Bitesize bundles F-actin and influences actin remodeling in syncytial Drosophila embryo development

  1. Anna R. Yeh
  2. Gregory J. Hoeprich
  3. Bruce L. Goode
  4. Adam C. Martin

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Abstract

Actin networks undergo rearrangements that influence cell and tissue shape. Actin network assembly and organization is regulated in space and time by a host of actin binding proteins. The Drosophila Synaptotagmin-like protein, Bitesize (Btsz), is known to organize actin at epithelial cell apical junctions in a manner that depends on its interaction with the actin-binding protein, Moesin. Here, we showed that Btsz functions in actin reorganization at earlier, syncytial stages of Drosophila embryo development. Btsz was required for the formation of stable metaphase pseudocleavage furrows that prevented spindle collisions and nuclear fallout prior to cellularization. While previous studies focused on Btsz isoforms containing the Moesin Binding Domain (MBD), we found that isoforms lacking the MBD also function in actin remodeling. Consistent with this, we found that the C-terminal half of BtszB cooperatively binds to and bundles F-actin, suggesting a direct mechanism for Synaptotagmin-like proteins regulating actin organization during animal development.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: Yeh et al., present novel findings that Bitesize (Btsz) a Synaptotagmin-like protein, helps organize actin during the syncytial blastoderm stages of Drosophila embryo development. Depleting Btsz leads to phenotypes in the syncytial cycles that mimic Drosophila mutants where actin or membrane trafficking is disrupted. Perhaps most interestingly, the authors show that a non-Moesin Binding Domain-containing isoform of Btsz is important for cytoskeletal regulation during syncytial cycles. The authors generated a BtszB-C terminal recombinant protein and showed using imaging and biochemistry that this conserved segment of Btsz (which is present in all isoforms) can bind to and bundle F-actin in vitro. Lastly, the authors show that Btsz localizes to the apical region of pseudocleavage furrows and cell interfaces at gastrulation, which is consistent with previous literature regarding its role in regulating adherens junctions.

    Major Concern: Both the imaging data, image analysis and biochemistry, are compelling. The findings regarding expression of alternative isoforms of Btsz are interesting within this developmental context. However, the final model is very simple and may benefit from the addition of experiments or at least further attention in the discussion. For example, it is not entirely clear what the division of labor may be between isoforms that do or do not bind Moesin. Do these isoforms work to accomplish a single function; or do they perform unique functions? Are the isoforms subject to similar or different regulation? At a minimum, the authors thoughts should be included in the Discussion, and a more integrated model presented. Relatedly, the authors mention the possibility that membrane trafficking may be impacted but end abruptly there. Additional experiments would obviously increase impact. If no experiments are added, the existing text should nonetheless be edited to include a more complete Discussion of the results.

    Specific Concerns:

    1. While the authors claim there is an actin defect, that defect is not readily revealed by a change in actin levels. Is the change perhaps in actin stability or in mechanical properties of the actin filaments (for example, if filaments can assemble but not be bundled or appropriately tethered to the plasma membrane in the mutant)? Have the authors tried either FRAP or laser cutting of furrows in mutant embryos?
    2. While prior publications mention the role for Btsz in building adherens junctions, it would still be useful to see an analysis of junction phenotypes in the hands of these authors. Also, where do junction components concentrate and what do they do, if known, in syncytial embryos? It would be helpful to include this information in the text.
    3. Does imaging of golgi or endosome markers reveal any differences in membrane compartments in Btsz mutant embryos? Even negative results would be interesting.
    4. In describing the Myosin network phenotype during cellularization, it is not clear what is meant by the statement that the network has "constricted" over the positions where nuclei were lost. That sounds like an active process. It seems equally possible that the Myosin is just coating the membrane that now fills the gaps where nuclei should be.
    5. Some aspects of Btsz gene expression are discussed and equated with a small number of previously described genes for cellularization. Are those genes only expressed during cellularization or beyond? It appears that Btsz is expressed beyond cellularization. Do those genes also have complex splicing patterns/multiple isoforms?
    6. Could the authors comment on why they chose to describe the syncytial phenotypes in Cycle 12 but not other syncytial cycles?

    Significance

    For strengths and limitations, see above.

    Advance: The authors advance the field of regarding Synaptotagmin-like proteins (Slps) by studying alternative isoforms of the proteins which lack a Moesin-binding domain (MBD). They find a novel function for Btsz isoforms that do not contain an MBD and show that a variant of these isoforms can directly bind to and bundle F-actin to regulate actin during syncytial nuclear divisions. Since the domain(s) they tested are conserved in all isoforms, this likely means that the actin binding function of Btsz could be conserved for most Slps, including Btsz isoforms which contain MBD.

    Audience: This work is of interest to cell and developmental biologists who study the regulation of actin cytoskeleton. The work as presented also has some relevance to those who study adherens junctions, membrane trafficking, and Synaptotagmin-like proteins. More broadly, this work may be of interest to those who study alternatively-spliced proteins in the context of development.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript by Yeh and colleagues examines the function of the Slp-family protein Bitesize in Drosophila. Btsz has been previously studied in the fly embryo and appreciated to regulate F-actin and adhesion-related properties in the formation of the epithelium, but in this manuscript Yeh et al. look at an earlier point of development (division in the early embryonic syncytium) and also examine the role of bitesize transcripts that lack the moesin-binding domain (MBD). The authors disrupt Btsz function and observe defects in actin-dependent structures, such as the apical actin cap and the pseudo-cleavage furrows, which leads to defect in the pseudo-cleavage divisions. They also perform actin-bundling assays with a btsz fragment that does not contain the MBD and see that btsz can still bundle actin, implicating the C2 domains in this function. The work appears well-done, with only one major area of concern (the imaging and analysis of actin caps, below). The manuscript is well-written, with a nice Introduction, and the Results are appropriately described and interpreted. The quantitation appears appropriate, and n number and the statistical tests used by the authors are consistently stated throughout the manuscript. A few more detailed comments are below:

    1. It does not appear that the actin caps are being measured and imaged. None of the usual internal structures of the caps are apparent, and instead it appears that what is presented are the apical margins of the pseudocleavage furrows (or the very edges of the caps).
    2. Along these lines, the argument that caps are smaller does not make much sense, since it appears that the "caps" are being measured late once the furrows have formed. Since these dimensions are set by the number of nuclei in the embryo, as long as the caps are growing large enough to get collisions between adjacent nuclei/caps, how can the caps be smaller unless there are fewer nuclei? These changes could also be secondary consequences of differences in nuclear distributions around the embryo periphery. For these reasons, and because of the close packing of nuclei together, usually cap growth rates are plotted in periods prior to cap collisions.
    3. Sorry if this was missed, but are the cycles at which measurements are made listed in each appropriate figure? I saw "cycle 12" listed in one figure legend, but not others.
    4. How do the authors know that the nuclear density defects in the CRISPR allele are due to the same mechanism? Could be through same mechanism, but could also be due to defect in nuclear anchoring, cortical portioning, etc...
    5. The schematics and illustrations are nicely done.

    Minor notes:

    • a) Should there be actin in the top row of 1A?

    Significance

    This manuscript by Yeh and colleagues examines the function of the Slp-family protein Bitesize in Drosophila. Btsz has been previously studied in the fly embryo and appreciated to regulate F-actin and adhesion-related properties in the formation of the epithelium, but in this manuscript Yeh et al. look at an earlier point of development (division in the early embryonic syncytium) and also examine the role of bitesize transcripts that lack the moesin-binding domain (MBD). The authors disrupt Btsz function and observe defects in actin-dependent structures, such as the apical actin cap and the pseudo-cleavage furrows, which leads to defect in the pseudo-cleavage divisions. They also perform actin-bundling assays with a btsz fragment that does not contain the MBD and see that btsz can still bundle actin, implicating the C2 domains in this function. The work should be of interest to a developmental community and those workers interested in Slp-family function.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Yeh and colleagues report the requirement of the Drosophila Synaptotagmin-like protein, Bitesize, for the proper formation of pseudocleavage furrows of the syncytial embryo (through shRNA affecting all Bitesize isoforms). Reduced sizes of the compartments for mitotic spindles and of the sizes of mitotic spindles are also quantified. Local losses of the furrows also correlated with collisions of neighbouring nuclei, and with loss of nuclei from the syncytial embryo periphery, consistent with the known role of the furrows. Bitesize has nine predicted splice isoforms. Some include a Moesin-binding domain, which has previously been implicated in Bitesize activity at post-syncytial developmental stages, and all include a shared C-terminus, which has been implicated in actin binding in a related vertebrate protein. Results suggest expression and functional involvement of isoforms with either potential links to the actin cortex, although definitive conclusions would require further analyses (below). In vitro assays showed the ability of the purified C-terminus to bind and to bundle F-actin. An isoform encoding the C-terminus, but not the Moesin-binding domain, localized to the pseudocleavage furrows, and displayed an internal punctate distribution. The effect of Btsz shRNA on F-actin was tested at cellularization, and no effect was observed by comparing F-actin levels at the apical end of the cell to that at furrow canals. One Btsz isoform lacking the Moesin-binding domain was shown to localize apically during cellularization.

    Major comments:

    • The phenotypic analyses of the Btsz shRNA embryos are clear.
    • The in vitro analyses of the F-actin binding and bundling of the Btsz C-terminus are clear.
    • Quantifications, statistics and explanations of methods are appropriate.
    • The analyses of isoform expression are a concern because it seems from Figure 1C that the primers to distinguish isoforms with and without the Moesin-binding domain could both be detecting isoform I. If this is the case, then primers to specifically detect "Non-MBD isoforms" should be used. If not, then the current primers for detecting "Non-MBD isoforms" should be clarified in relation to isoform I.
    • In the Abstract, Discussion, and Results it is concluded that isoforms lacking the Moesin-binding domain function in syncytial development, but this conclusion is not clearly supported by the data. An exon 4 deletion generating a premature stop was designed to disrupt a subset of isoforms lacking the Moesin-binding domain, but it also has the potential to disrupt isoform I which contains exon 4 and the Moesin-binding domain. RT-PCR should be able to detect isoform I specifically. If it is not expressed, then the conclusion would be strengthened. If it is expressed, then is seems difficult to make a specific conclusion about the role of the of non-MBD isoforms.
    • The authors say that the exon 4 deletion mutants and the Moesin-binding domain exon mutants have a weaker phenotype than Btsz shRNA embryos, but different markers were used and genetically encoded markers could contribute to the difference.
    • Additional analyses to pursue a possible defect in F-actin organization in Btsz shRNA embryos could better connect the in vitro and in vivo analyses.
    • That caveat that only one isoform was localized should be added to this sentence: "Unlike other actin cross-linkers involved in cellularization, BtszB did not localize to furrow canals, suggesting that the cellularization phenotypes we observed in Btsz mutants and Btsz RNAi (Figure 4D) were the result of prior syncytial division defects." The caveat also applies to this sentence in the Discussion: "Btsz is present uniquely in an apical-lateral compartment."

    Minor comments:

    • Within Fig 1A, the axes of the top image should be X and Z rather than X and Y.
    • The Arp3 RNAi data in Figure S1B isn't mentioned in the Results. I assume it is a positive control.
    • The internal punctate distribution BtszF in Fig 6A could be commented on in the Discussion paragraph about the possibility of Btsz also functioning in membrane trafficking.

    Significance

    • From the perspective of syncytial Drosophila development, a new factor is shown to be required for cortical reorganization.
    • From the perspective of Bitesize, an earlier role in development is shown.
    • From the perspective of Bitesize, an additional mechanism of action is implicated, F-actin binding and bundling, by which it could affect the cell cortex (although more work is needed to clarify this in vivo).
    • From the perspective of related vertebrate proteins, an F-actin binding activity found in one of these proteins seems to be conserved in Btsz.
    • The paper will be of interest to those studying Bitesize and orthologs, the cell cortex, the actin cytoskeleton, the morphogenesis of cells and tissues, and/or syncytial Drosophila development.
  5. Version published to 10.1101/2023.04.17.537198v1 on bioRxiv