Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

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1. 

   eLife

   Jun 28, 2021

   Author Response:

   Evaluation Summary:

   This manuscript is of interest to scientists within the fields of actin cytoskeleton, cellular neurobiology and neurodevelopment. It explores how actin regulators are coordinated to trigger the formation of branches in neuronal dendritic arbor. Experiments are very well performed. Conclusions of the manuscript are convincingly supported by the results, although strict dependence of Cobl and Cobl-like in dendritic branch formation should perhaps be confirmed with additional experiments or tuned down. Results concerning the spatiotemporal relationship between the molecular players involved are more preliminary and few findings already published by the same group in previous articles should be expunged from this manuscript.

   We thank the reviewers for the positive assessment of the quality and impact …

   Author Response:

   Evaluation Summary:

   This manuscript is of interest to scientists within the fields of actin cytoskeleton, cellular neurobiology and neurodevelopment. It explores how actin regulators are coordinated to trigger the formation of branches in neuronal dendritic arbor. Experiments are very well performed. Conclusions of the manuscript are convincingly supported by the results, although strict dependence of Cobl and Cobl-like in dendritic branch formation should perhaps be confirmed with additional experiments or tuned down. Results concerning the spatiotemporal relationship between the molecular players involved are more preliminary and few findings already published by the same group in previous articles should be expunged from this manuscript.

   We thank the reviewers for the positive assessment of the quality and impact of our work.

   The functional dependence of Cobl on Cobl-like and vice versa of Cobl-like on Cobl is explained in our comment to the general points raised by the reviewers and also is more clearly described and discussed in the revised manuscript.

   We acknowledge that analysis of the spatiotemporal relationship of molecular players involved in dendritic branch induction only is in its infancy, as at the current stage of research not even all important players of this process are known and this type of analysis is technically challenging to do in a quantitative manner in neurons.

   The revised manuscript does not only contain representative 3D-live imaging data but now also clearly demonstrates by quantitative evaluations of peak signal intensities that all four components studied (Cobl, Cobl-like, syndapin I and CaM) indeed show accumulations at branch induction sites prior to branch initiation (revised Figure 5C). These data are quite well in line with the relative accumulation data collected for two of the components at the 30 s time point prior to protrusion initiation for Cobl (Hou et al., 2015 PLoS Biol.) and for Cobl-like (Izadi et al., 2018 J. Cell Biol.).

   Furthermore the revised manuscript now contains a preliminary assessment of the average peak times of all four components studied here prior to dendritic branch induction (revised Figure 5D). The data highlights that they indeed do not only show spatial but also temporal overlap at branch initiation sites, as it can be expected from our finding that Cobl-like and Cobl can be interconnected by Cobl-like’s novel interaction partner syndapin I in a CaM-regulated mechanism converging on one particular of the three syndapin I binding motifs we identified in Cobl-like.

   Finally, the criticized side-by-side, software-based, detailed evaluation of Cobl and Cobl-like loss-of-function phenotypes during early dendritic arborization, has been moved to the Supplemental Material (Figure 1-Figure Supplement 1) in the revised manuscript, as one half of the data set of course indeed merely is a reproduction of the Cobl-like phenotype identified by the same method before (Izadi et al., 2018).

   However, the reviewers will acknowledge and readers will immediately understand that, without this comparison revealing the high degree of phenotypical copy, we would not have followed up and discovered the coordinated action of the two components powering actin filament formation during dendritic branch initiation we report here.

   Reviewer #1 (Public Review):

   This work investigates at the molecular and cellular levels the functional dependence of two actin filament nucleation factors, Cobl and Cobl-like proteins, in the formation of protrusive dendritic structures. Depletion of Cobl or Cobl-like lead to roughly similar phenotypes; overexpression of Cobl or Cobl-like induces excessive dendrite formation when the other protein is expressed at normal levels, but not when this other protein is depleted. Altogether, these observations lead the authors to conclude that these proteins work strictly interdependently. The authors then investigate how Cobl and Cobl-like are recruited, and identify syndapin as an essential component to bring Cobl and Cobl-like together at the membrane. This interaction is beautifully documented through a large number of pulldown experiments in vitro, and critical domains for these interactions are identified. These interactions are also confirmed in physiological conditions through ectopic localization experiments of those components to mitochondria. Syndapin I is identified as clusters at dendritic initiation sites by electron microscopy and all three components colocalize at the same nascent dendritic branch sites. In the last part of the manuscript, the authors further document the interaction between Cobl-like and syndapin, and find that calcium-dependent calmodulin binding to Cobl-like increases syndapin I's association through the first of the three KRAP's domains.

   Comments to be addressed in a revised manuscript:

   1. Some results appear inconsistent between different Figures. For example, in Figure 1D, Cobl RNAi shifts numbers of dendritic branch points from 10 to 6, while in Figure 2E, Cobl RNAi leaves numbers of dendritic branch points pretty much unchanged (around 7 or 8). Could the authors make sure that all data are consistent between Figures or explain apparent inconsistencies?

   We thank the reviewer for his/her careful evaluation of our data. The discrepancy noticed, however, is only an apparent inconsistency, as the experimental set-ups and purposes were different.

   It is correct that the both the absolute numbers of dendritic branches, terminal points and dendritic length and also the relative effects of Cobl and Cobl-like RNAi differ in particular between former Figure 1 and 2. Roughly, one can say that the RNAi effects in former Figure 1,9 and 10 are about twice as strong as in the former Figure 2. There are two simple reasons for this, which we unfortunately failed to communicate properly in our original manuscript

   i) time frame of phenotype development and suppression, respectively and

   ii) expression of only one versus two plasmids in the different types of experiments

   Concerning i): The former Figure 2 and actually also the former Figure 4 (suppression by syndapin I RNAi) both are suppressions of gain-of-function phenotypes, whereas the former Figures 1, 9 and 10 are loss-of-function experiments. Because the gain-of-function effects represent strong and fast inductions of dendritic arborization it suffices to do a short transfection (max. 34 h) and then evaluate. Loss-of-function effects in conditions using the RNAi tools alone are not very strong at such short times when compared to control (all three analyzed parameters are -15-25% for the stronger Cobl-like RNAi and 0 to -10% for the weaker Cobl RNAi at this short time; former Figure 1, please see Figure 1-Figure Supplement 2 of the revised manuscript).

   The loss-of-function experiments (former Figure 1, 9, 10) are different. The times need to be longer, as the phenotype is the normal growth of the dendritic arbor in controls vs. the putative suppression of this developmental process upon RNAi. Thus, transfections in these experiments usually need to be substantially longer (37-46 h) to show loss-of-function phenotypes compared to control - which then also may be more obvious (Cobl-like RNAi, -30 to -40 %; Cobl RNAi, -33% (*), -20% () and -10% (n.s.) (former Figure 1D-F – now Figure 1-Figure Supplement 2).

   Concerning ii): Suppression of gain-of-function experiments require the coexpression of two plasmids (one for the induction of the gain-of-function phenotype and the second for the RNAi (including reporter expression)), whereas we are able to drive loss-of-function/rescue experiments from only one plasmid driving both RNAi and the expression of a reporter or rescue mutant. Such transfections with two plasmids usually leads to gain-of-function but also suppression effects that are weaker than the effects of either overexpressing or knocking down proteins alone.

   The revised manuscript now provides information on the different time frames of transfection (see improved and expanded Figure legends and Material and Method section) and also briefly touches on the coexpression issue leading to different numbers in the different types of experiments.

   1. I find experiments of Figure 1 and 2 insufficient to conclude that Cobl and Cobl-like factors depend strictly on each other. One could imagine many scenarios where effects of Cobl or Cobl-like are highly concentration dependent, and lead to detectable effects in cells below or under certain thresholds (especially for multi-domain binding proteins such as Cobl and Cobl-like, which are likely to undergo complex phase transition behaviors when clustering at the membrane). Therefore I would recommend the authors to be very careful with wording and conclusions of their experiments, and stick to what can strictly be concluded.

   We share the reviewer’s concerns that suppression experiments are sometimes difficult to interpret. We hope the reviewer will be content with the revised version of our manuscript.

   We share the reviewer’s concerns that suppression experiments are sometimes difficult to interpret, if the experiments are not designed in a careful manner and/or show a complex outcome. This is not the case in our experiments, however (see details below).

   Of strong concern would be the following outcome: A presence of significant RNAi effect(s) alone compared to control and the results of the suppression attempt and the RNAi run for comparison are not equal but the effects of conducting RNAi alone are stronger. In this case of experimental outcome, one should rather abstain from any interpretation and try to adapt the experimental design to reach a clear conclusion. The reason is that, in this particular case, two processes (one positive, the other one negative) could simply operate in parallel, may not necessarily have anything to do with each other directly and may potentially be affected by unspecifiable dose effects as well – thus the experiment is not informative.

   In our experiments, the situation is different and the revised manuscript now contains an elucidation of the considerations required for a correct interpretation for the two vice versa suppression experiments we conducted and reported in the former Figure 2 (Figure 1C-P in the revised manuscript).

   In general, the reviewers will acknowledge that when component A is able to elicit a certain cell biological effect and this does not happen when component B is not present, then component A’s functions depend on B. This is a very classical experimental design and conclusion. The same can also be done with inhibitors - then A’s functions depend on B’s activity. However, it is absolutely crucial that the individual effects of the manipulations as well as the baseline control values are considered in the interpretation, too. If the suppression of the overexpression effect is larger than any putative RNAi effects compared to control or there is no such RNAi effect, the experiment and interpretation actually is very straight forward.

   In our study, this is the case for Cobl RNAi in the suppression of Cobl-like functions (Figure 1C-I in the revised manuscript): We observed complete suppression of Cobl-like’s effects with Cobl-like RNAi. Yet, the effects of GFP+Cobl RNAi expression are not distinguishable from control and the result thus is straight forward to interpret. We actually designed the experiment in a way that the individual RNAi conditions remained neglectable to reach this straight forward interpretation scenario.

   The same applies to the suppression of Cobl-like effects by syndapin I RNAi (Figure 3 in the revised manuscript). Under the conditions shown, syndapin I RNAi would not cause any phenotypes, yet, it completely suppressed the strong Cobl-like-mediated effects on all four parameters of dendritic arborization determined (former Figure 4; now Figure 3 in the revised manuscript).

   For the suppression of the Cobl gain-of-function phenotypes by Cobl-like RNAi (Figure 1J-P in the revised manuscript) the situation is a bit less obvious and we understand the concern of the reviewer that this may need a more detailed look. Here, in all three parameters shown, GFP+Cobl-like RNAi causes a relatively mild but significant phenotype when compared to GFP+Scrambled control. However, the reviewer will acknowledge that the RNAi effects deviating negatively from the GFP+Scrambled control are much smaller than the suppression of the Cobl-mediated effects on dendritic arborization, which are twice as high (branch points; total dendritic length) and three times as high (terminal branches), respectively. Thus, also here, we clearly observe a suppression of specifically Cobl functions and can exclude additive actions in opposite directions. Importantly, this conclusion is formally further underscored by the fact that in all three phenotypical analyses GFP-Cobl+Cobl-like RNAi and GFP+Cobl-like RNAi are not statistically different from one another but equal (Figure 1J-P in the revised manuscript). This makes the interpretation of the results of also this suppression experiment straight forward again.

   Other mentions such as (line 328) "their functions were cooperative", should also be avoided without any further explanations; Mentions such as (line 101) "Functional redundancy seemed unlikely, because both individual loss-of-function phenotypes were severe." should be explained so that readers can assess whether functional redundancy is indeed unlikely or not (for example by referencing a paper describing mild versus severe phenotypes).

   As already written in the Essential Revision list above, we apologize for the too much shortened argumentation in the original manuscript. This paragraph has been changed in the revised manuscript and now explains better why parallel action of Cobl and Cobl-like appeared unlikely and why we thus addressed the alternative hypothesis.

   1. One missing experiment in this story is whether this important effect of Ca2+/CaM signaling promoting syndapin I's association with the first of the three "KRAP" motifs is key to account for Cobl-like's clustering at the plasma membrane. Could the authors measure the effect of calcium for Cobl-like (KRAP1 deleted) clustering at the plasma membrane (as compared to wild-type Cobl-like)?

   We thank the reviewer for his/her suggestion of experiments suitable to significantly strengthen the manuscript.

   In order to address a putative impact of the first, Ca2+/CaM-regulated KRAP motif on the membrane recruitment of Cobl-like, we knocked-down endogenous Cobl-like and then quantified the membrane-association of reexpressed, RNAi-insensitive Cobl-like lacking KRAP1 at the plasma membrane of neurons in comparison to wild-type Cobl-like. Although KRAP1 is only one out of three identified syndapin I binding sites, we observed that deletion of merely this one site had a profound, statistically significant (p<0.0001; ****) impact on Cobl-like’s membrane localization in developing hippocampal neurons. This data obtained in our revision work is reported as Figure 9G-I in the revised manuscript.

   In brief, this type of experimentation was done as part of our revision efforts during the last weeks. It demonstrated a remarkable strong impact of deletion of KRAP1 on Cobl-like’s membrane localization in developing hippocampal neurons and is now reported in the newly added revised Figure 9G-I.

   1. I regret sometimes the lack of quantification for some experiments. For example, protein colocalization in cells should be quantified (for example by calculating Pearson's correlation coefficients of red and green signals at mitochondrial sites) because colocalization (or absence of) is not always obvious for non-expert eyes.

   It may have been overlooked that calculating Pearson's correlation coefficients is not useful in our case, as we are not addressing a correlation of the occurrence of individual signals of one type with another type but are addressing coaccumulations of components under a given condition versus a more diffuse localization under other condition.

   The original manuscript highlighted such coaccumulations by false-color heat map representations and marking sites of interest in two of our main figures.

   In order to also comply with the reviewer’s request concerning the other figures (the in vivo protein complex reconstitutions at mitochondrial membrane surfaces), we added high-magnification insets to all of these figures in the main manuscript and in the Supplementary information visualizing in a more easily accessible manner than in the small full-size images whether the respective mitochondrial patterns are occurring or only a diffuse localization pattern prevails. We furthermore conducted line scans to quantitative visualize coincidences of elevated or diminished signal intensities. We hope that the reviewer is content with these additional figure panels added to many of our revised figures.

   1. Figure 6 is beautiful, but I am wondering if these data could be exploited better. Is it possible to record data at shorter time intervals? It seems that Cobl-like appears before syndapin. Is that correct and if so, how is this coherent with a recruitement of Cobl-like through syndapin?

   We acknowledge that analysis of the spatiotemporal relationship of molecular players involved in dendritic branch induction only is in its infancy, as at the current stage of research not even all important players of this process are known and this type of analysis is technically challenging to do in a quantitative manner in neurons. The revised manuscript does now clearly demonstrate by quantitative evaluations of peak signal intensities that all four components studied (Cobl, Cobl-like, syndapin I and CaM) indeed show accumulation at branch induction sites prior to branch initiation. These data are quite well in line with the relative accumulation data collected for two of the components at the 30 s time point prior to protrusion initiation for Cobl (Hou et al., 2015 PLoS Biol.) and for Cobl-like (Izadi et al., 2018 J. Cell Biol.). Furthermore the revised manuscript now contains a preliminary assessment of the average peak times of all for components highlighting that they indeed do not only show spatial but also temporal overlap at branch initiation sites, as it can be expected from our finding that Cobl-like and Cobl can be interconnected by Cobl-like’s novel interaction partner syndapin I in a CaM-regulated mechanism converging on one particular of the three syndapin I binding motifs we identified in Cobl-like. The Cobl-like and the syndapin I data hereby showed significant variances and a surprisingly early appearance of both components together. The data obtained thus far do not suggest that Cobl-like is recruited before syndapin but in average showed the same peak time (please see revised Figure 5C,D (former Figure 6). Thus, while we honestly do not claim that we have detailed enough data on the different aspects of the spatiotemporal behaviors of all players in dendritic branch initiation and this will definitively require further studies focusing on these aspects specifically, there at least is no discrepancy with any of the molecular mechanisms involving Cobl-like and syndapin I, which we demonstrate in this manuscript.

   Reviewer #2 (Public Review):

   The manuscript by Izadi et al., "Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development" deals with the fundamental question of how actin regulators are orchestrated to control the formation of membranes protrusions during cells morphogenesis. In particular, the authors explored how actin nucleators are coordinated to trigger the formation of branches in neuronal dendritic arbor.

   In that context, Cobl have a crucial role in dendritic arbor formation in neuronal cells. Cobl contains a repeat of three WH2 domains interacting with actin and enabling nucleation of new actin filaments (F-actin). The initial idea was that tandem repeat of WH2 domains could be sufficient to trigger F-actin nucleation. However, other studies have shown that the WH2 repeat of Cobl has no nucleation activity of its own. Importantly, Cobl activity was shown to work in coordination with other actin regulators including the F-actin-binding protein Abp1 (Haag, J Neuro 2012) and the BAR domain protein syndapin (Schwintzer, EMBO J 2011).

   The manuscript of Izadi et al. builds on previous articles from the same group, in particular a study demonstrating that Cobl-like, an evolutionary ancestor of Cobl, is also crucial for dendritic branching (Izadi et al., 2018 JCB). This previous article showed that like Cobl (Haag, J Neuro 2012), Cobl-like protein works in coordination with the F-actin-binding protein Abp1 and Ca2+/CaM to promote dendritic branching through regulation of F-actin nucleation or/and assembly. In the current manuscript the authors showed that the two actin nucleators Cobl and Cobl-like proteins are interdependent to trigger dendritic branching.

   The authors used functional assays by quantifying the formation of dendritic branches in primary hippocampal neurons. Using fluorescence microscopy and siRNA-based knockdowns, the authors showed that Cobl and Cobl-like are functionally interdependent during dendritic branch formation in dissociated hippocampal neurons. They showed that siRNA decreasing Cobl or Cobl-like expression reduced the number of dendritic branch points to the same extent. Fluorescence time-lapses indicated that Cobl and Cobl-like proteins co-localized at abortive and effective branching points. Furthermore, they showed that the increase in branching induced by Cobl-like overexpression is reversed by using a siRNA that decreases Cobl expression, they also performed the reciprocal experiments. Using a variety of biochemistry assays (co-immunoprecipitation, in vitro reconstitutions with purified components…) the authors demonstrated that Cobl and Cobl-like do not interact directly, but that Cobl-like associates with syndapins, as previously shown for Cobl (Schwintzer et al., 2011; Hou et al., 2015). Thus, syndapin is the molecular and functional link between Cobl and Cobl-like proteins. The authors performed a very thorough characterisation of the biochemical interactions between the Cobl-like protein and syndapins. Syndapins and Cobl-like interactions were direct and based on SH3 domain/Prolin rich motif interactions respectively on syndapins and Cobl-like. The Prolin rich motifs were located in 3 KRAP domains at the Nter of Cobl-like proteins. The authors also showed that the interaction of the Nter proximal KRAP domain with syndapin is Ca2+/CaM dependent, and that this Ca2+/CaM dependent interaction is crucial for the function of the Cobl-like protein in the regulation of dendritic arbor formation. The authors confirmed most of their biochemical results by visualizing the formation of protein complexes on the surface of mitochondria in intact COS-7 cells. They also used time-lapse fluorescent microscopy to demonstrate that Syndapin and Cobl-like are co-localized at sites of dendritic branch induction. Importantly, the authors used Immunogold labeling of freeze-fractured plasma membranes combined with electron microscopy. Using this strategy, they showed that membrane-bound syndapin nanoclusters are preferentially located at the base of protrusive membrane topologies in developing neurons. Throughout the manuscript, the authors confronted their biochemistry experiments with functional assays quantifying the formation of dendritic branches.

   The overall conclusion of the manuscript is that a molecular complex involving Cobl, Cobl-like and syndapin and regulated by Ca2+/CaM, promotes the formation of actin networks leading to dendritic protrusions to initiate dendritic branches. Importantly, this manuscript demonstrated that multiple actin nucleators can be coordinated in neurons to trigger the formation of subcellular structures.

   The conclusions of the manuscript are, in most cases, convincingly supported by the results. In particular, the authors have performed a very comprehensive characterization of the biochemical interactions between Cobl, Cobl-like and syndapin, which are well supported by the functional results. However, the results found concerning the spatiotemporal relationship between Cobl, Cobl-like and syndapin during dendritic branch formation are more preliminary and do not take into account the roles of Ca2+/CaM. In addition, some of the findings presented in this manuscript have already been published by the same group, which diminishes the inherent originality of this manuscript. Apart from the main points raised above, the manuscript is experimentally solid and contains interesting results that are likely to stimulate further experiments in the fields of actin cytoskeleton but also in the fields of cellular neurobiology and neurodevelopment.

   We thank the reviewer for the positive assessment of the quality and impact of our work.

   As far as the first point of the reviewer is concerned, the spatiotemporal relationship between Cobl, Cobl-like and syndapin I.

   We acknowledge that analysis of the spatiotemporal relationship of molecular players involved in dendritic branch induction only is in its infancy, as at the current stage of research not even all important players of this process are known and this type of analysis is technically challenging to do in a quantitative manner in neurons.

   The revised manuscript does now clearly demonstrate by quantitative evaluations of peak signal intensities that all four components studied (Cobl, Cobl-like, syndapin I and CaM) indeed show accumulation at branch induction sites prior to branch initiation. These data are quite well in line with the relative accumulation data collected for two of the components at the 30 s time point prior to protrusion initiation for Cobl (Hou et al., 2015 PLoS Biol.) and for Cobl-like (Izadi et al., 2018 J. Cell Biol.).

   Furthermore the revised manuscript now contains a preliminary assessment of the average peak times of all for components highlighting that they indeed do not only show spatial but also temporal overlap at branch initiation sites, as it can be expected from our finding that Cobl-like and Cobl can be interconnected by Cobl-like’s novel interaction partner syndapin I in a CaM-regulated mechanism converging on one particular of the three syndapin I binding motifs we identified in Cobl-like. The Cobl-like and the syndapin I data hereby showed significant variances and a surprisingly early appearance of both components together. The data obtained thus far suggest that Cobl-like and syndapin I are in average recruited at the same peak time, whereas Cobl may perhaps peak a bit later (n.s.) and CaM overlaps with both (please see revised Figure 5C,D).

   However, even with these additional efforts we made during our revision work, one has to honestly admit that it is too early to claim that we have detailed enough data on the different aspects of the spatiotemporal behaviors of all players in dendritic branch initiation (which currently we may not all even have identified, yet). Although technically challenging to do at high enough resolution, with large enough time frames to capture the only relatively rare events of dendritic branch induction, at sufficient frame rates to not miss key events and with high enough numbers of transfected primary neurons of suitable developmental stages to reach sound quantitative data, this will require further comprehensive studies focusing on these aspects specifically.

   As far as the second point of the reviewer is concerned, the criticism that some of the findings presented in this manuscript have already been published.

   As all other points presented are novel, this probably refers to the side-by-side, software-based, detailed evaluation of Cobl and Cobl-like loss-of-function phenotypes during early dendritic arborization originally presented in Figure 1. This data has been moved to the Supplemental Material (Figure 1-Figure Supplement 1) in the revised manuscript, as one half of the data set of course indeed merely is a reproduction of the Cobl-like phenotype identified by the same method before (Izadi et al., 2018).

   However, the reviewers will acknowledge and readers will immediately understand that, without this comparison revealing the high degree of phenotypical copy, we would not have followed up and discovered the coordinated action of the two components powering actin filament formation during dendritic branch initiation we report here.

   Read the original source
2. 

   eLife

   Jun 28, 2021

   Evaluation Summary:

   This manuscript is of interest to scientists within the fields of actin cytoskeleton, cellular neurobiology and neurodevelopment. It explores how actin regulators are coordinated to trigger the formation of branches in neuronal dendritic arbor. Experiments are very well performed. Conclusions of the manuscript are convincingly supported by the results, although strict dependence of Cobl and Cobl-like in dendritic branch formation should perhaps be confirmed with additional experiments or tuned down. Results concerning the spatiotemporal relationship between the molecular players involved are more preliminary and few findings already published by the same group in previous articles should be expunged from this manuscript.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; …

   Evaluation Summary:

   This manuscript is of interest to scientists within the fields of actin cytoskeleton, cellular neurobiology and neurodevelopment. It explores how actin regulators are coordinated to trigger the formation of branches in neuronal dendritic arbor. Experiments are very well performed. Conclusions of the manuscript are convincingly supported by the results, although strict dependence of Cobl and Cobl-like in dendritic branch formation should perhaps be confirmed with additional experiments or tuned down. Results concerning the spatiotemporal relationship between the molecular players involved are more preliminary and few findings already published by the same group in previous articles should be expunged from this manuscript.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
3. 

   eLife

   Jun 28, 2021

   Reviewer #1 (Public Review):

   This work investigates at the molecular and cellular levels the functional dependence of two actin filament nucleation factors, Cobl and Cobl-like proteins, in the formation of protrusive dendritic structures. Depletion of Cobl or Cobl-like lead to roughly similar phenotypes; overexpression of Cobl or Cobl-like induces excessive dendrite formation when the other protein is expressed at normal levels, but not when this other protein is depleted. Altogether, these observations lead the authors to conclude that these proteins work strictly interdependently. The authors then investigate how Cobl and Cobl-like are recruited, and identify syndapin as an essential component to bring Cobl and Cobl-like together at the membrane. This interaction is beautifully documented through a large number of pulldown experiments …

   Reviewer #1 (Public Review):

   This work investigates at the molecular and cellular levels the functional dependence of two actin filament nucleation factors, Cobl and Cobl-like proteins, in the formation of protrusive dendritic structures. Depletion of Cobl or Cobl-like lead to roughly similar phenotypes; overexpression of Cobl or Cobl-like induces excessive dendrite formation when the other protein is expressed at normal levels, but not when this other protein is depleted. Altogether, these observations lead the authors to conclude that these proteins work strictly interdependently. The authors then investigate how Cobl and Cobl-like are recruited, and identify syndapin as an essential component to bring Cobl and Cobl-like together at the membrane. This interaction is beautifully documented through a large number of pulldown experiments in vitro, and critical domains for these interactions are identified. These interactions are also confirmed in physiological conditions through ectopic localization experiments of those components to mitochondria. Syndapin I is identified as clusters at dendritic initiation sites by electron microscopy and all three components colocalize at the same nascent dendritic branch sites. In the last part of the manuscript, the authors further document the interaction between Cobl-like and syndapin, and find that calcium-dependent calmodulin binding to Cobl-like increases syndapin I's association through the first of the three KRAP's domains.

   Comments to be addressed in a revised manuscript:

   1. Some results appear inconsistent between different Figures. For example, in Figure 1D, Cobl RNAi shifts numbers of dendritic branch points from 10 to 6, while in Figure 2E, Cobl RNAi leaves numbers of dendritic branch points pretty much unchanged (around 7 or 8). Could the authors make sure that all data are consistent between Figures or explain apparent inconsistencies?

   2. I find experiments of Figure 1 and 2 insufficient to conclude that Cobl and Cobl-like factors depend strictly on each other. One could imagine many scenarios where effects of Cobl or Cobl-like are highly concentration dependent, and lead to detectable effects in cells below or under certain thresholds (especially for multi-domain binding proteins such as Cobl and Cobl-like, which are likely to undergo complex phase transition behaviors when clustering at the membrane). Therefore I would recommend the authors to be very careful with wording and conclusions of their experiments, and stick to what can strictly be concluded.

   Other mentions such as (line 328) "their functions were cooperative", should also be avoided without any further explanations; Mentions such as (line 101) "Functional redundancy seemed unlikely, because both individual loss-of-function phenotypes were severe." should be explained so that readers can assess whether functional redundancy is indeed unlikely or not (for example by referencing a paper describing mild versus severe phenotypes).

   1. One missing experiment in this story is whether this important effect of Ca2+/CaM signaling promoting syndapin I's association with the first of the three "KRAP" motifs is key to account for Cobl-like's clustering at the plasma membrane. Could the authors measure the effect of calcium for Cobl-like (KRAP1 deleted) clustering at the plasma membrane (as compared to wild-type Cobl-like)?

   2. I regret sometimes the lack of quantification for some experiments. For example, protein colocalization in cells should be quantified (for example by calculating Pearson's correlation coefficients of red and green signals at mitochondrial sites) because colocalization (or absence of) is not always obvious for non-expert eyes.

   3. Figure 6 is beautiful, but I am wondering if these data could be exploited better. Is it possible to record data at shorter time intervals? It seems that Cobl-like appears before syndapin. Is that correct and if so, how is this coherent with a recruitement of Cobl-like through syndapin?

   Read the original source
4. 

   eLife

   Jun 28, 2021

   Reviewer #2 (Public Review):

   The manuscript by Izadi et al., "Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development" deals with the fundamental question of how actin regulators are orchestrated to control the formation of membranes protrusions during cells morphogenesis. In particular, the authors explored how actin nucleators are coordinated to trigger the formation of branches in neuronal dendritic arbor.

   In that context, Cobl have a crucial role in dendritic arbor formation in neuronal cells. Cobl contains a repeat of three WH2 domains interacting with actin and enabling nucleation of new actin filaments (F-actin). The initial idea was that tandem repeat of WH2 domains could be sufficient to trigger F-actin nucleation. However, other studies have shown that the WH2 repeat of Cobl has no …

   Reviewer #2 (Public Review):

   The manuscript by Izadi et al., "Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development" deals with the fundamental question of how actin regulators are orchestrated to control the formation of membranes protrusions during cells morphogenesis. In particular, the authors explored how actin nucleators are coordinated to trigger the formation of branches in neuronal dendritic arbor.

   In that context, Cobl have a crucial role in dendritic arbor formation in neuronal cells. Cobl contains a repeat of three WH2 domains interacting with actin and enabling nucleation of new actin filaments (F-actin). The initial idea was that tandem repeat of WH2 domains could be sufficient to trigger F-actin nucleation. However, other studies have shown that the WH2 repeat of Cobl has no nucleation activity of its own. Importantly, Cobl activity was shown to work in coordination with other actin regulators including the F-actin-binding protein Abp1 (Haag, J Neuro 2012) and the BAR domain protein syndapin (Schwintzer, EMBO J 2011).

   The manuscript of Izadi et al. builds on previous articles from the same group, in particular a study demonstrating that Cobl-like, an evolutionary ancestor of Cobl, is also crucial for dendritic branching (Izadi et al., 2018 JCB). This previous article showed that like Cobl (Haag, J Neuro 2012), Cobl-like protein works in coordination with the F-actin-binding protein Abp1 and Ca2+/CaM to promote dendritic branching through regulation of F-actin nucleation or/and assembly. In the current manuscript the authors showed that the two actin nucleators Cobl and Cobl-like proteins are interdependent to trigger dendritic branching.

   The authors used functional assays by quantifying the formation of dendritic branches in primary hippocampal neurons. Using fluorescence microscopy and siRNA-based knockdowns, the authors showed that Cobl and Cobl-like are functionally interdependent during dendritic branch formation in dissociated hippocampal neurons. They showed that siRNA decreasing Cobl or Cobl-like expression reduced the number of dendritic branch points to the same extent. Fluorescence time-lapses indicated that Cobl and Cobl-like proteins co-localized at abortive and effective branching points. Furthermore, they showed that the increase in branching induced by Cobl-like overexpression is reversed by using a siRNA that decreases Cobl expression, they also performed the reciprocal experiments. Using a variety of biochemistry assays (co-immunoprecipitation, in vitro reconstitutions with purified components...) the authors demonstrated that Cobl and Cobl-like do not interact directly, but that Cobl-like associates with syndapins, as previously shown for Cobl (Schwintzer et al., 2011; Hou et al., 2015). Thus, syndapin is the molecular and functional link between Cobl and Cobl-like proteins. The authors performed a very thorough characterisation of the biochemical interactions between the Cobl-like protein and syndapins. Syndapins and Cobl-like interactions were direct and based on SH3 domain/Prolin rich motif interactions respectively on syndapins and Cobl-like. The Prolin rich motifs were located in 3 KRAP domains at the Nter of Cobl-like proteins. The authors also showed that the interaction of the Nter proximal KRAP domain with syndapin is Ca2+/CaM dependent, and that this Ca2+/CaM dependent interaction is crucial for the function of the Cobl-like protein in the regulation of dendritic arbor formation. The authors confirmed most of their biochemical results by visualizing the formation of protein complexes on the surface of mitochondria in intact COS-7 cells. They also used time-lapse fluorescent microscopy to demonstrate that Syndapin and Cobl-like are co-localized at sites of dendritic branch induction. Importantly, the authors used Immunogold labeling of freeze-fractured plasma membranes combined with electron microscopy. Using this strategy, they showed that membrane-bound syndapin nanoclusters are preferentially located at the base of protrusive membrane topologies in developing neurons. Throughout the manuscript, the authors confronted their biochemistry experiments with functional assays quantifying the formation of dendritic branches.

   The overall conclusion of the manuscript is that a molecular complex involving Cobl, Cobl-like and syndapin and regulated by Ca2+/CaM, promotes the formation of actin networks leading to dendritic protrusions to initiate dendritic branches. Importantly, this manuscript demonstrated that multiple actin nucleators can be coordinated in neurons to trigger the formation of subcellular structures.

   The conclusions of the manuscript are, in most cases, convincingly supported by the results. In particular, the authors have performed a very comprehensive characterization of the biochemical interactions between Cobl, Cobl-like and syndapin, which are well supported by the functional results. However, the results found concerning the spatiotemporal relationship between Cobl, Cobl-like and syndapin during dendritic branch formation are more preliminary and do not take into account the roles of Ca2+/CaM. In addition, some of the findings presented in this manuscript have already been published by the same group, which diminishes the inherent originality of this manuscript. Apart from the main points raised above, the manuscript is experimentally solid and contains interesting results that are likely to stimulate further experiments in the fields of actin cytoskeleton but also in the fields of cellular neurobiology and neurodevelopment.

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5. 

   eLife

   Jun 28, 2021

   Reviewer #3 (Public Review):

   This manuscript by Izadi et al. explores the contribution of two actin nucleating proteins, Cobl and Cobl-like, to dendritic arborization. This work links CaCaM signaling with different post-translation modes of Cobl at the plasma membrane via a physical linkage between Cobl and Cobl-like proteins mediated by the F-BAR protein Syndapin I and coordination with the actin disassembly factor Cyclin-dependent kinase 1 (Srv2/CAP) to ultimately dictate actin-based neuromorphogenesis. The strength of this study includes a robust set of imaging and molecular biology analyses to show the localization and interaction of Cobl, Cobl-like, and Syndapin I. A potential weak point in this work is a lacking comparison between this actin nucleation mode and other neuronal actin nucleation proteins (i.e., Spire, Arp2/3 complex, …

   Reviewer #3 (Public Review):

   This manuscript by Izadi et al. explores the contribution of two actin nucleating proteins, Cobl and Cobl-like, to dendritic arborization. This work links CaCaM signaling with different post-translation modes of Cobl at the plasma membrane via a physical linkage between Cobl and Cobl-like proteins mediated by the F-BAR protein Syndapin I and coordination with the actin disassembly factor Cyclin-dependent kinase 1 (Srv2/CAP) to ultimately dictate actin-based neuromorphogenesis. The strength of this study includes a robust set of imaging and molecular biology analyses to show the localization and interaction of Cobl, Cobl-like, and Syndapin I. A potential weak point in this work is a lacking comparison between this actin nucleation mode and other neuronal actin nucleation proteins (i.e., Spire, Arp2/3 complex, or formin). This could allow readers to assess and/or compare the effectiveness of the Cobl and Cobl-like to previously discovered single actin-nucleation protein activities on neurogenesis.

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6. 

   Version published to 10.1101/2021.03.03.433715 on bioRxiv

   Mar 3, 2021