Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching

  1. Delia Ricolo
  2. Sofia J. Araújo

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Abstract

The establishment of branched structures by single cells involves complex cytoskeletal remodelling events. In Drosophila , epithelial tracheal system terminal cells (TCs) and dendritic arborisation neurons are models for these subcellular branching processes. During tracheal embryonic development, the generation of subcellular branches is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation where they act as microtubule organizing centres (MTOCs). However, not much is known on the events that lead to the growth of these subcellular luminal branches or what makes them progress through a particular trajectory within the cytoplasm of the TC. Here, we have identified that the spectraplakin Short-stop ( Shot ) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the TC cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal branching points in the embryonic and larval tracheal TC leading to cells with extra subcellular lumina. These data provide the first evidence for a role for spectraplakins in subcellular lumen formation and branching.

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    Reply to the reviewers

    We thank all reviewers for their comments and suggestions, which will make our manuscript a much better one. Accordingly, we have already made changes to the manuscript (marked in yellow) and we will perform all the experiments requested. Below, we answer the reviewers point by point.

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

    This study provides solid evidences showing a role for the spectraplakin Short-stop (Shot) in subcellular lumen formation in the Drosophila embryonic and larval trachea. This subcellular morphogenetic process relies on an inward membrane growth that depends on the proper organization of actin and microtubules (MTs) in terminal cells (TCs). Shot depletion leads to a defective or absent lumen while conversely, Shot overexpression promotes excessive branching, independently on the regulation of centrosome numbers previously shown to be important for the regulation of the lumen formation process (Ricolo, D., Deligiannaki, M., Casanova, J. & Araújo, S. J. Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells. Current Biology 26, 2805-2813 (2016)). Shot is rather important to regulate the organization of the cytoskeleton by crosslinking MTs and actin. Shot expression in TCs is controlled by the Drosophila Serum Response Factor (DSRF) transcription factor. Finally Shot functionally overlaps with the MT-stabilizing protein Tau to promote lumen morphogenesis.

    The figures are clear and the questions well addressed with carefully designed and controlled experiments. However, I would have few suggestions that will hopefully make some points clearer.

    **Major comments:**

    -Statistical analyses should be added for comparisons of proportions, including Fig. 1E, 1L, Fig. 2G-I, Fig. 6L, Fig. 7K, Fig. 8C-D and Fig. 9G.

    We agree with this and have now redone all graphs and revised all quantifications from this study. We have added error bars in all above mentioned graphs and have provided statistical analysis where appropriate. We have also redone all graphics and phenotype reporting, which is done now in relation to total TCs (rather than embryos or GBs and DBs TCs). This was suggested also by reviewer #2 and we agree because this is a more stringent and comparable way of quantifying our results.

    -It is not always clear what genotype has been used as the "wt" genotype, as in Fig. S2 or Fig. 3 for example, this should be added to figure legends.

    We have now clarified which flies are used as controls in each experiment throughout the paper. We have left wt where flies were wt, and changed all other cases to either the genotype or “control”.

    -Live imaging of Shot has been performed with ShotC-GFP, that cannot bind actin. Don't the authors think ShotA-GFP would reflect more accurately Shot endogenous behavior as it interacts both with actin and MTs? It would be better to show this, even if the results shown here tend to be consistent with Shot endogenous localization shown with Shot antibody staining.

    We agree and we will analyse movies with both ShotC and ShotA and present them in the revised version.

    -It is of course not possible to generate CRISPR mutant flies with mutations in putative DSRF binding sites in a reasonable amount of time, to confirm that Shot transcription is controlled by DSRF. It would thus be nice to reveal shot mRNA expression with in situ hybridization experiments in wt vs. bs embryos. This would confirm that Shot mRNA is downregulated upon DSRF inhibition and rule out a possible indirect effect on Shot protein stability for example.

    We believe the presented 3-way approach (in silico, protein quantification and phenotype rescue) is sufficient to show that Shot expression is regulated by DSRF. It is unlikely that we are dealing with protein stability or other issues, because we can rescue the lumen elongation phenotype by solely expressing Shot in TCs. However, we agree it would be nice to show this in an in situ hybridization experiment, and we will try to provide a conclusive one for resubmission. In situ detection methods, however, may not be accurate enough to detect such differences in single-cells.

    -In the same figure, it would also be interesting to show what happens to actin and MTs in bs TCs and to which extent their organization is rescued by Shot overexpression.

    We are working on this for resubmission. These experiments were frozen by the current COVID-19 pandemic and this is why they were not submitted with the first version.

    -UAS-EB1GFP does not seem to be an appropriate control in Figure 9 (A and B) since it can affect MT dynamics (Vitre, B. et al. EB1 regulates microtubule dynamics and tubulin sheet closure in vitro. Nat. Cell Biol. 10, 415-421 (2008)). Why not simply use an UAS-GFP?

    We have not detected any notorious larval TC phenotypes by overexpressing UASEB1GFP in TCs. Their branching is comparable to that in previous studies (for example Schotenfeld-Roames, et al Current Biology 2014) and there were no detectable luminal branching phenotypes. However, we agree it is more correct to analyse cells with a plain GFP and have repeated the controls for this experiment using DSRFGAL4UASGFP. This is now shown in figure 9.

    -Shot and probably Tau crosslinking activities are important for lumen morphogenesis with a striking increase in the number of embryos without lumen in shot3 and shot3 tauMR22 mutant embryos. The rescue experiments clearly show that Shot binding to both MT and actin is essential for efficient rescue. The same might apply to Tau since it is able to crosslink actin and MTs (Elie, A. et al. Tau co-organizes dynamic microtubule and actin networks. Sci Rep 5, 1-10 (2015)). I believe showing actin and MTs organization in these rescue experiments would be necessary.

    We agree and we will provide these experiments upon resubmission.

    Second, the overexpression experiments indicate that Shot is able to induce extra lumen formation even when unable to bind actin as shown with the increase in the number of supernumerary lumina (ESLs) under overexpression of ShotC and ShotCtail to a lesser extent. This phenotype is also observed under Tau overexpression. This suggest that not crosslinking anymore but rather making MTs more stable could be sufficient to promote extra lumen formation in a wt context. Stabilising MTs by treatment with Taxol might thus be sufficient to promote ESL formation. I am fully aware of the difficulty of treating Drosophila embryos with drugs, making this experiment hard to do, but I think this dual function of Shot and Tau (crosslinking actin and MTs to promote branching vs. stabilizing MTs leading to excessive branching) should be discussed.

    In Figure 2 we show not just that UASShotC is able to induce ESl but also that UAS-ShotCtail containing only the MT binding domain of Shot is enough to induce ESLs in TCs, whereas UAS-deltaCtail is not. We agree Taxol treatment would be a nice experiment to do, however we also think we provide enough evidence that MT stability is enough for ESL whereas de novo lumen formation requires crosslinking of MTs to actin. As advised, we will discuss better both Shot and Tau dual function in ESL generation and de novo lumen formation for resubmission.

    **Minor comments:**

    We have already addressed most these minor comments in the manuscript (text revised and changes in yellow). And we provide answers to some of the comments below.

    -p2 line 1: 'acentrosomal luminal branching points' may be better than 'acentrosomal branching points' to describe the phenotype.

    -p4, line 16: the reference 23 is not properly inserted (should be after 'closure').

    -p5, line 16: Please mention what the abbreviations Bnl and Btn stand for.

    -p5, line 20: these 80% of TCs cells with defects in subcellular lumen formation should appear on the graph in Fig. 1E (as shown in graph 1L).

    We have added shot RNAi results to graph E in figure 1.

    -p5, line 26: this 36% value does not seem to correspond to anything on the graph in Fig. 1N. According to the figure legend, 20% of TCs did not elongate at all and the lumen was completely absent (class IV), which is consistent with the result shown in Fig. 1L.

    Also, I am not sure why only 25 TCs were analysed in Fig. 1N while there are the data to analyse more as shown in Fig. 1E (400 TCs), this would make the graph more representative.

    Figure 1 N represents a detail of the different phenotypes present in shot mutant embryos. Whereas for most of the paper we consider only complete lack of TC lumen, here we show the different types of affected TCs and not just the ones with a complete lack of subcellular lumen. We apologise because it was not explained in the original manuscript that types III and IV are the “no lumen” class (they were subdivided into 2 classes because they have different cell enlongation phenotypes). 36% of the total of affected TCs displayed the lack of lumen phenotype (this means a 22,5 % of the total number of TCs, because total affected TCs are 62,5% only). Numbers are similar but not exactly the same because this analysis was done using confocal microscopy and cells analysed one by one in detail, which is not possible using colorimetric methods and only luminal markers. This is also the reason we only analysed 25 TCs in this case. We thank the reviewer for pointing this out and have better described it in the manuscript.

    -p6, line 8: ShotA-GFP is indeed a long isoform but is not the full-length Shot, as it does not contain the plakin repeat exon which would add another ~3000aa.

    We have corrected this.

    -p6, lines 21-23: ShotA-GFP localisation is not shown in FigS1. The authors should refer to Fig. 2. Enlarged areas/arrows might help the reader to better visualise the different localisations of ShotA-GFP and ShotC-GFP.

    We thank the reviewer for this request and we will change the figure providing enlarged areas upon resubmission. In this version of the manuscript we have already changed the error in figure referral in the text.

    -p7, line 23: Rca1 mutants should be better introduced here.

    We have added one sentence of introduction to the Rca1 phenotype.

    -p8, line 6: Shot colocalizes/associates with stable MTs and actin would be a more appropriate title for this paragraph.

    We thank the reviewer for this alternative, and we have changed this title in the manuscript.

    -p16, line 18: 'Shot is able to mediate crosstalk' would be better than 'Shot is able to crosstalk'.

    -p40, lines 6 and 7: L, M and N should be K', K' and K' respectively.

    -p41, Fig 10D: It is quite hard to see on the cartoon what the phenotype is for Shot OE.

    We will make this clearer for resubmission.

    -The following reference shows an important role for Shot in crosslinking actin and MTs during morphogenesis of the Drosophila embryo and should be cited in this manuscript (Booth, A. J. R., Blanchard, G. B., Adams, R. J. & Röper, K. A Dynamic Microtubule Cytoskeleton Directs Medial Actomyosin Function during Tube Formation. Developmental Cell 29, 562-576 (2014)).

    We thank the reviewer for pointing this out, because this is of course an important reference known to us, which we forgot to add. We have now added this to the manuscript.

    -FigS3. It would be good to add the labels on the figure (ShotC-GFP in green, and MoeRFP/lifeActinRFP in Magenta).

    We will do this for resubmission.

    Reviewer #1 (Significance (Required)):

    The findings shown in this manuscript shed an important light on the way subcellular morphogenesis occurs. It was known that both actin and MTs were required in this process, particularly during the formation of Drosophila trachea (JayaNandanan, N., Mathew, R. & Leptin, M. Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin. Nature Communications 1-10 (2019). doi:10.1038/ncomms4036; Gervais, L. & Casanova, J. In Vivo Coupling of Cell Elongation and Lumen Formation in a Single Cell. Current Biology 20, 359-366 (2010)). This work provides additional molecular insights into the way branching morphogenesis from a single cell occurs in vivo, clearly demonstrating a requirement for actin-MT crosslinking mediated by Shot and Tau.

    This could be of great interest in the field of branching morphogenesis and lumen formation, not only in invertebrates but also in vertebrates where such a crosslinking might occur in the vasculature, the lung, the kidney or the mammary gland for example (Ochoa-Espinosa, A. & Affolter, M. Branching Morphogenesis: From Cells to Organs and Back. Cold Spring Harb Perspect Biol 4, a008243-a008243 (2012)).

    *Field of expertise:* morphogenesis, Drosophila, cytoskeleton, microtubules.

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

    **Summary:**

    The development of branched structures with intracellular lumen is widely observed in single cells of circulatory systems. However the molecular and cellular mechanisms of this complex morphogenesis are largely unknown. In previous study, the authors revealed that centrosome as a microtubule organizing center (MTOC) located at the apical junction contributes subcellular lumen formation in the terminal cells of Drosophila tracheal system. The microtubule bundles organized by MTOC are suggested to serve as trafficking mediators and structural stabilizers for the newly elongated lumen.

    In this manuscript, they focused on a Drosophila spectraplakin, Shot, which have been reported to crosslink MT minus-ends to actin network, in the subcellular lumen formation. The paper started by description of lumen elongation defect of the tracheal terminal cells in the shot[3] null mutant. The overexpression of full-length and series of truncated form of shot exhibited extra-subcellular lumina (ESL) in TCs, suggesting that Shot is required for the lumen formation in dose dependent manner. They next addressed whether Shot overexpression induces ESL through the supernumerary centrosomes as in Rca1 mutant, however the number of centrosomes was not affected. Moreover, the ESL were sprouted distally from the apical junction, suggesting that Shot operate in different way from the Rca1-dependent microtubule organization. To get mechanistic insight of Shot in the luminal formation, they checked localization of the Shot and found it localized with stable MTs around the nascent lumen and with the F-actin at the tip of the cell during the cell elongation and subcellular lumen formation. In shot[3] mutant, the MT-bundles were no longer localized to apical region and the actin accumulation at the tip of the cell was also reduced. The rescue experiments using several truncated forms of Shot, and well-designed genetic analysis using various shot mutants revealed that both MT binding domain and actin binding domains are needed to develop the lumen. The expression of shot was under the regulation by terminal cell-specific transcription factor bs/DSRF, and the overexpression of shot in bs LOF mutant suppressed its phenotype, indicated that part of the luminal phenotype of bs mutant in terminal cells are due to lower levels of the activity of shot. Finally, they checked whether Tau can compensate the function of shot in the subcellular lumen formation. The lumen elongation defect in shot mutant was suppressed by tau expression, and tau overexpression phenocopied the shot overexpression-induced ESL. Although tau mutant did not show the lumen formation defects, the double mutant of shot and tau exhibited synergistic effect. Shot was also required for subcellular luminal branching at larval stages.

    Overall, this work highlighted the importance of Shot as a crosslinker between MT and actin that acts in downstream of the FGF signaling-induced bs/DSRF expression for the subcellular lumen formation. An excess of Shot is sufficient for ESL formation from ectopic acentrosomal branching points. Furthermore, the Tau protein can functionally replace Shot in this context.

    **Major comments:**

    *- Are the key conclusions convincing?* *- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?*

    The conclusions were basically supported by the set of data presented in this article, but following points need to be clarified.

    The truncated form ShotC lacks only half of calponin domain that are essential for the actin binding, thus it is still possible to bind actin to some extent. Although the actin binding activity is reported as "very weak" in the cited references, the quantitative analysis has not been done. Thus, the interpretation and claims based on the experiments using ShotC should be reviewed carefully.

    We agree with the reviewer and will revise all the text for resubmission in order to make this unambiguous. However, we would like to remark that our claims are not only based on UAS-ShotC but also in the shotkakP2 allele, which does not contain one of the calponin domains and in isoforms such UAS-Shot C-tail which do not have any ABD.

    Data set in some places seems fragmented. For example, overexpression study of shot constructs (Fig. 2) lacks phenotypic comparison of control (btl Gal4 driven control FP) to compare if phenotypes of shot constructs expression are different from control. Different methods of phenotypic quantification are employed. One was counting embryo number with at least one abnormality among 20 TCs of DB or GB, or the other counting every TC for the presence of lumen/branching conditions. The latter is more stringent measure and is more appropriate for the study of single cell morphogenesis.

    We totally agree with the reviewer. We have now revised all quantifications and graphs:

    1. We have used btl>GFP as control to all overexpression experiments in embryos and DSRFGAL4UASGFP in control larvae.

    2. We have made the paper uniform regarding quantifications, which are now all done in relation to total TCs and not embryos.

    For this reason, many of the graphs, figure legends and quantification values in the the manuscript text are now changed.

    *- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.*

    The all movies were using ShotC isoform which lacks half of the actin binding domain. The truncated isoform is not suitable to observe the localization, especially the colocalization with actin. The movies need to be retaken using full-length Shot at the dosage that does not interfere with normal TC development.

    We agree and we will analyse movies with both ShotC and ShotA for resubmission.

    Some statements on Moesin and Tau localization sound as if the authors studied Shot interaction with nascent Moe and Tau molecules. This is confusing because fragments of Moe and Tau, but not functional full length proteins, were used.

    We will revise the text to make this unambiguous fir resubmission.

    *- Are the suggested experiments realistic in terms of time and resources?* It would help if you could add an estimated cost and time investment for substantial experiments.

    Because the transgenic fly is already present, we assume it would be done in 4 weeks. However, it would be influnced under social circumstances whether the lab facilities are able to access or not.

    *- Are the data and the methods presented in such a way that they can be reproduced?* *- Are the experiments adequately replicated and statistical analysis adequate?*

    The methods provided seem to be sufficient for reproducing the data by competent researchers, and most of the data are solid and the sample numbers are sufficient for the claims. However, the criteria for phenotypic evaluation differs among graphs and figures, that possibly confuse the readers. Standardized measurement methods are desirable.

    **Minor comments:**

    *- Specific experimental issues that are easily addressable.*

    In the rescue experiments shown in Figure 6, only full-length Shot rescued the subcellular lumen formation, but either of truncated Shot did not. The localization study of MT and actin in those conditions will reveal whether proper localizations of actin and MT are critical for the lumen formation.

    We are working on this for resubmission. These experiments were stalled by the current COVID-19 pandemic and this is why they were not submitted with the first version. We will provide MT and actin localization for the rescue experiments with ShotA and ShotC.

    *- Are prior studies referenced appropriately?*

    The references are cited appropriately.

    *- Are the text and figures clear and accurate?*

    There are several typos: Remodelling -> remodeling, signalling -> signaling. In the figure 2, G and H seem redundant. Scale bars are missing in Fig1 F-K, Fig2 K-L, Fig6 A-I, Fig7 E-J and Fig8 E-J.

    We have changed the graphs in figure 2. Typos have been corrected. We will provide errors bars for resubmission.

    The author often called shot+ genotype as "wild type". They are transgenic strains with some mutations, and cannot be found in the wild. They should be simply called with genotype or "control" for experiments.

    We thank the reviewer for pointing these typos and incoherences with control genotypes. We have partly revise the text and figures and will finish for resubmission.

    *- Do you have suggestions that would help the authors improve the presentation of their data and conclusions?*

    In Figure 4, as the localization of Shot is difficult to see in detail, enlarged insets might help. In addition, the green and cyan in C'-E' is difficult to distinguish.

    We will change this for resubmission.

    With Figure 5, the authors claimed that Shot LOF leads to disorganized MT-bundles and actin localization. We feel this is an overstatement and the Figure should be backed up with better data, or removed. F-actin and microtubule localizations are highly dynamic and the snapshot pictures are insufficient for demonstrating defective localization. It is also possible that (potential) difference in the marker localization is due to indirect effect of Shot LOF in cell shape.

    We agree with the reviewer that fixed samples are not the best to analyse cytoskeletal components, but we observe clear differences in MT bundles and specially in actin localization in shot mutants as compared to controls and we believe it is important to show these results. Cell shape might of course alter the analysis which is why we present 3 different cell shapes in Figure 5. In addition, there are many previous studies where localization of MTs and actin was done in fixed mutant embryos, where cell shape is also affected, and revealed important steps in TC formation (Gervais and Casanova, 2010; JayanNadanan et al. 2014).Nonetheless, we have revised the text in order to avoid overstatements.

    Reviewer #2 (Significance (Required)):

    *- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.*

    *- Place the work in the context of the existing literature (provide references, where appropriate).*

    In blood capillary and insect trachea, the branching process of single vessel cells involves sprouting of cell protrusions, followed by the lumen extension from the main vessels. The lumen formation involves assembly of plasma membrane components inside of the cytoplasm. Since the luminal membrane is associated with protein complexes common to apical cell membrane, lumen formation is believed to involve redirection of apical trafficking of membranes to intracellular sites (Sigurbjörnsdóttir, Mathew, Leptin 2014, 10.1038/nrm3871). The authors previously demonstrated that centrosome is an important link of preexisting lumen to de novo lumen formation, leading to the hypothesis that centrosome-derived microtubules organize lumen membrane assembly.

    *- State what audience might be interested in and influenced by the reported findings.*

    In this manuscript, the authors addressed this issue by looking at the function of Shot/Plakin that has both microtubule and actin binding activities. Shot is an ideal candidate for linking actin-rich cell protrusions in the leading edge to centrosome- associated lumen tip. Indeed the authors clearly showed that shot is required for lumen extension and overexpressed shot protein associates with intracellular tract rich in microtubules and F-actin. Their findings are definitely a progress in the field of Drosophila tracheal development. Having said that, how Shot links leading edge protrusions and centrosomes, how it is organized into pre-lumen tract, and how it contribute to further assembly of luminal membrane and directed secretion, are not well understood yet. Without clues to those fundamental questions, I believe this paper is most appropriate for experts readers of Drosophila cell biology and tracheal development.

    Finally I feel that the paper include many data sets and some pictures are not easy to grasp essential points, such as three movies showing localization of overexpressed shot-C, RFP-moesin, and Lifeact.

    *- Define your field of expertise with a few keywords to help the authors contextualize your point of view.*

    Drosophila, tracheal cell biology.

    *- Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.*

    No

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

    **Summary**

    In their manuscript entitled "Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching" Ricolo and Araujo characterize the requirement for Short Stop (Shot) in the formation of subcellular tubes in tracheal terminal cells.

    The authors examined embryos homozygous for shot3, a presumed null allele of shot. They found an 80% penetrant defect in seamless tube formation or growth. The phenotype resembles that reported for mutations in blistered, which encodes the Drosophila SRF ortholog. The authors find that expression of SRF is not blocked by mutations in shot and later find that bs mutants have decreased levels of shot expression and that shot overexpression can partly suppress the bs tube formation defects.

    The authors then examine whether the requirement for shot is autonomous to the trachea and find that it is, as pan-tracheal shot RNAi replicates the seamless tube defects.

    The authors find that overexpression of various Shot isoforms results in the formation of ectopic seamless tubes within terminal cells. Using the various transgenic constructs available for shot, the authors show that the overexpression phenotype is dependent upon the interaction between Shot and microtubules, and is dose-dependent.

    Previous work had shown that ectopic terminal cell tubes also can arise due to increased centrosome number; the authors show that centrosome number is not altered in shot mutants.

    Shot has well characterized actin and microtubule binding functions, and the authors show that Shot localization overlaps both with microtubules and with actin, and that both cytoskeletal elements are aberrant in shot mutant cells. In a series of experiments utilizing various shot mutant backgrounds and shot transgenes, the authors identify requirements for both Shot-cytoskeleton interactions in the formation and branching of seamless tubes in terminal cells.

    Finally, the authors examine the requirement for Tau in the same processes. Tau and Shot had previously been found to work together in neurons, and this seems to be true in terminal cells as well. Tau overexpression induces ectopic seamless tubes and can partially suppress shot loss of function. Embryos mutant for tau showed seamless tube directionality defects, but not lumen formation or branching. Embryos doubly mutant for tau and shot showed a more severe seamless tube defect than shot mutants alone - an increase in terminal cells with no lumen from 22% to 85%.

    Authors also examined terminal cells in larval stages using dsrf-Gal4 to knockdown shot in terminal cells (rather than pan-tracheal knockdown with breathless).

    The authors conclude from their studies that Shot, through its interactions with microtubules and the actin cytoskeleton coordinate the outgrowth and branching of subcellular tubes. Overlapping function of Tau and possibly other additional MAPs also act in these processes.

    The work is largely well done and the conclusions are supported by the data.

    **Minor concerns:**

    -If one were to start this work today, crispr knockout and knockins would be preferred. While shot^3 is widely considered a null allele, there are indications that some shot function is still present in shot^3 embryos. This would also be relevant to the penetrance of the defects. The transgenes are useful, but given the dosage effects noted in various of the authors experiments, interpretation of some experiments is complicated as compared to a knockin. For overexpression experiments, landing site constructs would be preferable. I do not mean to suggest that the authors necessarily go this route, but am just pointing out a limitation of the approach.

    We agree, but we also think that with the amount of data and tools generated by other labs over recent years, regarding shot function in the nervous system (Voelzmann et al 2017), we are in a position to be able to take the conclusions of this work based on these transgenic and different shot alleles.

    -Insight into function at higher resolution than altered microtubule and actin organization would significantly increase the impact.

    -cell autonomy (line 19, p5) is not the correct term. Pan-tracheal knockdown tests tissue autonomy. Mosaic analysis or terminal cell specific knockdown would address cell autonomy.

    We have changed the manuscript accordingly.

    -line 14 p6 acting should be actin

    -dsrf-Gal4 transgenes were made by Mark Metzstein

    We have corrected these.

    -there also appears to be rescue of the fusion cell defects of shot by Tau overexpression. Authors should comment on this and what it means for the seamless tubulogenesis program in terminal cells vs fusion cells.

    We will reanalyse shot rescued with tau embryos focusing on fusion phenotypes and discuss this in the revised version.

    Reviewer #3 (Significance (Required)):

    The findings will be of interest to a broad cell biology community as they provide a conceptual advance and may help to focus future work on seamless tubulogenesis. The authors do a good job of placing the results in the context of previous studies.

    *Field of expertise:* Drosophila, tracheal tubulogenesis, developmental biology

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    In their manuscript entitled "Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching" Ricolo and Araujo characterize the requirement for Short Stop (Shot) in the formation of subcellular tubes in tracheal terminal cells.

    The authors examined embryos homozygous for shot3, a presumed null allele of shot. They found an 80% penetrant defect in seamless tube formation or growth. The phenotype resembles that reported for mutations in blistered, which encodes the Drosophila SRF ortholog. The authors find that expression of SRF is not blocked by mutations in shot and later find that bs mutants have decreased levels of shot expression and that shot overexpression can partly suppress the bs tube formation defects.

    The authors then examine whether the requirement for shot is autonomous to the trachea and find that it is, as pan-tracheal shot RNAi replicates the seamless tube defects.

    The authors find that overexpression of various Shot isoforms results in the formation of ectopic seamless tubes within terminal cells. Using the various transgenic constructs available for shot, the authors show that the overexpression phenotype is dependent upon the interaction between Shot and microtubules, and is dose-dependent.

    Previous work had shown that ectopic terminal cell tubes also can arise due to increased centrosome number; the authors show that centrosome number is not altered in shot mutants.

    Shot has well characterized actin and microtubule binding functions, and the authors show that Shot localization overlaps both with microtubules and with actin, and that both cytoskeletal elements are aberrant in shot mutant cells. In a series of experiments utilizing various shot mutant backgrounds and shot transgenes, the authors identify requirements for both Shot-cytoskeleton interactions in the formation and branching of seamless tubes in terminal cells.

    Finally, the authors examine the requirement for Tau in the same processes. Tau and Shot had previously been found to work together in neurons, and this seems to be true in terminal cells as well. Tau overexpression induces ectopic seamless tubes and can partially suppress shot loss of function. Embryos mutant for tau showed seamless tube directionality defects, but not lumen formation or branching. Embryos doubly mutant for tau and shot showed a more severe seamless tube defect than shot mutants alone - an increase in terminal cells with no lumen from 22% to 85%.

    Authors also examined terminal cells in larval stages using dsrf-Gal4 to knockdown shot in terminal cells (rather than pan-tracheal knockdown with breathless).

    The authors conclude from their studies that Shot, through its interactions with microtubules and the actin cytoskeleton coordinate the outgrowth and branching of subcellular tubes. Overlapping function of Tau and possibly other additional MAPs also act in these processes.

    The work is largely well done and the conclusions are supported by the data.

    Minor concerns:

    -If one were to start this work today, crispr knockout and knockins would be preferred. While shot^3 is widely considered a null allele, there are indications that some shot function is still present in shot^3 embryos. This would also be relevant to the penetrance of the defects. The transgenes are useful, but given the dosage effects noted in various of the authors experiments, interpretation of some experiments is complicated as compared to a knockin. For overexpression experiments, landing site constructs would be preferable. I do not mean to suggest that the authors necessarily go this route, but am just pointing out a limitation of the approach.

    -Insight into function at higher resolution than altered microtubule and actin organization would significantly increase the impact.

    -cell autonomy (line 19, p5) is not the correct term. Pan-tracheal knockdown tests tissue autonomy. Mosaic analysis or terminal cell specific knockdown would address cell autonomy.

    -line 14 p6 acting should be actin

    -dsrf-Gal4 transgenes were made by Mark Metzstein

    -there also appears to be rescue of the fusion cell defects of shot by Tau overexpression. Authors should comment on this and what it means for the seamless tubulogenesis program in terminal cells vs fusion cells.

    Significance

    The findings will be of interest to a broad cell biology community as they provide a conceptual advance and may help to focus future work on seamless tubulogenesis. The authors do a good job of placing the results in the context of previous studies.

    Field of expertise: Drosophila, tracheal tubulogenesis, developmental biology

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The development of branched structures with intracellular lumen is widely observed in single cells of circulatory systems. However the molecular and cellular mechanisms of this complex morphogenesis are largely unknown. In previous study, the authors revealed that centrosome as a microtubule organizing center (MTOC) located at the apical junction contributes subcellular lumen formation in the terminal cells of Drosophila tracheal system. The microtubule bundles organized by MTOC are suggested to serve as trafficking mediators and structural stabilizers for the newly elongated lumen.

    In this manuscript, they focused on a Drosophila spectraplakin, Shot, which have been reported to crosslink MT minus-ends to actin network, in the subcellular lumen formation. The paper started by description of lumen elongation defect of the tracheal terminal cells in the shot[3] null mutant. The overexpression of full-length and series of truncated form of shot exhibited extra-subcellular lumina (ESL) in TCs, suggesting that Shot is required for the lumen formation in dose dependent manner. They next addressed whether Shot overexpression induces ESL through the supernumerary centrosomes as in Rca1 mutant, however the number of centrosomes was not affected. Moreover, the ESL were sprouted distally from the apical junction, suggesting that Shot operate in different way from the Rca1-dependent microtubule organization. To get mechanistic insight of Shot in the luminal formation, they checked localization of the Shot and found it localized with stable MTs around the nascent lumen and with the F-actin at the tip of the cell during the cell elongation and subcellular lumen formation. In shot[3] mutant, the MT-bundles were no longer localized to apical region and the actin accumulation at the tip of the cell was also reduced. The rescue experiments using several truncated forms of Shot, and well-designed genetic analysis using various shot mutants revealed that both MT binding domain and actin binding domains are needed to develop the lumen. The expression of shot was under the regulation by terminal cell-specific transcription factor bs/DSRF, and the overexpression of shot in bs LOF mutant suppressed its phenotype, indicated that part of the luminal phenotype of bs mutant in terminal cells are due to lower levels of the activity of shot. Finally, they checked whether Tau can compensate the function of shot in the subcellular lumen formation. The lumen elongation defect in shot mutant was suppressed by tau expression, and tau overexpression phenocopied the shot overexpression-induced ESL. Although tau mutant did not show the lumen formation defects, the double mutant of shot and tau exhibited synergistic effect. Shot was also required for subcellular luminal branching at larval stages.

    Overall, this work highlighted the importance of Shot as a crosslinker between MT and actin that acts in downstream of the FGF signaling-induced bs/DSRF expression for the subcellular lumen formation. An excess of Shot is sufficient for ESL formation from ectopic acentrosomal branching points. Furthermore, the Tau protein can functionally replace Shot in this context.

    Major comments:

    - Are the key conclusions convincing? - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The conclusions were basically supported by the set of data presented in this article, but following points need to be clarified.

    The truncated form ShotC lacks only half of calponin domain that are essential for the actin binding, thus it is still possible to bind actin to some extent. Although the actin binding activity is reported as "very weak" in the cited references, the quantitative analysis has not been done. Thus, the interpretation and claims based on the experiments using ShotC should be reviewed carefully.

    Data set in some places seems fragmented. For example, overexpression study of shot constructs (Fig. 2) lacks phenotypic comparison of control (btl Gal4 driven control FP) to compare if phenotypes of shot constructs expression are different from control. Different methods of phenotypic quantification are employed. One was counting embryo number with at least one abnormality among 20 TCs of DB or GB, or the other counting every TC for the presence of lumen/branching conditions. The latter is more stringent measure and is more appropriate for the study of single cell morphogenesis.

    - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    The all movies were using ShotC isoform which lacks half of the actin binding domain. The truncated isoform is not suitable to observe the localization, especially the colocalization with actin. The movies need to be retaken using full-length Shot at the dosage that does not interfere with normal TC development.

    Some statements on Moesin and Tau localization sound as if the authors studied Shot interaction with nascent Moe and Tau molecules. This is confusing because fragments of Moe and Tau, but not functional full length proteins, were used.

    - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Because the transgenic fly is already present, we assume it would be done in 4 weeks. However, it would be influnced under social circumstances whether the lab facilities are able to access or not.

    - Are the data and the methods presented in such a way that they can be reproduced? - Are the experiments adequately replicated and statistical analysis adequate?

    The methods provided seem to be sufficient for reproducing the data by competent researchers, and most of the data are solid and the sample numbers are sufficient for the claims. However, the criteria for phenotypic evaluation differs among graphs and figures, that possibly confuse the readers. Standardized measurement methods are desirable.

    Minor comments:

    - Specific experimental issues that are easily addressable.

    In the rescue experiments shown in Figure 6, only full-length Shot rescued the subcellular lumen formation, but either of truncated Shot did not. The localization study of MT and actin in those conditions will reveal whether proper localizations of actin and MT are critical for the lumen formation.

    - Are prior studies referenced appropriately?

    The references are cited appropriately.

    - Are the text and figures clear and accurate?

    There are several typos: Remodelling -> remodeling, signalling -> signaling. In the figure 2, G and H seem redundant. Scale bars are missing in Fig1 F-K, Fig2 K-L, Fig6 A-I, Fig7 E-J and Fig8 E-J.

    The author often called shot+ genotype as "wild type". They are transgenic strains with some mutations, and cannot be found in the wild. They should be simply called with genotype or "control" for experiments.

    - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    In Figure 4, as the localization of Shot is difficult to see in detail, enlarged insets might help. In addition, the green and cyan in C'-E' is difficult to distinguish.

    With Figure 5, the authors claimed that Shot LOF leads to disorganized MT-bundles and actin localization. We feel this is an overstatement and the Figure should be backed up with better data, or removed. F-actin and microtubule localizations are highly dynamic and the snapshot pictures are insufficient for demonstrating defective localization. It is also possible that (potential) difference in the marker localization is due to indirect effect of Shot LOF in cell shape.

    Significance

    - Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    - Place the work in the context of the existing literature (provide references, where appropriate).

    In blood capillary and insect trachea, the branching process of single vessel cells involves sprouting of cell protrusions, followed by the lumen extension from the main vessels. The lumen formation involves assembly of plasma membrane components inside of the cytoplasm. Since the luminal membrane is associated with protein complexes common to apical cell membrane, lumen formation is believed to involve redirection of apical trafficking of membranes to intracellular sites (Sigurbjörnsdóttir, Mathew, Leptin 2014, 10.1038/nrm3871). The authors previously demonstrated that centrosome is an important link of preexisting lumen to de novo lumen formation, leading to the hypothesis that centrosome-derived microtubules organize lumen membrane assembly.

    - State what audience might be interested in and influenced by the reported findings.

    In this manuscript, the authors addressed this issue by looking at the function of Shot/Plakin that has both microtubule and actin binding activities. Shot is an ideal candidate for linking actin-rich cell protrusions in the leading edge to centrosome- associated lumen tip. Indeed the authors clearly showed that shot is required for lumen extension and overexpressed shot protein associates with intracellular tract rich in microtubules and F-actin. Their findings are definitely a progress in the field of Drosophila tracheal development. Having said that, how Shot links leading edge protrusions and centrosomes, how it is organized into pre-lumen tract, and how it contribute to further assembly of luminal membrane and directed secretion, are not well understood yet. Without clues to those fundamental questions, I believe this paper is most appropriate for experts readers of Drosophila cell biology and tracheal development.

    Finally I feel that the paper include many data sets and some pictures are not easy to grasp essential points, such as three movies showing localization of overexpressed shot-C, RFP-moesin, and Lifeact.

    - Define your field of expertise with a few keywords to help the authors contextualize your point of view.

    Drosophila, tracheal cell biology.

    - Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    No

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    Referee #1

    Evidence, reproducibility and clarity

    This study provides solid evidences showing a role for the spectraplakin Short-stop (Shot) in subcellular lumen formation in the Drosophila embryonic and larval trachea. This subcellular morphogenetic process relies on an inward membrane growth that depends on the proper organization of actin and microtubules (MTs) in terminal cells (TCs). Shot depletion leads to a defective or absent lumen while conversely, Shot overexpression promotes excessive branching, independently on the regulation of centrosome numbers previously shown to be important for the regulation of the lumen formation process (Ricolo, D., Deligiannaki, M., Casanova, J. & Araújo, S. J. Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells. Current Biology 26, 2805-2813 (2016)). Shot is rather important to regulate the organization of the cytoskeleton by crosslinking MTs and actin. Shot expression in TCs is controlled by the Drosophila Serum Response Factor (DSRF) transcription factor. Finally Shot functionally overlaps with the MT-stabilizing protein Tau to promote lumen morphogenesis.

    The figures are clear and the questions well addressed with carefully designed and controlled experiments. However, I would have few suggestions that will hopefully make some points clearer.

    Major comments:

    -Statistical analyses should be added for comparisons of proportions, including Fig. 1E, 1L, Fig. 2G-I, Fig. 6L, Fig. 7K, Fig. 8C-D and Fig. 9G.

    -It is not always clear what genotype has been used as the "wt" genotype, as in Fig. S2 or Fig. 3 for example, this should be added to figure legends.

    -Live imaging of Shot has been performed with ShotC-GFP, that cannot bind actin. Don't the authors think ShotA-GFP would reflect more accurately Shot endogenous behavior as it interacts both with actin and MTs? It would be better to show this, even if the results shown here tend to be consistent with Shot endogenous localization shown with Shot antibody staining.

    -It is of course not possible to generate CRISPR mutant flies with mutations in putative DSRF binding sites in a reasonable amount of time, to confirm that Shot transcription is controlled by DSRF. It would thus be nice to reveal shot mRNA expression with in situ hybridization experiments in wt vs. bs embryos. This would confirm that Shot mRNA is downregulated upon DSRF inhibition and rule out a possible indirect effect on Shot protein stability for example.

    -In the same figure, it would also be interesting to show what happens to actin and MTs in bs TCs and to which extent their organization is rescued by Shot overexpression.

    -UAS-EB1GFP does not seem to be an appropriate control in Figure 9 (A and B) since it can affect MT dynamics (Vitre, B. et al. EB1 regulates microtubule dynamics and tubulin sheet closure in vitro. Nat. Cell Biol. 10, 415-421 (2008)). Why not simply use an UAS-GFP?

    -Shot and probably Tau crosslinking activities are important for lumen morphogenesis with a striking increase in the number of embryos without lumen in shot3 and shot3 tauMR22 mutant embryos. The rescue experiments clearly show that Shot binding to both MT and actin is essential for efficient rescue. The same might apply to Tau since it is able to crosslink actin and MTs (Elie, A. et al. Tau co-organizes dynamic microtubule and actin networks. Sci Rep 5, 1-10 (2015)). I believe showing actin and MTs organization in these rescue experiments would be necessary.

    Second, the overexpression experiments indicate that Shot is able to induce extra lumen formation even when unable to bind actin as shown with the increase in the number of supernumerary lumina (ESLs) under overexpression of ShotC and ShotCtail to a lesser extent. This phenotype is also observed under Tau overexpression. This suggest that not crosslinking anymore but rather making MTs more stable could be sufficient to promote extra lumen formation in a wt context. Stabilising MTs by treatment with Taxol might thus be sufficient to promote ESL formation. I am fully aware of the difficulty of treating Drosophila embryos with drugs, making this experiment hard to do, but I think this dual function of Shot and Tau (crosslinking actin and MTs to promote branching vs. stabilizing MTs leading to excessive branching) should be discussed.

    Minor comments:

    -p2 line 1: 'acentrosomal luminal branching points' may be better than 'acentrosomal branching points' to describe the phenotype.

    -p4, line 16: the reference 23 is not properly inserted (should be after 'closure').

    -p5, line 16: Please mention what the abbreviations Bnl and Btn stand for.

    -p5, line 20: these 80% of TCs cells with defects in subcellular lumen formation should appear on the graph in Fig. 1E (as shown in graph 1L).

    -p5, line 26: this 36% value does not seem to correspond to anything on the graph in Fig. 1N. According to the figure legend, 20% of TCs did not elongate at all and the lumen was completely absent (class IV), which is consistent with the result shown in Fig. 1L.

    Also, I am not sure why only 25 TCs were analysed in Fig. 1N while there are the data to analyse more as shown in Fig. 1E (400 TCs), this would make the graph more representative.

    -p6, line 8: ShotA-GFP is indeed a long isoform but is not the full-length Shot, as it does not contain the plakin repeat exon which would add another ~3000aa.

    -p6, lines 21-23: ShotA-GFP localisation is not shown in FigS1. The authors should refer to Fig. 2. Enlarged areas/arrows might help the reader to better visualise the different localisations of ShotA-GFP and ShotC-GFP.

    -p7, line 23: Rca1 mutants should be better introduced here.

    -p8, line 6: Shot colocalizes/associates with stable MTs and actin would be a more appropriate title for this paragraph.

    -p16, line 18: 'Shot is able to mediate crosstalk' would be better than 'Shot is able to crosstalk'.

    -p40, lines 6 and 7: L, M and N should be K', K' and K' respectively.

    -p41, Fig 10D: It is quite hard to see on the cartoon what the phenotype is for Shot OE.

    -The following reference shows an important role for Shot in crosslinking actin and MTs during morphogenesis of the Drosophila embryo and should be cited in this manuscript (Booth, A. J. R., Blanchard, G. B., Adams, R. J. & Röper, K. A Dynamic Microtubule Cytoskeleton Directs Medial Actomyosin Function during Tube Formation. Developmental Cell 29, 562-576 (2014)).

    -FigS3. It would be good to add the labels on the figure (ShotC-GFP in green, and MoeRFP/lifeActinRFP in Magenta).

    Significance

    The findings shown in this manuscript shed an important light on the way subcellular morphogenesis occurs. It was known that both actin and MTs were required in this process, particularly during the formation of Drosophila trachea (JayaNandanan, N., Mathew, R. & Leptin, M. Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin. Nature Communications 1-10 (2019). doi:10.1038/ncomms4036; Gervais, L. & Casanova, J. In Vivo Coupling of Cell Elongation and Lumen Formation in a Single Cell. Current Biology 20, 359-366 (2010)). This work provides additional molecular insights into the way branching morphogenesis from a single cell occurs in vivo, clearly demonstrating a requirement for actin-MT crosslinking mediated by Shot and Tau.

    This could be of great interest in the field of branching morphogenesis and lumen formation, not only in invertebrates but also in vertebrates where such a crosslinking might occur in the vasculature, the lung, the kidney or the mammary gland for example (Ochoa-Espinosa, A. & Affolter, M. Branching Morphogenesis: From Cells to Organs and Back. Cold Spring Harb Perspect Biol 4, a008243-a008243 (2012)).

    Field of expertise: morphogenesis, Drosophila, cytoskeleton, microtubules.

  5. Version published to 10.1101/2020.05.09.085753 on bioRxiv