The dependence of shugoshin on Bub1-kinase activity is dispensable for the maintenance of spindle assembly checkpoint response in Cryptococcus neoformans

  1. Satya Dev Polisetty
  2. Krishna Bhat
  3. Kuladeep Das
  4. Ivan Clark
  5. Kevin G. Hardwick
  6. Kaustuv Sanyal

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Abstract

During chromosome segregation, the spindle assembly checkpoint (SAC) detects errors in kinetochore-microtubule attachments. Timely activation and maintenance of the SAC until defects are corrected is essential for genome stability. Here, we show that shugoshin (Sgo1), a conserved tension-sensing protein, ensures the maintenance of SAC signals in response to unattached kinetochores during mitosis in a basidiomycete budding yeast Cryptococcus neoformans . Sgo1 maintains optimum levels of Aurora B kinase Ipl1 and protein phosphatase 1 (PP1) at kinetochores. The absence of Sgo1 results in the loss of Aurora B Ipl1 with a concomitant increase in PP1 levels at kinetochores. This leads to a premature reduction in the kinetochore-bound Bub1 levels and early termination of the SAC signals. Intriguingly, the kinase function of Bub1 is dispensable for shugoshin's subcellular localization. Sgo1 is predominantly localized to spindle pole bodies (SPBs) and along the mitotic spindle with a minor pool at kinetochores. In the absence of proper kinetochore-microtubule attachments, Sgo1 reinforces the Aurora B kinase Ipl1 -PP1 phosphatase balance, which is critical for prolonged maintenance of the SAC response.

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    Referee #4

    Evidence, reproducibility and clarity

    Summary

    This study analyses the shugoshin gene (SGO1) of the single-celled basidiomycete Cryptococcus neoformans. The function of Sgo proteins has been studied in various organisms, including yeasts (budding and fission yeast, C. albicans), flies, frog eggs, mammals, and plants. In general, Sgo proteins function as adapter proteins that recruit activities, such as the PP2A phosphatase, the chromosomal passenger complex (CPC), kinesins, or condensin to the pericentromeric region and kinetochores. This is required for proper biorientation of sister kinetochores at metaphase, correction of erroneous microtubule-kinetochore attachments, signaling by the spindle-assembly checkpoint (SAC), and protection of centromeric cohesin from removal by separase at meiosis and a non-proteolytic pathway in mammalian mitosis. These processes are interconnected, which has made distinguishing different functions of Sgo proteins a challenging and ongoing task.

    The authors use plate assay to show that proliferation of the sgo1 mutant is sensitive to microtubule-depolymerizing drugs. Double mutants with a deletion of the SAC component MAD2 or a non-essential kinetochore subunit are even more sensitive. The sgo1 mutant fails to halt cell division, re-budding, and DNA replication in the presence of a MT drug, suggesting that it is defective in inducing or maintaining SAC activity.

    Live-cell imaging is used to analyze the kinetochores recruitment of several proteins involved in error correction and/or SAC activity in the presence of a MT drug. They conclude that sgo1 mutants fail to maintain the SAC kinase Bub1 at kinetochores. Furthermore, sgo1 mutants fail to maintain at kinetochores the Aurora B kinase, which is required for error correction and SAC activity. Conversely, sgo1 mutants recruit higher levels of the PP1 phosphatase, which is known to oppose Aurora B in several processes. The authors suggest that the sgo1 mutant is defective in SAC function because it shifts the balance between Aurora B and PP1 towards the latter.

    While the kinase activity of Bub1 promotes the SAC, it is not essential. However, kinase activity is required for recruitment of Sgo1 to centromeres/kinetochores. Bub1 phosphorylates histone H2A to which Sgo1 is thought to bind via its conserved SGO domain. The authors analyzed a BUB1 kinase-dead mutant and find it to be defective in SAC activity, while a mutation in Sgo1's SGO domain is SAC proficient. The authors conclude that Sgo1 recruitment in Cryptococcus differs from the conventional, Bub1-dependent mechanism.

    Finally, the author present experiments suggesting that Sgo1 localizes to spindle pole bodies (SPBs) and the spindle, whereas it accumulates at the pericentromere in other organisms.

    Major comments

    This work might be seen against the background of a large body of work on Sgo proteins in various organisms, including detailed and mechanistic studies in yeast and animals. The author should better explain what we might learn from a less-commonly studied microorganism in which detailed mechanistic studies are much harder. They briefly mention that Cryptococcus has an unconventional kinetochore but do not elaborate. Studying Sgo in such a context is certainly interesting and would give this work a unique angle.

    One of the challenges of working on Sgo function is to distinguish its functions in the interconnected processes of biorientation, error correction, and SAC activity. For instance, the authors show that sgo1 mutants fail to arrest at metaphase in response to microtubule depolymerization. Does this failure lead to the loss of Bub1/Aurora from kinetochores or is this loss the reason for the inability to arrest? Furthermore, error correction leads to SAC activation, which in turn leads to accumulation of proteins relevant to error correction. The authors should discuss these issues.

    The authors might want to refrain from making detailed claims about the mechanism of Sgo recruitment to subcellular structures, while the experiments presented are not detailed enough to reject more conventional models. The authors detect Sgo on SPBs and the spindle, which does not mean that it is absent from kinetochores or the pericentromere. I note that vertebrate Sgo1 has originally been identified as a microtubule-binding protein.

    Recruitment of Sgo to the pericentromere is more complex than implied by the authors. Bub1's kinase activity is important but not essential for SAC function. It is required for Sgo recruitment even in cells containing a phosphomimic version of histone H2A, and Sgo proteins have additional binding partners at the pericentromere, including cohesin and HP1.

    Minor comments

    The time course experiments are difficult to interpret. It would be preferably to show separate curves for large-budded cells and e.g. Bub1 or Aurora B at kinetochores. It is difficult to see what fraction of cells arrested at metaphase and what fraction recruited Bub1/Aurora B.

    I cannot judge the live imaging experiments. Materials and Methods mentions the removal of outliers and the bridging of gaps in the imaging but lacks information on how these procedures might affect the data presented. Furthermore, the graphs showing statistics are unconventional. They present the means of three experiments (open circles) and all the individual data points (colored circles), while the (very small) error bars refer to the means (that is what I assume). It would be preferable to separately show the individual data points and their respective means and then use an ANOVA to compare them.

    The live -cell imaging experiments could be presented as montages (or, indeed, movies) to capture changes over time. Also, the quantification might be presented as percentages or intensities over time - not just a single timepoint. After all, the authors claim that the relevant proteins are first recruited to but then lost from kinetochores in the sgo1 mutant.

    Referee Cross-Commenting

    I agree with the comments made by the other reviewers. In particular, all reviewers indicate that claims about a new mechanism (Bub1-independent role of Sgo1) should be toned down or backed-up by new experiments. Same for the relevance of the localization of Sgo1 to spindles and SPBs. In agreement with reviewer #2, I would strongly recommend describing and discussing the use of C. neoformans. I still feel that presentation, analysis, and statistics of the live-imaging experiments should be improved.

    Significance

    This study is interesting to researchers working on mechanisms of chromosome segregation in microorganisms and the functions of Sgo proteins. As stated above, the author could make their study more appealing if they explained the unique features of Cryptococcus with regards to chromosome segregation. This reviewer works on mechanisms controlling chromosome segregation including Sgo proteins but is not familiar with the Cryptococcus system.

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    Referee #3

    Evidence, reproducibility and clarity

    This study investigates the spindle assembly checkpoint (SAC) in a budding yeast Cryptococcus neoformans. The authors propose that Sgo1, the shugoshin homolog in the above species, maintains the SAC activity independently of the Bub1 kinase activity, which is usually critical for the full SAC activity in other species.

    Major comments:

    The main conclusion of the manuscript may not appear convincing because of the following reasons (1) The spotting assay in Fig. 5B can be interpreted as Sgo1 depends on the Bub1 kinase activity as in other species, but Sgo1 also has additional roles in chromosome segregation, which leads to the stronger sensitivity to TBZ in sgo1∆ compared to bub1-kd. (2) Both bub1-kd and sgo1∆ show defects in maintaining SAC arrest (Fig. 2D). If Sgo1 does not depend on the Bub1 kinase activity to maintain SAC arrest, Bub1 is maintaining the arrest through other pathways. Therefore, if the authors repeated this experiment with sgo1∆ bub1-kd double mutants, there would be an additive effect and the cells would arrest less efficient compared to single mutants. On the other hand, if Sgo1 depends on Bub1, the double mutant will behave similarly to single mutants. I strongly recommend the authors to perform this experiment. (3) Along the same line; if it is not through Sgo1, how does the Bub1 kinase activity support the SAC arrest? (4) The authors conclude that the SPB localization is critical for Sgo1 functions. However, there are some dotty Sgo1 signals between the SPBs in metaphase (Fig. 6A) that could be centromeric localization. I would recommend the authors to repeat this experiment in bub1-kd mutant to see if they detect similar centromeric enrichment of Sgo1. If not, this indicate that there is a Sgo1 pool at the centromere that depends on Bub1, similar to other species. (5) The only convincing supporting evidence is the Sgo1-K382A mutant showing no TBZ sensitivity, but this data is not sufficient to draw the conclusion (and having this conclusion as their tile) because the SGO motif is not so conserved and this mutation may not be completely abolishing the interaction with H2ApT120.

    Minor comments:

    Line 180-181: Mad2 is the master regulator of SAC, and mad2 mutants cancel the SAC completely in other systems. However, it appears that sgo1∆mad2∆ has slightly "stronger" SAC compared to mad2∆. This is an interesting observation, and I recommend the authors to speculate why deleting sgo1 made the SAC stronger in mad2∆ cells. Also, this contradicts with the TBZ sensitivity that sgo1∆mad2∆ shows higher sensitivity compared to mad2∆. This would also be a nice discussion point.

    Line 210: The evidence presented to make this point is flawed. Fig. 2D shows that the sgo1 mutant starts with a percent large-budded cells with signal below the other two (see 40 min). It would make sense then that the peak for sgo1∆ cells is lower at the inflection point the authors are referring to around 160min. A better argument is that bub1-kd and sgo1∆ act like each other, not that there is a particular difference. This again points out the possibility that Sgo1 and the Bub1 kinase activity is in the same pathway for the SAC signaling.

    Line 330: Is the centromeric localization actually killed in Sgo1-K382A mutant? Tagging the mutant protein with GFP would be informative (also see comments above regarding Sgo1 localization experiment in bub1-kd background).

    Line 385: The resolution used in this microscopy is not enough to tell whether or not Sgo1 localizes to centromeres in this species. Perhaps the kinetochore pool of Sgo1 is buried in the spindle signal the authors see. I understand the technical difficulty but an experimental system to completely dissociate kinetochores from SPB (like nda3-cs mutant in pombe) is required to make this conclusion.

    Line 418: Again, this conclusion is supported by weak evidence. Perhaps Sgo1 localization is more diffuse than expected along the spindle and concentrated at SPBs, but it is premature to conclude that there is not a pool of Sgo1 that does not localize in the proximity of centromeres in this particular species.

    • There are no error bars in the Figure 3F and Supp Figure 4B. Are these experiments repeated?
    • The quantification of the intensity of the fluorescent signals is performed on the maximum intensity Z-projected images, and it is recommended to use sum-projected images for accurate measurement.

    Significance

    The authors investigate fundamental mechanisms that support faithful chromosome segregation, using a non-typical model organism. It is very important to study diverse model systems to comprehensively understand what the conserved mechanisms are in each lineage.

    our expertise: mitosis and meiosis, mouse oocytes, spindle, chromosome segregation, centromere, confocal microscopy, genetics, cell biology

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    Referee #2

    Evidence, reproducibility and clarity

    The authors characterized the mitotic function of Shugoshin SGO1 in a basidiomycete budding yeast Cryptococcus neoformans. Sgo1 is well conserved protein, which ensures the maintenance of spindle assembly checkpoint signals in response to unattached kinetochore. Based on their results the authors propose that Shugoshin monitors the kinetochore-MT attachments by maintaining higher Aurora B and lower PP1 levels at kinetochores. Shugoshin localizes to centromeres/kinetochores during mitosis in most species, however Shugoshin in C. neoformans specifically localizes to SPBs and along the mitotic spindle. Two of the distinctive findings are the unique localization of Sgo1 and the Bub1 kinase independent checkpoint maintenance function of Sgo1.

    The experiments are performed thoroughly with appropriate controls. suitable statistical measures, and results are presented in a manner that is logical and easy to understand. Although results are interesting for the centromere and chromosome biology communities, mechanistic studies for localization of Sgo1 and how the unique Bub1 independent kinase function of Sgo1 mediates its function has not been adequately addressed. Also, I was not very clear of the unique advantage of pursuing studies with this yeast? How are these studies advancing our understanding of chromosome segregation in human health and disease? Are there other differences between findings from C. neoformans and budding/fission yeast wrt to chromosome segregation?

    Major Comments:

    1. Localization of Sgo1 to CEN or not? Authors report that Sgo1 is recruited to SPBs and along the mitotic spindle (lines 332-362). They have also observed Sgo1 signal at the periphery of the kinetochore. In other systems, Sgo1 has been associated with CEN and peri-CEN chromatin (Deng and Kuo 2018 G3 8:2901-2911; Garcia-Nieto et al. 2023 NSMB 30: 853-859). Cell biology is not most definitive to rule out kinetochore localization, ChIP experiments are most definitive can these be done?
    2. Localization of Sgo1 to SPB. As highlighted in summary of the review, recruitment of Sgo1 to the SPBs is an interesting observation for C. neoformans, however, the molecular mechanisms involved in this novel function of Sgo1 remains unknown. Which factors mediate the localization of Sgo1 to the SPBs during the cell cycle? The recruitment of Sgo1 to SPBs could be influenced or mediated by the geometric changes in the mitotic spindle that occur during the mitosis. It is possible that orientation of mitotic spindle relative to the kinetochore during the mitotic cell cycle may dictate the recruitment of Sgo1 to the SPBs. There is precedent from S. cerevisiae where kinetochore protein Ndc10 was found to be associated with SPBs and along mitotic spindle (Bouck and Bloom, 2005 PNAS 102: 5408-5413). It is likely that similar mechanisms might be involved for Sgo1. One of the candidate genes mediating localization of Sgo1 to the SPBs could be the components of CPCs (Abad et al. 2022 JCB 221:e202108156). Authors should at least examine one of them that will help provide a mechanistic insight into the novel role of Sgo1 at the SPBs.
    3. Bub1 independent function of Sgo1. It would be useful to provide the evolutionarily timescale for the gain or loss of Bub1-independent SAC function of Sgo1. An evolutionary comparison between basidiomycetes and ascomycetes would help readers understand the biological reasoning and significance of Bub1-independent SAC function of Sgo1.
    4. Bub1 independent function of Sgo1. What is the localization of Sgo1 in bub1-kd strain?
    5. Authors have established a relationship between Sgo1, Bub1 and Aurora B (lines 247-248). However, such observations could also be mediated by other molecular factors.. It will improve their manuscript and conclusions if they provide further evidence for in vivo interaction of Sgo1 with Bub1 or Aurora B in C. neoformans using Co-IP.

    Minor Comments:

    1. Figure 1D: The figure is very crowded and difficult to understand. Authors should use letters to show the statistical significance instead of drawing multiple lines.
    2. Figures 1B and 5D: The sgo1null strain is not sensitive to 3 ug of thiabendazole in Figure 1B but it is in figure 5D. Please explain.
    3. Figures 1 and 2: Authors have used 1 ug of nocodazole in figure 1 and it is 2.5 ug in figure 2. Please explain.
    4. Figure 2C-2E: Is GFP-Bub1 signals in sgo1∆ similar to GFP-bub1-kd signals? To clarify the GFP-bub1-kd signals in Figure 2, the authors should show pictures of GFP-bub1-kd signals in Figure 2C, as well as GFP-bub1-kd intensity in Figure 2E. They need to explain how they score the diffused GFP-Bub1 signals in the whole cells of 280 min post-nocodazole treatment.
    5. In line 253-256, the authors incubated the Aurora B-overexpressed cells (galactose medium) at permissive temperature, but Aurora B-depleted cells (glucose medium) at non-permissive temperature. What is the purpose for incubations at different temperatures? Do the Aurora B-overexpressed cells show a ts phenotype?
    6. Reproducibility of localization pattern of GFP-Sgo1 in Figure 6. Can the signals be quantified?
    7. Figure 5: Since Sgo1 in C. neoformans has unique localization pattern, it is possible that the target of phosphorylation by Bub1 is different, rather than binding to phosphorylated H2A. The authors should at least discuss the possibility.
    8. Authors have used a linker protein Bgi1 as a control (lines 148-150). However, there are other controls, which are more suitable than Bgi1. Perhaps authors should use a protein that interacts with microtubules. One of the candidates in this experiment would be evolutionarily conserved Ndc80.
    9. Bub1 independent function of Sgo1. It is unclear which factors are involved in Bub1-independent function of Sgo1.. It is possible that histone H3 or its variant CENP-A might have some role in Sgo1 recruitment as has been shown for S. cerevisiae and other systems (Luo et al. 2016 Genetics 204:1029-1043; Wu et al. 2023 JMCB, mjad061; Mishra et al. 2018 Cell Cycle 17:11-23). Authors can try to deplete CENP-A and examine the localization of Sgo1 at the kinetochore and at the SPBs in Bub1 and its mutant strains.

    Referee Cross-Commenting

    Summary: The data supporting an unusual role of Sgo1 as indicated in the title is not supported by the data. Two main conclusions of the paper are the unique localization of Sgo1 and the Bub1 kinase independent role of Sgo1. With respect to localization: Molecular evidence to rule out lack of Sgo1 at centromeric and pericentromeric regions is needed (ChIP, FRET) is needed. Same is true for SPB association is this affected in spb mutants?

    Bub1 kinase independent role of Sgo1 needs further experimentation.

    Discussion about the significance of studying C. neoformans needs to be included.

    Significance

    The experiments are performed thoroughly with appropriate controls. suitable statistical measures, and results are presented in a manner that is logical and easy to understand. Although results are interesting for the centromere and chromosome biology communities, mechanistic studies for localization of Sgo1 and how the unique Bub1 independent kinase function of Sgo1 mediates its function has not been adequately addressed. Also, I was not very clear of the unique advantage of pursuing studies with this yeast? How are these studies advancing our understanding of chromosome segregation in human health and disease? Are there other differences between findings from C. neoformans and budding/fission yeast wrt to chromosome segregation?

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    Referee #1

    Evidence, reproducibility and clarity

    The authors present a thorough characterization of the mitotic protein Shugoshin (Sgo1) in the context of Cryptococcus neoformans, an interesting and medically important fungal species. Sgo1 function in chromosome segregation has been well-studied in other eukaryotic species: it recruits the error correction kinase Aurora B and Protein Phosphatase 2 to the centromere and promotes the loading of cohesion at the centromeric locus. The primary function of these proteins is to enable the bipolar attachment of sister chromatids to the spindle apparatus. In this manuscript, Polysetty et al mainly investigate the "secondary" function of Sgo1 in Spindle Assembly Checkpoint (SAC) signaling. They show that: (1) sgo1Δ cells fail to maintain a prolonged SAC response to spindle poisons, because they cannot achieve or maintain normal Bub1 recruitment at unattached kinetochores. (2) The lower Bub1 recruitment is mainly due to the loss of centromeric Aurora B recruitment and increased PP1 recruitment to the kinetochore. There are some interesting departures from the behavior Sgo1 known in other eukaryotes. (3) Sgo1 localizes to the spindle poles in C neoformans with smaller amounts dispersed along the spindle. (4) Furthermore, Sgo1 localization at the centromeres does not require Bub1 kinase activity. Based on these and other observations, the authors advance a model for how Sgo1 is recruited to the centromeres and how it promotes strong SAC response to spindle poisons.

    Main comments:

    1. In interpreting the effects of thiabendazole on colony growth, the authors should also consider the role of Sgo1 in promoting bipolar attachments by establishing centromeric cohesion and proper geometry (Indjeian and Murray Current Biology 2007, Verzijlbergen et al. eLife 2014). In the absence of Sgo1, cells are more likely to divide with wrongly attached chromosomes and become highly aneuploid, which may promote mortality. The relative importance of Sgo1 function in biorientation and SAC signaling will have to be established using known separation of function mutations in Sgo1.
    2. Unless I am missing something, the phenotypes of Sgo1 related to the SAC are completely consistent with the established model of Aurora B and PP1 roles in regulating SAC signaling. Sgo1 promotes Aurora B activity at the kinetochore, and the increased Aurora B activity can promote SAC signaling by: (1) delaying SAC silencing by creating unattached kinetochores during error correction, (2) suppressing PP1 recruitment to the kinetochore, and (3) potentially phosphorylating MELT motif-proximal residues to promote Bub1 recruitment as has been observed in Drosophila (Audette et al MBoC 2021). Conversely, Sgo1 deletion will result in hyperstabilization of even wrong kinetochore-microtubule attachments, higher PP1 recruitment and, therefore, weaker SAC signaling. These points should be discussed when interpreting the results in this study.
    3. Phenotypes related to the sgo1-K382A mutations are interesting, and they suggest that Sgo1 recruitment may be independent of Bub1 kinase activity. However, the data need to be strengthened to fully support this conclusion. First, the multiple sequence alignment for Sgo1 shows lysine residues at position 381 and 382 in Cryptococcus. This makes me wonder if the point mutation is sufficient to abolish the Sgo1-pH2A interaction. Second, the expression levels of the mutant should be compared with the wild type to confirm that the phenotype is not due to over-expression/stabilization of the mutant protein. I understand that the thiabendazole resistance of Bub1-kd is quite interesting, but Haspin kinases are also involved in loading Sgo1 at the centromere. The authors could investigate their role in Crypotcoccus to define the alternative mechanism of centromere specific Sgo1 loading.
    4. In Figure 6, the authors find strong Sgo1 colocalization at the spindle poles and what they describe as "spindle-like location along the pole-to-pole axis" (lines 358-360). The authors should consider the possibility that this is the pool of centromere associated Sgo1 because it colocalizes reasonably well with CENPA. This interpretation obviates the necessity of the complex model in Figure 6E explaining Sgo1 loading at the centromeres. Given that there must be Sgo1 at the centromeres, the null hypothesis has to be that this Sgo1 is associated with centromeres rather than microtubules. The authors could use biochemical methods to test the hypothesis. Alternatively, BiFC ro FRET based assays may be useful if the authors want to persist with cell biology experiments.

    Minor points:

    1. The authors use gene repression and over-expression using the Gal1 promoter, but do not assay protein levels for the degree of over-expression. This is fine in many cases because the phenotypes are consistent with over-expression/repression. However, it needs to be confirmed when interpreting the effects of point mutations, e.g., Bub1-kd, Sgo1-K382, etc.
    2. I found the model in 5A confusing because the arrows are used to indicate both protein recruitment and promotion/expression of downstream proteins/events. Adding to the confusion is the inverted sgo1 → Aur B direction. The authors should simplify this important panel.
    3. The model in Figure 6E is missing some information: gradients in Sgo1 and Ipl1 pools are shown, but they don't line up with the spindle direction and are not otherwise indicated on the spindle. Importantly, this model is necessary only if the authors can conclusively show that the Sgo1 along the spindle axis is associated with microtubule and not the centromere.
    4. The authors examine Sgo1's role in loading cohesin at the centromere in Figure S6. An interesting experiment would be to test the thiabendazole sensitivity of Scc1 over-expressing cells to understand whether it can suppress the effects of sgo1Δ.

    Referee Cross-Commenting

    I think Reviewer #2's comment above summarizes the all the comments effectively. I don't have anything else to add.

    Significance

    This is a well-constructed and well-executed study. However, the interpretation of many of the results and how strongly these results depart from what's already known in other systems is debatable. Almost all the results describing CnSgo1 involvement in SAC signaling reinforce the established model that Sgo1 recruits Aurora B, which suppresses the recruitment of PP1, the main antagonist for Bub1 recruitment. Thus, Δsgo1 cells are unable to mount a strong SAC response because increased PP1 recruitment antagonizes SAC signaling. In my view, the novel findings are the Bub1 kinase activity-independent loading of Sgo1 at the centromere and the surprising Sgo1 localization at the spindle poles. The manuscript will generate wider interest if the authors dissected the molecular basis of these unexpected observations rather than its established functions. Doing so will require a functional and molecular dissection of Sgo1.

  6. Version published to 10.1101/2024.02.28.582509 on bioRxiv