Damak, nazofarenks ve maksiller sinüs tümörleri olan hastalar en çok trismus geliştirme riski taşırlar.

❸ If unmanaged, trismus makes eating and swallowing difficult and various dental clinical procedures almost impossible. ❸ Tedavi edilmezse, trismus yeme ve yutmayı zorlaştırır ve çeşitli diş klinik işlemlerini neredeyse imkânsız hale getirir.

❹ Trismus can be significant side effect of radiotherapy especially if the lateral pterygoid muscles are in the field. ❹ Trismus, özellikle lateral pterygoid kaslar radyoterapi alanı içindeyse, radyoterapinin önemli bir yan etkisi olabilir.

❺ In patients whom the pterygoid muscles were irradiated and not the temporomandibular joint 31% experienced trismus. ❺ Pterygoid kasları ışınlanan ancak temporomandibular eklemi ışınlanmayan hastalarda %31 oranında trismus görülmüştür.

❻ In addition, radiation to the temporomandibular joint also was associated with a decrease in maximum vertical opening of the mouth. ❻ Ayrıca, temporomandibular ekleme uygulanan radyasyon da ağızın maksimum dikey açılmasında azalmayla ilişkilidir.

① Strict daily oral hygiene that includes fluoride and meticulous plaque removal has been shown to prevent the development of caries. Florür içeren ve plak temizliğinin titizlikle yapıldığı sıkı günlük ağız hijyeninin, çürük gelişimini önlediği gösterilmiştir.

② Chlorhexidine gel has also been shown to clinically reduce caries risk. Klorheksidin jelinin de klinik olarak çürük riskini azalttığı kanıtlanmıştır.

③ The daily use of 4% stannous fluoride also can be effective. Günde %4 stannous (kalaylı) florür kullanımı da etkili olabilir.

④ Alcohol‑free formulation should be selected to reduce discomfort in patients with dry mouth. Kuru ağız sorunu olan hastalarda rahatsızlığı azaltmak için alkolsüz formülasyonlar tercih edilmelidir.

⑤ (Topical fluorides or chlorhexidine rinses may lead to reduced level of S. mutans but not of Lactobacilli. Due to the possibility of adverse drug interactions, fluoride and chlorhexidine dosing should be separated by several hours). (Topikal florürler veya klorheksidin gargara S. mutans seviyesini düşürebilir ancak Lactobacilli seviyesini düşürmez. Olumsuz ilaç etkileşimleri olabileceği için florür ve klorheksidin uygulamaları arasında birkaç saat ara verilmelidir.)

① Dental caries risk increases secondary to a number of factors including a cariogenic flora, reduced concentration of salivary, antimicrobial proteins and loss of mineralizing components. Diş çürüğü riski, kariyojenik florası, tükürükteki antimikrobiyal proteinlerin azalması ve mineralize edici bileşenlerin kaybı gibi bir dizi faktöre bağlı olarak artar.

② One of the most effective methods of treating this condition is through the daily use of topical application of fluoride. Bu durumun tedavisinde en etkili yöntemlerden biri, günlük topikal florür uygulamasının yapılmasıdır.

③ Both stannous and sodium fluoride have been used in a variety of forms with significant success. Stannous (kalaylı) ve sodyum florür, çeşitli formlarda ve önemli başarıyla kullanılmıştır.

④ After standard radiotherapy, there is a profound shift in the oral microflora to predominance of acidogenic microbes, primarily Streptococcus mutans and Lactobacilli coincident with a decrease in salivary flora and an increase in caries risk. Standart radyoterapiden sonra, oral mikrofloarada belirgin bir değişiklik olur; asit üreten mikroorganizmalar, özellikle Streptococcus mutans ve Lactobacilli baskın hale gelir; tükürük florası azalırken çürük riski artar.

⑤ Dental caries in irradiated patients may develop rapidly as early as 3 months after radiotherapy. Işın tedavisi gören hastalarda diş çürükleri, radyoterapiden sadece 3 ay sonra hızla gelişebilir.

⑥ Radiation caries are seen at the cusp tip and incisor teeth’s edge side. Radyasyon kaynaklı çürükler genellikle dişlerin tüberkül uçlarında ve kesici dişlerin kenarlarında görülür.

❶ As a result, there may be changes in eating patterns with a decrease in nutritional intake and weight loss. ❶ Sonuç olarak, beslenme alımında azalma ve kilo kaybıyla birlikte yeme alışkanlıklarında değişiklikler olabilir.

❷ Dry mucosa may also be more prone to bleeding, resulting in bleeding gums. ❷ Kuru mukoza ayrıca daha fazla kanamaya yatkın olabilir ve bu da diş eti kanamalarına yol açabilir.

❸ An alcohol‑free mouth rinse is recommended because the alcohol can be irritating and dehydrating to the oral mucosa. ❸ Ağız mukozasına tahriş edici ve kurutucu etkisi olabileceğinden, alkolsüz ağız gargarası önerilir.

❹ Sialogogic, cholinergic agents are used to stimulate saliva production from whatever salivary gland tissue remains. ❹ Kalan tükürük bezi dokusundan tükürük üretimini uyarmak için sialogogik, kolinerjik ajanlar kullanılır.

❺ Pilocarpine, a muscarinic‑cholinergic agonist, is a naturally occurring plant alkaloid that stimulates salivation. ❺ Pilokarpin, muskarinik-kolinerjik agonist olan, tükürük salgısını uyaran doğal olarak oluşan bir bitki alkaloididir.

❶ Significant increase in the concentration of salivary sodium chloride calcium magnesium and protein accompanied the decrease in salivary output, and a reduction of bicarbonate content is also seen. ❶ Tükürük akışındaki azalmaya eşlik eden tükürükte sodyum, klorür, kalsiyum, magnezyum ve protein konsantrasyonlarında belirgin bir artış görülür; ayrıca bikarbonat içeriğinde azalma da vardır.

❷ These changes make xerostomic saliva a much more saline rich and poorly buffered secretion than normal saliva. ❷ Bu değişiklikler, kuru ağız (kserostomi) tükürüğünü normal tükürüğe göre çok daha tuzlu ve zayıf tamponlanmış bir salgı haline getirir.

❶ The long‑term effects are due to changes in the vascularity and cellularity of soft tissue and bone, damage to salivary glands and increased collagen synthesis, leading to hypovascularity, hypocellularity, and hypoxia of the tissues: ❶ Uzun vadeli etkiler, yumuşak doku ve kemikteki damar yapısı ve hücresel yapının değişmesi, tükürük bezlerine zarar verilmesi ve artan kollajen sentezi nedeniyle oluşur; bu da dokularda azalmış damar yoğunluğu (hipovaskülarite), hücre sayısında azalma (hiposelülarite) ve oksijen yetersizliği (hipoksi) ile sonuçlanır:

❷ Common chronic complications are xerostomia, dental alteration, rampant caries, demineralization, alteration or loss of taste (dysgeusia), secondary opportunistic infection (e.g., candidiasis). Oral ulceration (soft tissue necrosis). Osteo radionecrosis, pain, temporomandibular joint disease, trismus, nutritional deficiencies, psychological impact. ❷ Yaygın kronik komplikasyonlar arasında kuru ağız (kserostomi), diş değişiklikleri, yaygın çürükler, mineral kaybı (demineralizasyon), tat değişikliği veya kaybı (disgeüzi), ikincil fırsatçı enfeksiyonlar (örneğin kandidiyazis), ağız ülserleri (yumuşak doku nekrozu), osteoradyonekroz, ağrı, temporomandibular eklem hastalığı, trismus, beslenme yetersizlikleri ve psikolojik etkiler yer alır.

❸ To a significant degree, the oral problem associated with RT can be prevented or minimized through optimal management ❸ Radyoterapiye bağlı ağız problemleri, uygun yönetimle önemli ölçüde önlenebilir veya azaltılabilir.

❶ Damage to the lining of the mouth and weakened immune system makes it easy for the infection to occur. ❶ Ağız astarının (mukozasının) zarar görmesi ve bağışıklık sisteminin zayıflaması, enfeksiyonun oluşmasını kolaylaştırır.

❷ Oral mucositis breaks down the lining of the mouth which lets, bacteria, viruses, and fungal get into the blood. ❷ Oral mukozit, ağız astarını bozar ve bu durum bakteri, virüs ve mantarların kana karışmasına neden olabilir.

❸ Dry mouth which is common during radiotherapy to head and neck may also raise the risk of infection in the mouth. ❸ Baş ve boyun radyoterapisi sırasında yaygın olan ağız kuruluğu da ağızda enfeksiyon riskini artırabilir.

❹ Infection may be caused by bacteria, virus or fungal. ❹ Enfeksiyon; bakteri, virüs veya mantar kaynaklı olabilir.

❺ A systemic review indicated that the weighted mean prevalence of clinical oral candidiasis during head and neck RT is 37.4%. ❺ Sistematik bir derleme, baş ve boyun radyoterapisi sırasında klinik oral kandidiyazis görülme sıklığının ağırlıklı ortalama %37.4 olduğunu göstermiştir.

❶ Common sites of oral cavity mucositis include the buccal, labial, and soft palate mucosa along with ventral surface of tongue, floor of the mouth. ❶ Oral kavite mukozitinin yaygın bölgeleri; bukkal (yanak), labial (dudak), yumuşak damak mukozası ile birlikte dilin alt yüzeyi ve ağız tabanıdır.

❷ If the pharynx is involved, the individual may have a sore throat and difficulties in swallowing and talking. ❷ Farenks etkilenmişse, kişide boğaz ağrısı ile birlikte yutma ve konuşmada güçlük görülebilir.

❸ Esophagitis is caused by damage to the mucosal lining and usually presents as dysphagia. ❸ Özofajit, mukoza tabakasının zarar görmesiyle oluşur ve genellikle disfaji (yutma güçlüğü) şeklinde kendini gösterir.

❹ Mucositis can cause severe pain and increase the risk of development of systemic infections from bacterial, fungal, or viral infections in the mouth. ❹ Mukozit şiddetli ağrıya neden olabilir ve ağızda bakteri, mantar veya virüs kaynaklı sistemik enfeksiyon gelişme riskini artırabilir.

❺ The hyperemic and edematous mucosa initially appears reddened. ❺ Hiperemik (aşırı kanlanmış) ve ödemli mukoza başlangıçta kızarık görünür.

❻ This erythema is due to a thinning of the epithelium and vascular dilation, inflammation and edema of the submucosa. ❻ Bu eritem (kızarıklık), epitelin incelmesi ve damar genişlemesi ile submukozanın iltihabı ve ödeminden kaynaklanır.

❼ With continued radiotherapy, the mucosa becomes denuded, ulcerated and covered with fibrinous exudates. ❼ Radyoterapi devam ettikçe mukoza soyulur, ülserleşir ve fibrinöz eksüdatlarla kaplanır.

❽ This is accompanied by pain, burning, and discomfort, which are greatly aggravated by contact with coarse or highly seasoned food. ❽ Bu duruma ağrı, yanma ve rahatsızlık eşlik eder; bunlar özellikle sert veya baharatlı yiyeceklerle temasla ciddi şekilde artar.

❾ Depending on the extent of the treatment field, involvement of the pharyngeal mucosa may produce difficulties in swallowing and speech. ❾ Tedavi alanının kapsamına bağlı olarak farengeal mukozanın tutulumu, yutma ve konuşma zorluklarına yol açabilir.

❿ This is often at its worst in 2–3 weeks of treatment, with symptoms usually diminishing thereafter completion of treatment. ❿ Bu durum genellikle tedavinin 2–3. haftasında en kötü halini alır ve belirtiler tedavi tamamlandıktan sonra genellikle azalır.

⓫ After 4 weeks of treatment, 90–95% of patients show complete resolution of mucositis and sore throat is absent or minimum. ⓫ Tedaviden 4 hafta sonra, hastaların %90–95'inde mukozit tamamen iyileşir ve boğaz ağrısı ya hiç kalmaz ya da minimum düzeye iner.

One of the earliest complications of RT is the development of mucositis. ❶ Radyoterapinin (RT) en erken komplikasyonlarından biri mukozitin gelişmesidir.

❷ The soft tissues in the treatment field after a week or two demonstrate a moderate amount of erythema. ❷ Tedavi alanındaki yumuşak dokular, bir veya iki hafta sonra orta derecede eritem (kızarıklık) gösterir.

❸ As the radiation continues, the mucosa may exhibit varying degree of desquamation and frank ulceration, resulting in pain and dysphagia which makes patients to eat a well-balanced diet leading to significant weight loss and malnutrition. ❸ Radyasyon devam ettikçe, mukozada değişen derecelerde soyulma ve belirgin ülserasyon görülebilir; bu durum ağrı ve yutma güçlüğüne yol açar, hastaların dengeli beslenmesini zorlaştırır ve bu da ciddi kilo kaybı ve malnütrisyona neden olur.

❶ The goal of this evaluation is to identify teeth at significant risk of infection, broken teeth that would optimally require aggressive or invasive dental treatment during and after the radiation that increases the risk of soft tissue necrosis and osteonecrosis. ❶ Bu değerlendirmenin amacı, enfeksiyon riski yüksek olan dişleri ve kırık dişleri tespit etmektir; çünkü bu dişler radyasyon sırasında veya sonrasında agresif ya da invaziv diş tedavileri gerektirebilir ve bu da yumuşak doku nekrozu ve osteonekroz riskini artırır.

Reviewer #1 (Public review):

Summary:

The manuscript focuses on the olfactory system of Pieris brassicae larvae and the importance of olfactory information in their interactions with the host plant Brassica oleracea and the major parasitic wasp Cotesia glomerata. The authors used CRISPR/Cas9 to knockout odorant receptor co-receptors (Orco), and conducted a comparative study on the behavior and olfactory system of the mutant and wild-type larvae. The study found that Orco-expressing olfactory sensory neurons in antennae and maxillary palps of Orco knockout (KO) larvae disappeared, and the number of glomeruli in the brain decreased, which impairs the olfactory detection and primary processing in the brain. Orco KO caterpillars show weight loss and loss of preference for optimal food plants; KO larvae also lost weight when attacked by parasitoids with the ovipositor removed, and mortality increased when attacked by untreated parasitoids. On this basis, the authors further studied the responses of caterpillars to volatiles from plants attacked by the larvae of the same species and volatiles from plants on which the caterpillars were themselves attacked by parasitic wasps. Lack of OR mediated olfactory inputs prevents caterpillars from finding suitable food sources and from choosing spaces free of enemies.

Strengths:

The findings help to understand the important role of olfaction in caterpillar feeding and predator avoidance, highlighting the importance of odorant receptor genes in shaping ecological interactions.

Weaknesses:

There are the following major concerns:

(1) Possible non-targeted effects of Orco knockout using CRISPR/Cas9 should be analyzed and evaluated in Materials and Methods and Results

(2) Figure 1E: Only one olfactory receptor neuron was marked in WT. There are at least three olfactory sensilla at the top of the maxillary palp. Therefore, to explain the loss of Orco expressing neurons in the mutant (Figure 1F), a more rigorous explanation of the photo is required.

(3) In Figure 1G, H, the four glomeruli circled by dotted lines: their corresponding relationship between the two figures needs to be further clarified.

(4) Line 130: Since the main topic in this study is the olfactory system of larvae, the experimental results of this part are all about antennal electrophysiological responses, mating frequency and egg production of female and male adults of wild type and Orco KO mutant, it may be considered to include this part in the supplementary files. It is better to include some data about the olfactory responses of larvae.

(5) Line 166: The sentences in the text is about the choice test between " healthy plant vs. infested plant", while in Fig 3C, it is "infested plant vs. no plant". The content in the text does not match the figure.

(6) Lines 174-178: Fig 3A showed that the body weight of Orco KO larvae in the absence of parasitic wasps also decreased compared with that of WT. Therefore, in the experiments of Fig 3A and E, the difference in the body weight of Orco KO larvae in the presence or absence of parasitic wasps without ovipositors should also be compared. The current data cannot determine the reduced weight of KO mutant is due to the Orco knockout or the presence of parasitic wasps.

(7) Lines 179-181: Fig 3F show that the survival rate of larvae of Orco KO mutant decreased in the presence of parasitic wasps, and the difference in survival rate of larvae of WT and Orco KO mutant in the absence of parasitic wasps should also be compared. The current data cannot determine whether the reduced survival of the KO mutant is due to the Orco knockout or the presence of parasitic wasps.

(8) In Figure 4B, why do the compounds tested had no volatiles derived from plants? Cruciferous plants have the well-known mustard bomb. In the behavioral experiments the larvae responses to ITC compounds were not included, which is suggested to be explained in the discussion section.

(9) The custom-made setup and the relevant behavioral experiments in Fig 4C needs to be described in detail (Line 545).

(10) Materials and Methods Line 448: 10 μL paraffin oil should be used for negative control.

Comments on revised version:

The authors have replied my concerns and made revisions accordingly.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript focuses on the olfactory system of Pieris brassicae larvae and the importance of olfactory information in their interactions with the host plant Brassica oleracea and the major parasitic wasp Cotesia glomerata. The authors used CRISPR/Cas9 to knockout odorant receptor coreceptors (Orco), and conducted a comparative study on the behavior and olfactory system of the mutant and wild-type larvae. The study found that Orco-expressing olfactory sensory neurons in antennae and maxillary palps of Orco knockout (KO) larvae disappeared, and the number of glomeruli in the brain decreased, which impairs the olfactory detection and primary processing in the brain. Orco KO caterpillars show weight loss and loss of preference for optimal food plants; KO larvae also lost weight when attacked by parasitoids with the ovipositor removed, and mortality increased when attacked by untreated parasitoids. On this basis, the authors further studied the responses of caterpillars to volatiles from plants attacked by the larvae of the same species and volatiles from plants on which the caterpillars were themselves attacked by parasitic wasps. Lack of OR-mediated olfactory inputs prevents caterpillars from finding suitable food sources and from choosing spaces free of enemies.

Strengths:

The findings help to understand the important role of olfaction in caterpillar feeding and predator avoidance, highlighting the importance of odorant receptor genes in shaping ecological interactions.

Weaknesses:

There are the following major concerns:

(1) Possible non-targeted effects of Orco knockout using CRISPR/Cas9 should be analyzed and evaluated in Materials and Methods and Results.

Thank you for your suggestion. In the Materials and Methods, we mention how we selected the target region and evaluated potential off-target sites by Exonerate and CHOPCHOP. Neither of these methods found potential off-target sites with a more-than-17-nt alignment identity. Therefore, we assumed no off-target effect in our Orco knockout. Furthermore, we did not find any developmental differences between wildtype and knockout caterpillars when these were reared on leaf discs in Petri dishes (Fig S4). We will further highlight this information on the off-target evaluation in the Results section.

(2) Figure 1E: Only one olfactory receptor neuron was marked in WT. There are at least three olfactory sensilla at the top of the maxillary palp. Therefore, to explain the loss of Orcoexpressing neurons in the mutant (Figure 1F), a more rigorous explanation of the photo is required.

Thank you for pointing this out. The figure shows only a qualitative comparison between WT and KO and we did not aim to determine the total number of Orco positive neurons in the maxillary palps or antennae of WT and KO caterpillars, but please see our previous work for the neuron numbers in the caterpillar antennae (Wang et al., 2024). We did indeed find more than one neuron in the maxillary palps, but as these were in very different image planes it was not possible to visualize them together. However, we will add a few sentences in the Results and Discussion section to explain the results of the maxillary palp Orco staining.

(3) In Figure 1G, H, the four glomeruli are circled by dotted lines: their corresponding relationship between the two figures needs to be further clarified.

Thank you for pointing this out. The four glomeruli in Figure 1G and 1H are not strictly corresponding. We circled these glomeruli to highlight them, as they are the best visualized and clearly shown in this view. In this study, we only counted the number of glomeruli in both WT and KO, however, we did not clarify which glomeruli are missing in the KO caterpillar brain. We will further clarify this in the figure legend.

(4) Line 130: Since the main topic in this study is the olfactory system of larvae, the experimental results of this part are all about antennal electrophysiological responses, mating frequency, and egg production of female and male adults of wild type and Orco KO mutant, it may be considered to include this part in the supplementary files. It is better to include some data about the olfactory responses of larvae.

Thank you for your suggestion. We do agree with your suggestion, and we will consider moving this part to the supplementary information. Regarding larval olfactory response, we unfortunately failed to record any spikes using single sensillum recordings due to the difficult nature of the preparation; however we do believe that this would be an interesting avenue for further research.

(5)Line 166: The sentences in the text are about the choice test between " healthy plant vs. infested plant", while in Fig 3C, it is "infested plant vs. no plant". The content in the text does not match the figure.

Thank you for pointing this out. The sentence is “We compared the behaviors of both WT and Orco KO caterpillars in response to clean air, a healthy plant and a caterpillar-infested plant”. We tested these three stimuli in two comparisons: healthy plant vs no plant, infested plant vs no plant. The two comparisons are shown in Figure 3C separately. We will aim to describe this more clearly in the revised version of this manuscript.

(6) Lines 174-178: Figure 3A showed that the body weight of Orco KO larvae in the absence of parasitic wasps also decreased compared with that of WT. Therefore, in the experiments of Figure 3A and E, the difference in the body weight of Orco KO larvae in the presence or absence of parasitic wasps without ovipositors should also be compared. The current data cannot determine the reduced weight of KO mutant is due to the Orco knockout or the presence of parasitic wasps.

Thank you for pointing this out. We did not make a comparison between the data of Figures 3A and 3E since the two experiments were not conducted at the same time due to the limited space in our BioSafety III greenhouse. We do agree that the weight decrease in Figure 3E is partly due to the reduced caterpillar growth shown in Figure 3A. However, we are confident that the additional decrease in caterpillar weight shown in Figure 3E is mainly driven by the presence of disarmed parasitoids. To be specific, the average weight in Figure 3A is 0.4544 g for WT and 0.4230 g for KO, KO weight is 93.1% of WT caterpillars. While in Figure 3E, the average weight is 0.4273 g for WT and 0.3637 g for KO, KO weight is 85.1% of WT caterpillars. We will discuss this interaction between caterpillar growth and the effect of the parasitoid attacks more extensively in the revised version of the manuscript.

(7) Lines 179-181: Figure 3F shows that the survival rate of larvae of Orco KO mutant decreased in the presence of parasitic wasps, and the difference in survival rate of larvae of WT and Orco KO mutant in the absence of parasitic wasps should also be compared. The current data cannot determine whether the reduced survival of the KO mutant is due to the Orco knockout or the presence of parasitic wasps.

We are happy that you highlight this point. When conducting these experiments, we selected groups of caterpillars and carefully placed them on a leaf with minimal disturbance of the caterpillars, which minimized hurting and mortality. We did test the survival of caterpillars in the absence of parasitoid wasps from the experiment presented in Figure 3A, although this was missing from the manuscript. There is no significant difference in the survival rate of caterpillars between the two genotypes in the absence of wasps (average mortality WT = 8.8 %, average mortality KO = 2.9 %; P = 0.088, Wilcoxon test), so the decreased survival rate is most likely due to the attack of the wasps. We will add this information to the revised version of the manuscript.

(8) In Figure 4B, why do the compounds tested have no volatiles derived from plants? Cruciferous plants have the well-known mustard bomb. In the behavioral experiments, the larvae responses to ITC compounds were not included, which is suggested to be explained in the discussion section.

Thank you for the suggestion. We assume you mean Figure 4D/4E instead of Figure 4B. In Figure 4B, many of the identified chemical compounds are essentially plant volatiles, especially those from caterpillar frass and caterpillar spit. In Figure 4D/4E, most of the tested chemicals are derived from plants. But indeed, we did not include ITCs, based on information from the EAG results in Figures 2A & 2B. Butterfly antennae did not respond strongly to ITCs, so we did not include ITCs in the larval behavioural tests. Instead, the tested chemicals in Figure 4D/4E either elicit high EAG responses of butterflies or have been identified as “important” by VIP scores in the chemical analyses. In the EAG results of Plutella xylostella (Liu et al., 2020), moths responded well to a few ITCs, the tested ITCs in our study are actually adopted from this study except for those that were not available to us. However, butterflies did not show a strong response to the tested ITCs; therefore, we did not include ITCs because we expected that Pieris brassicae caterpillars are not likely to show good responses to ITCs. We will add this explanation to the revised version of our manuscript.

(9) The custom-made setup and the relevant behavioral experiments in Figure 4C need to be described in detail (Line 545).

We will add more detailed descriptions for the setup and method in the Materials and Methods.

(10) Materials and Methods Line 448: 10 μL paraffin oil should be used for negative control.

Thank you for pointing this out. We used both clean filter paper and clean filter paper with 10 μL paraffin oil as negative controls, but we did not find a significant difference between the two controls. Therefore, in the EAG results of Figure 2A/2B, we presented paraffin oil as one of the tested chemicals. We will re-run our statistical tests with paraffin oil as negative control, although we do not expect any major differences to the previous tests.

Reviewer #2 (Public review):

Summary:

This manuscript investigated the effect of olfactory cues on caterpillar performance and parasitoid avoidance in Pieris brassicae. The authors knocked out Orco to produce caterpillars with significantly reduced olfactory perception. These caterpillars showed reduced performance and increased susceptibility to a parasitoid wasp.

Strengths:

This is an impressive piece of work and a well-written manuscript. The authors have used multiple techniques to investigate not only the effect of the loss of olfactory cues on host-parasitoid interactions, but also the mechanisms underlying this.

Weaknesses:

(1) I do have one major query regarding this manuscript - I agree that the results of the caterpillar choice tests in a y-maze give weight to the idea that olfactory cues may help them avoid areas with higher numbers of parasitoids. However, the experiments with parasitoids were carried out on a single plant. Given that caterpillars in these experiments were very limited in their potential movement and source of food - how likely is it that avoidance played a role in the results seen from these experiments, as opposed to simply the slower growth of the KO caterpillars extending their period of susceptibility? While the two mechanisms may well both take place in nature - only one suggests a direct role of olfaction in enemy avoidance at this life stage, while the other is an indirect effect, hence the distinction is important.

We do agree with your comment that both mechanisms may be at work in nature and we do address this in the Discussion section. In our study, we did find that wildtype caterpillars were more efficient in locating their food source and did grow faster on full plants than knockout caterpillars. This faster growth will enable wildtype caterpillars to more quickly outgrow the life-stages most vulnerable to the parasitoids (L1 and L2). The olfactory system therefore supports the escape from parasitoids indirectly by enhancing feeding efficiency directly.

Figure 3D shows that WT caterpillars prefer infested plants without parastioids to infested plants with parasitoids. In addition, we observed that caterpillars move frequently between different leaves. Therefore, we speculate that WT caterpillars make use of volatiles from the plant or from (parasitoid-exposed) conspecifics via their spit or faeces to avoid parts of the plant potentially attracting natural enemies. Knockout caterpillars are unable to use these volatile danger cues and therefore do not avoid plant parts that are most attractive to their natural enemies, making KO caterpillars more susceptible and leading to more natural enemy harassment. Through this, olfaction also directly impacts the ability of a caterpillar to find an enemy-free feeding site.

We think that olfaction supports the enemy avoidance of caterpillars via both these mechanisms, although at different time scales. Unfortunately, our analysis was not detailed enough to discern the relative importance of the two mechanisms we found. However, we feel that this would be an interesting avenue for further research. Moreover, we will sharpen our discussion on the potential importance of the two different mechanisms in the revised version of this manuscript.

(2) My other issue was determining sample sizes used from the text was sometimes a bit confusing. (This was much clearer from the figures).

We will revise the sample size in the text to make it more clear.

(3) I also couldn't find the test statistics for any of the statistical methods in the main text, or in the supplementary materials.

Thank you for pointing this out. We will provide more detailed test statistics in the main text and in the supplementary materials of the revised version of the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Abstract

Line 24: "optimal food plant" should be changed to "optimal food plants"

Thank you for the suggestion, we will revise it.

(2) Introduction

Lines 44-46: The sentence should be rephrased.

Thank you for the suggestion, we will revise it.

Line 50: "are" should be changed to "is".

Thank you for the suggestion, we will revise it.

Lines 57 and 58: Please provide the Latin names of "brown planthoppers" and "striped stem borer".

Thank you for the suggestion, we will revise it.

Line 85: "investigate the influence of odor-guided behavior by this primary herbivore on the next trophic levels"; similarly, Line 160: "investigate if caterpillars could locate the optimal host-plant when supplied with differently treated plants". These sentences are not very accurate in describing the relevant experiments. A: Thank you for the suggestion, we will revise them.

Reviewer #2 (Recommendations for the authors):

(1) L53 Remove the "the" from "Under the strong selection pressure"

Thank you for the suggestion, we will revise it.

(2) L80 I suggest adding a reference for the spitting behaviour, e.g. Muller et al 2003.

Thank you for the suggestion, we will add it.

(3) L89 establishing a homozygous KO insect colony.

Thank you for the suggestion, we will revise it.

(4) L107 perhaps this goes against the journal style but I always like to see acronyms explained the first time they are used.

Thank you for the suggestion, we will try to make it more understandable.

(5) L146-148 sentence difficult to read - consider rephrasing.

Thank you for the suggestion, we will revise it.

(6) L230 do you mean still produce? Rather than still reproduce?

Thank you for the suggestion, we will revise it.

(7) L233 missing an and before "a greater vulnerability to the parasitoid wasp".

Thank you for pointing this out, we will revise it.

(8) L238 malfunctional is a strange word choice.

Thank you for pointing this out, we will revise it.

(9) L181 - can the authors confirm that this lower survival was due to parasitism by the wasps?

This question is similar to Q(7) of Reviewer 1, so we quote our answer for Q(7) here:

When conducting these experiments, we selected groups of caterpillars and carefully placed them on a leaf with minimal disturbance of the caterpillars, which minimized hurting and mortality. We did test the survival of caterpillars in the absence of parasitoid wasps from the experiment presented in Figure 3A, although this was missing from the manuscript. There is no significant difference in the survival rate of caterpillars between the two genotypes in the absence of wasp (average mortality WT = 8.8 %, average mortality KO = 2.9 %; P = 0.088, Wilcoxon test), so the decreased survival rate is most likely due to the attack of the wasps. We will add this information to the revised version of the manuscript.

(10) L474 - has it been tested if wasps still behave similarly after their ovipositor has been removed?

Thank you for pointing out this issue. We did not strictly compare if disarmed and untreated wasps have similar behaviors. However, we did observe if disarmed wasps can actively move or fly after recovering from anesthesia before releasing into a cage, otherwise we would replace with another active one.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study aims to identify the proteins that compose the electrical synapse, which are much less understood than those of the chemical synapse. Identifying these proteins is important to understand how synaptogenesis and conductance are regulated in these synapses. The authors identified more than 50 new proteins and used immunoprecipitation and immunostaining to validate their interaction of localization. One new protein, a scaffolding protein, shows particularly strong evidence of being an integral component of the electrical synapse. However, many key experimental details are missing (e.g. mass spectrometry), making it difficult to assess the strength of the evidence.

Strengths:

One newly identified protein, SIPA1L3, has been validated both by immunoprecipitation and immunohistochemistry. The localization at the electrical synapse is very striking.<br /> A large number of candidate interacting proteins were validated with immunostaining in vivo or in vitro.

Weaknesses:

There is no systematic comparison between the zebrafish and mouse proteome. The claim that there is "a high degree of evolutionary conservation" was not substantiated.

We have added a table as supplementary figure 3 that shows a comparison of all candidates. While there are differences in both proteomes, components such as ZO proteins and the endocytosis machinery are clearly conserved.

No description of how mass spectrometry was done and what type of validation was done.

We have contacted the mass spec facility we worked with and added a paragraph explaining the mass spec. procedure in the material and methods section.

The threshold for enrichment seems arbitrary.

Yes, the thresholds are somewhat arbitrary. This is due to the fact that experiments that captured larger total amounts of protein (mouse retina samples) had higher signal-to-noise ratio than those that captured smaller total amounts of protein (zebrafish retina). This allowed us to use a more stringent threshold in the mouse dataset to focus on high probability captured proteins.

Inconsistent nomenclature and punctuation usage.

We have scanned through the manuscript and updated terms that were used inconsistently in the interim revision of the manuscript.

The description of figures is very sparse and error-prone (e.g. Figure 6).

In Figure 1B, there is very broad non-specific labeling by avidin in zebrafish (In contrast to the more specific avidin binding in mice, Figure 2B). How are the authors certain that the enrichment is specific at the electrical synapse?

The enrichment of the proteins we identified is specific for electrical synapses because we compared the abundance of all candidates between Cx35b-V5-TurboID and wildtype retinas. Proteins that are components of electrical synapses, will only show up in the Cx35b-V5-TurboID condition. The western blot (Strep-HRP) in figure 1C shows the differences in the streptavidin labeling and hence the enrichment of proteins that are part of electrical synapses. Moreover, while the background appears to be quite abundant in sections, biotinylation is a rare posttranslational modification and mainly occurs in carboxylases: The two intense bands that show up above 50 and 75 kDa. The background mainly originates from these two proteins. Therefore, it is easy to distinguish specific hits from non-specific background.

In Figure 1E, there is very little colocalization between Cx35 and Cx34.7. More quantification is needed to show that it is indeed "frequently associated."

We agree that “frequently associated” is too strong as a statement. We corrected this and instead wrote “that Cx34.7 was only expressed in the outer plexiform layer (OPL) where it was associated with Cx35b at some gap junctions” in line 151. There are many gap junctions at which Cx35b is not colocalized with Cx34.7.

Expression of GFP in HCs would potentially be an issue, since GFP is fused to Cx36 (regardless of whether HC expresses Cx36 endogenously) and V5-TurboID-dGBP can bind to GFP and biotinylate any adjacent protein.

Thank you for this suggestion! There should be no Cx36-GFP expression in horizontal cells, which means that the nanobody cannot bind to anything in these cells. Moreover, to recognize specific signals from non-specific background, we included wild type retinas throughout the entire experiments. This condition controls for non-specific biotinylation.

Figure 7: the description does not match up with the figure regarding ZO-1 and ZO-2.

It appears that a portion of the figure legend was left out of the submitted version of the manuscript. We have put the legend for panels A through C back into the manuscript in the interim revision.

Reviewer #2 (Public review):

Summary:

This study aimed to uncover the protein composition and evolutionary conservation of electrical synapses in retinal neurons. The authors employed two complementary BioID approaches: expressing a Cx35b-TurboID fusion protein in zebrafish photoreceptors and using GFP-directed TurboID in Cx36-EGFP-labeled mouse AII amacrine cells. They identified conserved ZO proteins and endocytosis components in both species, along with over 50 novel proteins related to adhesion, cytoskeleton remodeling, membrane trafficking, and chemical synapses. Through a series of validation studies¬-including immunohistochemistry, in vitro interaction assays, and immunoprecipitation - they demonstrate that novel scaffold protein SIPA1L3 interacts with both Cx36 and ZO proteins at electrical synapse. Furthermore, they identify and localize proteins ZO-1, ZO-2, CGN, SIPA1L3, Syt4, SJ2BP, and BAI1 at AII/cone bipolar cell gap junctions.

Strengths:

The study demonstrates several significant strengths in both experimental design and validation approaches. First, the dual-species approach provides valuable insights into the evolutionary conservation of electrical synapse components across vertebrates. Second, the authors compare two different TurboID strategies in mice and demonstrate that the HKamac promoter and GFP-directed approach can successfully target the electrical synapse proteome of mouse AII amacrine cells. Third, they employed multiple complementary validation approaches - including retinal section immunohistochemistry, in vitro interaction assays, and immunoprecipitation-providing evidence supporting the presence and interaction of these proteins at electrical synapses.

Weaknesses:

The conclusions of this paper are supported by data; however, some aspects of the quantitative proteomics analysis require clarification and more detailed documented. The differential threshold criteria (>3 log2 fold for mouse vs >1 log2 fold for zebrafish) will benefit from biological justification, particularly given the cross-species comparison. Additionally, providing details on the number of biological or technical replicates used in this study, along with analyses of how these replicates compare to each other, would strengthen the confidence in the identification of candidate proteins. Furthermore, including negative controls for the histological validation of proteins interacting with Cx36 could increase the reliability of the staining results.

While the study successfully characterized the presence of candidate proteins at the electrical synapses between AII amacrine cells and cone bipolar cells, it did not compare protein compositions between the different types of electrical synapses within the circuit. Given that AII amacrine cells form both homologous (AII-AII) and heterologous (AII-cone bipolar cell) electrical synapses-connections that serve distinct functional roles in retinal signaling processing-a comparative analysis of their molecular compositions could have provided important insights into synapse specificity.

Reviewer #3 (Public review):

Summary:

This study by Tetenborg S et al. identifies proteins that are physically closely associated with gap junctions in retinal neurons of mice and zebrafish using BioID, a technique that labels and isolates proteins proximal to a protein of interest. These proteins include scaffold proteins, adhesion molecules, chemical synapse proteins, components of the endocytic machinery, and cytoskeleton-associated proteins. Using a combination of genetic tools and meticulously executed immunostaining, the authors further verified the colocalizations of some of the identified proteins with connexin-positive gap junctions. The findings in this study highlight the complexity of gap junctions. Electrical synapses are abundant in the nervous system, yet their regulatory mechanisms are far less understood than those of chemical synapses. This work will provide valuable information for future studies aiming to elucidate the regulatory mechanisms essential for the function of neural circuits.

Strengths:

A key strength of this work is the identification of novel gap junction-associated proteins in AII amacrine cells and photoreceptors using BioID in combination with various genetic tools. The well-studied functions of gap junctions in these neurons will facilitate future research into the functions of the identified proteins in regulating electrical synapses.

Thank you for these comments.

Weaknesses:

I do not see major weaknesses in this paper. A minor point is that, although the immunostaining in this study is beautifully executed, the quantification to verify the colocalization of the identified proteins with gap junctions is missing. In particular, endocytosis component proteins are abundant in the IPL, making it unclear whether their colocalization with gap junction is above chance level (e.g. EPS15l1, HIP1R, SNAP91, ITSN in Figure 3B).

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) It would be helpful to include a comprehensive summary of the results from the quantitative proteomics analyses, such as the number of proteins detected in each species and the number of proteins associated with each GO term. Additionally, a clear figure or table highlighting the specific proteins conserved between zebrafish and mice would strengthen the evidence for evolutionary conservation of proteins at electrical synapses.

We have added the raw data we received from our mass spec facility including a comparison of all the candidates for different species. Supplementary figure 3.

(2) A more detailed description of the number of experimental and/or technical replicates would improve the technical rigor of the study. For example, what was the rationale for using different log2 fold-change cutoffs in mice versus zebrafish? Are the replicates consistent in terms of protein enrichment?

We have added raw data from individual experiments as a supplement (Excel spreadsheet). We have two replicates from zebrafish and two from mice. The first experiment in mice was conducted with fewer retinas and a different promoter (human synapsin promoter) and didn’t yield nearly as many candidates. We are currently running a third experiment with 35 mouse retinas which will most likely detect more candidates as we have identified currently. We can update the proteome in this paper once the analysis is complete. It is not feasible to conduct these experiments with multiple replicates at the same time, since the number of animals that have to be used is simply too high, especially since very specific genotypes are required that are difficult obtain.

(3) It would be interesting to determine whether there are differences in the presence of candidate proteins between AII-AII gap junctions and AII-cone bipolar cell gap junctions. Given that the subcellular localization of AII-AII gap junctions differs from that of AII-cone bipolar cell gap junctions (with most AII-AII gap junctions located below AII-cone ones), histological validations of the proteins shown in Figure 6 can be repeated for AII-AII gap junctions. This would help reveal similarities or differences in the protein compositions of these two types of gap junctions.

Thank you for this suggestion. We had similar plans. However, we realized that homologous gap junctions are difficult to recognize with GFP. The dense GFP labeling in the proximal IPL, where AII-AII gap junctions are formed, does not allow us to clearly trace the location of individual dendrites from different cells. Detecting AII-AII gap junctions would require intracellular dye Injections of neighboring AII cells. Unfortunately, we don’t have a set up that would allow this. Bipolar cell terminals, on the contrary, are a lot easier to detect with markers such as SCGN, which is why we decided to focus on AII/ONCB gap junctions.

(4) In Figures 1 and 2, it would be helpful to clarify in the figure legends whether the proteins in the interaction networks represent all detected proteins or only those selected based on log2 fold-change or other criteria.

Thank you for this suggestion! We have added a description in lines 643 and 662.

(5) In Figure 1A (bottom panel), please include a negative control for the Neutravidin staining result from the non-labeling group.

We only tested the biotinylation for wild type retinas in cell lysates and western blots as shown in figure 1C, which shows an entirely different biotinylation pattern.

(6) In Figure 2B, please include the results of Neutravidin staining for both the labeling and non-labeling groups.

Same comment: We see the differences in the biotinylation pattern on western blots, which is distinct for Cx36-EGFP and wild type retinas, although both genotypes were injected with the same AAV construct and the same dose of biotin. We hope that this provides sufficient evidence for the specificity of our approach.

(7) In Figure 5B, the sizes of multiple proteins detected by Western blotting are inconsistent and confusing. For example, the size of Cx36 in the "FLAG-SJ2BP" panel differs from that in the other three panels. Additionally, in the "Myc-SIPA1L3+" panel, the size of SIPA1l3 appears different between the input and IP conditions.

Thank you for pointing this out! The differences in the molecular weight can be explained by dimerization. We have indicated the position of the dimer and the monomer bands with arrows. Especially, when larger amounts of Cx36 are coprecipitated Cx36 preferentially occurs as a dimer. This can also be seen in our previous publication:

S. Tetenborg et al., Regulation of Cx36 trafficking through the early secretory pathway by COPII cargo receptors and Grasp55. Cellular and Molecular Life Sciences 81, 1-17 (2024). Figure 1D

The band that occurs above 150kDa in the SIPA1L3 input is most likely a non-specific product. The specific band for SIPA1L3 can be seen in the IP sample, which has the appropriate molecular weight. We often see much better immuno reactivity for the protein of interest in IP samples, because the protein is concentrated in these experiments which facilitates its detection.

(8) How specific are the antibodies used for validating the proteins in this study? Given that many proteins, such as EPS15l1, HIP1R, SNAP91, GPrin1, SJ2BP, Syt4, show broad distribution in the IPL (Figure 3B, 4A, 6D), it is important to validate the specificity of these antibodies. Additionally, including negative controls in the histological validation would strengthen the reliability of the results.

We carefully selected the antibodies based on western blot data, that confirmed that each antibody detected an antigen of appropriate size. Moreover, the distribution of the proteins mentioned is consistent with function of each protein described in the literature. EPS15L1 and GPrin1 for instance are both membrane-associated, which is evident in Hek cells. Figure 5C.

A true negative control would require KO tissue and we don’t think that this is feasible at this point.

(9) In Figure 7F, the model could be improved by highlighting which components may be conserved between zebrafish and mice, as well as which components are conserved between the AII-AII junction and AII-cone bipolar cell junction?

Thank you for this suggestion. However, we don’t think that this is necessary as our study primarily focuses on the AII amacrine cell.

Currently we are unable to distinguish differences in the composition of AII-AII and AII-ONCB junctions as described above.

(10) Are there any functional measurements that could support the conclusion that "loss of Cx36 resulted in a quantitative defect in the formation of electrical synapse density complex"?

The loss of electrical synapse density proteins is shown by these immunostaining comparisons. Functional measurements necessarily depend on the function of the electrical synapse itself, which is gone in the case of the Cx36 KO. It is not clear that a different functional measurement can be devised.

Reviewer #3 (Recommendations for the authors):

(1) It would be very helpful if there were page and line numbers on the manuscript.

Line and page numbers have been added.

(2) Typos in the 3rd paragraph, the sentence 'which is triggered by the influx of Calcium though non-synaptic NMDA...'

Should it read '... Calcium THROUGH non-synaptic NMDA'?

We have corrected this typo.

(3) Figure 1B: please add a description of the top panels, 'Cx36 S293'.

A description of the top panels has been added to the figure legend in line. Line 639.

(4) Figure 1C: what do the arrows indicate?

We apologize for the confusion. The arrows in the western blot indicate the position of the Cx35-V5-TurboID construct, which can be detected with streptavidin-HRP and the V5 antibody. We have added a description for these arrows to the figure legend. See line 641.

(5) Related to the point in the 'Weakness', there are some descriptions of how well some of the gap junction-associated proteins colocalize with Cx36 in immunostaining. For example, 'In comparison to the scaffold proteins, however, the colocalization of Cx36 with each of these endocytic components, was clearly less frequent and more heterogenous, which appears to reflect different stages in the life cycle of Cx36' and 'All of these proteins showed considerable colocalization with Cx36 in AII amacrine cell dendrites'. It would be nice to see quantification data to support these claims.

Thank you for this suggestion. We have added a colocalization analysis to figure 3 (C & D). We quantified the colocalization for the endocytosis proteins Eps15l1 and Hip1r. This quantification included a flipped control to rule out random overlap. For both proteins we confirmed true colocalization (Figure 3D).

(6) In Figure 5B, it would be helpful if there were arrows or some kind in western blottings to indicate which bands are supposed to be the targeted proteins.

We have added arrows in IP samples to indicate bands representing the corresponding protein.

(7) In the sentence including 'for the PBM of Cx36, as it is the case for ZO-1', what is PBM?

The PBM means PDZ binding motif. We have added an explanation for this abbreviation in line 244.

(8) Please add a description of the Cx35b promoter construct in the Method section.

The Cx35b Promoter is a 6.5kb fragment. We will make the clone available via Addgene to ensure that all details of the clone can be accessed via snapgene or alternative software.

Reviewer #3 (Public review):

Valencia et al. aim to elucidate the biochemical and cellular mechanisms through which the human formin FHOD3 drives sarcomere assembly in cardiomyocytes. To do so, they combined rigorous in vitro biochemical assays with comprehensive in vivo characterizations, evaluating two wild type FHOD3 isoforms and two function-separating mutants. Surprisingly, they found that both wild type FHOD3 isoforms can nucleate new actin filaments, as well as elongate existing actin filaments in conjunction with profilin following barbed-end capping. This is in addition to FHOD3's proposed role as an actin bundler. Next, the authors focused on the longer isoform FHOD3L due to its essential role in sarcomere assembly in cardiomyocytes. They asked whether FHOD3L promote sarcomere assembly through its activity in actin nucleation or rather elongation. To do so, the authors designed two function-separating mutants: the K1193L mutation in the FH2 domain, known for its importance in actin nucleation, and the glycine-serine linker substitution in the FH1 domain ("GS-FH1",) known for its requirement in actin elongation. They demonstrated that while K1193L maintains its elongation activity and greatly diminishes nucleation and bundling, in GS-FH1 keeps its nucleation activity while lose its capacity to drive elongation. Armed with these tools, the authors attempted to rescue FHOD3L siRNA-treated neonatal rat ventricular myocytes (NRVM) with transgenes carrying wild type, K1193L, or GS-FH1 mutant forms of human FHOD3. In each condition, they evaluated the numbers and morphology of sarcomeres, as well as their ability to beat and generate cardiac rhythm. The authors found that while the wild type FHOD3L and the K1193L mutant can rescue sarcomere morphology and physiology, the GS-FH1 mutant fails to do so. Given that in GS-FH1 mainly elongation activity is compromised, the authors concluded that the elongation activity of FHOD3 is essential for its role in sarcomere assembly in cardiomyocytes, while its nucleator activity is dispensable. Overall, this important study provided a broadened view on the biochemical activities of FHOD3, and a pioneering view on a possible cellular mechanism of how FHOD3L drives sarcomere assembly. If further validated, this can lead to new mechanistic models of sarcomere assembly and potentially new therapeutic targets of cardiomyopathy.

The conclusions of this paper are mostly well supported by the comprehensive biochemical analyses performed by the authors. In my original assessment, I raised the point that the extreme low level of GS-FH1 signal in transfected cells in Figure 6A may reflect a failure of actin-binding by this construct in vivo, rather than its inability of driving elongation. The authors have thoroughly addressed this concern by: 1) providing new images of the GS-FH1 rescue condition with HA-FHOD3L signal intensities matching that of the K1193L rescue condition, and 2) quantitatively demonstrating that the expression levels in the GS-FH1 rescue condition are comparable with that of wild type FHOD3L rescue condition. This is nicely complemented by the new phalloidin staining of the GS-FH1 rescue condition, which showcased additional details of actin puncta reminiscent of that present in muscle stress fibers or premyofibrils. Overall, I am now convinced that the GS-FH1 cannot rescue sarcomere formation even when expressed at comparable levels. Given that GS-FH1 demonstrates actin elongation defects in vitro, it is reasonable to conclude that the actin elongation function of FHOD3L is essential for sarcomere formation in vivo.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Formins are complex proteins with multiple effects on actin filament assembly, including nucleation, capping with processive elongation, and bundling. Determining which of these activities is important for a given biological process and normal cellular function is a major challenge.

Here, the authors study the formin FHOD3L, which is essential for normal sarcomere assembly in muscle cells. They identify point mutants of FHOD3L in which formin nucleation and elongation/bundling activities are functionally separated. Expression of these mutants in neonatal rat ventricular myocytes shows that the control of actin filament elongation by formin is the major activity required for the normal assembly of functional sarcomeres.

Strengths:

The strength of this work is to combine sensitive biochemical assays with excellent work in neonatal rat ventricular myocytes. This combination of approaches is highly effective for analyzing the function of proteins with multiple activities in vitro.

Weaknesses:

FHOD3L does not seem to be the easiest formin to study because of its relatively weak nucleation activity and the short duration of capping events. This difficulty imposes rigorous biochemical analysis and careful interpretation of the data, which should be improved in this work.

We thank the reviewer for their praise and appreciation of our work. Indeed, FHOD3L is a challenging formin to work with.

Important points are raised here and below regarding the brief elongation events we reported. As suggested, we performed more rigorous analysis of the data and present it in the revised manuscript. We now report that from 45 dim regions analyzed, in three independent experiments with wild type FHOD3L, we detected 40 bursts. (The remaining five could be formin falling off too quickly to detect or the dim spots could be regions of inhomogeneity in intensity, not due to formin.) For comparison to the presented data with FHOD3L-CT, we analyzed the filaments in TIRF assays with no formin present. As the reviewers point out, inhomogeneities in filament intensity are normal. Thus, we examined any dim spots for pauses and/or bursts. As is now reported in Figure 2G,H, the velocity of growth of these dim spots is indistinguishable from the velocity of the rest of the filament. We acknowledge that our numbers may not be perfectly accurate, due to the noise in our system, we believe that the difference of 3-4 fold increase versus no change in rate is substantial and convincing.

We also determined the number of dim spots per length of filament. We found a higher frequency when FHOD3L-CT or FHOD3S-CT was present vs no formin, as now shown in Figure 2 – supplements 1G and 2E.

We were asked about the pauses we observe before bursts of elongation and how we know they are functionally relevant. The short answer is that we do not know. We reported them because they were so common: Of the 40 bursts, pauses preceded the burst in 38 cases. We cannot rule out that this pause reflects an interaction with the surface but might expect the frequency to be lower if it were. We revise the text to make our conclusions about pauses more circumspect.

We are convinced that the brief dim events we observed in the presence of FHOD3L-CT, in fact, reflect formin-mediated elongation and worked hard to improve their presentation, in addition to the added analysis. We include new kymographs, including examples from FHOD3L, FHOD3S, K1193L, and actin alone. We hope that the reviewers are also convinced.

This does not preclude our interest in the microfluidics and two-color assays, which will be pursued in the future. We have reached out to a colleague who is set up to repeat these measurements with microfluidics-assisted TIRF. The noise should be greatly reduced and the system is also optimal for directly visualizing labeled FHOD3, as suggested. We expect these experimental approaches will provide additional insights.

Reviewer #2 (Public review):

This article elucidates the biochemical and cellular mechanisms by which the FHOD-family of formins, particularly FHOD3, contributes to sarcomere formation and contractility in cardiomyocytes. Formins are mainly known to nucleate and elongate actin filaments, with certain family members also exhibiting capping, severing, and bundling activities. Although FHOD3 has been well-established as essential for sarcomere assembly in cardiomyocytes, its precise biochemical functions and contributions to actin dynamics remain poorly understood.

In this study, the authors combine in vitro biochemical assays with cellular experiments to dissect FHOD3's roles in actin assembly and sarcomere formation. They demonstrate that FHOD3 nucleates actin filaments and acts as a transient elongator, pausing elongation after an initial burst of filament growth. Using separation-of-function mutants, they show thatFHOD3's elongation activity - rather than its nucleation, capping, or bundling capabilities - is key for its sarcomeric function.

The experiments have been conducted rigorously and well-analyzed, and the paper is clearly written. The data presented support the authors' conclusions. I appreciate the detailed description and rationale behind the FHOD3 constructs used in this study.

We are happy to hear others find paper to be clearly written and well described.

However, I was somewhat surprised and a bit disappointed that while the authors conducted single-color TIRF experiments to observe the effects of FHOD3 on single filaments, they did not use fluorescently labeled FHOD3 to directly visualize its behavior. Incorporating such experiments would significantly strengthen their conclusions regarding FHOD3's bursts of elongation interspersed with capping activity. While I understand this might require a few additional weeks of experiments, these data would add considerable value by directly testing the proposed mechanism.

We appreciate the suggestion and hope to incorporate a two-color approach soon. As noted, FHOD3L is not always easy to work with and we do not have a functional labeled copy of the protein at this time.

There is a typo in the word "required" in line number 30. The authors also use fit data to extract parameters in several panels (e.g., Figures 2b, 2d, 3a, and 3b). While these fit functions may be intuitive to actin experts, explicitly describing the fit functions in the figure legends or methods would greatly benefit the broader readership.

Thank you for these comments. We updated the indicated figures and described the analysis in greater detail.

Reviewer #3 (Public review):

Valencia et al. aim to elucidate the biochemical and cellular mechanisms through which the human formin FHOD3 drives sarcomere assembly in cardiomyocytes. To do so, they combined rigorous in vitro biochemical assays with comprehensive in vivo characterizations, evaluating two wild-type FHOD3 isoforms and two function-separating mutants. Surprisingly, they found that both wild-type FHOD3 isoforms can nucleate new actin filaments, as well as elongate existing actin filaments in conjunction with profilin following barbed-end capping. This is in addition to FHOD3's proposed role as an actin bundler. Next, the authors asked whether FHOD3L promotes sarcomere assembly in cardiomyocytes through its activity in actin nucleation or rather elongation. With two function-separating mutants, the authors evaluated the numbers and morphology of sarcomeres, as well as their ability to beat and generate cardiac rhythm. The authors found that while the wild-type FHOD3L and the K1193L mutant can rescue sarcomere morphology and physiology, the GS-FH1 mutant fails to do so. Given that in GS-FH1 mainly elongation activity is compromised, the authors concluded that the elongation activity of FHOD3 is essential for its role in sarcomere assembly in cardiomyocytes, while its nucleator activity is dispensable. Overall, this important study provided a broadened view on the biochemical activities of FHOD3, and a pioneering view on a possible cellular mechanism of how FHOD3L drives sarcomere assembly. If further validated, this can lead to new mechanistic models of sarcomere assembly and potentially new therapeutic targets of cardiomyopathy.

The conclusions of this paper are mostly well supported by the comprehensive biochemical analyses performed by the authors. However, the sarcomere assembly defect phenotype in the GS-FH1 rescue condition requires further investigation, as the extremely low level of GS-FH1 signal in transfected cells in Figure 6A may reflect a failure of actin-binding by this construct in vivo, rather than its inability to drive elongation. Though the authors do show in Figure 6 that GS-FH1 can bind to normal-looking sarcomeres when they are present, this may be due to a lack of siRNA activity in these cells, such that endogenous FHOD3L is still present. In this possible scenario, GS-FH1 may dimerize with endogenous FHOD3L. The authors should demonstrate that GS-FH1 alone can indeed interact with existing actin filaments in vivo. While this has been clearly demonstrated in vitro, given the more complex biochemical environment in vivo where additional unknown binding partners may present, cautions should be made when extrapolating findings from the former to the latter.

The reviewer is concerned about the low protein levels in the GS-FH1 rescue experiments as reflected in the HA fluorescence intensity distributions shown in Fig. 5 Supplement 2A. While the scenario proposed could explain our observations with the GSFH1 rescues it is quite complex. Nor does the scenario preclude the conclusion that the FH1 domain is critical. We agree that the observed sarcomeres are likely to be residual in cells with incomplete RNAi. We now include the image of a cell that is still full of sarcomeres and note that the GH-FH1 is expressed at a relatively high level and striated throughout the cell. We interpret this as evidence that GS-FH1 is stable when suitable binding sites are available. We cannot exclude that there is more GS-FH1 because there was more endogenous FHOD3L with which to heterodimerize. If the GS-FH1 heterodimer were simply poisoning the wild type protein, we do not expect that it would be bound correctly to sarcomeres. If, instead, heterodimers have some activity, it seems far from sufficient to rescue sarcomere formation, suggesting that two functional FH1 domains are critical.

Furthermore, we do not see evidence of correlation between protein levels and rescue at the level present in these cells (addressed below). Unfortunately, the proposed IP to test whether FHOD3L binds actin in vivo would only potentially report on filament side binding (both direct and indirect). It would not address whether the GS-FH1 mutant functions as a nucleator, elongator, bundler and/or capping protein in vivo.

The critical question that we can address is whether the phenotype is due to low protein levels, assuming the protein present is functional, or due to loss of elongation activity by FHOD3L. To address this question, we returned to our data.

First, we plotted the distributions of the intensities of the cells we analyzed further, in addition to the automated readout of all of the cells in the dish (Fig. 4 supplement 1). These cells were selected randomly and, as should be the case, the distributions of their intensities agree well with the original distributions for the three different rescue constructs: FHOD3L, K1193L, and GS-FH1 (Fig. 6 supplement 1). We then asked whether there was any correlation in HA intensities with the sarcomere metrics. As seen in our pilot data, no correlation is evident in any of the three cases across the range of intensities we collected (400 – 2700 a.u.) (old Fig. 6 supplement C,D,E). We now replace the data from pilot experiments with analysis of HA intensities and sarcomere metrics from the data sets included in the paper (new Fig 6. Supplement 1). Again, little to no correlation was observed (the single highest r-squared value is 0.2 and the remaining eight values are less than or equal to 0.08).

To more specifically address the question of whether low HA fluorescence intensity is likely to reflect sufficient protein levels to build sarcomeres we re-examined two data sets from the FHOD3L WT rescue data. We found that, by chance, the first replicate of data from the wild type rescue has a comparable intensity distribution to that of the GSFH1 rescues (580 +/- 261 / cell vs. 548 +/- 105 / cell). In addition, we collected all of the data from cells with intensity levels <720, designed to mimic the distribution of the GS-FH1 cells (Fig. 6 supplement 3). We then compared the sarcomere metrics (sarcomere number, sarcomere length, sarcomere width) between the full data set and the two low intensity subsets:

• Sarcomere number is the only non-normal metric. We therefore used the Mann Whitney U test, which shows no difference between all 3 WT distributions.

• We compared Z-line lengths by one-way ANOVA and Tukey's post hoc tests, again finding no significant difference for all distributions.

• Sarcomere length shows a weakly significant difference (p=0.038) between the whole WT data set and bio rep 1, but no difference between the whole WT data set and the HA<720 group.

Thus, cells expressing wild type FHOD3L at levels comparable to levels detected in GS-FH1 mutant rescues, are fully rescued. Based on these findings we conclude that the expression levels in the GS-FH1 are high enough to rescue the FHOD3 knock down, supporting our conclusion that the defect is due to loss of elongation activity. We have added this analysis and discussion to the revised manuscript.

Recommendations for the authors:

Reviewing Editor Comments:

You will see that the 3 reviewers are very positive about your work and appreciate the elegant combination of biochemical assays and functional tests in cardiomyocytes. We've had a long discussion with them and we all agree that two experiments deserve further effort to make the conclusions of your paper more convincing.

Thank you.

The first experiment is the TIRF elongation assay, where the two biochemist Reviewers remain doubtful that these short events are really due to the presence of a formin at the end of the filament. One of them suggests that two-color imaging with a labeled formin should clearly prove this point.

We agree that the elongation assays can be improved. Given the similarity of processivity of Fhod3L, Fhod3S and Drosophila FhodA (measured by a distinct method), we are inclined to believe them. However, the reviewer raises an excellent point about the accuracy of the measurements given the resolution (and noise) of the data. We are interested in the two-color imaging assay but do not believe it will necessarily simplify the analysis. We suspect that Fhod spends more time at/near the barbed end than is apparent based on elongation rates. The fact that we see repeated events on individual filaments at such low concentrations of FHOD3L (0.1 nM) supports this idea. Otherwise, the likelihood of FHOD3L finding barbed ends so often is really quite low.

We will return to these experiments, using alternate methods, curious to see what else we learn. In the meantime, we conducted more thorough analysis, including controls, and improved visualization of example traces. Data for elongation analysis and kymographs were acquired with Jfilament. We stretched the x-axis (time) in kymographs for FHOD3L-CT (Fig. 2F), FHOD3S-CT (Fig. 2, supplement 2C), FHOD3L-CT K1193L (Fig. 3, supplement 1A), and actin alone (Fig 2G), and highlighted regions of analysis. The slopes for these regions, separated based on intensity, were fit to the data in KaleidaGraph. The fits are offset from the data such that they do not obscure the filaments and corresponding rates are given. The fact that we never see fast dim regions when FHOD3 is not present, as shown in Fig. 2H and that the frequency of dim events is markedly increased (Fig. 2-supplements 1G and 2E) give us confidence that the events are real. We acknowledge in the text that the precise values of the short events may be inaccurate due to the resolution of our experiments. We hope the reviewers are convinced by the improved analysis.

The second experiment is the sarcomere assembly defect phenotype in the GS-FH1 rescue condition. This requires further investigation, as the extremely low level of GS-FH1 signal in transfected cells in Figure 6A may reflect a failure of actin-binding/nucleation in vivo, rather than its inability to elongate F-actin. Although you show that GS-FH1 can bind to sarcomeres when they are present, this may be due to a lack of siRNA activity in these cells, such that endogenous FHOD3L is still present. In this possible scenario, GS-FH1 could dimerize with endogenous FHOD3L.

We agree that the sarcomeres we see are likely to be residual and could reflect some remaining endogenous FHOD3. The reviewers are concerned about the low protein levels in the GSFH1 rescues. First, we do not agree that the levels are “extremely” low. Through careful analysis, we established that 3xHA-FHOD3L intensities between 300 and 3000 a.u./um² were sufficient for full rescue. The mean for the GSFH1 experiments is 533 +/- 93, which is well within this range. Furthermore, we did not observe correlation between sarcomere number, length, or width and HA intensity over the full range collected for wild type FHOD3L or within the GS-FH1 data. We previously showed pilot data but now show correlation analysis for every analyzed cell (Fig. 4 – figure supplement 1 D-F). We conducted this analysis on all of the mutant rescue experiments (Fig. 6-supplement 1). Finally, we identified two subpopulations of the wildtype rescue data. One is all of the cells with HA intensity < 720, which gives a distribution of mean 545 +/- 85. The second set is the first biological replicate of wild type rescue, which has a distribution of mean 560 +/- 160. Again correlation shows little relationship between HA levels and sarcomere metrics. Nevertheless, we show intensity level matched images in Fig 6, as opposed to images reflecting average intensities.

The critical question remains whether the phenotype is due to low protein levels or due to loss of elongation by FHOD3L. Notably, we now show a cell that is full of sarcomeres and has relatively high FHOD3L levels as well, consistent with available binding sites stabilizing mutant protein but not ruling out heterodimerization (Fig. 6 – figure supplement 2C). Others have expressed mutant FHOD3L in a wild type background in mice. They observed poisoning, consistent with heterodimerization. Thus, it is possible that, as suggested, the FHOD3L-GSFH1 detected in sarcomeres is in fact heterodimerized with residual endogenous FHOD3L. In this case, we would still conclude that the protein is not functional enough to rescue, supporting a role for the FH1 domain.

In the future, we plan to perform experiments with compromised, but not inactive, FH1 domains, as we discuss in the paper.

We hope that you will find these comments useful.

Yes, the comments were thoughtful and helped us write a better paper. Thank you.

Reviewer #1 (Recommendations for the authors):

Some experiments should be described and analyzed more carefully. This lack of clarity calls into question the interpretation of some experiments. Overall, this study is not yet as convincing as it should be.

Main recommendations:

(1) Formin elongation phases in the TIRF experiment are not convincing. They are rare and it is difficult to see any significant difference between the control movie without FHOD3L-CT and the movie with FHOD3L-CT. Filaments assembled in the absence of FHOD3L-CT also show some fluorescence inhomogeneity (which is normal), and measurements of formin elongation rates and capping times are not convincing (for example, the kymograph of the control profilin-actin situation in Figure 2F also shows a fast elongation phase on the right).

Please see response above. We conducted more thorough analysis and created improved visualizations. We hope the data are more convincing now.

It is also difficult to understand how an accurate measurement can be made from these noisy kymographs, and the method section should explain that precisely.

This is a valid point. We added details of analysis to the methods section and we discuss the fact that the measurements are at the limit of our resolution in the paper. We rely on the large (~3-fold) difference in elongation, more than specific elongation rates for our interpretation.

One of the problems is that these events are too transient to quantify well with noisy data. I noticed that the formin concentration used in these movies is quite low (0.1 nM FHOD3L-CT). Is there a reason for this? Is it possible to increase the formin concentration to increase the number of formin capping/elongation events and provide more convincing movies?

We acknowledge that the data are noisy. We felt that it was necessary to perform experiments with filaments only tethered at one end, leaving the growing end free. We did so, in part, because when we did experiments with biotinylated actin to anchor the filaments down, we observed pauses in the absence of formin. Ultimately, we compromised, using anchored seeds and a relatively low concentration of NEM-myosin to decrease motion of the actin filaments.

The experiments were performed with such low FHOD3L-CT because it was a potent nucleator in TIRF assays, making data analysis nearly impossible with more formin present. FHOD3S-CT and FHOD3L-CT K1193L behaved somewhat differently between these experiments and we were able to perform them with 1 nM formin.

Not seeing formin at the tip of the filaments is an additional difficulty because we do not know if these pauses occur because formin is stuck to the coverslips (which could very well happen with these sticky proteins) or freely bound at the end of a filament as the text suggests. Is there any argument in favor of one scenario over the other?

This will be an important experiment. As described above, we suspect that Fhod spends more time at/near the barbed end than is apparent based on elongation data. The fact that we see repeated events on individual filaments at such low concentrations of FHOD3L (0.1 nM) supports this idea. Otherwise, the likelihood of FHOD3L finding barbed ends so often is really quite low. In order to address the question about the cause of pauses, we reviewed our data, finding that 38 of 40 bursts were preceded by pauses. We do, however, discuss that we cannot rule out non-specific interactions with the surface.

(2) Pyrene elongation assays in the presence of profilin are actually more convincing to test the elongation ability of formins. However, such an assay is not presented for all mutants. It should be.

While we agree to some extent with this comment, we did not include the pyrene data for all of the mutants because the shapes of the curves were even more complicated than those seen with wild type FHOD3L-CT rendering them uninterpretable.

(3) Some experiments (e.g. in Figure 2E) are performed with yeast profilin, while others (e.g. in Figure 2F) are performed with human profilin. Obviously, both profilins could modulate formin activity differently and the side-by-side interpretation of both experiments is difficult. Could the authors stick to human profilin for all experiments?

We used to always perform pyrene assays with yeast profilin because it was known to be insensitive to pyrene. These data were collected before we realized that the affinity of human profilin for actin is so high that we could probably do everything with this profilin. We have compared the two profilins for other formins, e.g. Delphilin, Capu, and did not observe detectable differences.

Minor recommendations:

(1) The pyrene assays with the light blue colored curve choice are not ideal. I have difficulties seeing some of the curves.

Thank you. We added symbols to a subset of the traces to make them more visible.

(2) In the same curves, I can't understand what the +3.75 and 0.078 numbers mean. Could these results be plotted in a clearer way?

These values are the lowest concentrations in the range tests. They were matching light blue with black outline for visibility. We added symbols and changed the color of the numbering for improved visibility/understanding.

(3) In Figure 2D, is the Kd of I1163A really determined only from 2 experimental data points?

Of course not. We now show the figure with extended axes in Fig. 2 - figure supplement 1C.

(4) In Figure 2C, the shape of the curves suggests that this is not a pure capping assay, but a mix of capping and nucleation. It's not dramatic but could lead to an under-estimation of the capping efficiency.

We agree with the reviewer that the complicated shapes confound interpretation. Our analysis is based on the earliest slopes, in part, for this reason. We added discussion of this complication to the text.

Reviewer #3 (Recommendations for the authors):

Suggestions for additional experiments:

(1) To evaluate whether GS-FH1 alone can indeed interact with existing actin filaments in vivo, the authors may consider performing immunoprecipitation assays with GS-FH1 extracted from rescued NRVMs.

An IP of GS-FH1 from cells could show actin filament side binding but, unfortunately, will not provide any information about filament end binding, which is of much greater interest.

It will be helpful to show phalloidin staining in GS-FH1 rescues in a similar manner as in Figure 6-supplement 1, panel B, and compare that with mock rescue in Figure 4 panel D. It will be essential to prove this prior to concluding that actin elongation activity is essential for sarcomere assembly.

This is an excellent suggestion. We now include images of phalloidin stained cells from both K1193L and GS-FH1 rescues (Fig. 6A’ – supplement 2A,B). We were intrigued to see small actin punctae that were sometimes aligned. We speculate that these could be pre-premyofibrils and suggest that this is further evidence that the GS-FH1 protein is not completely unstable.

(2) Prior to sarcomere assembly, a-actinin is known to form short bundles with actin filaments (I-Z-I complex) without clearly defined periodicity. This semi-ordered state then transforms into the more ordered sarcomeres with periodic spacing. It will be valuable to show the phalloidin staining in addition to the a-actinin IF consistently across all conditions. This may lead to further insights into the defects of sarcomere assembly. Along the same vein, higher magnification images showcasing several sarcomeres will help the readers evaluate these defects.

We agree that there are additional valuable measurements to be made. In order to favor synchronized contraction, we plated the cells at too high a density to reliably identify IZI complexes. We have included some zoomed in images of the phalloidin staining.

Recommendations for improving the writing:

The authors mentioned the interaction between cardiac MyBP-C and FHOD3L as essential for the localization of FHOD3L to the C-line of the sarcomere. Can they discuss whether this interaction is important for the role of FHOD3L in sarcomere assembly? If so, how?

This is a very interesting question that we cannot answer at this time.

Minor corrections to the text and figures:

In the legend of Figure 2-Figure Supplement 1, the labels of (F) and (E) are swapped.

Thank you for catching this.

Author response:

eLife Assessment

This useful study presents Altair-LSFM, a solid and well-documented implementation of a light-sheet fluorescence microscope (LSFM) designed for accessibility and cost reduction. While the approach offers strengths such as the use of custom-machined baseplates and detailed assembly instructions, its overall impact is limited by the lack of live-cell imaging capabilities and the absence of a clear, quantitative comparison to existing LSFM platforms. As such, although technically competent, the broader utility and uptake of this system by the community may be limited.

We thank the reviewers and editors for their thoughtful evaluation of our work and for recognizing the technical strengths of the Altair-LSFM platform, including the custom-machined baseplates and detailed documentation provided to support accessibility and reproducibility. We respectfully disagree, however, with the assessment that the system lacks live-cell imaging capabilities. We are fully confident in the system’s suitability for live-cell applications and will demonstrate this by including representative live-cell imaging data in the revised manuscript, along with detailed instructions for implementing environment control. Moreover, we will expand our discussion to include a broader, more quantitative comparison to existing LSFM platforms—highlighting trade-offs in cost, performance, and accessibility—to better contextualize Altair’s utility and adaptability across diverse research settings.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The article presents the details of the high-resolution light-sheet microscopy system developed by the group. In addition to presenting the technical details of the system, its resolution has been characterized and its functionality demonstrated by visualizing subcellular structures in a biological sample.

Strengths:

(1) The article includes extensive supplementary material that complements the information in the main article.

(2) However, in some sections, the information provided is somewhat superficial.

Our goal was to make the supplemental content as comprehensive and useful as possible. In addition to the materials provided with the manuscript, our intention is for the online documentation (available at thedeanlab.github.io/altair) to serve as a living resource that evolves in response to user feedback. For this reason, we are especially interested in identifying and expanding any sections that are perceived as superficial, and we would greatly appreciate the reviewer’s guidance on which areas would benefit from further elaboration.

Weaknesses:

(1) Although a comparison is made with other light-sheet microscopy systems, the presented system does not represent a significant advance over existing systems. It uses high numerical aperture objectives and Gaussian beams, achieving resolution close to theoretical after deconvolution. The main advantage of the presented system is its ease of construction, thanks to the design of a perforated base plate.

We appreciate the reviewer’s assessment and the opportunity to clarify our intent. Our primary goal was not to introduce new optical functionality beyond that of existing high-performance light-sheet systems, but rather to reduce the barrier to entry for non-specialist labs.

(2) Using similar objectives (Nikon 25x and Thorlabs 20x), the results obtained are similar to those of the LLSM system (using a Gaussian beam without laser modulation). However, the article does not mention the difficulties of mounting the sample in the implemented configuration.

We agree that there are practical challenges associated with handling 5 mm diameter coverslips. However, the Nikon 25x can readily be replaced by a Zeiss W Plan-Apochromat 20x/1.0 objective, which eliminates the need for the 5 mm coverslip[1]. In the revised manuscript, we will more explicitly detail the practical challenges in handling a 5 mm coverslip and mention the alternative detection objective.

(3) The authors present a low-cost, open-source system. Although they provide open source code for the software (navigate), the use of proprietary electronics (ASI, NI, etc.) makes the system relatively expensive. Its low cost is not justified.

We understand the reviewer’s concern regarding the use of proprietary control hardware such as the ASI Tiger Controller and NI data acquisition cards. While lower-cost alternatives for analog and digital control (e.g., microcontroller-based systems) do exist, our choice was intentional. By relying on a unified and professionally supported platform, we minimize the complexity of sourcing, configuring, and integrating components from disparate vendors—each of which would otherwise demand specialized technical expertise. Moreover, in future releases, we aim to further streamline the system by eliminating the need for the NI card, consolidating all optoelectronic control through the ASI Tiger Controller. This approach allows users to purchase a fully assembled and pre-configured system that can be operational with minimal effort.

It is worth noting that the ASI components are not the primary cost driver. The full set—including XYZ and focusing stages, a filter wheel, a tube lens, the Tiger Controller, and basic optomechanical adapters—costs approximately $27,000, or ~18% of the total system cost. Additional cost reductions are possible. For example, replacing the motorized sample positioning and focusing stages with manual alternatives could reduce the cost by ~$12,000. However, this would eliminate key functionality such as autofocusing, 3D tiling, and multi-position acquisition. Open-source mechanical platforms such as OpenFlexure could in principle be adapted, but they would require custom assembly and would need to be integrated into our control software. Similarly, the filter wheel could be omitted in favor of a multi-band emission filter, reducing the cost by ~$5,000. However, this comes at the expense of increased spectral crosstalk, often necessitating spectral unmixing. An industrial CMOS camera—such as the Ximea MU196CR-ON, recently demonstrated in a Direct View Oblique Plane Microscopy configuration[2]—could substitute for the sCMOS cameras typically used in high-end imaging. However, these industrial sensors often exhibit higher noise floors and lower dynamic range, limiting sensitivity for low-signal imaging applications.

While a $150,000 system represents a significant investment, we consider it relatively cost-effective in the context of advanced light-sheet microscopy. For comparison, commercially available systems with similar optical performance—such as LLSM systems from 3i or Zeiss—are several-fold more expensive.

(4) The fibroblast images provided are of exceptional quality. However, these are fixed samples. The system lacks the necessary elements for monitoring cells in vivo, such as temperature or pH control.

We thank the reviewer for their positive comment regarding the quality of our fibroblast images. As noted, the current manuscript focuses on the optical design and performance characterization of the system, using fixed specimens to validate resolution and imaging stability. We acknowledge the importance of environmental control for live-cell imaging. Temperature regulation is routinely implemented in our lab using flexible adhesive heating elements paired with a power supply and PID controller. For pH stabilization in systems that lack a 5% CO₂ atmosphere, we typically supplement the imaging medium with 10–25 mM HEPES buffer. In the revised manuscript, we will introduce a modified sample chamber capable of maintaining user-specified temperatures, along with detailed assembly instructions. We will also include representative live-cell imaging data to demonstrate the feasibility of in vitro imaging using this system.

Reviewer #2 (Public review):

Summary:

The authors present Altair-LSFM (Light Sheet Fluorescence Microscope), a high-resolution, open-source microscope, that is relatively easy to align and construct and achieves sub-cellular resolution. The authors developed this microscope to fill a perceived need that current open-source systems are primarily designed for large specimens and lack sub-cellular resolution or are difficult to construct and align, and are not stable. While commercial alternatives exist that offer sub-cellular resolution, they are expensive. The authors' manuscript centers around comparisons to the highly successful lattice light-sheet microscope, including the choice of detection and excitation objectives. The authors thus claim that there remains a critical need for high-resolution, economical, and easy-to-implement LSFM systems.

Strengths:

The authors succeed in their goals of implementing a relatively low-cost (~ USD 150K) open-source microscope that is easy to align. The ease of alignment rests on using custom-designed baseplates with dowel pins for precise positioning of optics based on computer analysis of opto-mechanical tolerances, as well as the optical path design. They simplify the excitation optics over Lattice light-sheet microscopes by using a Gaussian beam for illumination while maintaining lateral and axial resolutions of 235 and 350 nm across a 260-um field of view after deconvolution. In doing so they rest on foundational principles of optical microscopy that what matters for lateral resolution is the numerical aperture of the detection objective and proper sampling of the image field on to the detection, and the axial resolution depends on the thickness of the light-sheet when it is thinner than the depth of field of the detection objective. This concept has unfortunately not been completely clear to users of high-resolution light-sheet microscopes and is thus a valuable demonstration. The microscope is controlled by an open-source software, Navigate, developed by the authors, and it is thus foreseeable that different versions of this system could be implemented depending on experimental needs while maintaining easy alignment and low cost. They demonstrate system performance successfully by characterizing their sheet, point-spread function, and visualization of sub-cellular structures in mammalian cells, including microtubules, actin filaments, nuclei, and the Golgi apparatus.

We thank the reviewer for their thoughtful summary of our work. We are pleased that the foundational optical principles, design rationale, and emphasis on accessibility came through clearly. We agree that the approach used to construct the microscope is highly modular, and we anticipate that these design principles will serve as the basis for additional system variants tailored to specific biological samples and experimental contexts. To support this, we provide all Zemax simulations and CAD files openly on our GitHub repository, enabling advanced users to build upon our design and create new functional variants of the Altair system.

Weaknesses:

There is a fixation on comparison to the first-generation lattice light-sheet microscope, which has evolved significantly since then:

(1) The authors claim that commercial lattice light-sheet microscopes (LLSM) are "complex, expensive, and alignment intensive", I believe this sentence applies to the open-source version of LLSM, which was made available for wide dissemination. Since then, a commercial solution has been provided by 3i, which is now being used in multiple cores and labs but does require routine alignments. However, Zeiss has also released a commercial turn-key system, which, while expensive, is stable, and the complexity does not interfere with the experience of the user. Though in general, statements on ease of use and stability might be considered anecdotal and may not belong in a scientific article, unreferenced or without data.

The referee is correct that our comparisons reference the original LLSM design, which was simultaneously disseminated as an open-source platform and commercialized by 3i. While we acknowledge that newer variants of LLSM have been developed—including systems incorporating adaptive optics[3] and the MOSAIC platform (which remains unpublished)—the original implementation remains the most widely described and cited in the literature. It is therefore the most appropriate point of comparison for contextualizing Altair’s performance, complexity, and accessibility. Importantly, this version of LLSM is far from obsolete; it continues to be one of the most commonly used imaging systems at Janelia Research Campus’s Advanced Imaging Center.

We acknowledge that more recent commercial implementation by Zeiss has addressed several of the practical limitations associated with the original design. In particular, we agree that the Zeiss Lattice Lightsheet 7 system, which integrates a meniscus lens to facilitate oblique imaging through a coverslip, offers a user-friendly experience—albeit with a modest tradeoff in resolution (reported deskewed resolution: 330 nm × 330 nm × 500–1000 nm).

While we recognize that statements on usability and stability can be subjective, one objective proxy for system complexity is the number of optical elements that require precise alignment during assembly. The original LLSM setup includes approximately 29 optical components that must each be carefully positioned laterally, angularly, and coaxially along the optical path. In contrast, the first-generation Altair system contains only 9 such elements. By this metric, Altair is considerably simpler to assemble and align, supporting our overarching goal of making high-resolution light-sheet imaging more accessible to non-specialist laboratories. In the revised manuscript, we will clarify the scope of our comparison and provide more precise language about what we mean by complexity (e.g., number of optical elements needed to align).

(2) One of the major limitations of the first generation LLSM was the use of a 5 mm coverslip, which was a hinderance for many users. However, the Zeiss system elegantly solves this problem, and so does Oblique Plane Microscopy (OPM), while the Altair-LSFM retains this feature, which may dissuade widespread adoption. This limitation and how it may be overcome in future iterations is not discussed.

We agree that the use of 5 mm diameter coverslips, while enabling high-NA imaging in the current Altair-LSFM configuration, may serve as an inconvenience for many users. We will discuss this more explicitly in the revised manuscript. Specifically, we note that changing the detection objective is sufficient to eliminate the need for a 5 mm coverslip. For example, as demonstrated in Moore et al., Lab Chip 2021, pairing the Zeiss W Plan-Apochromat 20x/1.0 objective with the Thorlabs TL20X-MPL allows imaging beyond the physical surfaces of both objectives, removing the constraint imposed by small-format coverslips[1]. In the revised manuscript, we will propose this modification as a straightforward path for increasing compatibility with more conventional sample mounting formats.

(3) Further, on the point of sample flexibility, all generations of the LLSM, and by the nature of its design, the OPM, can accommodate live-cell imaging with temperature, gas, and humidity control. It is unclear how this would be implemented with the current sample chamber. This limitation would severely limit use cases for cell biologists, for which this microscope is designed. There is no discussion on this limitation or how it may be overcome in future iterations.

We appreciate the reviewer’s emphasis on the importance of environmental control for live-cell imaging applications. It is worth noting that the original LLSM design, including the system commercialized by 3i, provided temperature control only, without integrated gas or humidity regulation. Despite this, it has been successfully used by a wide range of scientists to generate important biological insights.

We agree that both OPM and the Zeiss implementation of LLSM offer clear advantages in terms of environmental control, as we previously discussed in detail in Sapoznik et al., eLife, 2020[4]. However, assembly of high numerical aperture OPM systems is highly technical, and no open-source variant of OPM delivers sub-cellular scale resolution yet.

(4) The authors' comparison to LLSM is constrained to the "square" lattice, which, as they point out, is the most used optical lattice (though this also might be considered anecdotal). The LLSM original design, however, goes far beyond the square lattice, including hexagonal lattices, the ability to do structured illumination, and greater flexibility in general in terms of light-sheet tuning for different experimental needs, as well as not being limited to just sample scanning. Thus, the Alstair-LSFM cannot compare to the original LLSM in terms of versatility, even if comparisons to the resolution provided by the square lattice are fair.

We thank the reviewer for this comment. It is true that our discussion focused primarily on the square lattice implementation of LLSM. While this could be viewed as a subset of the system’s broader capabilities, we chose this focus intentionally, as the square lattice remains by far the most commonly used variant in practice. Even in the original LLSM publication, 16 out of 20 figure subpanels utilized the square lattice, with only one panel each representing the hexagonal lattice in SIM mode, a standard Bessel beam in incoherent SIM mode, a hex lattice in dithered mode, and a single Bessel in dithered mode. This usage pattern largely reflects the operational simplicity of the square lattice: it minimizes sidelobe growth and enables more straightforward alignment and data processing compared to hexagonal or structured illumination modes.

In 2019, we performed an exhaustive accounting of published illumination modes in LLSM and found that the SIM mode had only been used in two additional peer-reviewed publications at that time. We will consider updating this table in the revised manuscript and will expand our discussion to acknowledge the broader flexibility of the LLSM platform—including its capacity for structured illumination and alternative light-sheet geometries. However, we will also emphasize that, despite these advanced capabilities, the square lattice remains the dominant mode used by the community and therefore serves as a fair and practical benchmark for comparison.

(5) There is no demonstration of the system's live-imaging capabilities or temporal resolution, which is the main advantage of existing light-sheet systems.

In the revised manuscript, we will include a demonstration of live-cell imaging to directly validate the system’s suitability for dynamic biological applications. We will also characterize the temporal resolution of the system. As a sample-scanning microscope, the imaging speed is primarily limited by the performance of the Z-piezo stage. For simplicity and reduced optoelectronic complexity, we currently power the piezo through the ASI Tiger Controller. We will expand the supplementary material to describe the design criteria behind this choice, including potential trade-offs, and provide data quantifying the achievable volume rates under typical operating conditions.

While the microscope is well designed and completely open source, it will require experience with optics, electronics, and microscopy to implement and align properly. Experience with custom machining or soliciting a machine shop is also necessary. Thus, in my opinion, it is unlikely to be implemented by a lab that has zero prior experience with custom optics or can hire someone who does. Altair-LSFM may not be as easily adaptable or implementable as the authors describe or perceive in any lab that is interested, even if they can afford it. The authors indicate they will offer "workshops," but this does not necessarily remove the barrier to entry or lower it, perhaps as significantly as the authors describe.

We appreciate the reviewer’s perspective and agree that building any high-performance custom microscope—Altair-LSFM included—requires a baseline familiarity with optics and instrumentation. Our goal is not to eliminate this requirement entirely, but to significantly reduce the technical and logistical barriers that typically accompany custom light-sheet microscope construction.

Importantly, no machining experience or in-house fabrication capabilities are required—users can simply submit provided design files and specifications directly to the vendor. We will make this process as straightforward as possible by supplying detailed instructions, recommended materials, and vendor-ready files. Additionally, we draw encouragement from the success of related efforts such as mesoSPIM, which has seen over 30 successful implementations worldwide using a similar model of exhaustive online documentation, open-source control software, and community support through user meetings and workshops.

We recognize that documentation alone is not always sufficient, and we are committed to further lowering barriers to adoption. To this end, we are actively working with commercial vendors to streamline procurement and reduce the logistical burden on end users. Additionally, Altair-LSFM is supported by a Biomedical Technology Development and Dissemination (BTDD) grant, which provides dedicated resources for hosting workshops, offering real-time community support, and generating supplementary materials such as narrated video tutorials. We will expand our discussion in the revised manuscript to better acknowledge these implementation challenges and outline our ongoing strategies for supporting a broad and diverse user base.

There is a claim that this design is easily adaptable. However, the requirement of custom-machined baseplates and in silico optimization of the optical path basically means that each new instrument is a new design, even if the Navigate software can be used. It is unclear how Altair-LSFM demonstrates a modular design that reduces times from conception to optimization compared to previous implementations.

We appreciate the reviewer’s comment and agree that our language regarding adaptability may have been too strong. It was not our intention to suggest that the system can be easily modified without prior experience. Meaningful adaptations of the optical or mechanical design would require users to have expertise in optical layout, optomechanical design, and alignment.

That said, for labs with sufficient expertise, we aim to facilitate such modifications by providing comprehensive resources—including detailed Zemax simulations, CAD models, and alignment documentation. These materials are intended to reduce the development burden for those seeking to customize the platform for specific experimental needs.

In the revised manuscript, we will clarify this point and explicitly state in the discussion what technical expertise is required to modify the system. We will also revise our language around adaptability to better reflect the intended audience and realistic scope of customization.

Reviewer #3 (Public review):

Summary:

This manuscript introduces a high-resolution, open-source light-sheet fluorescence microscope optimized for sub-cellular imaging.

The system is designed for ease of assembly and use, incorporating a custom-machined baseplate and in silico optimized optical paths to ensure robust alignment and performance. The authors demonstrate lateral and axial resolutions of ~235 nm and ~350 nm after deconvolution, enabling imaging of sub-diffraction structures in mammalian cells.

The important feature of the microscope is the clever and elegant adaptation of simple gaussian beams, smart beam shaping, galvo pivoting and high NA objectives to ensure a uniform thin light-sheet of around 400 nm in thickness, over a 266 micron wide Field of view, pushing the axial resolution of the system beyond the regular diffraction limited-based tradeoffs of light-sheet fluorescence microscopy.

Compelling validation using fluorescent beads and multicolor cellular imaging highlights the system's performance and accessibility. Moreover, a very extensive and comprehensive manual of operation is provided in the form of supplementary materials. This provides a DIY blueprint for researchers who want to implement such a system.

Strengths:

(1) Strong and accessible technical innovation: With an elegant combination of beam shaping and optical modelling, the authors provide a high-resolution light-sheet system that overcomes the classical light-sheet tradeoff limit of a thin light-sheet and a small field of view. In addition, the integration of in silico modelling with a custom-machined baseplate is very practical and allows for ease of alignment procedures. Combining these features with the solid and super-extensive guide provided in the supplementary information, this provides a protocol for replicating the microscope in any other lab.

(2) Impeccable optical performance and ease of mounting of samples: The system takes advantage of the same sample-holding method seen already in other implementations, but reduces the optical complexity. At the same time, the authors claim to achieve similar lateral and axial resolution to Lattice-light-sheet microscopy (although without a direct comparison (see below in the "weaknesses" section). The optical characterization of the system is comprehensive and well-detailed. Additionally, the authors validate the system imaging sub-cellular structures in mammalian cells.

(3) Transparency and comprehensiveness of documentation and resources: A very detailed protocol provides detailed documentation about the setup, the optical modeling, and the total cost.

Weaknesses:

(1) Limited quantitative comparisons: Although some qualitative comparison with previously published systems (diSPIM, lattice light-sheet) is provided throughout the manuscript, some side-by-side comparison would be of great benefit for the manuscript, even in the form of a theoretical simulation. While having a direct imaging comparison would be ideal, it's understandable that this goes beyond the interest of the paper; however, a table referencing image quality parameters (taken from the literature), such as signal-to-noise ratio, light-sheet thickness, and resolutions, would really enhance the features of the setup presented. Moreover, based also on the necessity for optical simplification, an additional comment on the importance/difference of dual objective/single objective light-sheet systems could really benefit the discussion.

In the revised manuscript, we will expand our discussion to include a broader range of light-sheet microscope designs and imaging modes, including both single- and dual-objective configurations. We agree that highlighting the trade-offs between these approaches—such as working distance, sample geometry constraints, and alignment complexity—will enhance the overall context and utility of the manuscript.

To further aid comparison, we will include a summary table referencing key image quality parameters such as lateral and axial resolution, and illumination beam NA for Altair-LSFM. Where available, we will reference values from published work—such as the axial resolution reported in Valm et al. (Nature, 2017)—to provide a clearer benchmark. Because such comparisons can be technically nuanced, especially when comparing across systems with different geometries and sample mounting constraints, we will also include a supplementary note outlining the assumptions and limitations of these comparisons.

(2) Limitation to a fixed sample: In the manuscript, there is no mention of incubation temperature, CO₂ regulation, Humidity control, or possible integration of commercial environmental control systems. This is a major limitation for an imaging technique that owes its popularity to fast, volumetric, live-cell imaging of biological samples.

We thank the reviewer for highlighting this important consideration. In the revised manuscript, we will provide a detailed description of how temperature control can be implemented using flexible adhesive heating elements, a power supply, and a PID controller. Step-by-step assembly instructions and recommended components will be included to facilitate adoption by users interested in live-cell imaging. We also note that most light-sheet microscopy systems capable of sub-cellular resolution—including the original LLSM design, diSPIM, and ASLM—typically do not incorporate integrated CO₂ or humidity control. These systems often rely on HEPES-buffered media to maintain pH stability, which is generally sufficient for short- to intermediate-term imaging. While full environmental control may be necessary for extended time-lapse studies, it is not a prerequisite for high-resolution volumetric imaging in many applications. Nonetheless, we will include a discussion of the challenges associated with adding CO₂ and humidity control to open or semi-enclosed architectures like Altair-LSFM, and outline potential future paths for integration with commercial incubation systems.

(3) System cost and data storage cost: While the system presented has the advantage of being open-source, it remains relatively expensive (considering the 150k without laser source and optical table, for example). The manuscript could benefit from a more direct comparison of the performance/cost ratio of existing systems, considering academic settings with budgets that most of the time would not allow for expensive architectures. Moreover, it would also be beneficial to discuss the adaptability of the system, in case a 30k objective could not be feasible. Will this system work with different optics (with the obvious limitations coming with the lower NA objective)? This could be an interesting point of discussion. Adaptability of the system in case of lower budgets or more cost-effective choices, depending on the needs.

We thank the reviewer for raising this important point. First, we would like to clarify that the quoted $150k cost estimate includes the optical table and laser source. We apologize for any confusion and will communicate this more effectively in the revised manuscript.

We agree that adaptability is a key concern, especially in academic settings with limited budgets. The detection path can be readily altered depending on experimental needs and cost constraints. For example, in our discussion of alternatives to the 5 mm coverslip geometry, we will describe how switching to a Zeiss W Plan-Apochromat 20x/1.0 in combination with a compatible excitation objective allows high-resolution imaging while accommodating more conventional sample formats. We will expand this to include cost-effective alternatives as well.

We will also expand our discussion on cost-reduction strategies and the associated trade-offs. These include replacing motorized stages with manual ones, omitting the filter wheel in favor of a multi-band emission filter, or using industrial-grade cameras in place of scientific CMOS detectors. While each change entails some loss in functionality or sensitivity, such modifications allow users to tailor the system to their specific budget and application.

Finally, we recognize the challenge in communicating exact costs of commercial systems due to variability in configuration and pricing. Nonetheless, we will include approximate figures where possible and note that comparable commercial systems—such as LLSM platforms from 3i and Zeiss—are several-fold more expensive than the system presented here.

Last, not much is said about the need for data storage. Light-sheet microscopy's bottleneck is the creation of increasingly large datasets, and it could be beneficial to discuss more about the storage needs and the quantity of data generated.

Data storage is indeed a critical consideration in light-sheet microscopy. In the revised manuscript, we will provide a note outlining typical volume dimensions for live-cell imaging experiments along with the associated data overhead. This will include estimates for voxel counts, bit depth, time-lapse acquisitions, and multi-channel datasets to help users anticipate storage needs. We will also briefly discuss strategies for managing large datasets, file types and compression formats.

Conclusion:

Altair-LSFM represents a well-engineered and accessible light-sheet system that addresses a longstanding need for high-resolution, reproducible, and affordable sub-cellular light-sheet imaging. While some aspects-comparative benchmarking and validation, limitation for fixed samples-would benefit from further development, the manuscript makes a compelling case for Altair-LSFM as a valuable contribution to the open microscopy scientific community.

References

(1) Moore, R. P. et al. A multi-functional microfluidic device compatible with widefield and light sheet microscopy. Lab Chip 22, 136-147 (2021). https://doi.org/10.1039/d1lc00600b

(2) Lamb, J. R., Mestre, M. C., Lancaster, M. & Manton, J. D. Direct-view oblique plane microscopy. Optica 12, 469-472 (2025). https://doi.org/10.1364/OPTICA.558420

(3) Liu, T. L. et al. Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science 360 (2018). https://doi.org/10.1126/science.aaq1392

(4) Sapoznik, E. et al. A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics. eLife 9 (2020). https://doi.org/10.7554/eLife.57681

(5) Huisken, J. & Stainier, D. Y. Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt Lett 32, 2608-2610 (2007). https://doi.org/10.1364/ol.32.002608

(6) Ricci, P. et al. Removing striping artifacts in light-sheet fluorescence microscopy: a review. Prog Biophys Mol Biol 168, 52-65 (2022). https://doi.org/10.1016/j.pbiomolbio.2021.07.003

Reviewer #3 (Public review):

Summary:

This manuscript introduces a high-resolution, open-source light-sheet fluorescence microscope optimized for sub-cellular imaging.

The system is designed for ease of assembly and use, incorporating a custom-machined baseplate and in silico optimized optical paths to ensure robust alignment and performance. The authors demonstrate lateral and axial resolutions of ~235 nm and ~350 nm after deconvolution, enabling imaging of sub-diffraction structures in mammalian cells.

The important feature of the microscope is the clever and elegant adaptation of simple gaussian beams, smart beam shaping, galvo pivoting and high NA objectives to ensure a uniform thin light-sheet of around 400 nm in thickness, over a 266 micron wide Field of view, pushing the axial resolution of the system beyond the regular diffraction limited-based tradeoffs of light-sheet fluorescence microscopy.

Compelling validation using fluorescent beads and multicolor cellular imaging highlights the system's performance and accessibility. Moreover, a very extensive and comprehensive manual of operation is provided in the form of supplementary materials. This provides a DIY blueprint for researchers who want to implement such a system.

Strengths:

(1) Strong and accessible technical innovation:

With an elegant combination of beam shaping and optical modelling, the authors provide a high-resolution light-sheet system that overcomes the classical light-sheet tradeoff limit of a thin light-sheet and a small field of view. In addition, the integration of in silico modelling with a custom-machined baseplate is very practical and allows for ease of alignment procedures. Combining these features with the solid and super-extensive guide provided in the supplementary information, this provides a protocol for replicating the microscope in any other lab.

(2) Impeccable optical performance and ease of mounting of samples:

The system takes advantage of the same sample-holding method seen already in other implementations, but reduces the optical complexity. At the same time, the authors claim to achieve similar lateral and axial resolution to Lattice-light-sheet microscopy (although without a direct comparison (see below in the "weaknesses" section). The optical characterization of the system is comprehensive and well-detailed. Additionally, the authors validate the system imaging sub-cellular structures in mammalian cells.

(3) Transparency and comprehensiveness of documentation and resources:

A very detailed protocol provides detailed documentation about the setup, the optical modeling, and the total cost.

Weaknesses:

(1) Limited quantitative comparisons:

Although some qualitative comparison with previously published systems (diSPIM, lattice light-sheet) is provided throughout the manuscript, some side-by-side comparison would be of great benefit for the manuscript, even in the form of a theoretical simulation. While having a direct imaging comparison would be ideal, it's understandable that this goes beyond the interest of the paper; however, a table referencing image quality parameters (taken from the literature), such as signal-to-noise ratio, light-sheet thickness, and resolutions, would really enhance the features of the setup presented. Moreover, based also on the necessity for optical simplification, an additional comment on the importance/difference of dual objective/single objective light-sheet systems could really benefit the discussion.

(2) Limitation to a fixed sample:

In the manuscript, there is no mention of incubation temperature, CO₂ regulation, Humidity control, or possible integration of commercial environmental control systems. This is a major limitation for an imaging technique that owes its popularity to fast, volumetric, live-cell imaging of biological samples.

(3) System cost and data storage cost:

While the system presented has the advantage of being open-source, it remains relatively expensive (considering the 150k without laser source and optical table, for example). The manuscript could benefit from a more direct comparison of the performance/cost ratio of existing systems, considering academic settings with budgets that most of the time would not allow for expensive architectures. Moreover, it would also be beneficial to discuss the adaptability of the system, in case a 30k objective could not be feasible. Will this system work with different optics (with the obvious limitations coming with the lower NA objective)? This could be an interesting point of discussion. Adaptability of the system in case of lower budgets or more cost-effective choices, depending on the needs.

Last, not much is said about the need for data storage. Light-sheet microscopy's bottleneck is the creation of increasingly large datasets, and it could be beneficial to discuss more about the storage needs and the quantity of data generated.

Conclusion:

Altair-LSFM represents a well-engineered and accessible light-sheet system that addresses a longstanding need for high-resolution, reproducible, and affordable sub-cellular light-sheet imaging. While some aspects-comparative benchmarking and validation, limitation for fixed samples-would benefit from further development, the manuscript makes a compelling case for Altair-LSFM as a valuable contribution to the open microscopy scientific community.

DOI: 10.1038/s41593-025-01973-8

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What is this?

Reviewer #1 (Public review):

Summary:

The manuscript by Brothwell and colleagues describes a central role for hepatic cardiolipin deficiency in MASH. The authors identify cardiolipin as a mediator of two long-standing problems in the field: how dysregulated lipid metabolism relates to altered mitochondrial metabolism during MASLD, and what the innate changes are in the steatotic liver that cause the increased respiration. The authors identified reduced liver cardiolipin in humans with MASH and in a variety of mouse models with MASH. When they knocked out hepatic cardiolipin synthesis, mice developed steatosis and inflammation. These mice also recapitulated the elevated hepatic oxidative metabolism and oxidative stress found in obese humans with MASLD. Some of the in vivo functional data related to glucose homeostasis and substrate metabolism could be stronger, and interpretation of the in vitro flux data needs some clarification, but in both cases, the data are not essential to the main conclusions of the manuscript. Overall, the study offers compelling evidence that cardiolipin is reduced in MASLD and that impaired cardiolipin synthesis is sufficient to recapitulate many features of MASLD.

Strengths:

The main strengths of the study are:

(1) The identification of reduced cardiolipin levels in the liver of humans with MASLD and in a variety of mouse models of MASLD.

(2) The finding that loss of cardiolipin synthesis recapitulates steatosis and inflammation in MASH.

(3) The finding that loss of cardiolipin increases mitochondrial respiration, ROS production, and fat oxidation (in a separate hepatocyte cell line), again recapitulates several previous studies in obese humans with MASLD.

(4) Evidence, though less definitive, that cardiolipin deficiency promotes electron leak by disrupting respiratory supercomplexes and preventing CoQ reduction.

Weaknesses:

(1) Figure 3A-D tries to make the point that liver CLS KO causes defects in substrate handling in vivo, based on glucose and pyruvate tolerance tests. The KO mice have a blunted response to a glucose tolerance test, but the pyruvate tolerance test showed very little (almost no) effect on glucose levels in either WT or LKO mice. The small blunting of the response in the LKO is impossible to interpret (if it's real), since the ability to clear glucose is also increased, and no tracers were used. It might be useful to monitor pyruvate and lactate levels during the experiment. However, this reviewer doesn't think the data is essential to prove the authors' main points.

(2) After presenting convincing evidence that respiration is elevated in isolated mitochondria from CLS KO liver, the authors follow up the findings by investigating whether 13C-palmitate and 13C-glucose oxidation are altered by CLS knockdown in murine Hepa1-6 cells (Figure 4). A few comments are worth mentioning about Figure 4:

a. It is not clear why the authors chose to use a hepatoma cell line rather than primary hepatocytes from LKO mice. The latter would be more convincing, since there could be important differences in metabolism between hepatoma cells and hepatocytes (e.g., preference for fatty acids vs glucose). Nevertheless, I think the approach is sufficient to test the general effect of loss of CLS on substrate metabolism.

b. The authors use the M+2 enrichments of TCA cycle intermediates to infer rates of oxidation of [U-13C]palmitate or [U-13C]glucose. It is important to note that this kind of data reports fractional carbon sources (i.e., substrate preference) rather than rates of oxidation. For example, data from the 13C-palmitate experiment indicates that the CLS KD cells increase the fractional contribution from 13C palmitate (compared to glucose, for example) to the TCA cycle, but the actual rate of palmitate oxidation is not implicit in the data. However, it is reasonable to suggest that, in combination with the increased rates of O2 consumption observed in isolated mitochondria, this data supports increased fat oxidation.

c. I have some concern that the [U-13C]glucose experiment is more complicated to interpret than the description implies. I'm not sure what happens in this cell line, but in the liver, most labeling from pyruvate (i.e., originating from glucose in this case) enters the TCA cycle via pyruvate carboxylase, with smaller amounts entering via PDH (depending on the nutritional state). Since one could expect pyruvate carboxylase to contribute M+3 labeled TCA cycle intermediates initially, and M+2 on the first turn of the cycle, it's hard to conclude what the data indicates about glucose oxidation. The authors could generalize the conclusion by framing the TCA cycle enrichment data as the contribution of glucose carbons and noting in Figure 4A that pyruvate carbons can enter the TCA cycle via PDH or pyruvate carboxylase, without attempting to assign their relative contributions. There are better ways to do it, but it's a small nuance here since the authors aren't making a critical point about the pathways.

Reviewer #2 (Public review):

In this study, the authors show that alterations in the lipid composition of the inner mitochondrial membrane, particularly changes in cardiolipin (CL) content, lead to defects in electron transport, supercomplex formation, and oxidative stress. Using liver-specific CLS knockout mice, which are characterized by dysfunctional capacity for cardiolipin synthesis, the authors highlight an underappreciated role for CL in MASH pathology. Overall, this is an interesting study highlighting the importance of functional/physiological electron transport (and in this context, electron leakage) in MASH pathophysiology. Despite that, this manuscript has several weaknesses that require attention.

(1) For all LKO studies, it is stated that the decrease in hepatic CL is causal for the observed phenotype. However, it is evident that many other lipids are impacted by CLS KO, including a marked increase in hepatic PG. In this respect, the authors show no evidence that the observed metabolic phenotype is indeed due to the reduction in CL and not to other accompanying changes.

(2) In the results, the authors highlight that 'MASLD has been shown to alter the total cellular lipidome in liver.' Given that this study focused on CL, it would be useful to include specific studies that pointed to changes in hepatic CL content in MASLD/MASH/fibrosis.

(3) The initial human mitochondrial lipidomics studies show a reduction in mitochondrial CL and PG content. What was the content/expression of CL synthase and PGP synthase in these samples? If this cannot be assessed, is there any association of CLS or PGPS expression and MASLD/fibrosis (etc) in publicly available databases (e.g, GEP liver) that may explain the reduction in mitochondrial PG and CL content?

(4) The validation of MASH in patients (Figure 1B) is not convincing (ie., no quantification/scoring provided). NAS /fibrosis scoring (according to Kleiner) would help to define if all patients have indeed MASH, and what subset has fibrosis. Could the reduction in CL/PG content be (also) associated with fibrosis? In addition, Masson's Trichrome should be added to Figure 1B.

(5) In human lipidomics, the authors suggest that reductions are observed in tetralinoleoyl CL (Figure 1C). However, Figure 1C only shows the combined FA acyl chain length + unsaturation, therefore not allowing for FA-specific ID (unless such data are available from the LC/MS analysis).

(6) Figures 1 J/K/I. It is obvious that the background in all murine immunoblotting analysis has been altered. The authors should provide unaltered images for these immunoblots.

(7) For Figure 1, it is unclear what is meant by 'we performed all mitochondrial lipidomic analyses by quantifying lipids per mg of mitochondrial proteins'. Was the murine lipidomics carried out on fractionated mitochondria or whole liver? If whole liver, then how were the data corrected, particularly given that PG is not a mitochondria-specific lipid?

(8) While total CL content seems indeed decreased across the different mouse models, this is mostly due to 1-2 CL species showing a pronounced reduction, with the remainder being unaltered. This should at least be acknowledged in the results. This is similarly the case in the LKO livers.

(9) Figure 2. A secondary biochemical analysis of changes in lipid content should be provided, e.g., total triglyceride content, particularly given that the histology analysis does not show any major changes in hepatic lipid droplets/steatosis. In addition, the Masson's Trichrome staining shows almost no collagen deposition.

(10) Figure 3. 'CLS deletion modestly reduced glucose handling' should be reworded. The LKO mice show improved glucose tolerance (despite the MASH phenotype), which is not evident from the above wording.

(11) Looking at the mechanism behind the increase in hepatic steatosis, the authors state that lipid accumulation can occur due to increased lipogenesis, or dysfunctional VLDL secretion or beta oxidation, and subsequently assessed the relevant proteins/pathways. What about fatty acid uptake, which is also one of the four major pathways impacted in MASLD? This should be included in this assessment in Figure 3.

(12) For Figure 5A, it is simply stated 'CLS deletion promotes liver fibrosis in standard chow-fed condition', and it is unclear what is highlighted within the selected EM images and what the arrows refer to. The authors should clarify this within the text.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Mollá-Albaladejo et al. investigate the neurons downstream of GR64f and Gr66a, called G2Ns. They identify downstream neurons using trans-Tango labeling with RFP and then perform bulk RNA-seq on the RFP-sorted cells. Gene expression is up- or downregulated between the cell populations and between fed and starved states. They specifically identify Leukocinin as a neuropeptide that is upregulated in starved Gr66a cells. Leucokinin cells, identified by a GAL4 line indeed show higher expression when starved, especially in the SEZ. Furthermore, Leucokinin cells colocalize with the transTango signal from downstream neurons of both GRs. This connection is confirmed with GRASP. According to EM data, Leucokinin cells in the SEZ receive a lot of input and connect to many downstream neurons. In behavior experiments performed with flies lacking Leucokinin neurons, flies show reduced responsiveness to sugar and bitter mixtures when starved. The authors suggest that Leucokinin neurons integrate bitter and sugar tastes and that their output is modified by a hunger state.

Strengths:

The authors use a multitude of tools to identify SELK neurons downstream of taste sensory neurons and as starvation-sensitive cells. This study provides an example of how combining genetic labeling, RNA-seq, and EM analysis can be combined to investigate neural circuits.

Weaknesses:

The authors do not show a functional connection between sensory neurons and SELK neurons. Additionally, data from RNA seq, anatomical studies, and EM analysis are sometimes contradictory in terms of connectivity. GRASP signal is not foolproof that cells are synaptically connected.

We appreciate the reviewer’s comments. Unfortunately, we have not successfully demonstrated a functional response of SELK neurons using in vivo calcium imaging with UAS-GCaMP7 (we tried f, m, and s versions), primarily due to challenges in obtaining stable signals. We stimulated GRNs using sucrose, caffeine, or a mixture of both, and maybe even if the concentrations were high, they were not enough to induce a response.

Regarding GRASP, we acknowledge its limitations as a standalone technique for establishing genuine synaptic connections between neurons, as some signals may reflect false positives resulting from the mere proximity of the candidate neurons. To strengthen our findings, we complemented these results by demonstrating the positive colocalization of the Leucokinin antibody signal over the Gr66aGal4>trans-TANGO and Gr64f-Gal4>trans-TANGO (Figure 4), confirming that Leucokinin neurons are indeed postsynaptic to both sweet and bitter GRNs. Moreover, we incorporated BacTrace data to highlight the direct connectivity between sweet and bitter GRNs (now Figure 5E).

In the revised manuscript, we have introduced the active-GRASP technique (Macpherson et al., 2015). In this version of GRASP, the presynaptic half of GFP (GFP 1-10) is fused to synaptobrevin, which becomes accessible in the membrane of the presynaptic neuron within the synaptic cleft upon presynaptic stimulation (in our case, by stimulating with sucrose sweet Gr64f^GRNs and with caffeine the bitter Gr66a^GRNs). Utilizing this technique, we successfully demonstrated (see new Figure 5B and 5D) that when presented with water, no signal was detected in the Gr66a-LexA, Lk-Gal4 > active-GRASP, or Gr64f-LexA, Lk-Gal4 > active-GRASP transgene flies. However, in the presence of caffeine, Gr66aLexA, Lk-Gal4 > active-GRASP transgene flies exhibited a clear signal in the SEZ, and similarly, sucrose presentation to Gr64f-LexA, Lk-Gal4 > active-GRASP transgene flies yielded a detectable signal. The results obtained from active-GRASP provide additional evidence supporting the connectivity between SELK neurons and both Gr64f^GRNs and Gr66a^GRNs, further indicating the functional connectivity of the GRNs and SELK neurons.

The authors describe a behavioral phenotype when flies are starved, however, they do not use a specific driver for the described cell type, thus they should also tone down their claims.

We agree with the reviewer that the Lk-Gal4 driver line used labels SELK, LHLK, and ABLK neurons. The behavior examined in this paper, the Proboscis Extension Response (PER), measures the initiation of feeding. Although the neural circuit involved in this behavior is primarily confined to the SEZ where SELK neurons are located, we cannot rule out the possibility that other Lk neurons may also play a role in the process. To restrict expression of the Tetanus Toxin, we have utilized the tsh-Gal80 (Clyne et al., 2008) transgene in combination with the Lk-Gal4>UAS-TNT and Lk-Gal4>UAS-TNT^imp constructs to prevent the expression of the Tetanus Toxin in ABLK neurons, thereby restricting its expression to the SELK and LHLK neurons in the central brain. The new results (Sup Figure 7A) indicate that ABLK neurons do not play a role in integrating sweet and bitter information. However, we acknowledge the reviewer's point that we are still silencing LHLK neurons, so we have adjusted our claims to align more closely with our data

Generally, the authors do not provide a big advancement to the field and some of the results are contradictory with previous publications.

We believe our work does not contradict previous findings, nor does it invalidate the role of ABLK neurons in water homeostasis or the role of LHLK neurons in regulating sleep via starvation. We provide additional information on the possible role of SELK neurons in integrating gustatory information. The location of SELK neurons in the SEZ suggests that they may play a role in feeding behavior, and we have demonstrated that these neurons are indeed involved in integrating gustatory information to influence feeding decisions. We consider we have contributed by highlighting a new role for the Leucokinin neuropeptide in feeding behavior.

Reviewer #2 (Public review):

Summary:

A core task of the brain is processing sensory cues from the environment. The neural mechanisms of how sensory information is transmitted from peripheral sense organs to subsequent being processing in defined brain centers remain an important topic in neuroscience. The taste system hereby assesses the palatability of food by evaluating the chemical composition and nutrient content while integrating the current need for energy by assessing the satiation level of the organism. The current manuscript provides insights into the early circuits of gustatory coding using the fruit fly as a model. By combining trans-tango and FACS- based bulk RNAseq to assess the target neurons of sweet sensing (using Gr64fGal4) and bitter sensing (using Gr66a-Gal4) in a first set of experiments the authors investigate genes that are differentially expressed or co-expressed in normal and starved conditions. With a focus on neuropeptides and neurotransmitters, different expressions in the different conditions were assessed resulting in the identification of Leucokinin as a potentially interesting gene. The notion is further supported by RNAseq of Lk- Gal4>mCD8:GFP sorted cells and immunostainings. GRASP and BacTrace experiments further support that the two Lk- expressing cells in the SEZ should indeed be postsynaptic to both types of sensories. Using EM-based connectomics data (based on a previous publication by Engert et al.), the authors also look for downstream targets of the bitter versus sweet gustatory neurons to identify the Lk-neurons. Based on the morphology they identify candidates and further depict the potential downstream neurons in the connectome, which appears largely in agreement with GRASP experiments. Finally silencing the Lk- neurons shows an increased PER response in starved flies (when combined with bitter compounds) as well as increased feeding neurons shows an increased PER response in starved flies (when combined with bitter compounds) as well as increased feeding in a FlyPad assay. Strengths:

Overall this is an intriguing manuscript, which provides insight into the organization of 2nd order gustatory neurons. It specifically provides strong evidence for the Lk-neurons as a target of sweet and bitter GRNs and provides evidence for their role in regulating sweet vs bitter-based behavioral responses. Particularly the integration of different techniques and datasets in an elegant fashion is a strong side of the manuscript. Moreover to put the known LK-neurons into the context of 2nd order gustatory signalling is strengthening the knowledge about this pathway.

Weaknesses:

I do not see any major weakness in the current manuscript. Novelty is to some degree lessened by the fact, that the RNAseq approach did not identify new neurons but rather put the known LK-neurons as major findings. Similarly, the final behavioral section is not very deep and to some degree corroborates the previous publication by the Keene and Nässel labs - that said, the model they propose is indeed novel (but lacks depth in analyses; e.g. there is no physiology that would support the modulation of Lk neurons by either type of GRN). The connectomic section appears a bit out of place and after reading it it's not really clear what one should make of the potential downstream neurons (particularly since the Lk-receptor expression has been previously analyzed); here it might have been interesting to address if/how Lk-neurons may signal directly via a classical neurotransmitter (an information that might be found easily in the adult brain single-cell data).

We thank the reviewer for the comment. Indeed, we attempted in vivo Ca imaging but were unsuccessful. We have rewritten the connectomic section to better integrate it with the rest of the text and have reanalyzed the data obtained. We considered gathering data from the single-cell adult dataset, but this dataset includes the entire adult fly brain, encompassing SELK and LHLK neurons, making it impossible to differentiate between the two types of Lk neurons. Any further analysis will require transcriptomic analysis of SELK via scRNAseq under the different metabolic conditions tested in this study work.

Reviewer #3 (Public review):

Summary:

To make feeding decisions, animals need to process three types of information: positive cues like sweetness, negative cues like bitterness, and internal states such as hunger or satiety. This study aims to identify where the information is integrated into the fruit fly brain. The authors applied RNA sequencing on second-order gustatory neurons responsible for sweet and bitter processing, under fed and starved conditions. The sequencing data reveal significant changes in gene expression across sweet vs. bitter pathways and fed vs. starved states. The authors focus on the neuropeptide Leucokinin (Lk), whose expression is dependent on the starvation state. They identify a pair of neurons, named SELK neurons, which express Lk and receive direct input from both sweet and bitter gustatory neurons. These SELK neurons are ideal candidates to integrate gustatory and internal state information. Behavioral experiments show that blocking these neurons in starved flies alters their tolerance to bitter substances during feeding.

Strengths:

(1) The study employs a well-designed approach, targeting specific neuronal populations, which is more efficient and precise compared to traditional large-scale genetic screening methods.

(2) The RNAseq results provide valuable data that can be utilized in future studies to explore other molecules beyond Lk.

(3) The identification of SELK neurons offers a promising avenue for future research into how these neurons integrate conflicting gustatory signals and internal state information.

Weaknesses:

(1) Unfortunately, due to technical challenges, the authors were unable to directly image the functional activity of SELK neurons.

(2) In the behavioral experiments, tetanus toxin was used to block SELK neurons. Since these neurons may release multiple neurotransmitters or neuropeptides, the results do not specifically demonstrate that Leucokinin (Lk) is the critical factor, as suggested in Figure 8. To address this, I recommend using RNAi to inhibit Lk expression in SELK neurons and comparing the outcomes to wild-type controls via the PER assay.

We appreciate the author's comments and suggestions. As noted, Tetanus Toxin silences the neuron’s activity, affecting the functioning of various neurotransmitters and neuropeptides released by the targeted neuron. In response to the reviewer's recommendation, we employed an RNAi line specifically designed to silence Leucokinin production in Lk-expressing neurons.

The results presented in Supplementary Figure 7B demonstrate that knocking down Leucokinin in Lk neurons significantly reduces the flies' tolerance to caffeine in sweet food.

It is crucial to highlight that the sucrose concentration used in Figure 7C was 50mM, whereas in Supplementary Figure 7B, it was increased to 100mM. This adjustment was necessary because the Lk-Gal4, UAS-RNAi, and Lk-Gal4>UAS-RNAi transgenic lines exhibited reduced sensitivity to sucrose compared to the Lk-Gal4>UAS-TNT or Lk-Gal4>UAS-TNT^imp lines. We aimed to establish a sucrose concentration that would elicit a 50% Proboscis Extension Response (PER) without adding any other compound, thereby allowing us to evaluate the additional effect of caffeine in the food.

However, according to the data derived from the connectome, SELK neurons might be cholinergic, and this neurotransmitter might be involved in controlling also the behavior of the flies.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

To get more evidence for connections between sensory cells and SELK neurons, could the authors also analyze a second available EM data set? Would setting a different threshold (>5 synapses) reveal connections to both sensories? Comparisons between SELK in- and outputs from EM data and Tango labeling also seem to differ quite a lot based on provided images - can the authors count cell bodies in the stainings? Further proof would be to provide functional imaging data that shows that SELK neurons respond to sugar and bitter compounds.

In this study, we utilized the recently published EM dataset for the Drosophila central brain connectome (Dorkenwald et al., 2024; Flywire.ai). Changing the number of synapses affects the counts of pre- and postsynaptic neurons. We set a threshold of more than five synapses, as recommended by Flywire, to avoid false positives (Dorkenwald et al., 2024). This threshold has been widely used in recent papers (Engert et al., 2022; Shiu et al., 2022; Walker et al., 2025).

The neuron counts in the connectomic data differ from those in the trans- and retro-TANGO experiments. In our initial trans-TANGO experiment, which labeled postsynaptic neurons in the Gr64fGal4 and Gr66a-Gal4 transgenic lines, we counted the labeled neurons (see Supplementary Figure 1C) and observed considerable variability between different brains. Due to anticipated variability, we did not count the labeled neurons from trans-TANGO and retro-TANGO techniques in the Leucokinin neurons. Furthermore, neither technique labels all postsynaptic or presynaptic neurons, respectively. A recent study on the retro-TANGO technique (Sorkac et al., 2023) found a minimum threshold: the presynaptic neuron must form a certain number of synapses with the neuron of interest to be adequately labeled. According to this paper, the established threshold is 17 synapses. It is likely that the trans-TANGO technique also has a threshold relating to the number of labeled neurons, contingent on the synapse count. This would explain the discrepancy between the two results.

Unfortunately, we have not been able to provide functional data pointing to the activation of SELK neurons by sucrose or caffeine. However, our active-GRASP data indicates that the connectivity between Gr64f^GRNs and Gr66a^GRNs with SELK neurons is present and functional.

How many Leucokinin-positive cells are in the SEZ? Does the RNA-seq data provide further information about the SELK neurons? Potential receptor candidates for how they integrate hunger signals? AMPKa was described to be required in LHLK neurons.

There are two SELK neurons in the SEZ. Due to the nature of our bulk RNA sequencing (RNAseq), we cannot link any additional gene expressions detected in our transcriptomic analysis specifically to the SELK neurons regarding the integration of various signaling processes. Furthermore, the single-cell RNA sequencing (scRNAseq) data available from the Drosophila brain, as reported by Li et al. (2022), does not allow accurate differentiation between SELK and LHLK neurons. To understand how these neurons integrate both metabolic and sensory information, it is crucial to conduct a focused RNAseq study specifically on the SELK neurons to understand how these neurons integrate both metabolic and sensory information. This targeted analysis would provide the necessary insights to elucidate their functional roles better. However, according to the data derived from the connectome, SELK neurons might be cholinergic, and this neurotransmitter might be involved in controlling also the behavior of the flies.

According to previous studies (Yurgel et al., 2019), the Lk-GAL4 line is also expressed in the VNC, thus the authors could make use of the tsh-GAL80 tool to clean up the line. This study also performed GCaMP imaging in fed and 24h starved animals in SELK and couldn't find a difference, can the authors explain this discrepancy?

We thank the reviewer for this suggestion. We have now added a new piece of data using the tsh-Gal80 transgene in our PER experiments (Supplementary Figure 7A). Blocking the expression of TNT in the ABLK neurons does not affect the main conclusion of the behavioral results. As stated previously, we were unable to obtain in vivo Ca imaging responses in SELK neurons upon exposure to sucrose, caffeine, or mixtures of sucrose and caffeine. We do not believe this is a discrepancy with previous works like Yurgel et al., 2019. It is likely that we faced technical issues regarding expression stability and that the stimulation was possibly too weak to detect changes in GFP levels

Reviewer #2 (Recommendations for the authors):

As mentioned above I do not have any major comments on the manuscript, but there are a few points that I feel should be considered:

(1) The identification of the Lk-candidate neurons in the connectome remains a bit mysterious. In the method sections, this reads as follows "manual and visual criteria were applied to identify the neurons of interest ". a) What precisely was done to get to the candidates?b) Are there alternative candidates that may be Lk-neurons? c) How would another neuron affect the conclusion of the downstream analysis?

We thank the reviewer for this comment. We have now modified and added new information in the connectomic section, reinforcing our conclusions and correcting the results obtained.

Our GRASP, BacTRace, and immunohistochemistry experiments pointed to SELK neurons as postsynaptic to both Gr64f^GRNs (sweet) and Gr66a^GRNs (bitter). To identify which neurons in the connectome could be the SELK neurons, we utilized a previously described set of GRNs already identified in the connectome (Shiu et al., 2022). We extracted all postsynaptic neurons to the sweet and bitter GRNs identified and intersected both datasets, retaining only those candidate hits receiving simultaneous input from sweet and bitter GRNs. This process yielded a total of 333 hits. Through visual inspection, we discarded all hits that were merely neuronal fragments or neurons that clearly were not our candidates. We narrowed the list down to a final set of 17 candidate neurons whose arborization was located in the SEZ. We reduced the candidates to two final entries from this list: ID 720575940623529610 (GNG.276) and ID 720575940630808827 (GNG.685). The GNG.276 neuron had a counterpart in the SEZ identified as GNG.246. Both of these neurons were annotated as DNg70 in the Flywire database. GNG.685 had a counterpart identified as GNG.595, and these two neurons were classified as DNg68. In both cases, the neuronal candidates, DNg70 and DNg68, were classified as descending neurons, a characteristic of previously described SELK neurons (Nässel et al., 2021). In our initial analysis published in bioRxiv and sent for revision, we identified DNg70 as potentially the SELK neurons based solely on the morphology of the neurons via visual inspection. However, we employed a better method to determine which candidate is more likely to be the SELK neurons, concluding that DNg68, rather than DNg70, represents the SELK neurons. Briefly, we performed an immunohistochemistry for GFP in the Lk-Gal4>UAS-CD8:GFP flies. We aligned the resulting image in a Drosophila reference brain (JRC2018 U) using the CMTK Registration plugin in ImageJ. The resulting image was skeletonized using the Single Neurite Tracer plugin in ImageJ and later uploaded to the Flywire Gateway platform to compare the structure of the aligned and skeletonized SELK neurons to our candidates. This comparison clearly indicated that the DNg68 neurons are the best candidates for representing the SELK neurons, rather than DNg70. We have updated the text and Figures 6 and Supplementary Figure 6 to reflect the new results. These new results do not alter the conclusions of the paper.

(2) In the transcriptomic experiments It seems that the raw transcripts are reporters, rather than normalised data. Why?

All transcriptomic data is normalized. In Figure 1 the differential expression was calculated using Deseq2 normalized counts. In Figure 2, Transcripts Per Million (TPM) were calculated using the Salmon package and normalized for the gene length.

(3) The expression of nAChRbeta1 in the transcriptomic data is rather striking. However, this remains currently not addressed: is this expression real?

We have not confirmed the upregulation or downregulation in gene expression for other but for Leucokinin, which is our main interest. We found the presence of nAChRbeta1 interesting, as GRNs are cholinergic (Jaeger et al., 2018), suggesting that it would make sense to find cholinergic receptors in G2Ns. However, it is possible that these receptors are expressed in all G2Ns and serve as a common means of communication.

(4) The description of the behavioural experiments in the results section is rather brief. I had a hard time following it since the genotypes are not repeated nor is it stated what is different in the experimental group vs control (but instead simply what changes in the experimental group, in a rather discussion-like fashion).

We thank the reviewer for the comment, we have rewritten this section to improve its clarity.

(5) If I understand the genetics for the behavioural experiments correctly it addresses the entire Lk-Gal4 expressing population, thus it is not possible to describe the role of the two SEZ neurons, but rather LkGal4 neurons. This should be clarified.

We thank the reviewer for this comment. Indeed, the Lk-Gal4 driver we used drives expression in all Leucokinin neurons, making it impossible to distinguish between the SELK, LHLK, or ABLK neurons. We have added a new piece of behavioral data by using the tsh-Gal80 transgene to prevent the expression of TNT in the ABLK neurons (Supplementary Figure 7A), but still we cannot distinguish between SELK and LHLK. We have rewritten the text to clarify this fact.

Reviewer #3 (Recommendations for the authors):

Overall, the manuscript is well-written, I only have one minor suggestion for improvement. In Figure 8C, please clarify the use of TNT to block Lk release.

We thank the reviewer for the comment, we have clarified the use of TNT in the text.

References Clyne, J. D. & Miesenböck, G. Sex-Specific Control and Tuning of the Pattern Generator for Courtship Song in Drosophila. Cell 133, 354–363 (2008).

Dorkenwald, S. et al. Neuronal wiring diagram of an adult brain. Nature 634, 124–138 (2024).

Engert, S., Sterne, G. R., Bock, D. D. & Scott, K. Drosophila gustatory projections are segregated by taste modality and connectivity. Elife 11, e78110 (2022).

Jaeger, A. H. et al. A complex peripheral code for salt taste in Drosophila. Elife 7, e37167 (2018).

Macpherson, L. J. et al. Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation. Nat Commun 6, 10024 (2015).

Nässel, D. R. Leucokinin and Associated Neuropeptides Regulate Multiple Aspects of Physiology and Behavior in Drosophila. Int J Mol Sci 22, 1940 (2021).

Shiu, P. K., Sterne, G. R., Engert, S., Dickson, B. J. & Scott, K. Taste quality and hunger interactions in a feeding sensorimotor circuit. eLife 11, e79887 (2022).

Walker, S. R., Peña-Garcia, M. & Devineni, A. V. Connectomic analysis of taste circuits in Drosophila. Sci. Rep. 15, 5278 (2025).

Author response:

Reviewer #1:

As this code was developed for use with a 4096 electrode array, it is important to be aware of double-counting neurons across the many electrodes. I understand that there are ways within the code to ensure that this does not happen, but care must be taken in two key areas. Firstly, action potentials traveling down axons will exhibit a triphasic waveform that is different from the biphasic waveform that appears near the cell body, but these two signals will still be from the same neuron (for example, see Litke et al., 2004 "What does the eye tell the brain: Development of a System for the Large-Scale Recording of Retinal Output Activity"; figure 14). I did not see anything that would directly address this situation, so it might be something for you to consider in updated versions of the code.

We thank the reviewer for this insightful comment. We agree that signals from the same neuron may be collected by adjacent channels. To address this concern in our software, we plan to add a routine to SpikeMAP that allows users to discard nearby channels where spike count correlations exceed a pre-determined threshold. Because there is no ground truth to map individual cells to specific channels on the hd-MEA, a statistical approach is warranted.

Secondly, spike shapes are known to change when firing rates are high, like in bursting neurons (Harris, K.D., Hirase, H., Leinekugel, X., Henze, D.A. & Buzsáki, G. Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells. Neuron 32, 141-149 (2001)). I did not see this addressed in the present version of the manuscript.

This is a valid concern. To ensure that firing rates are relatively constant over the duration of a recording, we will plot average spike rates using rolling windows of a fixed duration. We expect that population firing rates will remain relatively stable across the duration of recordings.

Another area for possible improvement would be to build on the excellent validation experiments you have already conducted with parvalbumin interneurons. Although it would take more work, similar experiments could be conducted for somatostatin and vasoactive intestinal peptide neurons against a background of excitatory neurons. These may have different spike profiles, but your success in distinguishing them can only be known if you validate against ground truth, like you did for the PV interneurons.

We agree that further cycles of experiments could be performed with SOM, VIP, and other neuronal subtypes, and we hope that researchers will take advantage of SpikeMAP too. We will clarify this possibility in the Discussion section of the manuscript.

Reviewer #2:

Summary:

While I find that the paper is nicely written and easy to follow, I find that the algorithmic part of the paper is not really new and should have been more carefully compared to existing solutions. While the GT recordings to assess the possibilities of a spike sorting tool to distinguish properly between excitatory and inhibitory neurons are interesting, spikeMAP does not seem to bring anything new to state-of-the-art solutions, and/or, at least, it would deserve to be properly benchmarked. I would suggest that the authors perform a more intensive comparison with existing spike sorters.

We thank the reviewer for this comment. As detailed in Table 1, SpikeMAP is the only method that performs E/I sorting on large-scale multielectrodes, hence a comparison to competing methods is not currently possible. That being said, many of the pre-processing steps of SpikeMAP (Figure 1) involve methods that are already well-established in the literature and available under different packages. To highlight the contribution of our work and facilitate the adoption of SpikeMAP, we plan to provide a “modular” portion of SpikeMAP that is specialized in performing E/I sorting and can be added to the pipeline of other packages such as KiloSort more clearly.  This modularized version of the code will be shared freely along with the more complete version already available.

Weaknesses:

(1) The global workflow of spikeMAP, described in Figure 1, seems to be very similar to that of Hilgen et al. 2020 (10.1016/j.celrep.2017.02.038). Therefore, the first question is what is the rationale of reinventing the wheel, and not using tools that are doing something very similar (as mentioned by the authors themselves). I have a hard time, in general, believing that spikeMAP has something particularly special, given its Methods, compared to state-of-the-art spike sorters.

We agree with the reviewers that there are indeed similarities between our work and the Hilgen et al. paper. However, while the latter employs optogenetics to stimulate neurons on a large-scale array, their technique does not specifically target inhibitory (e.g., PV) neurons as described in our work. We will clarify our paper accordingly.

This is why, at the very least, the title of the paper is misleading, because it lets the reader think that the core of the paper will be about a new spike sorting pipeline. If this is the main message the authors want to convey, then I think that numerous validations/benchmarks are missing to assess first how good spikeMAP is, with reference to spike sorting in general, before deciding if this is indeed the right tool to discriminate excitatory vs inhibitory cells. The GT validation, while interesting, is not enough to entirely validate the paper. The details are a bit too scarce for me, or would deserve to be better explained (see other comments after).

The title of our work will be edited to make it clear that while elements of the pipeline are well-established and available from other packages, we are the first to extend this pipeline to E/I sorting on large-scale arrays.

(2) Regarding the putative location of the spikes, it has been shown that the center of mass, while easy to compute, is not the most accurate solution [Scopin et al, 2024, 10.1016/j.jneumeth.2024.110297]. For example, it has an intrinsic bias for finding positions within the boundaries of the electrodes, while some other methods, such as monopolar triangulation or grid-based convolution, might have better performances. Can the authors comment on the choice of the Center of Mass as a unique way to triangulate the sources?

We agree with the reviewer and will point out limits of the center-of-mass algorithm based on the article of Scopin et al (2024). Further, we will augment the existing code library to include monopolar triangulation or grid-based convolution as options available to end-users.

(3) Still in Figure 1, I am not sure I really see the point of Spline Interpolation. I see the point of such a smoothing, but the authors should demonstrate that it has a key impact on the distinction of Excitatory vs. Inhibitory cells. What is special about the value of 90kHz for a signal recorded at 18kHz? What is the gain with spline enhancement compared to without? Does such a value depend on the sampling rate, or is it a global optimum found by the authors?

We will clarify these points. Specifically, the value of 90kHz was chosen because it provided a reasonable temporal characterization of spikes; this value, however, can be adjusted within the software based on user preference.

(4) Figure 2 is not really clear, especially panel B. The choice of the time scale for the B panel might not be the most appropriate, and the legend filtered/unfiltered with a dot is not clear to me in Bii.

We will re-check Fig.2B which seems to have error in rendering, likely due to conversion from its original format.

In panel E, the authors are making two clusters with PCA projections on single waveforms. Does this mean that the PCA is only applied to the main waveforms, i.e. the ones obtained where the amplitudes are peaking the most? This is not really clear from the methods, but if this is the case, then this approach is a bit simplistic and does not really match state-of-the-art solutions. Spike waveforms are quite often, especially with such high-density arrays, covering multiple channels at once, and thus the extracellular patterns triggered by the single units on the MEA are spatio-temporal motifs occurring on several channels. This is why, in modern spike sorters, the information in a local neighbourhood is often kept to be projected, via PCA, on the lower-dimensional space before clustering. Information on a single channel only might not be informative enough to disambiguate sources. Can the authors comment on that, and what is the exact spatial resolution of the 3Brain device? The way the authors are performing the SVD should be clarified in the methods section. Is it on a single channel, and/or on multiple channels in a local neighbourhood?

Here, the reviewer is suggesting that it may be better to perform PCA on several channels at once, since spikes can occur at several channels at the same time. To address this concern, small routine will be written allowing users to choose how many nearby channels to be selected for PCA.

(5) About the isolation of the single units, here again, I think the manuscript lacks some technical details. The authors are saying that they are using a k-means cluster analysis with k=2. This means that the authors are explicitly looking for 2 clusters per electrode? If so, this is a really strong assumption that should not be held in the context of spike sorting, because, since it is a blind source separation technique, one cannot pre-determine in advance how many sources are present in the vicinity of a given electrode. While the illustration in Figure 2E is ok, there is no guarantee that one cannot find more clusters, so why this choice of k=2? Again, this is why most modern spike sorting pipelines do not rely on k-means, to avoid any hard-coded number of clusters. Can the authors comment on that?

It is true that k=2 is a pre-determined choice in our software. In practice, we found that k>2 leads to poorly defined clusters. However, we will ensure that this parameter can be adjusted in the software. Furthermore, if the user chooses not to pre-define this value, we will provide the option to use a Calinski-Harabasz criterion to select k.

(6) I'm surprised by the linear decay of the maximal amplitude as a function of the distance from the soma, as shown in Figure 2H. Is it really what should be expected? Based on the properties of the extracellular media, shouldn't we expect a power law for the decay of the amplitude? This is strange that up to 100um away from the soma, the max amplitude only dropped from 260 to 240 uV. Can the authors comment on that? It would be interesting to plot that for all neurons recorded, in a normed manner V/max(V) as function of distances, to see what the curve looks like.

We share the reviewer’s concern and will add results that include a population of neurons to assess the robustness of this phenomenon.

(7) In Figure 3A, it seems that the total number of cells is rather low for such a large number of electrodes. What are the quality criteria that are used to keep these cells? Did the authors exclude some cells from the analysis, and if yes, what are the quality criteria that are used to keep cells? If no criteria are used (because none are mentioned in the Methods), then how come so few cells are detected, and can the authors convince us that these neurons are indeed "clean" units (RPVs, SNRs, ...)?

We applied stringent criteria to exclude cells, and we will revise the main text to be clear about these criteria, which include a minimum spike rate and the use of LDA to separate out PCA clusters. For the cells that were retained, we will include SNR estimates.

(8) Still in Figure 3A, it looks like there is a bias to find inhibitory cells at the borders, since they do not appear to be uniformly distributed over the MEA. Can the authors comment on that? What would be the explanation for such a behaviour? It would be interesting to see some macroscopic quantities on Excitatory/Inhibitory cells, such as mean firing rates, averaged SNRs... Because again, in Figure 3C, it is not clear to me that the firing rates of inhibitory cells are higher than Excitatory ones, whilst they should be in theory.       

We will include a comparison of firing rates for E and I neurons. It is possible that I cells are located at the border of the MEA due to the site of injections of the viral vector, and not because of an anatomical clustering of I cells per se. We will clarify the text accordingly.

(9) For Figure 3 in general, I would have performed an exhaustive comparison of putative cells found by spikeMAP and other sorters. More precisely, I think that to prove the point that spikeMAP is indeed bringing something new to the field of spike sorting, the authors should have compared the performances of various spike sorters to discriminate Exc vs Inh cells based on their ground truth recordings. For example, either using Kilosort [Pachitariu et al, 2024, 10.1038/s41592-024-02232-7], or some other sorters that might be working with such large high-density data [Yger et al, 2018, 10.7554/eLife.34518].

As mentioned previously, Kilosort and related approaches do not address the problem of E/I identification (see Table 1). However, they do have pre-processing steps in common with SpikeMAP. We will add some specific comparison points – for instance, the use of k-means and PCA (which is more common across packages) and the use of cubic spline interpolation (which is less common). Further, we will provide a stand-alone E/I sorting module that can be added to the pipeline of other packages, so that users can use this functionality without having to migrate their entire analysis.

(10) Figure 4 has a big issue, and I guess the panels A and B should be redrawn. I don't understand what the red rectangle is displaying.

We apologize for this issue. It seems there was a rendering problem when converting the figure from its original format. We will address this issue in the revised version of the manuscript.

(11) I understand that Figure 4 is only one example, but I have a hard time understanding from the manuscript how many slices/mice were used to obtain the GT data? I guess the manuscript could be enhanced by turning the data into an open-access dataset, but then some clarification is needed. How many flashes/animals/slices are we talking about? Maybe this should be illustrated in Figure 4, if this figure is devoted to the introduction of the GT data.

We will mention how many flashes/animals/slices were employed in the GT data and provide open access to these data.

(12) While there is no doubt that GT data as the ones recorded here by the authors are the most interesting data from a validation point of view, the pretty low yield of such experiments should not discourage the use of artificially generated recordings such as the ones made in [Buccino et al, 2020, 10.1007/s12021-020-09467-7] or even recently in [Laquitaine et al, 2024, 10.1101/2024.12.04.626805v1]. In these papers, the authors have putative waveforms/firing rate patterns for excitatory and inhibitory cells, and thus, the authors could test how good they are in discriminating the two subtypes.

We thank the reviewer for the suggestion that SpikeMAP could be tested on artificially generated spike trains and will add the citation of the two papers mentioned. We hope future efforts will employ SpikeMAP on both synthetic and experimental data to explore the neural dynamics of E and I neurons in healthy and pathological circuits of the brain.

Reviewer #2 (Public review):

Summary:

In this paper, entitled "SpikeMAP: An unsupervised spike sorting pipeline for cortical excitatory and inhibitory 2 neurons in high-density multielectrode arrays with ground-truth validation", the authors present spikeMAP, a pipeline for the analysis of large-scale recordings of in vitro cortical activity. According to the authors, spikeMAP not only allows for the detection of spikes produced by single neurons (spike sorting), but also allows for the reliable distinction between genetically determined cell types by utilizing viral and optogenetic strategies as ground-truth validation. While I find that the paper is nicely written and easy to follow, I find that the algorithmic part of the paper is not really new and should have been more carefully compared to existing solutions. While the GT recordings to assess the possibilities of a spike sorting tool to distinguish properly between excitatory and inhibitory neurons are interesting, spikeMAP does not seem to bring anything new to state-of-the-art solutions, and/or, at least, it would deserve to be properly benchmarked. I would suggest that the authors perform a more intensive comparison with existing spike sorters.

Strengths:

The GT recordings with optogenetic activation of the cells, based on the opsins, is interesting and might provide useful data to quantify how good spike sorting pipelines are, in vitro, to discriminate between excitatory and inhibitory neurons. Such an approach can be quite complementary to artificially generated ground truth.

Weaknesses:

(1) The global workflow of spikeMAP, described in Figure 1, seems to be very similar to that of Hilgen et al. 2020 (10.1016/j.celrep.2017.02.038). Therefore, the first question is what is the rationale of reinventing the wheel, and not using tools that are doing something very similar (as mentioned by the authors themselves). I have a hard time, in general, believing that spikeMAP has something particularly special, given its Methods, compared to state-of-the-art spike sorters. This is why, at the very least, the title of the paper is misleading, because it lets the reader think that the core of the paper will be about a new spike sorting pipeline. If this is the main message the authors want to convey, then I think that numerous validations/benchmarks are missing to assess first how good spikeMAP is, with reference to spike sorting in general, before deciding if this is indeed the right tool to discriminate excitatory vs inhibitory cells. The GT validation, while interesting, is not enough to entirely validate the paper. The details are a bit too scarce for me, or would deserve to be better explained (see other comments after).

(2) Regarding the putative location of the spikes, it has been shown that the center of mass, while easy to compute, is not the most accurate solution [Scopin et al, 2024, 10.1016/j.jneumeth.2024.110297]. For example, it has an intrinsic bias for finding positions within the boundaries of the electrodes, while some other methods, such as monopolar triangulation or grid-based convolution,n might have better performances. Can the authors comment on the choice of the Center of Mass as a unique way to triangulate the sources?

(3) Still in Figure 1, I am not sure I really see the point of Spline Interpolation. I see the point of such a smoothing, but the authors should demonstrate that it has a key impact on the distinction of Excitatory vs. Inhibitory cells. What is special about the value of 90kHz for a signal recorded at 18kHz? What is the gain with spline enhancement compared to without? Does such a value depend on the sampling rate, or is it a global optimum found by the authors?

(4) Figure 2 is not really clear, especially panel B. The choice of the time scale for the B panel might not be the most appropriate, and the legend filtered/unfiltered with a dot is not clear to me in Bii. In panel E, the authors are making two clusters with PCA projections on single waveforms. Does this mean that the PCA is only applied to the main waveforms, i.e. the ones obtained where the amplitudes are peaking the most? This is not really clear from the methods, but if this is the case, then this approach is a bit simplistic and does not really match state-of-the-art solutions. Spike waveforms are quite often, especially with such high-density arrays, covering multiple channels at once, and thus the extracellular patterns triggered by the single units on the MEA are spatio-temporal motifs occurring on several channels. This is why, in modern spike sorters, the information in a local neighbourhood is often kept to be projected, via PCA, on the lower-dimensional space before clustering. Information on a single channel only might not be informative enough to disambiguate sources. Can the authors comment on that, and what is the exact spatial resolution of the 3Brain device? The way the authors are performing the SVD should be clarified in the methods section. Is it on a single channel, and/or on multiple channels in a local neighbourhood?

(5) About the isolation of the single units, here again, I think the manuscript lacks some technical details. The authors are saying that they are using a k-means cluster analysis with k=2. This means that the authors are explicitly looking for 2 clusters per electrode? If so, this is a really strong assumption that should not be held in the context of spike sorting, because, since it is a blind source separation technique, one can not pre-determine in advance how many sources are present in the vicinity of a given electrode. While the illustration in Figure 2E is ok, there is no guarantee that one can not find more clusters, so why this choice of k=2? Again, this is why most modern spike sorting pipelines do not rely on k-means, to avoid any hard-coded number of clusters. Can the authors comment on that?

(6) I'm surprised by the linear decay of the maximal amplitude as a function of the distance from the soma, as shown in Figure 2H. Is it really what should be expected? Based on the properties of the extracellular media, shouldn't we expect a power law for the decay of the amplitude? This is strange that up to 100um away from the soma, the max amplitude only dropped from 260 to 240 uV. Can the authors comment on that? It would be interesting to plot that for all neurons recorded, in a normed manner V/max(V) as function of distances, to see what the curve looks like.

(7) In Figure 3A, it seems that the total number of cells is rather low for such a large number of electrodes. What are the quality criteria that are used to keep these cells? Did the authors exclude some cells from the analysis, and if yes, what are the quality criteria that are used to keep cells? If no criteria are used (because none are mentioned in the Methods), then how come so few cells are detected, and can the authors convince us that these neurons are indeed "clean" units (RPVs, SNRs, ...)?

(8) Still in Figure 3A, it looks like there is a bias to find inhibitory cells at the borders, since they do not appear to be uniformly distributed over the MEA. Can the authors comment on that? What would be the explanation for such a behaviour? It would be interesting to see some macroscopic quantities on Excitatory/Inhibitory cells, such as mean firing rates, averaged SNRs... Because again, in Figure 3C, it is not clear to me that the firing rates of inhibitory cells are higher than Excitatory ones, whilst they should be in theory.

(9) For Figure 3 in general, I would have performed an exhaustive comparison of putative cells found by spikeMAP and other sorters. More precisely, I think that to prove the point that spikeMAP is indeed bringing something new to the field of spike sorting, the authors should have compared the performances of various spike sorters to discriminate Exc vs Inh cells based on their ground truth recordings. For example, either using Kilosort [Pachitariu et al, 2024, 10.1038/s41592-024-02232-7], or some other sorters that might be working with such large high-density data [Yger et al, 2018, 10.7554/eLife.34518].

(10) Figure 4 has a big issue, and I guess the panels A and B should be redrawn. I don't understand what the red rectangle is displaying.

(11) I understand that Figure 4 is only one example, but I have a hard time understanding from the manuscript how many slices/mices were used to obtain the GT data? I guess the manuscript could be enhanced by turning the data into an open-access dataset, but then some clarification is needed. How many flashes/animals/slices are we talking about? Maybe this should be illustrated in Figure 4, if this figure is devoted to the introduction of the GT data.

(12) While there is no doubt that GT data as the ones recorded here by the authors are the most interesting data from a validation point of view, the pretty low yield of such experiments should not discourage the use of artificially generated recordings such as the ones made in [Buccino et al, 2020, 10.1007/s12021-020-09467-7] or even recently in [Laquitaine et al, 2024, 10.1101/2024.12.04.626805v1]. In these papers, the authors have putative waveforms/firing rate patterns for excitatory and inhibitory cells, and thus, the authors could test how good they are in discriminating the two subtypes.

https://reddit.com/user/colombocollection/comments/1ldes6b/to_all_my_north_georgia_peeps_i_will_be_at_my/

Jennifer Colombo of Colombo Collection has ties to Ellijay, GA.

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Reply to the reviewers

We appreciated the constructive suggestions from the reviewers, and the explanation of the contribution of the manuscript. We have revised the manuscript in accordance with their suggestions, as discussed below.

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ This manuscript presents a computational analysis of PRC1, a passive microtubule crosslinker important for cell division, with a focus on its role in resisting force generation within antiparallel bundles, whose sliding is promoted by active kinesin motors. Using a previously developed simulator and several assumptions, the authors successfully recapitulated the two modes of PRC1 sliding resistance - coasting and braking - that were previously observed in in vitro reconstruction assays. The simulation also reproduces the redistribution of PRC1 within the overlap region as microtubules transition into the braking mode, a phenomenon also observed experimentally. An interesting outcome of the simulation is the change in spacing between the microtubules: The distance narrows as the sliding polymers switch from the coasting to the breaking mode, associated with an increased tilt of PRC1.

Major comments: I find that this manuscript makes a valuable contribution to the cytoskeletal community, as the role of interfilament spacing in polymer assembly has been relatively underexplored, except for more classic studies such as those on muscle contraction and flagellar beating. What I had difficulty fully visualizing the model was the behavior of PRC1 during the coasting and braking modes. In my understanding, if individual heads of PRC1 bind and unbind to and from microtubules while microtubules that they crosslink slide apart, PRC1 should experience greater stretching and thus tilt more at higher sliding speeds. When the sliding slows down, the relative polymer position changes less within a given time, and PRC1 unbinding and re-binding would more easily reset their tilt to an equilibrium angle. However, the authors' simulation shows the opposite: PRC1 exhibits a greater tilt during the braking mode. This seems counterintuitive and a more detailed description and interpretation would worth. I suggest that the authors include a schematic illustrating the configuration of individual PRC1 molecules (e.g., angle and stretch) within the ensemble, particularly during their transition phase. This would greatly help readers grasp how this important protein ensemble switches its mechanical mode depending on polymer sliding and geometry.

We thank the reviewer for the comments on the contribution of the results of the manuscript. Braking typically initiates at higher sliding speeds, when PRC1 do experience greater stretching and tilt more as the reviewer writes. As sliding slows down, the ability of PRC1 to unbind, re-bind, and rest their tilt to the equilibrium angle is restricted by the small distance between the microtubules: PRC1 binding will tend to occur tilted in the direction of sliding, and molecules tilted in this direction promote close separations, keeping overlaps braking. To clarify why braking overlaps are stable we added text and figure 4H. Steric interactions within the clusters at the overlap edges also restrict rebinding. To illustrate the behavior of PRC1 molecules during the transition from coasting to braking , we have added in figure 4A a schematic derived from simulation data of the microtubule and PRC1 positions, separations, and tilts during the transition from coasting to braking.

Minor comments: 1. How was the bimodal velocity distribution (Fig. 1D) obtained experimentally? Were the individual data averaged over time from the start to the end of individual sliding events? If so, does mode switching within a pair lead to under/over-estimate of the coasting and braking speeds?

These data are reproduced from Alfieri et al. Current Biology 2021. In that paper, we acquired this data by observing the sliding separation of PRC1-crosslinked microtubule pairs and recorded two distinct velocities for each pair: the “bundled” velocity when overlap>0 and PRC1 was engaged in crosslinking and then the “escaped” velocity once the two microtubules had separated. In the vast majority of cases (>90%) each of these velocities was well measured by fitting a slope to the kymograph, as there were only very minor deviations from a linear position-versus-time relationship (e.g. we rarely saw acceleration or deceleration within an individual pair). In the rare (

Line 158 includes typo.

We thank the reviewer for pointing out this typo, which has been corrected.

The fixed-separation simulation in Fig. 3D is important for demonstrating the causality. How was the average speed (V_avg) calculated in this case? Specifically, do microtubule pairs that slide at coasting mode maintain a high speed over the entire sliding event when the inter-filament spacing is fixed at a large distance?

We thank the reviewer for raising this point, which was not clear in the original manuscript. In the fixed-separation simulation of Fig 3D the average speed is calculated for the whole simulation. We have clarified this in the figure 3 caption. We have also added a supplementary figure showing the velocity distribution. The coasting pairs do maintain high speed over the event.

In my understanding, the attractive and repulsive lateral forces exerted by PRC1 with positive and negative tilts arise because PRC1 has a natural tilt relative to the perpendicular. Is this correct? It would be helpful to illustrate this assumption in a figure to clarify the molecular behavior being modelled.

The reviewer raises an important point that we have clarified in the revised manuscript. The linear (spring stretch/compression) force is the primary contributor to the attractive lateral force in both braking and coasting states. The torsional force that arises from the natural tilt of PRC1 does contribute significantly to repulsion between microtubules in the coasting state. We have clarified this in the text and added a supplementary figure showing the energy and forces from PRC1 molecules as a function of angle.

In the paragraph starting from line 258, the authors discuss Ase1 and the yeast spindles. What is the relevance to PRC1 particularly in considering that Ase1 exerts an entropic force within the confined microtubule bundles to resist sliding (e.g., Lasky et al., 2015)?

We thank the reviewer for raising this important point. It is true that Ase1 has been shown to generate entropic forces that work to push against microtubule sliding, while this specific behavior has not been observed for PRC1. We believe that such forces are likely to arise when Ase1 is in a coasting-like mode and the individual crosslinkers are free to diffuse within the confines of the overlap, which is the mechanism Lansky et al. propose. In this paragraph of the discussion, we are highlighting the experimental observation that microtubule-microtubule spacing significantly reduces as a yeast cell proceeds from metaphase to anaphase, with late anaphase MT separations measured to be ~15nm, similar to what we predict for microtubule pairs that have engaged in a braking mode. We therefore speculate that a coasting-to-braking transition may be more generally applicable across different spindle types, at least when involving MAP65 family members such as Ase1 and PRC1. In the yeast spindle, then, we speculate that when microtubule separation is larger, Ase1 would be arranged in a coasting-like mode of binding, capable of generating entropic forces. Later, it is possible the molecules switch to a more braking-like mode, where MT-MT spacing reduces significantly as shown in EM data from yeast spindles. It will be useful in the future to acquire similar data from mammalian spindles to determine if late anaphase midzone separation also compacts when PRC1 is present, which would further validate our predictions. We have clarified the discussion of this point in the revision.

Fig. 1B, C would benefit from additional labels, as the colors in the images do not match those in the accompanying cartoon.

We thank the reviewer for the suggestion, and have added additional labels.

Reviewer #1 (Significance (Required)):

As in my major comments above. My expertise is experimental biophysics on microtubules and motors.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __The paper presents simulations of sliding antiparallel microtubules linked by PRC1 crosslinking proteins. It aims to reproduce and explain experimental observations by Alfieri et al. that suggested that PRC1 could adopt two distinct modes of resistive force production against kinesin-driving sliding forces.

The model which the authors propose is that antiparallel sliding leads to the accumulation of PRC1 at the edges, which results in higher tilt angles of PRC1 molecules and consequently smaller microtubule separation. In the higher tilt regime PRC1 can exert more braking forces since, its angle with the Microtubules is smaller. To my understanding the key parameters for this model to work it the spontaneous tilt angle, and torsional spring that PRC-1's structure encodes. The authors demonstrates that for reasonable values very good agreement with experimental observations can be reached. The simulations are done in the CYlAks framework, which the Betterton group developed and validated in earlier work. The discussion is clear and readable

Major Comment: While the paper goes at great length to successfully reproduce experiments, it is not discussed how sensitive the model is to changes in parameters. In particular it remains unclear how sensitive the model is to changes in the torsional spring that is being used to model PRC-1. Given that this is key to the findings presented here, I would have hoped for a more extensive discussion of the relevant physics. In particular It should be discussed how non-linearities and asymmetries in the torsional spring would affect the phenomenon identified here.

We appreciate the reviewer’s suggestion to examine sensitivity to variation in model parameters. We note that we do present in Figure 2 a smaller exploration of parameter space; when key values are modulated by an order of magnitude, we find differences in the simulated outputs (e.g. enhanced or reduced tip clustering in response to changes in MAP diffusion or end binding). We also note the supplementary information includes the effect of varying parameters including the strength and asymmetry of the torsional spring, which addresses the specific concern noted. Given the length of the current manuscript, we propose to delay a more extensive study of parameter sensitivity to future work.

(Very) Minor remark: the orientation of PRC-1 molecules is inconsistent between figures.

We thank the reviewer for pointing this out. We have edited the figures to make the orientation consistent.

__Reviewer #2 (Significance (Required)): __ PRC-1 is an important cross-linking protein in cell division, and its mechanics is at the center of much current research interest. As such this paper is timely. The key physics that is interesting here is the link between geometry, PRC1-arrangements and geometry of the MT network. The authors reproduce successfully the experimental observations, with reasonable parameters. But a parameter study that exposes the physics at play, and would help the reader generalize the concepts at play is missing.

In its current state the paper will be of interest to experimentalists and theoreticians working on cytoskeletal filament networks. But it could be even more so, if the authors sought to generalize beyond the experiment at hand.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The manuscript by Steckhahn and colleagues is a computational study of the mechanics of microtubule interactions with an essential mitotic crosslinker, PRC1. PRC1 is known to act as a molecular clutch, resisting the sliding of antiparallel microtubules in order to maintain mitotic spindle integrity. The present study aims to explain the recently discovered two modes of action of this clutch: a weakly resistant 'coasting' mode and a highly resistant 'braking' mode. The authors employ their previously developed Cytoskeleton Lattice-based Kinetic Simulator (CyLaKS) model to carry out Monte Carlo/Langevin dynamics simulations of microtubule sliding, driven by a mitotic kinesin and resisted by an ensemble of PRC1 crosslinkers, with explicit account of their diffusion, binding-unbinding kinetics, stretching-compression, and volume-exclusive interactions. Their reasonable model successfully reproduces the bimodal distribution of microtubule sliding rates, and offers a simple explanation of the two modes of action of the crosslinkers. According to the authors' conclusion, in the coasting mode PRC1 molecules are almost perpendicular to the microtubules, while the microtubules are separated by about 30 nm (close to the rest length of PRC1). When the overlap between the sliding microtubules shrinks, the PRC1 molecules cluster, which facilitates their tilting. This has two effects: a projection of force bringing microtubules closer together appears, and a projection of resistive force along the microtubule axis becomes substantial, enabling more efficient 'braking'.

The key conclusions are convincing, clearly stated, and supported by data. The simulation techniques are justified and well described. I have no concerns about the technical side of this study.

We thank the reviewer for their clear summary of the results of the paper.

Reviewer #3 (Significance (Required))

I believe this is a useful piece of work, which clarifies some important aspects of the PRC1 mechanism of action by showing that a simple but rigorous mechanical consideration is sufficient to explain the observed bimodal behavior of the mitotic crosslinkers. The findings could be interesting to biophysicists and cells biologists, interested in cytoskeleton and cell division.

Data and data discrepancies

Data discrepancies

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The Authors investigated the anatomical features of the excitatory synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of the synapse, the macular or the perforated appearance and the size of the synaptic active zone, the number and volume of the mitochondria, the number of the synaptic and the dense core vesicles, also differentiating between the readily releasable, the recycling and the resting pool of synaptic vesicles. The coverage of the synapse by astrocytic processes was also assessed, and all the above parameters were compared to other layers of the human temporal neocortex. The Authors conclude that the subcellular morphology of the layer 1 synapses is suitable for the functions of the neocortical layer, i.e. the synaptic integration within the cortical column. The low glial coverage of the synapses might allow the glutamate spillover from the synapses enhancing synaptic crosstalk within this cortical layer.

Strengths:

The strengths of this paper are the abundant and very precious data about the fine structure of the human neocortical layer 1. Quantitative electron microscopy data (especially that derived from the human brain) are very valuable, since this is a highly time- and energy consuming work. The techniques used to obtain the data, as well as the analyses and the statistics performed by the Authors are all solid, strengthen this manuscript, and support the conclusions drawn in the discussion.

Comments on latest version:

The third version of this paper has been substantially improved. The English is significantly better, there are only few paragraphs and sentences which are hard to understand (see my comments and suggestions below). Almost all of my suggestions were incorporated.

We would like to thank the reviewer for the comments and incorporated the suggestions within the latest version of the manuscript.

Remaining minor concerns:

About epileptic and non-epileptic (non-affected) tissue. I am aware that temporal lobe neocortical tissue derived from epileptic patients is regarded as non-affected by many groups, and they are quite similar to the cortex of non-epileptic (tumour) patients in their electrophysiological properties and synaptic physiology. But please, note, that one paper you cited did not use samples from epileptic patients, but only tissue from non-epileptic tumor patients (Molnár et al. PLOS 2008).

When you look deeper, and make thorough comparison of tissues derived from epileptic and non-epileptic patients, there are differences in the fine structure, as well as in several electrophysiological features. See for example Tóth et al., J Physiol, 2018, where higher density of excitatory synapses were found in L2 of neocortical samples derived from epileptic patients compared to non-epileptic (tumor) patients. Furthermore, the appearance of population bursts is similar, but their occurrence is more frequent and their amplitude is higher in tissue from epileptic compared to non-epileptic patients. So, I still cannot agree, that temporal neocortex of epileptic patients with the seizure focus in the hippocampus would be non-affected. Therefore I suggested to use the term biopsy tissue.

We are thankful for this comment on using non-epileptic tissue also by others. We are also aware that Molnár et al. 2008 worked with tumor tissue.

It is still not emphasized in the first paragraph of the Discussion, that only excitatory axon terminals were investigated.

We now mentioned in the first paragraph of the discussion that only excitatory synaptic boutons were investigated.

The text in the Results and the Discussion are somewhat inconsistent.

The last two paragraphs of the Results section ends with several sentences which should be part of the discussion, such as line 328: This finding strongly supports multivesicular release... or line 344: --- pointing towards a layer-specific regulation of the putative RRP. Moreover, the results suggest that... and line 370: ... it is most likely... Please, correct this.

We disagree with the reviewer on these points because these sentences summarizes the findings.

The first paragraph of the Discussion summarizes the work of the quantitative EM work and gives one conclusion about the astrocytic coverage. This last sentence is inconsistent with the other parts of the paragraph. I would either write that "astrocytic coverage was also investigated" (or something similar), or move this sentence to the paragraph which discusses the astrocytic coverage.

Results line 180-183. "Special connections" between astrocytic processes and synaptic boutons are mentioned, but not shown. Either show these (but then prove with staining!), or leave out this paragraph.

We deleted this paragraph as suggested.

Reviewer #2 (Public review):

Summary:

The study of Rollenhagen et al examines the ultrastructural features of Layer 1 of human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as further from the epilepsy focus, and as such considered to be non-epileptic. The analyses has included 4 patients with different age, sex, medication and onset of epilepsy. The manuscript is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:

Layer 4 - Yakoubi et al 2019 eLife

Layer 5 - Yakoubi et al 2019 Cerebral Cortex,

Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex

They find, the L1 synaptic boutons mainly have single active zone a very large pool of synaptic vesicles and are mostly devoid of astrocytic coverage.

Strengths:

The MS is well written easy to read. Result section gives a detailed set of figures showing many morphological parameters of synaptic boutons and surrounding glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in human brain are still very limited, the current MS has substantial relevance. The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analyses is clear and precise.

Weaknesses:

The authors made all the corrections required and answered all of my concerns, included additional data sets, and clarified statements where needed.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Minor suggestions:

Synaptic density, lines 189-193. If you say "comparatively" high, then compare to something (cite your own work for the other layers, and tell the approximative values for the other layers). Same in line 194 comparably high to what? Other option: say "relatively high".

We corrected the sentences as suggested by the reviewer.

Line 206: When present, mitochondria (comma missing)

Corrected as suggested by the reviewer.

Line 265: Dot is missing at the end of the sentence (after Shapira et al. 2003)

Corrected as suggested by the reviewer.

Lines 300-301: Check the English for this sentence: significant difference BETWEEN TWO sublaminae and not significant difference for both sublaminae.

Corrected as suggested by the reviewer.

Lines 304-305: Check the sentence, please, it is not understandable without the text in parenthesis.

Corrected as suggested by the reviewer.

Line 354 Dot missing at the end of the sentence (after Figure 6A, B)

Corrected as suggested by the reviewer.

Line 354-358: Please rephrase this sentence (too complicated, not understandable). I do not understand why results of the L4, L5, L6 are described here. What does it mean "Astrocytes and their fine processes formed a relatively dense, but a comparably loose network within the neuropil in L1"? Dense or loose?

In the experiment measuring the volume fraction of astrocytic processes (Figure 6C), all six cortical layers were analyzed, thus we compared the values obtained for L1 with the results for L4, L5 and L6. For more clarity, we rephrased the sentence: “Astrocytes and their fine processes formed a relatively dense network in L4 and L5, but a comparably loose one within the neuropil in L1…” We also rephrased other sentences in this paragraph (as also suggested below).

Lines 359-369: Please rephrase this paragraph. The sentences are too complicated, have too many parentheses, and are not understandable. I suggest to write first how many synapses were examined in L1 and L4, then how many of them were on spine and on dendrites (either n or %). Then give the values how many (n or %) of them were "tripartite synapses", out of spine synapses and of dendritic synapses in both layers. How many of them were partially covered in both layers. Please, write the data in a systematic way. The best would be to give the values in a table as well. This way it will be more understandable (now, it is chaotic, hard to follow).

We rephrased the paragraph and added a new table (3).

Line 383: Dot missing from the end of the sentence.

Corrected as suggested by the reviewer.

Line 436: Reconsider "comparably low compared to". The comparably means what in this case? The whole paragraph is hard to understand, please, check and review for improvements to the use of English or use chatGPT to check it.

We corrected the sentence according to the reviewer’s suggestion.

Line 487: Same thing again: "The comparably largest size of the RP in L1 when compared..." What would you like to say with "comparably"? Check the meaning of this word in a dictionary, please. I have the feeling that you are using this word instead of "relatively".

Corrected as suggested by the reviewer.

Line 488 "and TO that found fot L4 and L5 in rodents..."

Corrected as suggested by the reviewer.

Line 493-495: Same again, comparably when compared, correct, please.

Corrected as suggested by the reviewer.

Supplemental figures: Now I do understand why Hu-01 and Hu-02 are twice, and I think, 3 patients were examined for L1a and three for L1b. But which side is which on the subfigures? Left side (Hu-01, 02 03) was used for L1a, or L1b? Could you write this in the legend, or mark on the figure (at least at one subfigure), please?

We implemented a comment for clarity.

Try to give a child a good environment 🧠 Jean Piaget (Cognitive Constructivism) Focus: Stages of cognitive development 🧱

Method: Child learns independently, discovering via interaction with the environment.

Key Idea: Development precedes learning.

Classroom: Structured tasks matched to cognitive stage.

🔍 Source: Piaget, The Origins of Intelligence in Children (1952). Credibility: 9/10 (peer-reviewed, foundational)

🧠 John Dewey (Pragmatism & Experiential Learning) Focus: Learning by doing 🛠️

Method: Social interaction + real-world experience.

Key Idea: Learning and development are continuous and intertwined.

Classroom: Project-based, democratic, socially interactive.

🔍 Source: Dewey, Experience and Education (1938). Credibility: 9/10 (peer-reviewed, seminal work)

⚔️ Key Differences Concept Piaget Dewey Child's role Little scientist 🧪 Social learner 🤝 Teacher's role Guide for stage-appropriate tasks Facilitator of real experiences Development vs Learn Develop → Learn Develop while learning Social learning Secondary Core principle

este valor hubiese estado bien informarlo

el perfil de campo magnético, la apalabra distribución se reserva para cuando hablamos de distribuciones de probabilidad

dicho así parece que el objetivo del experimento era determinar que el montaje era fiable, y esa no era la idea

se extendió significa que se hizo otro ajuste? o es el mismo ajuste con otro modelo, distinto a la ecuación 2.2, que contempla dos solenoides superpuestos?, no queda claro.

había que presentar un solo gráfico haciendo el promedio de las dos direcciones, gente

aclarar que se esta hablando de la componente vertical

Tienen que explicar mejor como obtuvieron esos valores, fue promediando? tomaron el valor en el máximo de las curvas? es ese el valor total o una componente?. Convenia antes de presentar el resultado, presentar las curvas, y a partir de la descripción de estas contar como obtuvieron los valores.

justamente el problema con V0 es que no se mantiene constante, puede depender de temperatura por eso uno quiere sacarselo de encima rápido en al medición. Lo que habia que hacer era fijar la sonda en una posición medir B_0 con la corriente para un lado, medir B_180 con la corriente para el otro lado, y hacer (B_0- B_180)/2 para obtener el valor del campo, de esa manera se sacaban de encima V0. No era la idea calcular V0

adjuntamos es más para un mail, "en la figura 1 se muestra un esquema del dispositivo experimental"

"hizo circular". Para evitar confusiones, reservemos la palabra inducir para el fenómeno de inducción.

ya lo habían definido antes

usar la letra V para el campo magnético no es una notación muy feliz

los resultados obtenidos son para describir físicamente el fenómeno no para mostrar la efectividad de las técnicas utilizadas

?

esto no es para el resumen, fíjense que no definieron todavía que es una configuración invertida

esta explicación no se pone en la parte de resumen

Erstaunlich wegen dem Wallis? Sonst ja nicht. Schon seit Engels ist klar, dass die Urgesellschaften egalitär waren.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Firstly, we would like to thank the reviewers for their time and efforts in critiquing this paper. The reviewers addressed our study to be significant, but also presented great suggestions to improve our manuscript, mainly the comparison of mRNA and eRNA for predicting subtype specificity and prognosis, the integration with independent validation datasets, etc. Our preliminary analyses showed that our classified mRNAs can predict subtypes better which is not surprising, as these subtypes were initially discovered using mRNA differences. Hence, we employed a novel approach of associating these classified mRNA and eRNA with distance and identified 71% classified eRNAs are associated with classified mRNAs. We also propose to integrate the datasets with PEGS (Briggs et al 2021) to achieve better mRNA-eRNA association and Perturb-seq validated regions to achieve functional validation of the eRNA loci. We believe that our potential improved integrative analyses will improve the novelty and power of our findings, as this is an unique approach which is employed in patient samples-based high resolution eRNA atlas for the first time. We have addressed most of the other major and minor comments of the reviewers and have provided the preliminary revised manuscript.

Reviewer #1

Evidence, reproducibility and clarity

Summary<br /> This study assesses eRNA activity as a classifier of different subtypes of breast cancer and as a prognosis tool. The authors take advantage of previously published RNA-seq data from human breast cancer samples and assess it more deeply, considering the cancer subtype of the patient. They then apply two machine learning approaches to find which eRNAs can classify the different breast cancer subtypes. While they do not find any eRNA that helps distinguish ductal vs. lobular breast cancers, their approach helps identify eRNAs that distinguish luminal A, B, basal and Her2+ cancers. They also use motif enrichment analysis and ChIP-seq datasets to characterize the eRNA regions further. Through this analysis, they observe that those eRNAs where ER binds strongest are associated with a poor patient prognosis.

Major comments:

Part of the rationale for this study is the previous observation that eRNAs are less associated with the prognosis of breast cancer patients in comparison to mRNAs and they claim that the high heterogeneity between breast cancer subtypes would mask the importance of eRNAs. In this study, the authors solely focus on eRNAs as a classification of breast cancer subtypes and prognostic tool and do not answer whether eRNAs or mRNAs are a better predictor of cancer subtypes and of prognosis. Since the answer and the tools are already in their hands, it would be important to also see a comparative analysis where they assess which of the two (mRNAs or eRNAs) is a better predictor.

Response: We appreciate the reviewer for this valid point about comparing the prognostic eRNAs vs mRNAs. Our study doesn’t imply that eRNA markers are better than mRNAs in predicting subtype specificity and/or prognosis, but our motivation for working with eRNAs is that they can be used to define relevant transcriptional regulators and prognosis generally if they are subtyped. As the molecular subtypes in breast cancers were established using gene expression datasets, mRNAs would perform better as predictors of subtypes and or prognosis. However, identifying regulatory networks with emphasis on transcription factor binding motif analyses is not achievable using mRNA datasets. Analysing the active enhancer regions with eRNA transcription will provide high resolution landscape of TF and epigenetic networks. These sorts of analyses usually require ATAC-seq or H3K27ac datasets, but these assays need fresh frozen tissue material and laborious experimental designs compared to RNA-seq datasets. Furthermore, eRNA-transcribing enhancers represent highly active enhancers, while ATAC and H3K27ac datasets can identify all enhancers, which can be inactive or poised, but captured due to the dynamic nature of enhancers. We demonstrate that traditional RNA-seq datasets mapped on active enhancer regions showing eRNA transcription would be sufficient to identify the highly active TF network and gene-enhancer regulatory frameworks in a subtype-specific manner, hence emphasising the potential of eRNA studies.

Hence, the scope of our study is not to establish which RNA can predict subtype and survival, but to demonstrate the potential of studying eRNAs in patient samples using traditional RNA-seq assays. This study would be beneficial for epigenetics biologists of how enhancer transcription can be associated with gene regulation through deregulated transcription factor networks in patients. The above section had been included in the discussion in the revised manuscript.

As the comparative analyses suggested by the reviewer will substantiate the potential of eRNAs being studied as cancer prognostic markers, we performed identical methodologies with our machine learning approaches on the published TCGA mRNA-seq datasets, identify the subtype-specific mRNAs as well as prognostic mRNAs and perform the comparative analyses of eRNAs and mRNAs. As we expected, mRNAs indeed perform better in associating with subtype specificity than eRNAs as we could identify more subtype-specific mRNAs with better statistics metrics. The results exhibit great separation across subtypes (Basal, Her2, LumA/B) as well as Ductal vs Lobular.

We believe that eRNA and mRNA are complementary but not comparative to predict subtype-specific survival. To address this in the revised manuscript, we performed an initial selection of the eRNAs associated with their corresponding subtype-specific mRNAs within 50 kb distance which can be integrated with the above analyses, based on the suggestion from reviewer 3. In our preliminary analysis, around 71% of eRNAs are associated with the subtype-specific mRNAs and we also observed an observable separation of ductal and lobular subtypes using this method.

Furthermore, we integrated our enhancer RNAs with the key enhancer regions which show significant impact on gene transcription, as shown in single cell CRISPRi screens (Perturb-seq) datasets derived from ATAC-matched H3K27ac datasets verified on one ER+ and one ER- breast cancer cell lines (Wang et al., Genome Biology 2025, https://genomebiology.biomedcentral.com/articles/10.1186/s13059-025-03474-0) . Our initial analyses identified at least 29 regions from the Perturb-seq datasets overlapping with 72 and 5 eRNAs of subtype classification and Her2 survival respectively.

For the revised manuscript, we will perform the mRNA-eRNA association in a detailed manner and include the data. We will also employ our well-established tool for associating mRNAs and noncoding elements, Peak set Enrichment in Gene Sets (PEGS, Briggs et al., F1000 research, 2021 https://f1000research.com/articles/10-570/v2 ). We hypothesise that this will improve the power of the classification models used in the study and will also provide gene-enhancer RNA interaction landscape in patient samples for the first time. Furthermore, we will integrate the activity of these eRNA-mRNA pairs with chromatin accessibility and enhancer activity using ATAC-seq and H3K27ac ChIP-seq datasets to establish more robust active regulatory networks in patient samples. We will also perform motif analyses on the published ATAC-seq peaks (performed on TCGA-BRCA patient samples, Corces et al., 2018) close to the eRNA loci to identify the TF networks with better precision, hopefully unravelling novel and relevant subtype-specific TFs in an efficient manner, better than our original work. Furthermore, as an experimental functional validation of our classified eRNAs, we will investigate the regulatory effect of 29 Perturb-seq overlapped regions. Hence, our revised manuscript will potentially provide a comprehensive validated list of enhancer RNA regions which are highly active, actively transcribing, subtype and survival specific regulatory networks in breast cancer patients for the first time.

The authors run the umaps of Fig. 1C only taking the predictor eRNAs. It is then somewhat expected to observe a separation. Coming from a single-cell omics field, what I would suggest is to take the eRNA loci and compute a umap with the highly variable regions, perform clustering on it and assess how the cancer subtypes are structured within the data. This would give a first overview of how much segregation and structure one can have with this data. Having a first step of data exploration would also strengthen the paper. If the authors have tried it, could the authors comment on it?

Response: We appreciate the reviewer for sharing their experience from single cell omics analysis. In our case, following the scRNA like pipeline is not appropriate, given the focus of our study on identifying markers on the already annotated subtypes. Basically, we aim to assess the quality of the identified markers (the quality is quantified by the statistics provided for random forest classification), and we see that the data is well-separated in PCA using only PC1 and PC2. We showed the umap (using PC1 and PC2) for better visualization in the original manuscript and we included the PCA plots in the revised manuscript.

'neither measures could classify any distinct eRNAs for invasive ductal vs lobular cancer samples' S1B. Just by eye, I can see a potential enrichment of ductal on the left and on the right while lobular stays in the center. This suggests to me that, while perhaps each eRNA alone does not have the power to classify the lobular vs ductal subtype, perhaps there is a difference - which could result from a cooperative model of eRNA influence - that would need further exploration. Would a PCA also show enrichments of ductal vs. lobular in specific parts of the plot? It may be worth exploring the PC loadings to see which eRNAs could play an influence. In this regard, a more unbiased visual examination, as suggested in my previous point, could help clarify whether there could be an association of certain eRNAs that cannot be captured by ML.

Response: The subtypes of cancer patients (Basal, Her2, LumA/B) possess clear differences in mRNA expression in breast cancer studies. Given the fixed annotations of the subtypes in the patient datasets, we applied our methodologies on mRNA datasets, and the results exhibited great separation across subtypes (Basal, Her2, LumA/B) as well as Ductal vs Lobular. In addition, 70% of subtype-specific eRNAs are located next to mRNA. This ensures that we detected proper eRNA markers. Furthermore, Random Forest is the standard and powerful non-linear classifier for these types of classifying questions. Therefore, we hypothesized that the data which can distinguish Ductal vs Lobular does not exist in the used eRNA dataset. We only detected 38 subtype-specific mRNAs using information gain with standard cutoff 0.05 which they have classifying power across ductal-lobular. With this standard cutoff only one eRNA-associated gene was detected. To explore more, we used low cutoff for information gain (0.01) and then took only the eRNAs which are located near classified mRNAs (up to 50KB). In this way, we detected 96 eRNA candidates linked to 8 classified mRNAs. These 96 eRNAs could, to some extent, classify ductal vs lobular (PCA plots attached above). This observation can further verify that if a more comprehensive eRNA dataset exists, we could detect better eRNA markers and cover more (probably all) mRNA markers. Hence, cooperative model of eRNA as suggested by the reviewer can't be achieved and random forest is one of the efficient tools to decipher the cooperation if it exists. Besides, as we demonstrated in this paper that eRNA is a complementary dataset to mRNA which can assist in the identification of regulatory networks. For the revision, we will provide more detailed eRNA-mRNA associations using integration with PEGS and Perturb-seq validated regions, in both subtype classification and survival and will motivate the potential similar studies for ductal vs lobular in the discussion.

"we employed machine learning approaches on 302,951 eRNA loci identified from RNA-seq datasets from 1,095 breast cancer patient samples from previous studies" - the previous studies from which the authors take the data [11,12] highlight the presence of ~60K enhancers in the human genome and they use less than that in their analysis. Could the authors please clarify the differences in numbers with previous studies and give a reasoning?

Response: ~300K enhancers are derived from ENCODE H3K27ac datasets which represents all active enhancer regions marked by H3K27ac (Hnisz et al., 2013). This is a high-resolution map of eRNA loci ever presented. In Chen et al 2020, 1,531 superenhancers representing 30K eRNA loci was utilised for exploratory analysis, and the findings were generalised back to the 300K set. 65K enhancer loci covers tissue-specific enhancers initially identified by FANTOM CAGE datasets and this subset provide limited regions of eRNA expression. Hence, our analyses on ~300K eRNA loci provide unbiased information on subtype specificity and gene-TF regulatory networks. The differences had been highlighted in the methods and results in the revised manuscript.

Also, from the methods section, they discard many patient samples due to low QC, so, from what I understand, the number of samples analyzed in the end is 975 and not 1,095.

Response: We thank the reviewer for pointing this out and we have updated the numbers in the revised manuscript.

Minor comments:

• Can the authors please state the parameters of the umap in methods? Although it could be intrinsic to the dataset, data points are grouped in a way that makes me think that the granularity is too forced. Could the authors please show how the umap would behave with more lenient parameters? Or even with PCA?

Response: We used ‘umap’ function from umap package (with default parameters) in R using only PC1 and PC2, hence the granularity is not forced. As suggested by the reviewer, we have now added PCA plots in the main figures (Fig. 1E) and moved all the umap plots to the Supplementary figures (Fig.S1B) in the revised manuscript.

• 'Majority of the basal' -> The majority of the basal.

Response: We thank the reviewers for noticing the typo and we corrected this in the revised manuscript.

Significance

This is a paper relevant in the cancer field, particularly for breast cancer research. The significance of the paper lies in digging into the breast cancer samples, taking the different existing subtypes into account to assess the contribution of eRNAs as a classifier and as a prognostic tool. The data is already available but it has not been studied to this degree of detail. It highlights the importance of characterizing cancer samples in more depth, considering its intrinsic heterogeneity, as averaging across different subtypes would mask biology. My expertise lies in gene regulation and single-cell omics. My contribution will therefore be more focused on the analysis and extraction of biological information. The extent of its specific relevance in cancer research falls beyond my expertise.

Response: We appreciate the reviewer for understanding our efforts to bring out the importance of subtyping and to explore the association of eRNA in breast cancer transcriptional gene regulatory networks.

Reviewer #2

Evidence, reproducibility and clarity

Summary<br /> Enhancer RNAs (eRNAs) are early indicators of transcription factor (TF) activity and can identify distinct molecular subtypes and pathological outcomes in breast cancer. In this study, Patel et al. analysed 302,951 polyadenylated eRNA loci from 1,095 breast cancer patients using RNA-seq data, applying machine learning (ML) to classify eRNAs associated with specific molecular subtypes and survival. They discovered subtype-specific eRNAs that implicate both established and novel regulatory pathways and TFs, as well as prognostic eRNAs -specifically, LumA and HER2-survival- that distinguish favorable from poor survival outcomes. Overall, this ML-based approach illustrates how eRNAs reveal the molecular grammar and pathological implications underlying breast cancer heterogeneity.

Major comments

1. The authors define 302,951 eRNA loci based on RNA-seq data, yet it is widely known that many enhancers reside in proximity to promoters or within intronic regions (examples presented in Fig. 3B and S3). Consequently, it seems likely that reads mapped to these regions might not truly represent eRNA signals but include mRNA contamination. Could the authors clarify how they ensured that the identified eRNAs were not confounded by mRNA reads? What fraction of these enhancer loci is promoter proximal or intronic? How does H3K4me3, a well-established and standardized active promoter histone mark, behave on these loci? The reviewer considers it important to confirm that the identified eRNAs are indeed of enhancer origin rather than promoter transcripts.

Response: For this study, we utilised pan cancer atlas-based published work (Chen et al 2018 and 2020) where the abundant RNA signals on intronic and intergenic regions are included, and promoter-based signals are excluded. These studies utilise the advantage of identifying eRNAs on large sample size and the possibility of mRNA being on introns in 1000s of patient samples is very low. A clarification of this concern had been discussed in the Introduction of these studies as follows: “because eRNA reads associated with real enhancer activity recurrently accumulate, whereas background transcription noise tends to occur stochastically. The large number of RNA-seq reads obtained would compensate for the statistical power compromised by the low eRNA expression level typically observed in a single sample.” We included clarification of this concern in the discussion. Furthermore, as per the reviewer’s suggestion, we examined the distribution of the eRNA loci across the genome and found that majority of eRNA regions are located on introns and intergenic regions. This figure had been included in the Supplementary Fig. S6A.

2. In Fig. 1B, the F measure (0.540) of the Basal subtype using the Logmc method contradicts its extremely high precision (1.000) and sensitivity (0.890). The authors need to clarify the exact formula or method used to compute F1 and the discrepancy in the reported metrics for this subtype and perhaps other subtypes as well.

Response: We apologise for the mistake in this section and thank the reviewer for pointing this out. We included the formulas for each statistical metric in the method section of the manuscript. The F-measure was mentioned wrong which led to the confusion here. The figure had been corrected with the F-measure of 0.94 in the revised manuscript.

3. As shown in Fig. 4C, S4B, and most, if not all, tracks of Fig. S3, ER binding regions are not annotated as eRNA loci. It seems, in this reviewer's opinion, very unlikely that this is because they generally lack eRNA expression, but rather they do not express polyadenylated eRNA (typically 1D eRNA), which is captured in this dataset. The reviewer posits that these enhancers produce more transient, non-polyadenylated 2D eRNA. It has been widely documented in prior studies that ER-bound enhancers exhibit bimodal eRNA expression patterns [e.g., Li, W. et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498, 516-520 (2013)]. Could the authors address this opinion and elaborate on how the restriction to polyadenylated transcripts might underrepresent enhancers regulated by ER and other TFs and whether this bias impacts the overall findings?

Response: The authors appreciate the reviewer’s suggestion to address the caveats of using polyadenylated eRNAs to identify the ER binding patterns. TCGA eRNA atlas with polyadenylated eRNAs indeed possesses this disadvantage of using polyadenylated eRNAs for this study, however currently there are no data available with bidirectional transcripts in any breast cancer patient samples. The tools to profile these RNAs are not robust enough to be performed on frozen cancer tissue samples which are extremely limited in their size and availability. By utilising the polyadenylated eRNA-seq datasets, we might not only lose the accuracy of ER binding patterns, but also for other transcription factors which activate/associate with bimodal expression around enhancers. However, our integrative analysis on stable polyadenylated eRNA loci can still identify the most-relevant TF networks of each subtype.

Furthermore, we validated this finding by analysing our own datasets of KAS-seq which represents any active transcribing bidirectional enhancers from MCF7 cell line. Independently, we also incorporated ATAC-seq, H3K27ac ChIP-seq, CAGE and GRO-seq data on the gene profiles in Fig. S3 to associate the eRNA regions identified in polyadenylated RNA datasets with ER binding sites in patients and published bidirectional transcripts in the preliminarily revised manuscript. We observed that all the ER binding sites are accompanied by open and active enhancer marks with bidirectional transcription (either GRO- or CAGE positive) but they are not on the exact location of eRNA regions. Subtype-specific eRNA regions close to genes like MLPH and XBP1 possess both active bidirectional transcribing ER bound sites far away (around 1.5 kb) from subtype-specific eRNA loci and bidirectional transcribing ER unbound sites. However, these distal ER binding sites are close to the regions from the list of 300K eRNA loci and they were simply not identified as subtype-specific regions. Hence, it can be true that the occupancy of ER might not be present on all subtype-specific eRNA loci, but our subtype-specific eRNA sites are representative of bidirectional transcription.

Upon the suggestion from the reviewer, we discussed the potential of identifying TF networks by analysing the 1D eRNAs, in the revised manuscript.

4. Despite the unsatisfied performance of the ML approach on classifying Her2 subtypes, the hierarchical clustering performed in Fig. 2A and S2A appears to show a reasonable separation of Her2 subtypes, showing as a clustered green band. Could the authors quantitatively assess how effective this clustering results and compare that to the ML outcome? (OPTIONAL)

Response: The authors acknowledge this interpretation from the reviewers. Using both the measures, our ML platform can identify markers for Her2 subtype but some of the statistical metrics are poor. As the heatmaps were performed based on these identified Her2 markers, a separate analysis on this cluster would not be much informative. The poor metrics for Her2 classification was already justified, partly due to the low number of Her2+ patients in the cohort.

5. In Fig. 4 and S4, the authors reported to have enriched binding or motif of TFs, e.g., FOXA1, AP-2, and E2A, specifically at enhancer loci with low eRNA level, which conflicts with their established roles as transcriptional activators. The reviewer asks for an address as to why these factors would be associated with basal low-eRNA regions and whether any additional data might clarify their functional role in these contexts.

Response: The authors appreciate the reviewer’s concern, but we would like to clarify that eRNAs which are less expressed in basal subtype are classified as basal low. These regions show high expression in luminal patients. Hence, there is a strong overlap of basal low and luminal high regions. FOXA1 and AP2 factors are strongly established coactivators in luminal ER+ transcriptional signaling, hence they are associated with basal low eRNA regions. We clarified this in the discussion and provided more literature evidence in the revised manuscript to demonstrate the strong role of FOXA1 and AP2 factors in ER+ luminal breast cancer transcriptional response.

6. Regarding Fig. 4B, the authors state that "ER binding occupies only the strongest ssDNA and GRO-seq-positive sites". Firstly, the GRO-seq data quality is poor with indiscernible peaks. This may be insufficient for a qualified representation of nascent eRNA expression. More importantly, it appears each heatmap is ranked independently, so top loci for ssDNA are not necessarily top loci for GRO-seq, ER, Pol-II, or H3K27ac. The reviewer requests clarification on how the authors plot these heatmaps and questions whether the statement is supported by the analysis as presented.

Response: We acknowledge the reviewer’s concern and based on their suggestion, we utilised another set of GRO-seq datasets which is more deeply sequenced and published by the same lab. The average plot from these new datasets showed better profile. We also apologize for not providing enough details of how we generated the heatmaps in Fig. 4B. The heatmaps were made separately for each profile to auto scale with their own intensity levels but the order of the regions is based on KAS-seq intensity. The order of these regions was kept the same between each profile. Hence, top loci of ssDNA are not exact top loci of GRO, ER, H3K27ac and Polymerase but top loci of ssDNA also show similar high intensity in GRO, ER, H3K27ac and Polymerase, hence correlated. We also removed regions which belong to blacklisted regions of hg38 and the regions which were over-sequenced due to amplifications and showed weird signals. We provided the new heatmaps and profile plots in the revised manuscript with different clusters of KAS-seq intensity. We also updated the methods section to clarify how these heatmaps were made.

7. In Fig. S4B and the third plot of 4C, the averaged histogram of ER binding appears in multiple sharp peaks with drastic asymmetric positioning around the enhancer centre, which is highly atypical of most published ER ChIP-seq profiles. Could the authors discuss possible "spatial syntax" or directional patterns of ER binding in relation to eRNA loci and cite any literature showing a similar pattern? Further evidence is required to substantiate these observations, as they are remarkably unique.

Response: The authors agree with the reviewer’s point about asymmetric peaks of ER on the luminal specific eRNA regions. Due to the nature of the average profile plots and the number of regions explored here are so low, the profiles look asymmetrical and different than the published literature. Heatmaps lose their resolution when made on a very low number of regions. The focus of this analysis is to highlight that the ER is not binding to the centre of eRNA loci which is contradictory to the published findings from in vitro studies, but further away on these subtype-specific regions. We don’t have any solid evidence to demonstrate the directional patterns of ER binding related to this data. To avoid any confusion, we removed these average plots but focused on the already existing single gene profiles in Fig. S3 and discussed our interpretations in detail.

Minor comments<br /> 1. When introducing eRNAs, the reviewer recommends mentioning that 1) eRNA levels correlate with enhancer activity and 2) eRNA expression precedes target gene transcription, thus reflecting upstream regulatory events. Relevant references include: Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010-1014 (2015); Carullo, N. V. N. et al. Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems. Nucleic Acids Res. 48, 9550-9570 (2020); Kaikkonen, Minna U. et al. Remodeling of the Enhancer Landscape during Macrophage Activation Is Coupled to Enhancer Transcription. Mol. Cell 51, 310-325 (2013).

Response: These are great recommendations from the reviewer, and we included the suggested publications in the Introduction section of the revised manuscript.

2. H3K27ac is used initially to define these regulatory loci, and like eRNAs, H3K27ac also varies among patients. Which H3K27ac dataset(s) were used initially, and could this approach potentially overlook patient-specific enhancers? (OPTIONAL)

Response: This is a totally valid point from the reviewer. The idea of this project is to define common subtype-specific enhancers which can be regulatory and prognostic, hence can be developed further as biomarkers providing benefit for more patients in the future. Hence, investigating the common enhancers which are activated in multiple normal and cancer cell lines defined by ENCODE is more valid than patient-specific enhancers whose activity might be influenced by specific genetic alterations. There is very limited availability of H3K27ac ChIP-seq datasets from cancer patients to explore the patient-specific enhancers, and our analyses were totally based on the published work, hence not possible to fully address this concern. The source of the H3K27ac ENCODE datasets (from 86 human cell lines and tissue samples) is clarified in the revised manuscript.

3. In addition to the overall metrics displayed in Fig. 2B, could the authors provide precision and sensitivity values for LumA and LumB separately under the Logmc method, given the observation in Fig. 2E that LumA and LumB are not well separated in the UMAP projection?

Response: The authors appreciate the suggestion from the reviewer. We have included the metrics separately for LumA and LumB in the revised manuscript in Fig. S1D.

4. Could the author elaborate, in the discussion section, on why there is a substantial difference in ML performance depending on whether InfoGain or Logmc is used?

Response: We have included the following text in the discussion to explain the differences between these two measures.

“InfoGain measure work with the approach of binarization with k-means (k=2). It has the potential to capture both strongly expressed eRNAs which are differential between subtypes as well as low expressed sparser on and off eRNAs. In the first case, although eRNA is highly expressed in all patients, the higher expression mode becomes 1 and the lower expressed mode become 0. However, in case of low expression, more on and off expression, recentered logmc would not generate a striking high value. Furthermore, binarization is also a strong process to perform better clustering and classification, as distinguishing between data points gets better and clearer. “

5. How does the expression pattern of Basal high, Basal low, Her2, and Lum eRNA clusters behave differentially in Basal, Her2, and LumA/B subtypes? Are Basal high eRNAs downregulated in Her2 or Lum subtypes, and vice versa? Since many downstream analyses rely on these eRNA clusters, it is suggested to include a heatmap and/or boxplot that displays how each eRNA category is expressed in each subtype to confirm that these definitions are consistent.

Response: We thank the reviewers for this suggestion and apologise for not providing enough clarification on the expression of eRNAs in other subtypes. Indeed, Basal high expressed eRNA are expressed low in LumA and LumB and Basal low expressed eRNAs are expressed higher in lumA and lumB. Her2 subtype-specific eRNAs has a trend of expression between Basal and Lum, as it can be seen in the umap and PCA. Basically, the Basal high expressed eRNAs are Lum lower expressed eRNAs, and the Basal low expressed markers are Lum higher expressed markers. As per the suggestion from the reviewer, we provided heatmaps on eRNA expression of each subtype-specific with regulation in other subtype patients in figure S2F-K.

Referee cross-commenting

I share Reviewer #1's opinion that the manuscript should assess whether mRNA or eRNA is the stronger predictor of breast cancer subtypes and clinical outcomes. It will greatly improve the novelty if eRNA is shown to be a better indicator for cancer characterization.

Also, I strongly concur with Reviewer #3 that the current informatics approach is superficial and that several conclusions are contentious. The authors need to resolve the inconsistencies in their ML statistics and the potentially misleading interpretations of the ChIPseq and motif enrichment results.

It is further recommended that, building Reviewer #3's comment, the study integrate eRNA signatures with their proximal genes to address 1) whether genes located near these enhancers are differentially expressed-and correlated with enhancer activity-across cancer subtypes, and 2) whether it provides insights into understanding the enhancer-gene regulatory architecture in a subtype-specific context.

Response: We thank reviewer 2 for cross-commenting on reviewer 1 and 3’s suggestions. Indeed, these are interesting points to cover and will increase the novelty of the study. Based upon these suggestions and discussed earlier for reviewer 1’s comments, we will explore the comparison of mRNAs vs eRNAs as predictor of cancer subtypes and prognosis and the association of genes-eRNAs in cis as discussed in other reviewer’s comments. Our preliminary analyses show a strong association of eRNA and mRNA specific to subtypes and an observable separation on subtypes which were harder to classify markers using eRNAs alone. Hence, we will improve these analyses, and the manuscript further as discussed above in the final revision.

Significance

General Assessment

This study provides insights into the potential use of eRNA to classify breast cancer subtypes and refine prognostic markers. A strength is the integration of large-scale RNA-seq data with machine learning to identify eRNA signatures in biologically-meaningful patient samples, revealing both established and novel TF networks. The study also discovered eRNA clusters that correlate with the survival of patients, thus providing strong clinical implications. However, the ML approach yields several inconsistencies-for instance, unsatisfactory classification results for the Her2 subtype as well as the confused statistical metrics in the results. Furthermore, the ML model struggles to differentiate more nuanced molecular classes (e.g., LumA vs. LumB) and higher-level histological subtypes (e.g., lobular vs. ductal), thus limiting its power to dissect more delicate pathological and molecular mechanisms. Another limitation worth noting of this ML approach is the exclusive use of only polyadenylated eRNAs via RNA-seq, which excludes perhaps the more prominent 2D eRNA expressed in regulatory enhancers. Moreover, certain datasets appear to be of suboptimal quality, leading to assertions that would benefit from additional supporting evidence. Altogether, while the study offers a promising angle on eRNA-based tumor stratification, more robust experimental validations are needed to resolve inconsistencies and clarify the mechanistic underpinnings.

Advance<br /> Conceptually, the study highlights the potential for eRNA-based signatures to capture regulatory variation beyond classical markers. However, the utility of these signatures is constrained by the focus on polyadenylated transcripts alone, likely underrepresenting key enhancer regions, and certain evidence presented in this study is not substantial enough to support some statements. While the work adds an important dimension to the understanding of enhancer biology in breast cancer, the resulting insights are partly hampered by limitations in data coverage and quality.

Audience<br /> The primary audience includes cancer epigenetics, functional genomics, and bioinformatics researchers who are interested in leveraging eRNAs as biomarkers and dissecting complex regulatory networks in breast cancer. Clinically oriented scientists focusing on molecular diagnostics may also find relevance in the authors' approach to stratify subtypes and outcomes. The research is most relevant to a specialized audience within basic and translational cancer genomics, as well as computational biology groups interested in eRNA analysis.

Field of Expertise

I evaluate this manuscript as a researcher specializing in cancer epigenetics, functional genomics, and NGS-based data analysis. Parts of the manuscript touching on clinical outcome measures may require additional review from practicing oncologists.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This study aims to classify prognostic and subtype-specific eRNAs in breast cancer, highlighting their potential as biomarkers.<br /> Data was analysed using existing machine learning algorithms,<br /> Data analysis is superficial and it is hard to understand the key significant findings.

This is an important topic and a highly relevant approach to identifying RNA-based biomarkers.<br /> They analyse published RNAseq datasets by focusing on molecular subtype-specific eRNAs, enhancing clinical relevance and thereby addressing the heterogeneity of the cancer type (strength of the study).

Weaknesses include: Most of the findings are purely correlation-based and also based on a reanalysis of published datasets; it would benefit from experimental validation to support their findings. Differential expression analysis of large datasets likely yields some differences in the transcriptome. How significant are these changes?<br /> Does the expression of eRNAs affect the expression of genes in cis? Although this analysis would provide some associated gene expression differences, it can also provide some insights into subtype-specific differences in gene expression programs.<br /> If the authors find experimental validations are not feasible, I recommend validating the eRNA signature in an independent dataset.

Response: We acknowledge the weaknesses noticed by the reviewer from this study about the correlation-based analyses of published datasets. While the TCGA eRNA atlas datasets are reanalysed, these are the high-resolution maps ever published on eRNA expression on cancer patient samples, and our study is the first to establish the subtype specific classification of eRNAs. We believe that the eRNAs are biologically relevant, as they are strongly associated with the subtype-specific pathways and epigenetic regulators. Upon suggestion from the reviewers, we will explore the association of mRNAs and eRNAs in cis to establish further significance and relevance of the eRNAs we identified (discussed earlier in reviewer 1 comments).

We would like to focus on studying the functional relevance of eRNAs as a separate project. In vitro studies to establish the knockdown of eRNAs are not straightforward due to the toxicity and non-specific targeting of the locked nucleic acids approach or Cas13-based RNA targeting. siRNA-based approaches don't target the nuclear eRNAs effectively, even though they were widely used by other labs to target eRNAs. Hence, a lot of effort on optimisations are needed to establish functional validation of our eRNAs, hence not under the scope and time frame of this study/revision. To provide validation and significance using independent datasets, we will explore the association of these factors with the expression of subtype-specific eRNAs further in our final revised manuscript using the tools explained above for reviewer 1 (PEGS and Perturb-seq integration). Integration of our classified eRNAs with the published Perturb-seq validated regions from ER+ and ER- breast cancer cell lines will provide the functional validation of patient-associated classified enhancer/eRNAs. Hence, our study would be the first to demonstrate the validated gene-enhancer regulatory networks from breast cancer patient datasets.

Furthermore, we included the single gene visualisation profiles of independent datasets of ER ChIP-seq from different patients (Ross-Innes et al., 2012), ATAC-seq from TCGA patients (Corces et al., 2018), H3K27ac ChIP-seq datasets from cell lines (Theodorou et al., 2013 and Hickey et al., 2021) and GRO-seq and CAGE data published in MCF7 cells close to the eRNA regions and discussed their overlap with the eRNA regions in the revised manuscript. In the final revision, we will perform further detailed integration of all these profiles. Overall, our study will provide the integratory analysis of various independent epigenetic and functional profiles to validate our classified subtype and survival-specific eRNA regions.

Here are major points; addressing these points in the revised version is important.

From Figure 1B, what eRNAs were identified for LumB using log2MC?

Response: The authors acknowledge the lack of analyses on LumB eRNAs in the original version of the manuscript. In the final revised manuscript after associating with mRNAs, we will provide the heatmaps, pathway analyses and other functional annotations for LumB specific eRNAs.

Page 8 However, sensitivity and F-measure .... It would help to include the metrics for the number of patients in each subtype. The ratio of eRNAs/number of cases in each subtype would inform if the number of eRNAs is an outcome of no. of cases or subgroup-specific.

Response: This is a great suggestion from the reviewer, and we included the number of patients for each subtype in the table in Fig. 1D. We observed that the basal patients are low in number, but we identified more basal eRNAs. Hence, the number of eRNAs identified in subtype-specific manner is not correlated to the number of patients in the cohort.

Page 9 "Altogether, both measurements classify eRNAs efficiently based on subtypes, InfoGain allowed us to distinguish further samples based on high and low expression of eRNAs for basal subtype and performed better in statistical metrics" Based on statistical metrics, both models seem to be performing similarly except for Her2.

Response: We apologise for this wrong interpretation. We corrected this in the revised manuscript at page 9.

In Fig. 1B, the F-measure metrics are wrong for basal LogMC, as it is 0.94 rather than 0.54, which could lead to a misinterpretation of the model.

Response: We apologise for the mistake in this figure, and we included the corrected heatmap in the revised manuscript.

Many genome browser figures, including Figure S3. TFBS is not at the same site as eRNAs detected. Is there CAGE data to show that binding these TFs at these sites leads to the expression of eRNAs? That will give direct evidence that the eRNAs are transcribed due to these TFs

Response: This is a great suggestion from the reviewer. We incorporated ATAC-seq, H3K27ac ChIP-seq, CAGE and GRO-seq data on the gene profiles in Fig. S3 to validate the activity of these ER binding sites in the preliminarily revised manuscript. We observed that all the ER binding sites are accompanied by open and active enhancer marks with bidirectional transcription (either GRO- or CAGE positive) but they are not on the exact location of eRNA regions (250-1000 bps away from the centre of ER binding site). Subtype-specific eRNA regions close to genes like MLPH and XBP1 possess active bidirectional transcribing ER binding sites far away from subtype-specific eRNA loci and also ER unbound sites. However, these distal ER binding sites are close to the regions from the list of 300K eRNA loci and they were simply not identified as subtype-specific regions.

Page 10, There were 30 Her2-specific eRNA regions.... Do the same enhancers also regulate these genes as those from which eRNAs are transcribed? Is it cis-effect, or could these affect the trans-regulating of other genes?

Response: We acknowledge the concern from the reviewer, however this is hard to be validated, as functional experiments to explore the 3D interactions of enhancers and gene promoters are not robust enough to be performed in patient samples and can't be performed within the revision time frame. In the final revised manuscript, we will explore the association of enhancers and promoters of ERBB2 with PEGS association as discussed above and with available HiC datasets in Her2+ cell lines (HCC1954, GSE167150, Kim et al., 2022 https://pubmed.ncbi.nlm.nih.gov/35513575/ )

Minor comments:

Page 8 "InfoGain meausure..." Fig. S2A also shows high and low expressed eRNAs for the basal group

Response: We apologise for the lack of clarity here. InfoGain measure identifies both high and low expressed eRNAs in all patients showing similar pattern of regulation among patients. However, logmc derived eRNAs are highly expressed in most patients. Low expressed eRNAs could not be identified in logmc measure as strong as InfoGain regions. The text in the results had been edited in the revised manuscript to reflect better clarity on this point.

Page 11, Our analyses also identified the role of another..... The statement is misleading as it is the enrichment of these TFs with the eRNAs<br /> Response: We included the word “enrichment” to clarify this statement.

Page 13, "Around 90% of eRNAs are bidirectional and non-polyadenylated [53]. TCGA expression datasets are based on RNA-seq assays, which capture only non-polyadenylated RNAs. Thus, analysing the expression of eRNAs on mRNA-seq datasets might not be adequate". It is very confusing, please check<br /> Response: We apologise for the mistake, and this has been corrected in the revised manuscript.

Reviewer #3 (Significance (Required)):

This is an important topic and a highly relevant approach to identifying RNA-based biomarkers.<br /> They analyse published RNAseq datasets by focusing on molecular subtype-specific eRNAs, enhancing clinical relevance and thereby addressing the heterogeneity of the cancer type (strength of the study).

medimos la distancia

La palabra simular se reserva para procedimientos numéricos mas complejos, ustedes graficaron un función solamente

el exponente puesto así queda desprolijo. Cualquier editor de texto hoy en día les ofrece la posibilidad de escribir bien un exponente. Lo mismo con el "+-" utilizado mas abajo en este párrafo.

Tienen que priorizar claridad en la escritura. Lo conveniente en este caso es hacer una tabla con todos los valores medidos e informar el calculo del modulo al final, no alcanza solo con mostrar capturas de pantalla que ni siquiera están discutidas en el texto principal.

nunca se refieran a una figura como "la de abajo", "la de arriba", "la del costado", etc. Lo correcto es "Una captura de pantalla de lo obtenido puede observarse en la figura 2"

la sonda no es un dispositivo móvil

este esquema es confuso, parece que la sonda se utiliza por afuera del cilindro.

Redacción poco clara. Yo lo entiendo por que conozco el experimento, pero a un lector que no sabe lo que hicieron se le puede complicar entender. No es conveniente usar oraciones tan largas. Les sugiero realizar oraciones cortas y concisas, priorizando claridad en la transmisión de información

"Finalizada la caracterización del instrumental..."

Nassim - 16/06 - La société a été racheté l'année dernière

Al medir el campo en una dirección B_0 y en la otra B_180, los módulos deberían ser parecidos pero lo que cambia es el signo. Supongamos que B_180<0, entonces al tomar el promedio B_0 - (-B_180)/ 2 estos en realidad se sumaban. Por lo que vi en el cuaderno, Clase 20/05/25, pareciera ser que se quedaron solo con el modulo sin tener en cuenta el signo. Por suerte, cuando hicieron el exp de la bobina si lo tuvieron en cuenta.

redacción poco clara

Hay que poner la referencia acá

usar tiempos verbales en presente o pasado, evitar los futuros. Por ejemplo "En este trabajo medimos la intensidad del campo magnético ..." o "En este trabajo de midió la intensidad .."

la ecuación 3

Erwartung, dass wir solche Art von Hausarbeiten mit eigener Argumentation liefern, kann dann nur erfolgen, wenn Studis schon von zu Beginn an beigebracht wird, wie Texte zu bearbeiten und zu hinterfragen sind. Ich finde - und das ist selbst bei uns in der Sozialen Arbeit der Fall - Textarbeit findet sehr selten statt. Texte werden ja meistens nur als Hausaufgabe aufgegeben und dann als grundlegendes Wissen vorausgesetzt.

Nassim - 16/06 - Est fermé à toute proposition

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Reply to the reviewers

Manuscript number: RC-2025-03004

Corresponding author(s): Kentaro Furukawa and Tomotake Kanki

1. General Statements [optional]

We would like to thank the reviewers for their constructive and positive feedback. We are encouraged that all three reviewers consider the identification of Mfi2 as an outer mitochondrial membrane fission factor required for mitophagy to be a significant and important contribution to the research field. We acknowledge the concerns raised and propose the following plan to address them through additional experiments and clarifications. We believe that these revisions will further strengthen the manuscript and enhance its impact.

2. Description of the planned revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Furukawa and colleagues identified Mfi2 as novel factor that promotes fragmentation and removal of damaged mitochondria by mitophagy. They report that parallel loss of Dnm1 and Mfi2 blocks mitophagy. Mfi2 acts on the outer membrane, while the previous found Atg44 functions in the intermembrane space. How the proteins cooperate remains unknown. This is an elegant study with high-quality data. The findings are interesting for a broad readership. There are some issues as outline below that should be solved.

Response:

We would like to thank Reviewer #1 for their thoughtful evaluation of our manuscript and for recognizing the interest and quality of the study.

1. It remains unclear how Mfi2 is anchored into the outer mitochondrial membrane. Does it contain a transmembrane domain? The carbonate resistance indicates the presence of such transmembrane domain. However, the presented structures lack such membrane-spanning segment. This point should be clarified.

Response:

We performed an in silico topology prediction of Atg44 and Mfi2 using TMHMM. This tool identified a weakly hydrophobic region of Mfi2 near the N-terminus but did not predict a definitive transmembrane domain (see new Fig. EV1E) (Page 6, lines 8-9). This result implies that Mfi2 interacts with the outer membrane in a monotopic or peripheral manner, rather than as a classical transmembrane protein. Such proteins may remain in the membrane pellet after carbonate treatment due to their strong hydrophobic insertion into the lipid bilayer (e.g., yeast tafazzin/Taz1; Brandner et al., Mol. Biol. Cell, 2005; DOI: 10.1091/mbc.E05-03-0256). We will incorporate this interpretation in the revised manuscript.

How does Mfi2 cooperate with Dnm1? Is there any interaction between these proteins? Some further information could provide mechanistic insights into the function of Mfi2.

Response:

While our study does not explicitly suggest that Mfi2 cooperates with Dnm1, we plan to investigate whether these proteins physically associate. We will perform co-immunoprecipitation experiments under growing and mitophagy-inducing conditions to examine potential interactions between Mfi2 and Dnm1. Further insights into their interaction could help clarify the mechanistic role of Mfi2 in mitochondrial fission and mitophagy.

The authors report a CL-dependent binding of Mfi2 to liposomes. Is the recruitment of Mfi2 to mitochondria impaired when CL-synthesis is blocked, e.g. in crd1delta mitochondria?

Response:

To assess the role of cardiolipin in Mfi2 localization, we will compare the efficiency of mitochondrial targeting of endogenous Mfi2 in WT and crd1Δ cells. Additionally, as mentioned in Reviewer #3's comment, we plan to perform coarse-grained molecular dynamics simulations to further investigate the interaction between Mfi2 and cardiolipin. The results of these simulations will be incorporated into the discussion to provide deeper mechanistic insights.

Figure 4B: a wild-type control should be added.

Response:

We appreciate Reviewer #1’s suggestion to include a WT control in Figure 4B. However, given the focus of this figure on the rescue of mitophagy defects in the mfi2Δ dnm1Δ strain, we believe that adding a WT control is not essential for the analysis. The key comparison here is between the mfi2Δ dnm1Δ strain and the rescue conditions, and statistical analysis was performed to support the conclusions. We hope this clarifies our approach, but we will make adjustments if necessary.

Reviewer #1 (Significance (Required)):

The reported findings are interesting for a broad readership.

Response:

We appreciate Reviewer #1’s recognition of the relevance of our findings to a broad readership.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this study, the authors discover a mitochondrial fission factor, termed Mfi2, that promotes mitophagy efficiency and that functions in a partially redundant way with Dnm1 for the fission of mitochondrial outer membranes during mitophagy. The discovery helps to clarify why Dnm1 does not appear to be essential for fission mediated mitophagy by Dnm1. Mfi2 is structurally similar to the inner membrane fission factor Atg44 which is consistent with Mfi2's fission activity. The authors show that Mfi2 has membrane fission activity towards nanotubes in vitro, and that membrane binding is dependent of high levels of cardiolipin, a mitochondrially enriched lipid. In summary, the authors show that Mfi2 mediates mitochondrial outer membrane fission together with Drp1, whereas Atg44 mediates inner membrane fission, that together are necessary for mitophagy.

Response:

We thank Reviewer #2 for the positive assessment and for clearly summarizing the main contributions of our work.

Major: 1. Figure 2: How do the expression levels of the Mfi2 constructs compare to the endogenous levels of the protein? This will help to gauge to what degree Mfi2 N66 overexpression is needed to achieve mitochondrial fragmentation in Atg44 delta cells and also the low level of mitophagy rescue that was observed.

Response:

We used the TDH3 promoter for the expression of Mfi2 in Figures 2D and 2E. Unfortunately, our Mfi2 antibody only detects full-length Mfi2, as it recognizes a C-terminal region of the protein. This means we cannot directly compare the expression levels of Mfi2(N66) to those of endogenous full-length Mfi2. To clarify the expression levels, we will provide the following data:

(1) Mfi2 antibody: Endogenous Mfi2(Full) and overexpressed Mfi2(Full)

(2) FLAG antibody: Overexpressed Mfi2(Full)-FLAG and overexpressed Mfi2(N66)-FLAG

Figure 3A-B: The cardiolipin binding results in vitro are interesting but the concentration of cardiolipin is much lower on the outer membrane versus the inner membrane. Can the authors comment on whether the cardiolipin levels used on the nanotubes are relevant to that of the mitochondrial outer membrane where Mfi2 is located? Can the authors provide quantitative data for these experiments to help strengthen their conclusions?

Can the authors also use purified MBP alone or a form of Mfi2 that cannot bind to membrane e.g. Mfi2-C33) as a control?

Response:

We thank the reviewer for raising this important point regarding our cardiolipin-dependent in vitro data. In our experiments, we used 20 mol% cardiolipin (CL), a concentration higher than the typical levels in the mitochondrial outer membrane, which contains less than 5% CL. However, it is known that CL translocates to the outer membrane under mitophagy-inducing conditions (e.g., Chu et al., Nat Cell Biol, 2013; Kagan et al., Cell Death Differ, 2016). Our use of elevated CL levels aligns with standard practices in in vitro reconstitution assays to ensure adequate membrane curvature and charge density, which are necessary for robust and reproducible protein-membrane interaction assessments.

To strengthen our conclusions, we will provide a quantitative analysis of the nanotube fission experiments. This will include the percentage of severed tubes under each condition, the total number of tubes analyzed (n), and the relationship between tube diameter and fission efficiency. These additional data will allow for a more thorough evaluation of the membrane fission activity of Mfi2.

Furthermore, we will include control experiments using purified MBP alone and a membrane-binding-deficient mutant of Mfi2 (C33), as suggested by the reviewer.

Figure 4D: The protrusions are very difficult to visualize. Can the authors also provide zoomed in regions. Is the data representative from 3 or more independent experiments? Can the authors provide a graph of the quantitation to aid readers with analysis of the data?

Response:

We thank the reviewer for this helpful suggestion. In the revised manuscript, we will provide higher magnification images to improve the visibility of mitochondrial protrusions. We confirm that the presented images are representative of results obtained from three independent experiments. Additionally, as requested, we will include a graph quantifying the frequency and morphology of protrusions to facilitate data interpretation.

Figure 4D: It is fascinating to see the mitochondrial protrusion formation being dependent on autophagy factors but not mitochondrial fission factors. To help visualize this, can the authors image one of either Atg1, Atg8 to address whether phagophores are forming on the protrusions and if so where they are positionally located on the protrusion in control and/or mfi2,dnm1,atg44 triple mutant cells?

Response:

We thank the reviewer for this insightful comment. In our previous study (Fukuda et al., Mol Cell, 2023), we demonstrated that Atg proteins, such as Atg8, accumulate at mitochondrial protrusions formed in atg44Δ cells, suggesting that these structures can serve as sites for phagophore assembly. However, as in our previous microscopy analysis, the resolution limitations of our imaging system make it difficult to precisely determine the exact location of phagophores on the protrusions.

Whether similar recruitment occurs in the absence of both Mfi2 and Dnm1 remains untested. To address this, we will perform fluorescence imaging of fluorescent protein tagged Atg proteins, such as GFP-Atg8, in mfi2Δ dnm1Δ atg44Δ triple mutant cells to examine whether phagophores form on the mitochondrial protrusions under these conditions. This will help us determine whether phagophore formation requires mitochondrial fission or occurs independently of it.

Minor: 1. Is it possible to target Atg44 to the mitochondrial outer membrane, either by attaching an OM anchor or using part of the N-terminus of Mfi2? This will help elucidate how Mfi2 reaches the outer membrane and whether Atg44 can be just as active on the outer membrane as long as it can access it.

Response:

We thank the reviewer for this suggestion. We will construct chimeric proteins between Atg44 and Mfi2 and examine where such proteins are localized. Additionally, we will assess whether these chimeric proteins have the functional activity of Mfi2, as this will help determine if Atg44 can be active on the mitochondrial outer membrane when properly targeted.

Are microtubules or actin required for the protrusions to form? Using the triple mutant cells that have a high proportion of protrusions, it could be tried to add cytoskeletal depolymerizing drugs such as nocodazole for microtubules or Latrunculin A or Latrunculin B for actin.

Response:

We thank the reviewer for this suggestion. We will test the effect of cytoskeletal depolymerizing drugs on protrusion formation in the mfi2Δ dnm1Δ atg44Δ triple mutant cells.

Reviewer #2 (Significance (Required)):

Significance: The discovery of Mfi2 as an outer membrane mitophagy fission factor is an exciting, and very important and significant contribution to the field. The data are in this study are clear and the conclusions are generally well supported by the experiments. This study appears to be suitable as a report style manuscript given that there is limited mechanistic analysis of Mfi2 activity. This does not affect the importance of the work, it just means that it is suited as a report of a significant discovery. Overall, this fills an important knowledge gap in solving the mystery behind which factors are involved in mitochondrial outer membrane fission during mitophagy, and provides a clarification why Dnm1 loss alone minimally affects mitophagy. This work will appeal to researchers interested in mitochondrial biology, the autophagy field, and cell biologists interested in organelle membrane dynamics, and is also broadly important and interesting to all cell biologists.

Reviewer expertise: mitophagy mechanisms, cell biology of mitophagy, autophagy and autophagosome formation, mitochondrial biology including OXPHOS and mitochondrial dynamics

Response:

We appreciate Reviewer #2’s comments on the importance and potential impact of our discovery for the mitophagy and cell biology fields.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The manuscript by Furukawa et al. presents a well-structured and thorough study identifying Mfi2 as a novel mitochondrial outer membrane-resident fission factor required for mitophagy in Saccharomyces cerevisiae. The authors demonstrate that Mfi2, together with the inner membrane mitofissin Atg44 and the dynamin-related GTPase Dnm1, contributes to mitochondrial fragmentation during mitophagy. Importantly, they show that while Dnm1 is dispensable on its own, Mfi2 and Dnm1 act redundantly from the outer membrane to support Atg44-mediated fission. The data are robust, the figures are clear, and the mechanistic insight into how mitophagy-specific fission is achieved is of high relevance to the field of mitochondrial quality control.

Overall, this is a logically constructed and convincing study with important implications for understanding compartment-specific mechanisms of mitochondrial fission during selective autophagy. The conclusions are largely well supported by the data. However, a few issues and points of clarification should be addressed before publication.

Response:

We thank Reviewer #3 for the careful and constructive review and for acknowledging the logical structure and robustness of our data.

Major Comments

1. The observation that both Mfi2 and Atg44 require high cardiolipin (CL) content for membrane binding and fission in vitro is intriguing, especially given that CL is enriched in the inner membrane. The authors mention CL externalisation during mitophagy, but this connection could be made more explicit earlier in the manuscript. Furthermore, since the molecular mechanism of membrane interaction remains unresolved, I would strongly encourage the authors to undertake coarse-grained molecular dynamics simulations to explore how Mfi2 might interact with lipid bilayers of differing composition. This could clarify the role of CL and the potential structural contribution of the disordered C-terminal region. Response:

We thank the reviewer for highlighting the need to clarify the connection between CL externalization and the observed CL-dependent membrane binding and fission activity of Mfi2 and Atg44. While we briefly mentioned CL externalization during mitophagy in the Discussion, we agree that this connection should be made more explicit earlier in the manuscript. In the revised version, we will incorporate a brief rationale in the Results section to clarify that CL translocates to the mitochondrial outer membrane under mitophagy-inducing conditions (e.g., Chu et al., Nat Cell Biol 2013). This will provide a physiological basis for our in vitro reconstitution assays using CL-containing liposomes.

We also appreciate the reviewer’s suggestion to explore the molecular basis of Mfi2-lipid interaction through coarse-grained molecular dynamics (CGMD) simulations. In collaboration with Dr. Yuji Sakai, we will perform coarse-grained molecular dynamics (CGMD) simulations to investigate how Mfi2 interacts with lipid bilayers of varying compositions, focusing particularly on the role of cardiolipin and the structural contribution of the disordered C-terminal region. If successful, we will include the results in the revised manuscript.

While the genetic and phenotypic data indicate that Mfi2 and Dnm1 act independently to support mitochondrial fission, the spatial and temporal organisation of their activity during mitophagy remains unclear. Do Mfi2 and Dnm1 colocalise at fission sites, or do they act at separate subdomains of the outer membrane? Live-cell imaging with fluorescently tagged Mfi2 and Dnm1, particularly during mitophagy induction, could help clarify whether these factors act in concert or at distinct locations and time points. This would also help determine whether their apparent redundancy reflects parallel mechanisms or functional compensation at shared sites. It would also be interesting to combine this with Atg44.

Response:

We thank the reviewer for this insightful comment. We plan to perform co-localization analysis of Mfi2 and Dnm1 during mitophagy induction to clarify whether these proteins colocalize at fission sites or act at separate subdomains of the outer membrane. Additionally, we will conduct co-immunoprecipitation experiments of Mfi2 and Dnm1 (see also Response to Reviewer #1’s major comment 2) to further investigate their potential interaction. It is challenging to analyze Mfi2, Dnm1, Atg44, and mitochondrial fission sites simultaneously, as fluorescence-tagged Atg44 has been shown to lose its function (Fukuda et al., Mol Cell, 2023).

Minor Comments

1. The sodium carbonate extraction and proteinase K assays (Figure 1E-F) are standard but may not be familiar to all readers. A brief explanatory sentence clarifying what these methods reveal about membrane topology would improve accessibility. Response:

We thank the reviewer for this helpful comment. We have added a brief explanatory sentence in the revised manuscript to clarify the principles and interpretation of the sodium carbonate extraction and proteinase K assays (Page 5, lines 23-25; Page 6, lines 1-3).

While immunoblot quantifications are shown throughout, it would be helpful to include statistical analysis where appropriate, especially in cases where differences between genotypes or constructs are modest.

Response:

Statistical analyses have been added for immunoblot quantifications where appropriate, particularly in cases where differences between genotypes or constructs are modest.

The naming of Mfi2 as a mitofissin is consistent with previous terminology introduced for Atg44, but the term remains relatively new. A brief clarification distinguishing "mitofissin" from the better-known "mitofusin" family in mammals would help avoid confusion for readers less familiar with yeast-specific nomenclature.

Response:

We have added a brief explanation of the term "mitofissin" to distinguish it from the mammalian "mitofusin" family in Introduction (Page 3, line 26-Page 4 line 1).

Reviewer #3 (Significance (Required)):

This is a strong and well-executed study that provides mechanistic insight into how mitochondrial fission is coordinated during mitophagy in yeast. A major strength is the identification and characterisation of Mfi2 as a previously unrecognised outer membrane fission factor acting in parallel with Dnm1 and in coordination with the intermembrane space protein Atg44. The genetic, imaging, and in vitro biochemical data are carefully integrated, and the authors are transparent about limitations, including open questions around the C-terminal domain of Mfi2, CL dependence, and the evolutionary conservation of mitofissins.

The work makes a conceptual advance by showing that mitophagy-specific mitochondrial fission requires the cooperation of spatially separated factors acting from both the inside and outside of mitochondria, a mechanism that had not been fully appreciated. This study helps resolve previous contradictions regarding the dispensability of Dnm1 in mitophagy, thereby filling a gap in our understanding of organelle-specific fission. While the findings are focused on yeast, they raise broader questions about whether similar principles apply to higher eukaryotes (historically yeast research was always at the forefront of autophagy field).

The study will be of interest to specialists in autophagy, mitochondrial dynamics, and yeast cell biology, as well as researchers working on membrane remodelling and organelle quality control. While the audience is primarily specialised, the conceptual insights will resonate more broadly in the cell biology community.

I am an expert in mitophagy mechanisms in mammalian cells, and while not a specialist in yeast models, I found the study logical, rigorous, and of clear relevance to the broader autophagy field.

Response:

We are grateful for Reviewer #3’s recognition of the conceptual advance provided by our study and its relevance beyond yeast biology.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Responses to Reviewer #1:

・We performed in silico topology prediction of Atg44 and Mfi2 using TMHMM. This tool identified a weakly hydrophobic region of Mfi2 near the N-terminus but did not predict a definitive transmembrane domain (new Fig. EV1E) (Page 6, lines 8-9).

Responses to Reviewer #3:

・We have added a brief explanatory sentence in the revised manuscript to clarify the methods and interpretation of the sodium carbonate extraction and proteinase K assays (Page 5, lines 23-25; Page 6, lines 1-3).

・Statistical analyses have been added for immunoblot quantifications where appropriate, particularly in cases where differences between genotypes or constructs are modest.

・We have added a brief explanation of the term "mitofissin" to distinguish it from the mammalian "mitofusin" family in Introduction (Page 3, line 26-Page 4, line 1).

4. Description of analyses that authors prefer not to carry out

Response to Reviewer #1 (Major 4):

We will not include the WT strain as a control. See our response.

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Referee #2

Evidence, reproducibility and clarity

In this study, the authors discover a mitochondrial fission factor, termed Mfi2, that promotes mitophagy efficiency and that functions in a partially redundant way with Dnm1 for the fission of mitochondrial outer membranes during mitophagy. The discovery helps to clarify why Dnm1 does not appear to be essential for fission mediated mitophagy by Dnm1. Mfi2 is structurally similar to the inner membrane fission factor Atg44 which is consistent with Mfi2's fission activity. The authors show that Mfi2 has membrane fission activity towards nanotubes in vitro, and that membrane binding is dependent of high levels of cardiolipin, a mitochondrially enriched lipid. In summary, the authors show that Mfi2 mediates mitochondrial outer membrane fission together with Drp1, whereas Atg44 mediates inner membrane fission, that together are necessary for mitophagy.

Major:

1. Figure 2: How do the expression levels of the Mfi2 constructs compare to the endogenous levels of the protein? This will help to gauge to what degree Mfi2 N66 overexpression is needed to achieve mitochondrial fragmentation in Atg44 delta cells and also the low level of mitophagy rescue that was observed.
2. Figure 3A-B: The cardiolipin binding results in vitro are interesting but the concentration of cardiolipin is much lower on the outer membrane versus the inner membrane. Can the authors comment on whether the cardiolipin levels used on the nanotubes are relevant to that of the mitochondrial outer membrane where Mfi2 is located? Can the authors provide quantitative data for these experiments to help strengthen their conclusions? Can the authors also use purified MBP alone or a form of Mfi2 that cannot bind to membrane e.g. Mfi2-C33) as a control?
3. Figure 4D: The protrusions are very difficult to visualize. Can the authors also provide zoomed in regions. Is the data representative from 3 or more independent experiments? Can the authors provide a graph of the quantitation to aid readers with analysis of the data?
4. Figure 4D: It is fascinating to see the mitochondrial protrusion formation being dependent on autophagy factors but not mitochondrial fission factors. To help visualize this, can the authors image one of either Atg1, Atg8 to address whether phagophores are forming on the protrusions and if so where they are positionally located on the protrusion in control and/or mfi2,dnm1,atg44 triple mutant cells?

Minor:

1. Is it possible to target Atg44 to the mitochondrial outer membrane, either by attaching an OM anchor or using part of the N-terminus of Mfi2? This will help elucidate how Mfi2 reaches the outer membrane and whether Atg44 can be just as active on the outer membrane as long as it can access it.
2. Are microtubules or actin required for the protrusions to form? Using the triple mutant cells that have a high proportion of protrusions, it could be tried to add cytoskeletal depolymerizing drugs such as nocodazole for microtubules or Latrunculin A or Latrunculin B for actin.

Significance

The discovery of Mfi2 as an outer membrane mitophagy fission factor is an exciting, and very important and significant contribution to the field. The data are in this study are clear and the conclusions are generally well supported by the experiments. This study appears to be suitable as a report style manuscript given that there is limited mechanistic analysis of Mfi2 activity. This does not affect the importance of the work, it just means that it is suited as a report of a significant discovery. Overall, this fills an important knowledge gap in solving the mystery behind which factors are involved in mitochondrial outer membrane fission during mitophagy, and provides a clarification why Dnm1 loss alone minimally affects mitophagy. This work will appeal to researchers interested in mitochondrial biology, the autophagy field, and cell biologists interested in organelle membrane dynamics, and is also broadly important and interesting to all cell biologists.

Reviewer expertise: mitophagy mechanisms, cell biology of mitophagy, autophagy and autophagosome formation, mitochondrial biology including OXPHOS and mitochondrial dynamics

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors describe a good-quality ancient maize genome from 15th-century Bolivia and try to link the genome characteristics to Inca influence. Overall, the manuscript is below the standard in the field. In particular, the geographic origin of the sample and its archaeological context is not well evidenced. While dating of the sample and the authentication of ancient DNA have been evidenced robustly, the downstream genetic analyses do not support the conclusion that genomic changes can be attributed to Inca influence. Furthermore, sections of the manuscript are written incoherently and with logical mistakes. In its current form, this paper is not robust and possibly of very narrow interest.

Strengths:

Technical data related to the maize sample are robust. Radiocarbon dating strongly evidenced the sample age, estimated to be around 1474 AD. Authentication of ancient DNA has been done robustly. Spontaneous C-to-T substitutions, which are present in all ancient DNA, are visible in the reported sample with the expected pattern. Despite a low fraction of C-to-T at the 1st base, this number could be consistent with the cool and dry climate in which the sample was preserved. The distribution of DNA fragment sizes is consistent with expectations for a sample of this age.

Weaknesses:

(1) Archaeological context for the maize sample is weakly supported by speculation about the origin and has unreasonable claims weighing on it. Perhaps those findings would be more convincing if the authors were to present evidence that supports their conclusions: i) a map of all known tombs near La Paz, ii) evidence supporting the stone tomb origins of this assemblage, and iii) evidence supporting non-Inca provenance of the tomb.

(2) Dismissal of the admixture in the reported samples is not evidenced correctly. Population f3 statistic with an outgroup is indeed one of the most robust metrics for sample relatedness; however, it should not be used as a test of admixture. For an admixture test, the population f3 statistic should be used in the form: i) target population, ii) one possible parental population, iii) another possible parental population. This is typically done iteratively with all combinations of possible parental populations. Even in such a form, the population f3 statistic is not very sensitive to admixture in cases of strong genetic drift, and instead population f4 statistic (with an outgroup) is a recommended test for admixture.

(3) The geographic placement of the sample based on genetic data is not robust. To make use of the method correctly, it would be necessary to validate that genetic samples in this region follow the assumption of the 'isolation-by-distance' with dense sampling, which has not been done. Additionally, the authors posit that "This suggests that aBM might not only be genetically related to the archaeological maize from ancient Peru, but also in the possible geographic location." The method used to infer the location is based on pure genetic estimation. The above conclusion is not supported by this method, and it directly contradicts the authors' suggestion that the sample comes from Bolivia.

(4) The conclusion that Ancient Andean maize is genetically similar to European varieties and hence shares a similar evolutionary history is not well supported. The PCA plot in Figure 4 merely represents sample similarity based on two components (jointly responsible for about 20% of the variation explained), and European samples could be very distant based on other components. Indeed, the direct test using the outgroup f3 statistic does not support that European varieties are particularly closely related to ancient Andean maize. Perhaps these are more closely related to Brazil? We do not know, as this has not been measured.

(5) The conclusion that long branches in the phylogenetic tree are due to selection under local adaptation has no evidence. Long branches could be the result of missing data, nucleotide misincorporations, genetic drift, or simply due to the inability of phylogenetic trees to model complex population-level relationships such as admixture or incomplete lineage sorting. Additionally, captions to Figure S3, do not explain colour-coding.

(6) The conclusion that selection detected in aBM sample is due to Inca influence has no support. Firstly, selection signature can be due to environmental or other factors. To disentangle those, the authors would need to generate the data for a large number of samples from similar cultural contexts and from a wide-ranging environmental context, followed by a formal statistical test. Secondly, allele frequency increase can be attributed to selection or demographic processes, and alone is not sufficient evidence for selection. The presented XP-EHH method seems more suitable. Overall, methods used in this paper raise some concerns: i) how accurate are allele-frequency tests of selection when only single individual is used as a proxy for a whole population, ii) the significance threshold has been arbitrary fixed to an absolute number based on other studies, but the standard is to use, for example, top fifth percentile. Finally, linking selection to particular GO terms is not strong evidence, as correlation does not imply causation, and links are unclear anyway.

In sum, this manuscript presents new data that seems to be of high quality, but the analyses are frequently inappropriate and/or over-interpreted.

Reviewer #3 (Public review):

Summary:

The authors seek to place archaeological maize samples (2 kernels) from Bolivia into genetic and geographical context and to assess signatures of selection. The kernels were dated to the end of the Incan empire, just prior to European colonization. Genetic data and analyses were used to characterize the distance from other ancient and modern maize samples and to predict the origin of the sample, which was discovered in a tomb near La Paz, Bolivia. Given the conquest of this region by the Incan empire, it is possible that the sample could be genetically similar to populations of maize in Peru, the center of the Incan empire. Signatures of selection in the sample could help reveal various environmental variables and cultural preferences that shaped maize genetic diversity in this region at that time.

Strengths:

The authors have generated substantial genetic data from these archaeological samples and have assembled a data set of published archaeological and modern maize samples that should help to place these samples in context. The samples are dated to an interesting time in the history of South America during a period of expansion of the Incan empire and just prior to European colonization. Much could be learned from even this small set of samples.

Weaknesses:

(1) Sample preparation and sequencing:<br /> Details of the quality of the samples, including the percentage of endogenous DN,A are missing from the methods. The low percentage of mapped reads suggests endogenous DNA was low, and this would be useful to characterize more fully. Morphological assessment of the samples and comparison to morphological data from other maize varieties is also missing. It appears that the two kernels were ground separately and that DNA was isolated separately, but data were ultimately pooled across these genetically distinct individuals for analysis. Pooling would violate assumptions of downstream analysis, which included genetic comparison to single archaeological and modern individuals.

(2) Genetic comparison to other samples:<br /> The authors did not meaningfully address the varying ages of the other archaeological samples and modern maize when comparing the genetic distance of their samples. The archaeological samples were as old as >5000 BP to as young as 70 BP and therefore have experienced varying extents of genetic drift from ancestral allele frequencies. For this reason, age should explicitly be included in their analysis of genetic relatedness.

(3) Assessment of selection in their ancient Bolivian sample:<br /> This analysis relied on the identification of alleles that were unique to the ancient sample and inferred selection based on a large number of unique SNPs in two genes related to internode length. This could be a technical artifact due to poor alignment of sequence data, evidence supporting pseudogenization, or within an expected range of genetic differentiation based on population structure and the age of the samples. More rigor is needed to indicate that these genetic patterns are consistent with selection. This analysis may also be affected by the pooling of the Bolivian archaeological samples.

(4) Evidence of selection in modern vs. ancient maize: In this analysis, samples were pooled into modern and ancient samples and compared using the XP-EHH statistic. One gene related to ovule development was identified as being targeted by selection, likely during modern improvement. Once again, ancient samples span many millennia and both South, Central, and North America. These, and the modern samples included, do not represent meaningfully cohesive populations, likely explaining the extremely small number of loci differentiating the groups. This analysis is also complicated by the pooling of the Bolivian archaeological samples.

Ha de dir: Benvinguda a la plataforma participativa Decidim CoARA-UOC. Construïm un sistema d’avaluació seguint els principis CoARA. Uneix-te, participa i decideix.

Constituția de la Weimar a fost legea fundamentală a Republicii de la Weimar, statul german care a existat între 1919 și 1933, după Primul Război Mondial și prăbușirea Imperiului German.

Context istoric: După înfrângerea Germaniei în Primul Război Mondial (1918), împăratul Wilhelm al II-lea a abdicat.

Germania a fost transformată într-o republică parlamentară.

La 31 iulie 1919, Adunarea Națională Constituantă, întrunită la Weimar (nu la Berlin, din motive de securitate), a adoptat noua constituție. A intrat în vigoare pe 11 august 1919.

ha take that (wait I need to ask my brother)

https://patents.google.com/patent/USD114262

Westclox Style 5 and 5a Big Ben Alarm Clocks by [[Bill Stoddard]]

I have a problem with my USB-A mouse too. Befoer my Mac was uprade to Apple Chips, the same mouse was stable on the Intel-based Mac. Now it's a frequent issue. The mouse if fully charged (using an AA battery).

I don't have the "jittery" problem as shown by the OP though. It is more of issue of the mouse pointer sometimes not moving, not activated, connection or response failure type of situation.

ich würde sagen, ja - und zwar ab dem Punkt, wo das Output als wissenschaftlich valide behandelt wird-

hier wäre Hinweis auf FAIR Prinzipien schön - die ja Operationalsierungen ganz praktisch leiten

da scheint mir zentral und eine wichtige Beobachtung des Artikels! Könnt Ihr ein Beispiel geben?

ja, aber ohne Beispiel ist das insbesondere für Neulinge im Feld schwer einzuordnen. Am Beispiel könnte gezeigt werden: Wie soll was gemessen werden? Gibt es dabei unterschiedliche und ggf. parallele Wege zum Ziel? (Stichwort Triangulation; Converging Evidence). Und wie geht man mit unterschiedlichen Operationalisierungen vpn Konstrukten nebeneinander um? Wichtig wohl auch Vorläufigkeit, Kontingenz von Operationalisierungen, und Falsifierbarkeit von dazugehörigen Annahmen/Hypothesen, die ggf. zu Neuoperationalisierungen führen.

Brauchen wir hier in diesem Glossar so viel zu Bridgman? Was davon ist tatächlich relevant für die heutige Verwendung und die Praktiken, die wir unter Operationalisierung verstehen? Wichtig fände ich in jedem Fall, auf die Dimensionen von Standardisierung (ggf. der Meter, das Kilo etc) und die damit einhergehende Überprüfbarkeit und Reproduzierbarkeit von Forschung (auch in den DH) einzugehen - also das "physikalische Erbe" des Begriffs deutlicher anzuschliessen an die hier relevante Perspektive der digitalen GW.

das ist ja richtig, aber es wäre m.E. für die LEswer*innenschaft besser, etwas allgemeiner anzusetzen und den Begriff zunächst in den empirischen Wissenschaften generell einzuordnen, und darauf hinzuweisen, dass er in algorithmischen, formalen (formalisierenden) und quantifizierenden Verfahren der DH eine Schlüsselrolle spielt und hier wohl am wirkmächtigsten von Moretti zuerst verwendet wurde. Aber nicht nur von ihm, siehe Ansätze der empirischen Literaturwissenschaft, z.B. bei Groeben, Norbert. 2013. “Was kann/soll ›Empirisierung (in) der Literaturwissenschaft‹ heißen?” In Empirie in der Literaturwissenschaft, 47–74. Brill mentis. https://doi.org/10.30965/9783957439710_005. Siehe auch Forschung aus der kognitiven Korpuslinguistik/-stilistik zur Metapher als semiotisch-konzeptuelles und potenziell kognitives Schema mit sprachlichen Indikatoren : Herrmann, J. Berenike. 2022. “Operationalisierung der Metapher zur quantifizierenden Untersuchung deutschsprachiger literarischer Texte im Übergang vom Realismus zur Moderne.” In Digitale Literaturwissenschaft, edited by Fotis Jannidis, 629–62. Germanistische Symposien. Stuttgart: J.B. Metzler. https://doi.org/10.1007/978-3-476-05886-7_25.

m.E. wäre es für das Verständnis hilfreich, hier und im Weiteren deutlicher auf die Messbarmachung, den Prozess für die Herbeiführung von Beobachtbarkeit abzuheben. Das steht zwar drin, könnte aber noch mehr akzentuiert werden. Wichtiger ist dabei wohl, dass die Bestimmung von "Indikatoren" in der Regel als Teil der "Operationalisierung" begriffen wird. Indikatoren sind also nicht vorgelagert (so könnte die Definition im jetzigen Wortlaut missverstanden werden), sondern werden im Prozess konstruiert bzw. festgelegt. D.h. "identifiziert werden können" ist also missverständlich und sollte m.E. in "festgelegt" o.ä. verändert werden. Auch das "werden können" sollte verändert werden in "werden". Das wird ja tatsächlich so gemacht. :-)

Too historical, it is not a primary goal of today's EA as I understand it. I would simply remove or rephrase this sentence.

Ca also junction have a name? I expect so.

reply to u/TypewriterJustice at https://old.reddit.com/r/typewriters/comments/1lbjr5f/sorry_to_say/mxunsb6/

I think the real crime here was the quote of $200 for all this work. $200 should just barely cover the recovered platen, rollers, and new feet with any margin. The full clean, oil, and adjust is a few more hours at $40-75/hour and that's not even getting to the parts or labor on the tougher troubleshooting and repair portions. With this rough diagnosis and potential issues, I (and many others I'm sure) would be quoting closer to $500-600 for a refurbish job at this level.

Living in the LA area, I'm blessed to have 7 shops within a reasonable drive, but if I put a machine into the queue at most of them it'd be a two or three month's wait time at the very best. Most of them have been at the game for decades much less in the midst of also recently setting up a brick and mortar shop.

As a point of comparison, Lucas Dul publishes his wait list on his website (currently 84 people) where he states "Average repair cost is $300-350 for general cleaning, service, and minor repairs. Average turnaround time is 2-3 weeks." Perhaps Charlie might benefit from creating a wait list and not taking machines into the shop until his time and attention can turn directly to them?

It's not often addressed here in this fora how much one should expect to either pay or wait for repair services which aren't evenly distributed across the United States and likely even less so in many other countries. In the broader scheme of things, I think that you get a far better deal at professional shops than you're going to find within the broader public of so-called typewriter sellers (antique shops, thrift stores, etc.)

As a point of reference, I'm an advanced hobbyist with my own garage-based shop for my personal collection and even I get one or two queries a week about repairing or restoring the machines of others, so I'm at least reasonably aware of what some of the wait times can look like. I wish I had the time or stock of parts machines to do more than a handful of friends and family repairs on top of my own personal repair work.

Sadly, at the end of the day, it sounds like both sides were potentially not good at communicating expectations about how long repairs would take. If nothing else we should all be sharing more details about these issues to help level set how this all works for the broader typewriter community.

DOI: 10.1172/JCI187323

Resource: RRID:Addgene_62988

Curator: @olekpark

SciCrunch record: RRID:Addgene_62988

What is this?

DOI: 10.1128/jvi.02234-24

Resource: RRID:Addgene_22575

Curator: @olekpark

SciCrunch record: RRID:Addgene_22575

What is this?

DOI: 10.1038/s41467-025-59886-w

Resource: RRID:Addgene_48138

Curator: @olekpark

SciCrunch record: RRID:Addgene_48138

What is this?

DOI: 10.1038/s41401-025-01477-y

Resource: RRID:Addgene_10878

Curator: @olekpark

SciCrunch record: RRID:Addgene_10878

What is this?

DOI: 10.1038/s41386-025-02065-8

Resource: RRID:Addgene_121539

Curator: @olekpark

SciCrunch record: RRID:Addgene_121539

What is this?

DOI: 10.1101/2025.05.09.653147

Resource: (BD Biosciences Cat# 553028, RRID:AB_394566)

Curator: @scibot

SciCrunch record: RRID:AB_394566

What is this?

DOI: 10.1093/jbmrpl/ziaf093

Resource: (Thermo Fisher Scientific Cat# A-11057, RRID:AB_2534104)

Curator: @scibot

SciCrunch record: RRID:AB_2534104

What is this?

Je ne comprends pas très bien ce qui est signifié ici ?

Je trouve cette idée de plateformisation très intéressante. Cela m'évoque des discussions que nous avons eues en interne autour du livre de Colin & Verdier "L'âge de la multitude". La plateformisation ne concerne pas seulement la délinéarisation des structures internes mais implique aussi une ouverture sur l'hypertexte du web et l'insertion dans son écosystème par l'interaction avec les usagers (notamment sur les réseaux sociaux comme vous le soulignez). C'est aussi une remédiation qui déplace le centre de l'attention vers les usagers, d'une façon très différente de la période précédente. La bibliothèque numérique post-PLAO avait déjà une dimension de médiation et d'éditorialisation vers le grand public (avec les dossiers thématiques) mais cela se trouve renforcé dans ces années 2000-2010 avec l'effort d'ouverture sur les réseaux sociaux, et le "pivot documentaire" allait aussi dans ce sens.

Tout dépend ce qu'on entend par infrastructure, les 2 partagent tout de même un système d'information commun, avec le catalogue comme socle. De fait, la circulation des documents s'ancre au même réservoir de données dans les deux "bibliothèques".

C'est un peu implicite ici, mais en lien avec mon commentaire ci-dessus : cette tension notamment autour de la question de la conservation des collections conduit, dans cette 2e étape de remédiation, à privilégier quand même la numérisation de documents hors collections patrimoniales. Ainsi, si l'audience visée change, on reste sur un modèle hybride qui vise à donner accès à des documents en général, pas nécessairement ceux de la bibliothèque, dans une logique de corpus. Le questionnement associé et notamment ses implications techniques et en matière de structuration des documents est bien décrit par JD Wagneur ici : https://books.openedition.org/bibpompidou/1500

Je trouve significatif, par rapport à la théorie que vous formulez ici, que les documents ou corpus numérisés ne soient pas les collections elles-mêmes de la bibliothèque (comme cela avait pu être envisagé par le politique au départ) mais des documents déjà reproduits, ou des doublons acquis ad hoc, ou encore des textes obtenus par d'autres voies (comme le partenariat avec l'Inist sur Frantext). On voit bien que la bibliothèque numérique, à ce moment-là, est envisagée comme une collection de textes pour la lecture savante, sans lien direct avec les collections physiques. Ce modèle de collection est décrit dans (Richard, 1993).

Il ne s'agit pas ici de la BPI, mais bien du Centre Pompidou : le projet de Centre Pompidou Virtuel englobait le Musée national d'Art Moderne, les productions événementielles et audiovisuelles du Centre (expositions, spectacle vivant, conférences) et les 2 bibliothèques (bibliothèque Kandinsky et BPI).

Document de Synthèse : L'Emprise et ses Implications Médico-Légales

Ce briefing synthétise les points clés de la conférence intitulée "Conférence CRIAVS - Emprise", abordant la nature complexe de la relation d'emprise, son exploration psychiatrique et ses implications juridiques.

L'intervenant, un psychiatre, met en lumière les désaccords avec les approches précédentes, soulignant l'importance d'une compréhension holistique du lien auteur-victime.

1. Préambule : Distinction et Compréhension

L'orateur introduit son propos en soulignant une divergence d'approche avec la loi actuelle. Tandis que le droit distingue clairement les victimes des auteurs, la psychiatrie s'intéresse à la dynamique du lien entre les deux.

• L'approche juridique vs. psychiatrique : "d'un côté il y a la loi qui aujourd'hui distingue de façon très claire les victimes d'un côté les auteurs de l'autre... moi je me place du côté de la psychiatrie et du côté de la psychiatrie il y a un intérêt à aller chercher à renseigner ce qui se passe dans le lien entre une victime et un auteur."

• Objectif de la compréhension : Comprendre les mécanismes ne signifie ni excuser l'auteur, ni blâmer la victime, mais "identifier mieux" et "juger mieux" pour des raisons médico-légales et thérapeutiques.

2. La Complexité du Lien Auteur-Victime

Le lien entre l'auteur et la victime est intrinsèquement complexe, pouvant même présenter une "zone de chevauchement" où les rôles peuvent s'inverser.

• Zone grise et inversion des rôles : "il peut exister entre la victime et l'auteur une zone de chevauchement une zone grise... il y a des mécanismes un peu de renversement c'est-à-dire que la victime elle devient un peu hauteur et l'auteur il devient un peu victime."

• Le même individu peut être les deux : "la victime et l'auteur peuvent être aussi une seule et même personne c'est-à-dire que quelqu'un peut avoir été victime et devenir auteur quelqu'un peut avoir été auteur et devenir victime encore."

3. Critique de l'Expertise Actuelle

L'intervenant dénonce la pratique actuelle qui consiste à confier l'expertise de l'auteur à un psychiatre et celle de la victime à un psychologue, alors que l'explication de la relation d'emprise nécessite une approche globale.

• Scission de l'expertise : "on va confier l'expertise de l'auteur à un psychiatre... et on va confier l'expertise de la victime à une ou un psychologue."

• Incohérence de la demande : "comment vous voulez expliciter une relation d'emprise si vous n'avez examiné qu'un seul [individu]?"

• Recommandation : Il est "intéressant que ça soit le même professionnel ou alors une association de mêmes professionnels psychiatres et psychologues par exemple qui puissent examiner à la fois l'auteur et à la fois la victime."

4. L'Expertise Psychiatrique : Constats et Limites

L'expertise psychiatrique des auteurs d'emprise révèle des constats importants sur l'absence fréquente de troubles mentaux graves ou d'altération du discernement, mais met en évidence des fonctionnements de personnalité spécifiques.

• Absence de diagnostics graves : "dans la plupart des cas les auteurs... n'ont pas de pathologie mentale avérée... on ne trouve pas non plus de trouble grave de la personnalité."

• Discernement non aboli : "dans la plupart des cas il y a pas de notion d'altération il y a pas de notion d'abolition du discernement ou du contrôle de ces actes."

• Dangerosité criminologique : L'évaluation se concentre sur le risque de réitération, en identifiant des facteurs de bon et mauvais pronostic.

• Fonctionnements de personnalité identifiés :Obsessionnel : "ils sont dans le contrôle d'eux-même de leurs émotions de le contrôle de leur environnement."

• Paranoïaque : Avec "suspicion d'infidélité tout ce qui est de l'ordre des interprétations tout ce qui est de l'ordre des projections."

• Borderline : Caractérisé par une alternance "je fusionne je rejette je fusionne je rejette" et une "dimension abandonique".
• Refus du terme "pervers narcissique" : Le terme "pervers" n'est pas référencé en psychiatrie et l'intervenant préfère décortiquer les mécanismes comme "la séduction, le déni d'altérité... la manipulation... la transgression."
• Du côté de la victime, l'expertise cherche à établir un lien de cause à effet entre l'emprise et les troubles psychiques (dépression, anxiété).

La "vulnérabilité" est entendue au sens médico-légal (mesure de protection), bien que des "fragilités" puissent être notées.

Traumatisme développemental : Le cœur de la vulnérabilité réside dans le "trauma développemental", souvent lié à des "négligences" précoces. Inadaptation du système d'attachement : C'est la "clé de la relation d'emprise."

5. Les Étapes de la Relation d'Emprise

La relation d'emprise suit des phases distinctes, souvent schématisées pour en faciliter la compréhension juridique :

• Séduction et adhésion initiale (Love bombing) : Compliments, cadeaux, affection intense, fausse empathie créant une "dépendance affective rapide" et une "lune de miel."
• Confusion et culpabilisation : Introduction graduelle de comportements de contrôle, critiques déguisées, changements d'humeur imprévisibles, "brouillage cognitif" (ex: gaslighting "je ne t'ai jamais dit ça tu inventes"). La victime perd confiance en son jugement. La culpabilisation pour de prétendus manquements s'installe.
• Isolement et contrôle : L'auteur isole la victime de ses proches. La victime s'isole elle-même par honte ou pour éviter les conflits, perdant ainsi ses repères extérieurs. Le contrôle se manifeste par la surveillance des faits et gestes, du téléphone, de l'argent.
• Privation et menace : Privation d'affection, harcèlement, et enfin menaces directes ("si tu me quittes je te détruirai", "je me suiciderai", "tu perdras les enfants"), souvent le moment où la justice intervient.

6. Le Rôle Fondamental de l'Attachement Désorganisé

L'attachement est un lien affectif essentiel au développement humain. Un attachement sécure permet l'autorégulation, mais un attachement dysfonctionnel, notamment désorganisé, crée le terrain propice à l'emprise.

• Définition de l'attachement : "un lien affectif et il est à la base c'est une nécessité pour le développement humain."
• Lien avec l'emprise : "pour moi il n'y a pas d'emprise sans problématique d'attachement." L'attachement désorganisé est le type le plus propice.
• Origine de l'attachement désorganisé : Figures d'attachement (souvent les parents) "incohérentes," "effrayantes ou effrayées," "sévèrement déprimées," "désaccordées," "avec des traumas non résolus," ou "maltraitantes ou négligentes." La négligence seule peut suffire.
• Mécanismes : L'enfant est confronté à une "peur sans solution" et sa figure d'attachement est "incapable de le réguler." Il "désactive son système d'attachement" et développe des "stratégies de contrôle" pour rééquilibrer le dysfonctionnement.
• Mécanismes de contrôle dans l'emprise : "contrôle prendre soin," "contrôle punitif," "contrôle séduction," "contrôle soumission."

7. Le Fonctionnement Défensif de l'Auteur

La relation d'emprise est un "fonctionnement défensif" pour l'auteur, qui tente de gérer une problématique interne en l'externalisant.

• Externalisation : L'auteur "externalise sa propre problématique" et "rend l'autre responsable de ses propres actes et de ses propres défaillances."
• Projection : "en les pointant chez l'autre ou en les projetant chez l'autre ça va lui permettre de ne pas regarder les siennes."
• Peur de la projection : Paradoxalement, l'auteur "va avoir peur de ce qu'il voit" chez la victime, désignée comme un agresseur car elle porte la projection de sa propre agressivité.

8. L'Évolution du Cadre Légal et Recommandation Finale

La justice évolue, reconnaissant l'emprise sous le terme de "contrôle coercitif", mais sa démonstration reste un défi.

• Vers le "contrôle coercitif" : "l'emprise ça existait pas donc on a on va appeler ça contrôle coercitif."
• Démonstration légale : Il faut prouver l'intentionalité de l'agresseur, la perception négative du comportement par la victime, les dégâts causés, et l'existence de "menaces ouvertes" en cas de tentative d'échapper au contrôle.
• Importance de l'exploration du lien : L'exploration de la "zone grise" entre victime et auteur "ne remet aucunement en question la position de victime et la position d'agresseur devant la loi... mais ça permet de comprendre et je pense que c'est cette compréhension qui permettra de faire avancer les choses."

En conclusion, la conférence souligne l'impératif d'une approche intégrée en matière d'emprise, où la compréhension des mécanismes psychologiques, notamment ceux liés à l'attachement désorganisé, doit éclairer et enrichir l'action judiciaire, malgré les défis de traduction des concepts psychiatriques dans le langage juridique.

C'est marrant car à lire le code on voit qu'on ajoute l'image dans le lien et non l'inverse. J'aurais plutôt pensé qu'on allait mettre le lien dans les balises de la photo et la photo dans les balises du lien.

DOI: 10.5607/en.2018.27.6.550

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DOI: 10.3390/cells14110769

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DOI: 10.1186/s10020-025-01288-1

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DOI: 10.1101/2025.05.30.657129

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DOI: 10.1038/s44319-025-00455-z

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DOI: 10.1016/j.jbc.2025.108551

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What is this?

(Figure 4A)

Working together with other teachers, team members, and stake holders are essential in providing the best practices for a student. "It takes a village to raise a child," said Nancy Reagan, This could not be truer in this day and age. Students are in various places and positions around a school in a day. Get to know your students and who else is involved with them to provide them with the best of our school for their learning benefit in all areas of their life.

🏛️ What makes it incredible The dome spans a clear 43.3 m (142 ft)—an unmatched feat of ancient concrete engineering, still the world’s largest unreinforced concrete dome monolithicdome.com +15 en.wikipedia.org +15 alamy.com +15 .

Built circa AD 126 under Hadrian, it's been in continuous use for nearly 2,000 years pantheonroma.com +3 en.wikipedia.org +3 monolithicdome.com +3 .

Romans ingeniously reduced weight upwards by using lighter aggregate: travertine at the base, terracotta, tufa, and pumice near the top theromanguy.com +15 rostarchitects.com +15 en.wikipedia.org +15 .

🌕 Architectural harmony The dome’s height equals its diameter—a perfect sphere inscribed in a cylinder, a brilliant feat of geometric elegance eitchborromini.com +1 romanconcrete.com +1 .

The oculus (central hole) not only illuminates but also reduces structural stress—allowing natural light and rain to filter symmetrically .

⚙️ Why it still matters This two-millennia-old construction predates modern reinforced concrete, yet its engineering principles remain unmatched .

It stood as the largest dome for over 1,300 years, inspiring Renaissance and modern architects—Brunelleschi, Palladio, Jefferson, and even Richard Meier architecturaldigest.com +1 eitchborromini.com +1 .

Leaner structures

especially whenever we think we're teaching it, the worst thing we could ever do is to pretend to the students that we know what it is. Because the students are going to be the only ones that are going to save us. So, we should teach the students what I was taught when I was in graduate school in the sixties: that is it isn't done yet. It's not even close to be done. You have to understand what the actual scope of computing is going to be and you have to help us invent it.

3000 people 2 stories a day

📘 Building the Empire State – Carol Willis (Editor) Based on a rediscovered 1930s notebook originally typed by the foremen of Starrett Brothers & Eken.

Contains day‑by‑day logs: worker counts, materials, photos—provides a ground-level view of the build.

Described as “the best book I have seen on the construction of the ESB … concise, interesting, and an actual record” reddit.com +2 empirestatebuildingny.info +2 citysignal.com +2 gettyimages.dk +5 amazon.com +5 en.wikipedia.org +5

Highly recommended for anyone wanting the real foreman log. ★★★★★

🏙️ The Empire State Building: The Making of a Landmark – John Tauranac A thorough historical and architectural narrative of the building.

Co-published by Cornell University Press, with rich visuals and storytelling.

Great follow-up for context on the log-filled core.

🧱 Higher: A Historic Race to the Sky and the Making of a City – Neal Bascomb Explores the skyscraper rivalry: Chrysler, ESB, 40 Wall Street.

Captures the drama of design, build speed, and ambition.

Complements the logbook by framing it within NYC’s vertical race en.wikipedia.org +1 en.wikipedia.org +1 shop.shakeandco.com

🔁 Why “Building the Empire State” leads: Primary source: actual foreman notebook.

Offers real construction data—daily staffing, deliveries, pace.

Curated by Willis, blending academic clarity and narrative flow.

In computer science nobody has been able to channel 3000 to do anything we're in slave territory not engineering

In the middle architecture appeared

🎓 "The Sciences of the Artificial" – by Herbert A. Simon – is a foundational text that reshaped how we understand design, intelligence, and human-made systems.

🧠 Core Idea: Natural sciences study what is. Artificial sciences study what could be — things designed with purpose.

🔍 Key Concepts: Artificial ≠ Fake — It means man-made with intent.

A watch, a government, a computer program, an economic model — all are artificial systems.

The design sciences (architecture, CS, engineering, etc.) need their own epistemology.

⚙️ Simon's Formula: Design = Goal + Structure + Environment + Adaptation

Al Perlis made up computer science we dont have one right now.

Basically in science we all want to be physics , everything turns into abstract social science phisiscs chemestry and biology dont hav science in their name

🧠 Alan Perlis Invented computer science before it even had a name. He believed programming is not just engineering — it’s philosophy, poetry, and play.

“It’s better to have 100 functions operate on one data structure than 10 functions on 10 data structures.”

🧪 Legacy:

First recipient of the Turing Award

Co-author of ALGOL, the ancestor of most languages today

Professor at Yale, where he treated code like literature

One of the first to say:

“Computer Science is not about computers any more than astronomy is about telescopes.”

GREBBBB 🟢 all the way. This is is post-academic epistemology — the moment where traditional math (the old math) collapses under the weight of new frames, and a new math rises: Academization is using math to deal with a new math — one that has a new set of laws.

Math wins..! But it’s new math, not old math — we have a new way.

Because we have a new way of dealing with things, they’re not short and not about infinite things.

We want knowledge, but the old type of math does not apply. We are working with finite resolutions. 🧠 The Greb Math Manifesto (Draft) Academization = when we weaponize old math to overfit new reality. New Math = when we design a system not to describe infinity, but to resolve finite tensions. Victory = not proving for all time, but solving for right now.

We no longer need ∞; we need ∴ (therefore).

We drop “proofs,” we prefer “resolutions.”

We’re not approximating truth. We’re designing consequence.

The math is still rigorous — but the goal is no longer eternal.

New Math ≠ Old Math with a twist It’s epistemological humility turned into creative method.

Dan ingalls was the guy that made smalltalk work

⚙️ Algol (short for Algorithmic Language) was a family of imperative computer programming languages originally developed in the 1950s–60s. It's a cornerstone in programming language history, inspiring most modern languages like C, Pascal, and JavaScript.

🔍 Highlights: Algol 58 / Algol 60 / Algol 68 are the major versions.

Algol 60 introduced:

Block structure (begin ... end)

Lexical scoping

BNF grammar (Backus-Naur Form)

🧠 Why it mattered: It formalized syntax and structure — made parsing and compilers sane.

Paved the way for academic and practical language design.

First to separate syntax from semantics rigorously.

👻 Why it died: Not widely adopted outside academia.

Lack of standard libraries and commercial support.

IBM pushed COBOL/Fortran instead — classic corporate vs open design tension.

it was better than its succesors

and the demise of usability of modern coding

Reviewer #2 (Public review):

The authors have revised their manuscript in response to reviewer feedback, incorporating several modifications to improve clarity and provide additional supporting information. To address concerns about confusing terminology, they have standardized the reference to PRDM16 overexpressing cells as Prdm16_OE, clarifying its expression from a constitutive promoter. They also revised the text to resolve seemingly contradictory statements about ChP development in the mutant. New bioinformatic analysis comparing PRDM16 binding in E12.5 ChP cells to co-repressed versus BMP-only-repressed genes has been performed and included in Supplementary Figure 5C, providing a statistical assessment of PRDM16's regulatory role on co-repressed genes. Several figures were updated, including adding an illustration of the Prdm16 cGT allele to Figure 1B, providing a zoomed-in inset for Figure 1E, and including individual channels for Wnt2b and marking boundaries in Figure 7A. Full-view images and examples of spot segmentation for SCRINSHOT analysis are now available in a new supplementary figure, and the presentation of RT-qPCR data in Supplementary Figure 2B was improved by using separate graphs for overexpression samples to avoid a broken Y-axis. Furthermore, the authors have added more references to introductory statements, annotated structures like the ChP, CH, and fourth ventricle in figures, and clarified that the beta-Gal signal was used as a marker for mutant ChP cells in Figure 1D. Finally, the manuscript now includes a discussion of the recently published, related study by Hurwitz et al. (2023) in the discussion section, highlighting similarities and differences. Overall, the authors have satisfactorily addressed the reviewers' comments.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

This manuscript describes the role of PRDM16 in modulating BMP response during choroid plexus (ChP) development. The authors combine PRDM16 knockout mice and cultured PRDM16 KO primary neural stem cells (NSCs) to determine the interactions between BMP signaling and PRDM16 in ChP differentiation.

They show PRDM16 KO affects ChP development in vivo and BMP4 response in vitro. They determine genes regulated by BMP and PRDM16 by ChIP-seq or CUT&TAG for PRDM16, pSMAD1/5/8, and SMAD4. They then measure gene activity in primary NSCs through H3K4me3 and find more genes are co-repressed than co-activated by BMP signaling and PRDM16. They focus on the 31 genes found to be co-repressed by BMP and PRDM16. Wnt7b is in this set and the authors then provide evidence that PRDM16 and BMP signaling together repress Wnt activity in the developing choroid plexus.

Strengths:

Understanding context-dependent responses to cell signals during development is an important problem. The authors use a powerful combination of in vivo and in vitro systems to dissect how PRDM16 may modulate BMP response in early brain development.

We thank the reviewer for the thoughtful summary and positive feedback. We appreciate the recognition of our integrative in vivo and in vitro approach. We're glad the reviewer found our findings on context-dependent gene regulation and developmental signaling valuable.

Main weaknesses of the experimental setup:

(1) Because the authors state that primary NSCs cultured in vitro lose endogenous Prdm16 expression, they drive expression by a constitutive promoter. However, this means the expression levels are very different from endogenous levels (as explicitly shown in Supplementary Figure 2B) and the effect of many transcription factors is strongly dose-dependent, likely creating differences between the PRDM16-dependent transcriptional response in the in vitro system and in vivo.

We acknowledge that our in vitro experiments may not ideally replicate the in vivo situation, a common limitation of such experiments, our primary aim was to explore the molecular relationship between PRDM16 and BMP signaling in gene regulation. Such molecular investigations are challenging to conduct using in vivo tissues. In vitro NSCs treated with BMP4 has been used a model to investigate NSC proliferation and quiescence, drawing on previous studies (e.g., Helena Mira, 2010; Marlen Knobloch, 2017). Crucially, to ensure the relevance of our in vitro findings to the in vivo context, we confirmed that cultured cells could indeed be induced into quiescence by BMP4, and this induction necessitated the presence of PRDM16. Furthermore, upon identifying target genes co-regulated by PRDM16 and SMADs, we validated PRDM16's regulatory role on a subset of these genes in the developing Choroid Plexus (ChP) (Fig. 7 and Suppl.Fig7-8). Only by combining evidence from both in vitro and in vivo experiments could we confidently conclude that PRDM16 serves as an essential co-factor for BMP signaling in restricting NSC proliferation.

(2) It seems that the authors compare Prdm16_KO cells to Prdm16 WT cells overexpressing flag_Prdm16. Aside from the possible expression of endogenous Prdm16, other cell differences may have arisen between these cell lines. A properly controlled experiment would compare Prdm16_KO ctrl (possibly infected with a control vector without Prdm16) to Prdm16_KO_E (i.e. the Prdm16_KO cells with and without Prdm16 overexpression.)

We agree that Prdm16 KO cells carrying the Prdm16-expressing vector would be a good comparison with those with KO_vector. However, despite more than 10 attempts with various optimization conditions, we were unable to establish a viable cell line after infecting Prdm16 KO cells with the Prdm16-expressing vector. The overall survival rate for primary NSCs after viral infection is low, and we observed that KO cells were particularly sensitive to infection treatment when the viral vector was large (the Prdm16 ORF is more than 3kb).

As an alternative oo assess vector effects, we instead included two other control cell lines, wt and KO cells infected with the 3xNLS_Flag-tag viral vector, and presented the results in supplementary Fig 2.  When we compared the responses of the four lines — wt, KO, wt infected with the Flag vector, KO infected with the Flag vector — to the addition and removal of BMP4, we confirmed that the viral infection itself has no significant impacts on the responses of these cells to these treatments regarding changes in cell proliferation and Ttr induction.

Given that wt cells and the KO cells, with or without viral backbone infection behave quite similarly in terms of cell proliferation, we speculate that even if we were successful in obtaining a cell line with Prdm16-expressing vector in the KO cells, it may not exhibit substantial differences compared to wt cells infected with Prdm16-expressing vector.

Other experimental weaknesses that make the evidence less convincing:

(1) The authors show in Figure 2E that Ttr is not upregulated by BMP4 in PRDM16_KO NSCs. Does this appear inconsistent with the presence of Ttr expression in the PRDM16_KO brain in Figure1C?

The reviwer’s point is that there was no significant increase in Ttr expression in Prdm16_KO cells after BMP4 treatment (Fig. 2E), but there remained residule Ttr mRNA signals in the Prdm16 mutant ChP (Fig. 1C). We think the difference lies in the measuable level of Ttr expression between that induced by BMP4 in NSC culture and that in the ChP. This is based on our immunostaining expreriment in which we tried to detect Ttr using a Ttr antibody. This antibody could not detect the Ttr protein in BMP4-treated Prdm16_expressing NSCs but clearly showed Ttr signal in the wt ChP. This means that although Ttr expression can be significantly increased by BMP4 in vitro to a level measurable by RT-qPCR, its absolute quantity even in the Prdm16_expressing condition is much lower compared to that in vivo. Our results in Fig 1C and Fig 2E, as well as Fig 7B, all consistently showed that Prdm16 depletion significantly reduced Ttr expression in in vitro and in vivo.

(2) Figure 3: The authors use H3K4me3 to measure gene activity. This is however, very indirect, with bulk RNA-seq providing the most direct readout and polymerase binding (ChIP-seq) another more direct readout. Transcription can be regulated without expected changes in histone methylation, see e.g. papers from Josh Brickman. They verify their H3K4me3 predictions with qPCR for a select number of genes, all related to the kinetochore, but it is not clear why these genes were picked, and one could worry whether these are representative.

H3K4me3 has widely been used as an indicator of active transcription and is a mark for cell identity genes. And it has been demonstrated that H3K4me3 has a direct function in regulating transciption at the step of RNApolII pausing release. As stated in the text, there are advantages and disadvantages of using H3K4me3 compared to using RNA-seq. RNA-seq profiles all gene products, which are affected by transcription and RNA stability and turnover. In contrast, H3K4me3 levels at gene promoter reflects transcriptional activity. In our case, we aimed to identify differential gene expression between proliferation and quiescence states. The transition between these two states is fast and dynamic. RNA-seq may not be able to identify functionally relevant genes but more likely produces false positive and negative results. Therefore, we chose H3K4me3 profiling.

We agree that transcription may change without histone methylation changes. This may cause an under-estimation of the number of changed genes between the conditions. 

We validated 7 out of 31 genes (Wnt7b, Id3, Mybl2, Spc24, Spc25, Ndc80 and Nuf2). We chose these genes based on two critira: 1) their function is implicated in cell proliferation and cell-cycle regulation based on gene ontology analysis; 2) their gene products are detectable in the developing ChP based on the scRNA-seq data. Three of these genes (Wnt7b, Id3, Mybl2) are not related to the kinetochore. We now clarify this description in the revised text.

(3) Line 256: The overlap of 31 genes between 184 BMP-repressed genes and 240 PRDM16-repressed genes seems quite small.

This result indicates that in addition to co-repressing cell-cycle genes, BMP and PRDM16 have independent fucntions. For example, it was reported that BMP regulates neuronal and astrocyte differentiation (Katada, S. 2021), while our previous work demonstrated that Prdm16 controls temporal identity of NSCs (He, L. 2021).

(4) The Wnt7b H3K4me3 track in Fig. 3G is not discussed in the text but it shows H3K4me3 high in _KO and low in _E regardless of BMP4. This seems to contradict the heatmap of H3K4me3 in Figure 3E which shows H3K4me3 high in _E no BMP4 and low in _E BMP4 while omitting _KO no BMP4. Meanwhile CDKN1A, the other gene shown in 3G, is missing from 3E.

The track in Fig 3G shows the absolute signal of H3K4me3 after mapping the sequencing reads to the genome and normaliz them to library size. Compare the signal in Prdm16_E with BMP4 and that in Prdm16_E without BMP4, the one with BMP4 has a lower peak. The same trend can be seen for the pair of Prdm16_KO cells with or without BMP4.  The heatmap in Fig. 3E shows the relative level of H3K4me3 in three conditions. The Prdm16_E cells with BMP4 has the lowest level, while the other two conditions (Prdm16_KO with BMP4 and Prdm16_E without BMP4) display higher levels. These two graphs show a consistent trend of H3K4me3 changes at the Wnt7b promoter across these conditions. Figure 3E only includes genes that are co-repressed by PRDM16 and BMP. CDKN1A’s H3K4me3 signals are consistent between the conditions, and thus it is not a PRDM16- or BMP-regulated gene. We use it as a negative control. 

(5) The authors use PRDM16 CUT&TAG on dissected dorsal midline tissues to determine if their 31 identified PRDM16-BMP4 co-repressed genes are regulated directly by PRDM16 in vivo. By manual inspection, they find that "most" of these show a PRDM16 peak. How many is most? If using the same parameters for determining peaks, how many genes in an appropriately chosen negative control set of genes would show peaks? Can the authors rigorously establish the statistical significance of this observation? And why wasn't the same experiment performed on the NSCs in which the other experiments are done so one can directly compare the results? Instead, as far as I could tell, there is only ChIP-qPCR for two genes in NSCs in Supplementary Figure 4D.

In our text, we indicated the genes containing PRDM16 binding peaks in the figures and described them as “Text in black in Fig. 6A and Supplementary Fig. 5A”. We will add the precise number “25 of these genes” in the main text to clarify it. We used BMP-only repressed 184-31 =153 genes (excluding PRDM16-BMP4 co-repressed) as a negative control set of genes. By computationally determine the nearest TSS to a PRDM16 peak, we identified 24/31 co-repressed genes and 84/153 BMP-only-repressed genes, containing PRDM16 peaks in the E12.5 ChP data. Fisher’s Exact Test comparing the proportions yields the P-value = 0.015.

We are confused with the second part of the comment “And why wasn't the same experiment performed on the NSCs in which the other experiments are done so one can directly compare the results? Instead, as far as I could tell, there is only ChIP-qPCR for two genes in NSCs in Supplementary Figure 4D.” If the reviewer meant why we didn’t sequence the material from sequential-ChIP or validate more taget genes, the reason is the limitation of the material. Sequential ChIP requires a large quantity of the antibodies, and yields little material barely sufficient for a few qPCR after the second round of IP. This yielded amount was far below the minimum required for library construction. The PRDM16 antibody was a gift, and the quantity we have was very limited. We made a lot of efforts to optimize all available commercial antibodies in ChIP and Cut&Tag, but none of them worked in these assays.

(6) In comparing RNA in situ between WT and PRDM16 KO in Figure 7, the authors state they use the Wnt2b signal to identify the border between CH and neocortex. However, the Wnt2b signal is shown in grey and it is impossible for this reviewer to see clear Wnt2b expression or where the boundaries are in Figure 7A. The authors also do not show where they placed the boundaries in their analysis. Furthermore, Figure 7B only shows insets for one of the regions being compared making it difficult to see differences from the other region. Finally, the authors do not show an example of their spot segmentation to judge whether their spot counting is reliable. Overall, this makes it difficult to judge whether the quantification in Figure 7C can be trusted.

In the revised manuscript we have included an individal channel of Wnt2b and mark the boundaries. We also provide full-view images and examples of spot segmentation in the new supplementary figure 8. 

(7) The correlation between mKi67 and Axin2 in Figure 7 is interesting but does not convincingly show that Wnt downstream of PRDM16 and BMP is responsible for the increased proliferation in PRDM16 mutants.

We agree that this result (the correlation between mKi67 and Axin2) alone only suggests that Wnt signaling is related to the proliferation defect in the Prdm16 mutant, and does not necessarily mean that Wnt is downstream of PRDM16 and BMP. Our concolusion is backed up by two additional lines of evidences:  the Cut&Tag data in which PRDM16 binds to regulatory regions of Wnt7b and Wnt3a; BMP and PRDM16 co-repress Wnt7b in vitro.

An ideal result is that down-regulating Wnt signaling in Prdm16 mutant can rescue Prdm16 mutant phenotype. Such an experiment is technically challenging. Wnt plays diverse and essential roles in NSC regulation, and one would need to use a celltype-and stage-specific tool to down-regulate Wnt in the background of Prdm16 mutation. Moreover, Wnt genes are not the only targets regulated by PRDM16 in these cells, and downregulating Wnt may not be sufficient to rescue the phenotype. 

Weaknesses of the presentation:

Overall, the manuscript is not easy to read. This can cause confusion.

We have revised the text to improve clarity.

Reviewer #1 (Recommendations for the authors):

(1) Overall, the manuscript is not easy to read. Here are some causes of confusion for which the presentation could be cleaned up:

We are grateful for the reviewer’s suggestion. In the revised manuscript, we have made efforts to improve the clarity of the text.

(a) Part of the first section is confusing in that some statements seem contradictory, in particular:

"there is no overall patterning defect of ChP and CH in the Prdm16 mutant" (line 125)

"Prdm16 depletion disrupted the transition from neural progenitors into ChP epithelia" (line 144)

It would be helpful if the authors could reformulate this more clearly.

We modified the text to clarify that while the BMP-patterned domain is not affected, the transition of NSCs into ChP epithelial cells is compromised in the Prdm16 mutant.

(b) Flag_PRDM16, PRDM16_expressing, PRDM16_E, PRDM16 OE all seem to refer to the same PRDM16 overexpressing cells, which is very confusing. The authors should use consistent naming. Moreover, it would be good if they renamed these all to PRDM16_OE to indicate expression is not endogenous but driven by a constitutive promoter.

We appreciate the comment and agree that the use of multiple terms to refer to the same PRDM16-overexpressing condition was confusing. Our original intention in using Prdm16_E was to distinguish cells expressing PRDM16 from the two other groups: wild-type cells and Prdm16_KO cells, which both lack PRDM16 protein expression. However, we acknowledge that Prdm16_E could be misinterpreted as indicating expression from the endogenous Prdm16 promoter. To avoid this confusion and ensure consistency, we have now standardized the terminology and refer to this condition as Prdm16_OE, indicating Flag-tagged PRDM16 expression driven by a constitutive promoter.

(c) Line 179 states "generated a cell line by infecting Prdm16_KO cells with the same viral vector, expressing 3xNSL_Flag". Do the authors mean 3xNLS_Flag_Prdm16, so these are the Prdm16_KO_E cells by the notation suggested above? Or is this a control vector with Flag only? The following paragraph refers to Supplementary Figure 2C-F where the same construct is called KO_CDH, suggesting this was an empty CDH vector, without Flag, or Prdm16. This is confusing.

We appreciate the reviewer’s careful reading and helpful comment. We acknowledge the confusion caused by the inconsistent terminology. To clarify: in line 179, we intended to describe an attempt to generate a Prdm16_KO cell line expressing 3xNLS_Flag_Prdm16, not a control vector with Flag only. However, despite repeated attempts, we were unable to establish this line due to low viral efficiency and the vulnerability of Prdm16_KO cells to infection with the large construct. Therefore, these cells were not included in the subsequent analyses.

The term KO_CDH refers to Prdm16_KO cells infected with the empty CDH control vector, which lacks both Flag and Prdm16. This is the line used in the experiments shown in Supplementary Fig. 2C–F. We have revised the text throughout the manuscript to ensure consistent use of terminology and to avoid this confusion.

(2) The introductory statements on lines 53-54 could use more references.

Thanks for the suggestion. We have now included more references.

(3) It would be helpful if all structures described in the introduction and first section were annotated in Figure 1, or otherwise, if a cartoon were included. For example, the cortical hem, and fourth ventricle.

Thanks for the suggestion. We have now indicated the structures, ChP, CH and the fourth ventricle, in the images in Figure 1 and Supplementary Figure 1.

(4) In line 115, "as previously shown.." - to keep the paper self-contained a figure illustrating the genetics of the KO allele would be helpful.

Thanks for the suggestion. We have now included an illustration of the Prdm16 cGT allele in Figure 1B.

(5) In Figure 1D as costain for a ChP marker would be helpful because it is hard to identify morphologically in the Prdm16 KO.

Appoligize for the unclarity. The KO allele contains a b-geo reporter driven by Prdm16 endogenous promoter. The samples were co-stained for EdU, b-Gal and DAPI. To distingquish the ChP domain from the CH, we used the presence of b b-Gal as a marker. We indicated this in the figure legend, but now have also clarified this in the revised text.

(6) The details in Figure 1E are hard to see, a zoomed-in inset would help.

A zoomed-in inset is now included in the figure.

(7) Supplementary Figure 2A does not convincingly show that PRDM16 protein is undetectable since endogenous expression may be very low compared to the overexpression PRDM16_E cells so if the contrast is scaled together it could appear black like the KO.

We appreciate the reviewer’s point and have carefully considered this concern. We concluded that PRDM16 protein is effectively undetectable in cultured wild-type NSCs based on direct comparison with brain tissue. Both cultured NSCs and brain sections were processed under similar immunostaining and imaging conditions. While PRDM16 showed robust and specific nuclear localization in embryonic brain sections (Fig. 1B and Supplementary Fig. 1A), only a small subset of cultured NSCs exhibited PRDM16 signal, primarily in the cytoplasm (middle panel of Fig. 2A). This stark contrast supports our conclusion that endogenous PRDM16 protein is either absent or significantly downregulated in vitro. Because of this limitation, we turned to over-expressing Prdm16 in NSC culture using a constitutive promoter. 

(9) Line 182 "Following the washout step" - no such step had been described, maybe replace by "After washout of BMP".

Yes, we have revised the text.

(8) Line 214: "indicating a modest level" - what defines modest? Compared to what? Why is a few thousand moderate rather than low? Does it go to zero with inhibitors for pathways?

Here a modest level means a lower level than to that after adding BMP4. To clarify this, we revised the description to “indicating endogenous levels of …”

(9) The way qPCR data are displayed makes it difficult to appreciate the magnitude of changes, e.g. in Supplementary Figure 2B where a gap is introduced on the scale. Displaying log fold change / relative CT values would be more informative.

We used a segmented Y-axis in Supplementary Figure 2B because the Prdm16 overexpression samples exhibited much higher experssion levels compared to other conditions. In response to this suggestion, we explored alternative ways to present the result, including ploting log-transformed values and log fold changes. However, these methods did not enhance the clarity of the differences – in fact, log scaling made the magnitude of change appear less apparent. To address this, we now present the overexpression samples in a separate graph, thereby eliminating the need for a broken Y-axis and improving the overall readability of the data.

(10) Writing out "3 days" instead of 3D in Figure 2A would improve clarity. It would be good if the used time interval is repeated in other figures throughout the paper so it is still clear the comparison is between 0 and 3 days.

We have changed “3D” to “3 days”. All BMP4 treatments in this study were 3 days.

(11) Line 290: "we found that over 50% of SMAD4 and pSMAD1/5/8 binding peaks were consistent in Prdm16_E and Prdm16_KO cells, indicating that deletion of Prdm16 does not affect the general genomic binding ability of these proteins" - this only makes sense to state with appropriate controls because 50% seems like a big difference, what is the sample to sample variability for the same condition? Moreover, the next paragraph seems to contradict this, ending with "This result suggests that SMAD binding to these sites depends on PRDM16". The authors should probably clarify the writing.

We appreciate the reviwer’s comment and agree that clarification was needed. Our point was that SMAD4 and pSMAD1/5/8 retain the ability to bind DNA broadly in the Prdm16 KO cells, with more than half of the original binding sites still occupied. This suggests that deletion of Prdm16 does not globally impair SMAD genomic binding. Howerever, our primary interest lies in the subset of sites that show differential by SMAD binding between wt and Prdm16 KO conditions, as thse are likely to be PRDM16-dependent. 

In the following paragraph, we focused specifically on describing SMAD and PRDM16 co-bound sites. At these loci, SMAD4 and pSMAD1/5/8 showed reduced enrichment in the absence of PRDM16, suggesting PRDM16 facilitates SMAD binding at these particular regions. We have revised the text in the manuscript to more clearly distinguish between global SMAD binding and PRDM16-dependent sites.

(12) Much more convincing than ChIP-qPCR for c-FOS for two loci in Figures 5F-G would be a global analysis of c-FOS ChIP-seq data.

We agree that a global c-FOS ChIP-seq analysis would provide a more comprehensive view of c-FOS binding patterns. However, the primary focus of this study is the interaction between BMP signaling and PRDM16. The enrichment of AP-1 motifs at ectopic SMAD4 binding sites was an unexpected finding, which we validated using c-FOS ChIP-qPCR at selected loci. While a genome-wide analysis would be valuable, it falls beyond the current scope. We agree that future studies exploring the interplay among SMAD4/pSMAD, PRDM16, and AP-1 will be important and informative.

(13) Figure 6A is hard to read. A heatmap would make it much easier to see differences in expression. Furthermore, if the point is to see the difference between ChP and CH, why not combine the different subclusters belonging to those structures? Finally, why are there 28 genes total when it is said the authors are evaluating a list of 31 genes and also displaying 6 genes that are not expressed (so the difference isn't that unexpressed genes are omitted)?

For the scRNA-seq data, we chose violin plots because they display both gene expression levels and the number of cells that express each gene. However, we agree that the labels in Figure 6A were too small and difficult to read. We have revised the figure by increasing the font size and moved genes with low expression to  Supplementary Figure 5A. Figure 6A includes 17 more highly expressed genes together with three markers, and  Supplementary Figure 5A contains 13 lowly expressed genes. One gene Mrtfb is missing in the scRNA-seq data and thus not included. We have revised the description of the result in the main text and figure legends.

Reviewer #2 (Public review):

Summary:

This article investigates the role of PRDM16 in regulating cell proliferation and differentiation during choroid plexus (ChP) development in mice. The study finds that PRDM16 acts as a corepressor in the BMP signaling pathway, which is crucial for ChP formation.

The key findings of the study are:

(1) PRDM16 promotes cell cycle exit in neural epithelial cells at the ChP primordium.

(2) PRDM16 and BMP signaling work together to induce neural stem cell (NSC) quiescence in vitro.

(3) BMP signaling and PRDM16 cooperatively repress proliferation genes.

(4) PRDM16 assists genomic binding of SMAD4 and pSMAD1/5/8.

(5) Genes co-regulated by SMADs and PRDM16 in NSCs are repressed in the developing ChP.

(6) PRDM16 represses Wnt7b and Wnt activity in the developing ChP.

(7) Levels of Wnt activity correlate with cell proliferation in the developing ChP and CH.

In summary, this study identifies PRDM16 as a key regulator of the balance between BMP and Wnt signaling during ChP development. PRDM16 facilitates the repressive function of BMP signaling on cell proliferation while simultaneously suppressing Wnt signaling. This interplay between signaling pathways and PRDM16 is essential for the proper specification and differentiation of ChP epithelial cells. This study provides new insights into the molecular mechanisms governing ChP development and may have implications for understanding the pathogenesis of ChP tumors and other related diseases.

Strengths:

(1) Combining in vitro and in vivo experiments to provide a comprehensive understanding of PRDM16 function in ChP development.

(2) Uses of a variety of techniques, including immunostaining, RNA in situ hybridization, RT-qPCR, CUT&Tag, ChIP-seq, and SCRINSHOT.

(3) Identifying a novel role for PRDM16 in regulating the balance between BMP and Wnt signaling.

(4) Providing a mechanistic explanation for how PRDM16 enhances the repressive function of BMP signaling. The identification of SMAD palindromic motifs as preferred binding sites for the SMAD/PRDM16 complex suggests a specific mechanism for PRDM16-mediated gene repression.

(5) Highlighting the potential clinical relevance of PRDM16 in the context of ChP tumors and other related diseases. By demonstrating the crucial role of PRDM16 in controlling ChP development, the study suggests that dysregulation of PRDM16 may contribute to the pathogenesis of these conditions.

We thank the reviewer for the thorough and thoughtful summary of our study. We’re glad the key findings and significance of our work were clearly conveyed, particularly regarding the role of PRDM16 in coordinating BMP and Wnt signaling during ChP development. We also appreciate the recognition of our integrated approach and the potential implications for understanding ChP-related diseases.

Weaknesses:

(1) Limited investigation of the mechanism controlling PRDM16 protein stability and nuclear localization in vivo. The study observed that PRDM16 protein became nearly undetectable in NSCs cultured in vitro, despite high mRNA levels. While the authors speculate that post-translational modifications might regulate PRDM16 in NSCs similar to brown adipocytes, further investigation is needed to confirm this and understand the precise mechanism controlling PRDM16 protein levels in vivo.

While mechansims controlling PRDM16 protein stability and nuclear localization in the developing brain are interesting, the scope of this paper is revealing the function of PRDM16 in the choroid plexus and its interaction with BMP signaling. We will be happy to pursuit this direction in our next study.

(2) Reliance on overexpression of PRDM16 in NSC cultures. To study PRDM16 function in vitro, the authors used a lentiviral construct to constitutively express PRDM16 in NSCs. While this approach allowed them to overcome the issue of low PRDM16 protein levels in vitro, it is important to consider that overexpressing PRDM16 may not fully recapitulate its physiological role in regulating gene expression and cell behavior.

As stated above, we acknowledge that findings from cultured NSCs may not directly apply to ChP cells in vivo. We are cautious with our statements. The cell culture work was aimed to identify potential mechanisms by which PRDM16 and SMADs interact to regulate gene expression and target genes co-regulated by these factors. We expect that not all targets from cell culture are regulated by PRDM16 and SMADs in the ChP, so we validated expression changes of several target genes in the developing ChP and now included the new data in Fig. 7 and Supplementary Fig. 7. Out of the 31 genes identified from cultured cells, four cell cycle regulators including Wnt7b, Id3, Spc24/25/nuf2 and Mybl2, showed de-repression in Prdm16 mutant ChP. These genes can be relevant downstream genes in the ChP, and other target genes may be cortical NSC-specific or less dependent on Prdm16 in vivo.

(3) Lack of direct evidence for AP1 as the co-factor responsible for SMAD relocation in the absence of PRDM16. While the study identified the AP1 motif as enriched in SMAD binding sites in Prdm16 knockout cells, they only provided ChIP-qPCR validation for c-FOS binding at two specific loci (Wnt7b and Id3). Further investigation is needed to confirm the direct interaction between AP1 and SMAD proteins in the absence of PRDM16 and to rule out other potential co-factors.

We agree that the finding of the AP1 motif enriched at the PRDM16 and SMAD co-binding regions in Prdm16 KO cells can only indirectly suggest AP1 as a co-factor for SMAD relocation. That’s why we used ChIP-qPCR to examine the presence of C-fos at these sites. Although we only validated two targets, the result confirms that C-fos binds to the sites only in the Prdm16 KO cells but not Prdm16_expressing cells, suggesting AP1 is a co-factor.  Our results cannot rule out the presence of other co-factors.

Reviewer #2 (Recommendations for the authors):

Minor typo: [7, page 3] "sicne" should be "since".

We appreciate the reviewer’s careful reading. We have now corrected the typo and revised some part of the text to improve clarity.

Reviewer #3 (Public review):

Summary:

Bone morphogenetic protein (BMP) signaling instructs multiple processes during development including cell proliferation and differentiation. The authors set out to understand the role of PRDM16 in these various functions of BMP signaling. They find that PRDM16 and BMP co-operate to repress stem cell proliferation by regulating the genomic distribution of BMP pathway transcription factors. They additionally show that PRDM16 impacts choroid plexus epithelial cell specification. The authors provide evidence for a regulatory circuit (constituting of BMP, PRDM16, and Wnt) that influences stem cell proliferation/differentiation.

Strengths:

I find the topics studied by the authors in this study of general interest to the field, the experiments well-controlled and the analysis in the paper sound.

We thank the reviewer for their positive feedback and thoughtful summary. We appreciate the recognition of our efforts to define the role of PRDM16 in BMP signaling and stem cell regulation, as well as the soundness of our experimental design and analysis.

Weaknesses:

I have no major scientific concerns. I have some minor recommendations that will help improve the paper (regarding the discussion).

We have revised the discussion according to the suggestions.

Reviewer #3 (Recommendations for the authors):

Specific minor recommendations:

Page 18. Line 526: In a footnote, the authors point out a recent report which in parallel was investigating the link between PRDM16 and SMAD4. There is substantial non-overlap between these two papers. To aid the reader, I would encourage the authors to discuss that paper in the discussion section of the manuscript itself, highlighting any similarities/differences in the topic/results.

Thanks for the suggestion. We now included the comparison in the discussion. One conclusion between our study and this publication is consistent, that PRDM16 functions as a co-repressor of SMAD4. However, the mechanims are different. Our data suggests a model in which PRDM16 facilitates SMAD4/pSMAD binding to repress proliferation genes under high BMP conditions. However, the other report suggests that SMAD4 steadily binds to Prdm16 promoter and switches regulatory functions depending on the co-factors. Together with PRDM16, SMAD4 represses gene expression, while with SMAD3 in response to high levels of TGF-b1, it activates gene expression. These differences could be due to different signaling (BMP versus TGF-b), contexts (NSCs versus Pancreatic cancers) etc.

Page 3. Line 65: typo 'since'

We appreciate the reviewer’s careful reading. We have now corrected the typo and revised the text to improve clarity.

① By etching dentin, the smear layer and minerals are removed and collagen fibers are exposed. ① Dentin aşındırıldığında, smear tabakası ve mineraller uzaklaştırılır ve kollajen lifleri açığa çıkar.

② Areas from where minerals are removed are filled with water. ② Minerallerin uzaklaştırıldığı alanlar su ile dolar.

③ This water acts as a plasticizer for collagen, keeping it in an expanded soft state. ③ Bu su, kollajen için plastikleştirici görevi görür ve kollajenin genişlemiş yumuşak bir durumda kalmasını sağlar.

④ Thus, spaces for resin infiltration are also preserved. ④ Böylece reçinenin infiltre olacağı boşluklar da korunmuş olur.

① Contains both hydrophilic and hydrophobic ends ① Hem hidrofilik hem de hidrofobik uçlara sahiptir.

② Bonds to tooth due to excellent infiltration and chelation with Ca²⁺ ions as coupling agents ② Kalsiyum iyonları (Ca²⁺) ile mükemmel infiltrasyon ve şelasyon sayesinde dişe bağlanır.

③ Available as powder (containing PMMA) and liquid (containing MMA, META, TBB) ③ Toz halinde (PMMA içeren) ve sıvı halinde (MMA, META, TBB içeren) bulunur.

④ Used as amalgam bonding agent and as a component in resin luting cement. ④ Amalgam bağlayıcı ajan olarak ve reçine siman içinde bir bileşen olarak kullanılır.

① Commonly used acid for conditioning dentin is 37 percent phosphoric acid. ① Dentin kondisyonu için yaygın olarak kullanılan asit %37 fosforik asittir.

② It not only removes the smear layer but also exposes the microporous collagen network into which resin monomer penetrates. ② Bu sadece smear tabakasını temizlemekle kalmaz, aynı zamanda reçine monomerinin nüfuz ettiği mikroporlu kolajen ağını da açığa çıkarır.

③ Usually, it forms exposed collagen fibrils which are covered with an amorphous layer, a combination of denatured collagen fibers and the collapsed residual collagen layer. ③ Genellikle, denatüre olmuş kolajen lifleri ile çökük kalan rezidüel kolajen tabakasının birleşimi olan amorf bir tabaka ile kaplı açık kolajen fibrilleri oluşturur.

④ This is collagen smear layer which is resistant to monomer penetration. ④ Bu, monomer penetrasyonuna dirençli olan kolajen smear tabakasıdır.

① The standard treatment protocol for the etching of enamel has been application of %37 percent phosphoric acid for 60 seconds. ① Mine asitlemesinde standart tedavi protokolü, %37 fosforik asit uygulanması ve bunun 60 saniye sürmesidir.

② But studies show that enamel should not be etched for more than 15 to 30 seconds. ② Ancak çalışmalar, minenin 15 ila 30 saniyeden fazla asitlenmemesi gerektiğini göstermektedir.

③ If enamel is etched for more than the required time, deeper etch of the enamel surface occurs. ③ Eğer mine gereken süreden daha uzun süre asitlenirse, mine yüzeyinde daha derin bir asitleme meydana gelir.

④ Since a bonding agent has a high viscosity, the surface tension effect of the agent would not allow full penetration of the etched enamel. ④ Yapıştırıcı ajan yüksek viskoziteye sahip olduğundan, yüzey gerilimi etkisi bu ajanın asitlenmiş mineye tam nüfuz etmesine izin vermez.

⑤ This will result in a ‘dead space’ beyond the bonded area. ⑤ Bu durum, bağlanmış alanın ötesinde bir “ölü boşluk” oluşmasına neden olur.

⑥ When enamel bends, or the weak resin based bond breaks off, the dead space becomes exposed to oral fluids which has lower surface tension and thus penetrates the dead space. ⑥ Mine eğildiğinde ya da zayıf reçine bazlı bağ koptuğunda, düşük yüzey gerilimine sahip olan ağız sıvıları bu ölü boşluğa ulaşır ve buraya nüfuz eder.

⑦ This may result in secondary caries or discoloration of the margins. ⑦ Bu durum, ikincil çürüğe veya kenar bölgelerde renk değişimine neden olabilir.

① If the concentration is greater than 50 percent, then monocalcium phosphate monohydrate may get precipitated ① Konsantrasyon %50'den büyükse, monokalsiyum fosfat monohidrat çökelebilir.

② while at concentrations lower than 30 percent, dicalcium phosphate monohydrate is precipitated which interferes with adhesion ② Konsantrasyon %30'dan düşük olduğunda ise dikalsiyum fosfat monohidrat çökelir ki bu da adezyona engel olur.

les petits pas permettent de reduire la difficulté, l'enjeu et le risque d'échec, et permette ainsi d'abaisser les résistances, pour passer à l'action

Reviewer #1 (Public review):

Summary:

In the manuscript submission by Zhao et al. entitled, "Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila" the authors assert that cardiac arrhythmias in Drosophila on a high fat diet is due in part to adipokinetic hormone (Akh) signaling activation. High fat diet induces Akh secretion from activated endocrine neurons, which activate AkhR in posterior cardiac neurons. Silencing or deletion of Akh or AkhR blocks arrhythmia in Drosophila on high fat diet. Elimination of one of two AkhR expressing cardiac neurons results in arrhythmia similar to high fat diet.

Strengths:

The authors propose a novel mechanism for high fat diet induced arrhythmia utilizing the Akh signaling pathway that signals to cardiac neurons.

Comments on revisions:

The authors have addressed my other concerns. The only outstanding issue is in regard to the following comment:

The authors state that "HFD led to increased heartbeat and an irregular rhythm." In representative examples shown, HFD resulted in pauses, slower heart rate, and increased irregularity in rhythm but not consistently increased heart rate (Figures 1B, 3A, and 4C). Based on the cited work by Ocorr et al (https://doi.org/10.1073/pnas.0609278104), Drosophila heart rate is highly variable with periods of fast and slow rates, which the authors attributed to neuronal and hormonal inputs. Ocorr et al then describe the use of "semi-intact" flies to remove autonomic input to normalize heart rate. Were semi-intact flies used? If not, how was heart rate variability controlled? And how was heart rate "increase" quantified in high fat diet compared to normal fat diet? Lastly, how does one measure "arrhythmia" when there is so much heart rate variability in normal intact flies?

- The authors state that 8 sec time windows were selected at the discretion of the imager for analysis. I don't know how to avoid bias unless the person acquiring the imaging is blinded to the condition and the analysis is also done blind. Can you comment whether data acquisition and analysis was done in a blinded fashion? If not, this should be stated as a limitation of the study.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In the manuscript submission by Zhao et al. entitled, "Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila" the authors assert that cardiac arrhythmias in Drosophila on a high-fat diet are due in part to adipokinetic hormone (Akh) signaling activation. High-fat diet induces Akh secretion from activated endocrine neurons, which activate AkhR in posterior cardiac neurons. Silencing or deletion of Akh or AkhR blocks arrhythmia in Drosophila on a high-fat diet. Elimination of one of two AkhR-expressing cardiac neurons results in arrhythmia similar to a high-fat diet.

Strengths:

The authors propose a novel mechanism for high-fat diet-induced arrhythmia utilizing the Akh signaling pathway that signals to cardiac neurons.

Weaknesses:

Major comments:

(1) The authors state, "Arrhythmic pathology is rooted in the cardiac conduction system." This assertion is incorrect as a blanket statement on arrhythmias. There are certain arrhythmias that have been attributable to the conduction system, such as bradycardic rhythms, heart block, sinus node reentry, inappropriate sinus tachycardia, AV nodal reentrant tachycardia, bundle branch reentry, fascicular ventricular tachycardia, or idiopathic ventricular fibrillation to name a few. However the etiological mechanism of many atrial and ventricular arrhythmias, such as atrial fibrillation or substrate-based ventricular tachycardia, are not rooted in the conduction system. The introduction should be revised to reflect a clear focus (away from?) on atrial fibrillation (AF). In addition, AF susceptibility is known to be modulated by autonomic tone, which is topically relevant (irrelevant?) to this manuscript.

Thank you for the helpful comment. We rephrased the sentence as “Arrhythmic pathology is often rooted in the cardiac conduction system”.

(2) The authors state that "HFD led to increased heartbeat and an irregular rhythm." In representative examples shown, HFD resulted in pauses, slower heart rate, and increased irregularity in rhythm but not consistently increased heart rate (Figures 1B, 3A, and 4C). Based on the cited work by Ocorr et al (https://doi.org/10.1073/pnas.0609278104), Drosophila heart rate is highly variable with periods of fast and slow rates, which the authors attributed to neuronal and hormonal inputs. Ocorr et al then describe the use of "semi-intact" flies to remove autonomic input to normalize heart rate. Were semi-intact flies used? If not, how was heart rate variability controlled? And how was heart rate "increase" quantified in high-fat diet compared to normal-fat diet? Lastly, how does one measure "arrhythmia" when there is so much heart rate variability in normal intact flies?

We also observed that fly heart rate is highly variable with periods of fast and slow rates. To control heart rate variability, Ocorr et al. used semi-intact flies to record the heartbeat  (https://doi.org/10.1073/pnas.0609278104). We consider it a rigorous method to get highly consistent results with high quality videos/images. Since our work has a focus on the neuronal inputs to the heart, we did not use the semi-intact method. Our concern is that it is likely to disrupt the neuronal processes during the dissection. Using OCT, we recorded the heartbeat of intact flies in an 8 s time window, when the heartbeat was relatively stable. The different groups of flies, which were fed on a high-fat diet or a normal-fat diet, were recorded using the same method. Thus, we could compare the differences in heart rate.

(3) The authors state, "to test whether the HFD-induced increase in Akh in the APC affects APC neuron activity, we used CaLexA (https://doi.org/10.3109/01677063.2011.642910)." According to the reference, CaLexA is a tool to map active neurons and would not indicate, as the authors state, whether Akh affects APC neuron activity specifically. It is equally possible that APC neurons may be activated by HFD and produce more Akh. Please clarify this language.

Thank you for clarifying the calcium reporter, CaLexA. We rephrased this sentence to “to test whether HFD affects APC neuron activity, we used CaLexA”.

(4) Are the AkhR+ neurons parasympathetic or sympathetic? Please provide additional experimentation that characterizes these neurons. The AkhR+ neurons appear to be anti-arrhythmic. Please expand the discussion to include a working hypothesis of the overall findings on Akh, AkhR, and AkhR+ neurons.

Noyes et al. showed that Akh treatment increases heartbeat (Noyes, B. E., F. N. Katz, and M. H. Schaffer. 1995. “Identification and Expression of the Drosophila Adipokinetic Hormone Gene.” Molecular and Cellular Endocrinology 109 (2): 133–41.), suggesting that AkhR+ neurons are sympathetic. We showed that high-fat diet induced Akh expression and secretion, which led to stimulation of AkhR+ neuron and increased heart rate, supporting the sympathetic role of the AkhR+ neurons. Additional explanation on the sympathetic & anti-arrhythmic role of the Akh, AkhR, and AkhR+ neurons were added to the discussion.

(5) The authors state, "Heart function is dependent on glucose as an energy source." However, the heart's main energy source is fatty acids with minimal use of glucose (doi: 10.1016/j.cbpa.2006.09.014). Glucose becomes more utilized by cardiomyocytes under heart failure conditions. Please amend/revise this statement.

Thank you for pointing this out and providing the reference. We rephrased this sentence “Heart function is dependent on continuous ATP production. Cardiac ATP in Drosophila might come from fatty acids, glucose, and lactate (Kodde et al., 2007), as well as trehalose.”

Reviewer #2 (Public Review):

This manuscript explores mechanisms underlying heart contractility problems in metabolic disease using Drosophila as a model. They confirm, as others have demonstrated, that a high-fat diet (HFD) induces cardiac problems in flies. They showed that a high-fat diet increased Akh mRNA levels and calcium levels in the Akh-producing cells (APC), suggesting there is increased production and release of this hormone in a HFD context. When they knock down Akh production in the APCs using RNAi they see that cardiac contractility problems are abolished. They similarly show that levels of the Akh receptor (Akhr) are increased on a HFD and that loss of Akhr also rescues contractility problems on a HFD.

One highlight of the paper was the identification of a pair of neurons that express a receptor for the metabolic hormone Akh, and showing initial data that these neurons innervate the cardiac muscle. They then overexpress cell death gene reaper (rpr) in all Akhr-positive cells with Akhr-GAL4 and see that cardiac contractility becomes abnormal.

However, this paper contains several findings that have been reported elsewhere and it contains key flaws in both experimental design and data interpretation. There is some rationale for doing the experiments, and the data and images are of good quality. However, others have shown that HFD induces cardiac contractility problems (Birse 2010), that Akh mRNA levels are changed with HFD (Liao 2021) that Akh modulates cardiac rhythms (Noyes 1995), so Figures 1-4 are largely a confirmation of what is already known. This limits the overall magnitude of the advances presented in these figures. Overall, the stated concerns limit the impact of the manuscript in advancing our understanding of heart contractility.

We thank the reviewer for the positive comments and appreciate the reviewer for the instructive suggestions. Birse 2010 (PMID: 21035763) was cited in our manuscript. Liao 2021 showed that Akh mRNA levels are changed with HFD. We added the reference to the revised manuscript and modified the text as: “In consistent with a previous work (Liao et al., 2020), we showed that the expression of Akh was significantly up-regulated in the flies fed a HFD, compared to NFD-fed flies (Figure 2B)”. Our qPCR verified Liao’s results. On top of this, we investigated the calcium levels in the Akh producing cells (APCs) and showed elevated calcium levels in the APC in HFD fed flies. In the revised version, we added more data to show that Akh protein levels were increased with HFD (Figure 2E-F). In line with Noyes' discovery, which showed that Akh injection caused cardioaccelation in prepupae, we showed that genetic manipulation of Akh expression affected heartbeat in the adults.   

Reviewer #3 (Public Review):

Zhao et al. provide new insights into the mechanism by which a high-fat diet (HFD) induces cardiac arrhythmia employing Drosophila as a model. HFD induces cardiac arrhythmia in both mammals and Drosophila. Both glucagon and its functional equivalent in Drosophila Akh are known to induce arrhythmia. The study demonstrates that Akh mRNA levels are increased by HFD and both Akh and its receptor are necessary for high-fat diet-induced cardiac arrhythmia, elucidating a novel link. Notably, Zhao et al. identify a pair of AKH receptor-expressing neurons located at the posterior of the heart tube. Interestingly, these neurons innervate the heart muscle and form synaptic connections, implying their roles in controlling the heart muscle. The study presented by Zhao et al. is intriguing, and the rigorous characterization of the AKH receptor-expressing neurons would significantly enhance our understanding of the molecular mechanism underlying HFD-induced cardiac arrhythmia.

Many experiments presented in the manuscript are appropriate for supporting the conclusions while additional controls and precise quantifications should help strengthen the authors' augments. The key results obtained by loss of Akh (or AkhR) and genetic elimination of the identified AkhR-expressing cardiac neurons do not reconcile, complicating the overall interpretation.

It is intriguing to see an increase in Akh mRNA levels in HFD-fed animals. This is a key result for linking HFD-induced arrhythmia to Akh. Thus, demonstrating that HFD also increases the Akh protein levels and Akh is secreted more should significantly strengthen the manuscript.

Thank you for the positive comments and the instructive suggestions. We performed immunostaining to show that Akh protein levels increased, which is consistent with elevated Akh mRNA expression in HFD-fed flies. The data was added to Figure 2, panels E and F. Akh secretion from the APCs is regulated by APC activity (https://doi.org/10.1038/s41586-019-1675-4). We used a calcium reporter CaLexA (https://doi.org/10.3109/01677063.2011.642910) to monitor APC activity and showed that HFD increased APC activity (Figure 2, C-D).