Dynamic protein assembly and architecture of the large solitary membraneless organelle during germline development in the wasp Nasonia vitripennis

  1. Kabita Kharel
  2. Samuel J. Tindell
  3. Allie Kemph
  4. Ryan Schmidtke
  5. Emma Alexander
  6. Jeremy A. Lynch
  7. Alexey L. Arkov

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Abstract

Germ cells in different animals assemble characteristic membraneless organelles referred to as germ granules, which contain RNA and proteins required for germline development. Typically, the germ granules are small spherical or amorphous cytoplasmic granules and often, they assemble around membrane-bound organelles such as nuclei, mitochondria and endoplasmic reticulum. In particular, in egg chambers of the fruit fly Drosophila , nurse cells assemble perinuclear granules, referred to as nuage, along with multiple small germ granules formed at the posterior pole of the oocyte (polar granules). Nuage is assembled in a very similar way in the wasp Nasonia vitripennis , despite the long evolutionary distance from Drosophila. In contrast, Nasonia forms a very different single germ granule, called the oosome, at the posterior, which is about 40 times larger than a homologous Drosophila polar granule. Here, using molecular and super-resolution imaging approaches, we provide insights into protein assembly and architecture of the oosome during germline development. Interestingly, unlike the fly, the wasp utilizes alternatively spliced RNA-helicase Vasa isoforms during germline development and oosome formation. The isoforms differ by an unstructured region, containing repeats of phenylalanine and glycine, that is similar to functional domains characteristic of nucleoporins. In addition, while other conserved components of germ granules, such as Oskar, Aubergine and Tudor proteins are recruited to the oosome, these polypeptides show a distinct and specific localization within the oosome. Of particular note, Tudor protein forms a shell encapsulating the oosome, while small Oskar/Vasa/Aubergine granules occur inside the oosome core. Also, in surprising contrast to Drosophila egg chambers, we found that a subset of the wasp nurse cells located in anterior show dramatic DNA damage and assemble higher levels of nuage than their posterior counterparts. The characteristics of two distinct nurse cell populations suggest a mechanistic link between the higher amounts of nuage assembled in anterior nurse cells and their need to silence transposable elements in the presence of double-strand DNA breaks. Our results point to the high degree of plasticity in the assembly of membraneless organelles, which adapt to specific developmental needs of different organisms, and suggest that novel molecular features of conserved proteins result in the unique architecture of the oosome in the wasp.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript the authors study a unique membraneless organelle (MLO) in the developing germline of the wasp (Nasonia vitripennis) and highlights the differences with polar granules, homologous membraneless assemblies formed in the Drosophila germline. They identify that in contrast to Drosophila, the wasp utilises an alternatively spliced isoform of the conserved RNA Helicase, Vasa, where the longer isoform harbours FG-repeats characteristic of nucleoporins. Additionally, they observe striking differences in the assembly of the perinucelar 'nuage' where the nuage components are heavily enriched at the anterior half as a mechanism to effectively silence transposon activity in the anterior nurse cells which are characterised by a high degree of DNA double strand breaks. In the course of oogenesis to embryogenesis, the authors observe that the oosome is dynamic and conserved germ plasm proteins (Osk, Vas, Tud) transition from diffuse distribution (in the oocytes) to a dense Tud shell surrounding there oosome filled with Osk-Vas granules (in the embryo).
    While the study provides insights into a novel germline condensate, there are some key questions which need to addressed to support publication.

    Major Comments:

    1. Nasonia expresses two distinct Vasa isoforms differing by 96 amino acids close to the N-terminus. The authors claim that the 96 amino acid insertion is FG-rich and intrinsically disordered providing experimental evidence with Circular Dichroism of the purified 96-amino acid fragment. However the amino acid sequence of the remaining N-terminal region upstream of the folded RecA domain is low complexity with an apparent over-representation of G, R, D, N as well as several FG repeats. Any computational disorder prediction tool (such as, IUPred, D2P2, etc) can be used to predict the sequence disorder of the entire N-terminus. Therefore, it is unclear why the authors claim that the 96 amino acid insertion exclusively confers special advantages and contribute to mesh-like properties to the oosome. Does the Long isoform provide specific advantages? This needs to be addressed. "Computational analysis predicted two alternatively spliced Nv-vas mRNAs, that should result in 92.3 kD and 82 kD proteins ": Please explain the analysis and tools used in methods. Fig. 1d : Please discuss the source and identity of the multiple non-specific bands of the RT-PCR experiment in the figure legends. Fig. 1f : For ease of readers, it is recommended to label the figure panels with stages as well as proteins probed.
    2. Are there sequence similarities between the novel 57-residue NTD of Nv-Osk and the Drosophila NTD (138 amino acid long) present exclusively in Drosophila Long Oskar?
      • Fig. 2c : Consider including corresponding micrographs of the oocyte oosome imaged with AiryScan.
      • Fig 2d,e : Is Osk-GFP cytoplasmic or form peri-nuclear condensates? Is Vasa-mCherry nuclear or cytoplasmic or both? Please include DAPI channels and consider drawing outlines of the cell membrane.
      • Are these S2 cell images completely representative of the localisation patterns of Osk and Vas in the cells or do the authors only observe other phenotypic classes as well? If yes, please include the different classes observed with relevant statistics.
    3. The differential assembly of the 'nuage' between anterior and posterior nurse cells is intriguing and well addressed. The higher degree of nuage assembly coincides with higher amount of DSBs in anterior nurse cells. Is this a cause or a consequence? Exposure to mutagens can induce DSBs uniformly in all nurse cells; will this lead to up regulation of nuage assembly uniformly in all nurse cells?
    4. Dynamic sub-compartmentalization of Tudor to the periphery of the embryonic oosome suggests remodelling of the proteins components post-fertilization.
      • Can the authors perform live-imaging to track oosome dynamics from oogenesis to embryogenesis using FP tagged-germ plasm components? This would provide valuable insights into the assembly mechanism of this giant membraneless organelle.
      • Fig. 4 a, b: Line profiles required to show redistribution of Tudor. What do the three panels per condition indicate? What makes the authors conclude that the Tudor shell is "fibrillar" in nature? Is there any evidence?
    5. Maternal mRNAs form homotypic clusters in Drosophila germ granules (Treck et al., 2015). Where do the maternal mRNAs localise in the oosome? In case they are recruited by Oskar (in line with Drosophila germ granules assembly model), are they diffusely distributed in the oocyte oosome and do they redistribute in the Osk-Vas granules that form at the core of the embryonic oosome? RNA in situ hybridization with a few maternal mRNAs can be done to understand how RNAs are distributed within this giant condensate.
    6. The 'dense-shell liquid core' architecture of the oosome as proposed by the authors lacks any concrete proof. The migration of the nuclei (lines 342-345) can be facilitated by changes in physical properties of the Tudor shell in that particular embryonic stage, promoted in turn by key PTMs, for instance. Moreover, there is no evidence that the core oosome has liquid-like properties. In absence of live imaging and FRAP data, the 'dense-shell liquid core' architecture can not be addressed.

    Minor Comments:

    Line 40: "small spherical or amorphous cytoplasmic granules"; the terms "spherical" and "amorphous" have very different implications-one is for shape and the other indicates molecular organization. Consider re-phrasing.

    Lines 55-57: mention embryonic stages as the Tud re-organization within oosome is a dynamic process.

    Line 63: Is this really addressed in the manuscript?

    Lines 95-97: What about Long Oskar in Drosophila? There is a 138 amino acid extension 5' of the LOTUS domain.

    Line126: "proteome of the oosome". Proteome analysis would require isolation of the oosome followed by mass spectrometric identification of constituent proteins. Here the authors investigate the conserved germ plasm proteins and not the whole proteome. Please re-phrase accordingly.

    Lines 308-310: "protein-free channels or cavities"- how do the authors know that they are protein free by probing for only three proteins? Consider re-phrasing.

    Significance

    The study describes a giant membraneless organelle in the developing wasp germline by developing an important set of tools and reagents necessary to study this novel organelle in greater depths in future. Experiments are well designed and validations of the tools developed are adequately carried out. However, the study is largely descriptive and the suggested experiments need to be performed to provide deeper insights into the significance of the work.

    Considering the existing resources on assembly of germ granules by liquid-liquid phase separation, the 'oosome' represents another class of germ granules whose mechanism of assembly, dynamics and physical properties appear to be distinct from Drosophila polar granules as well as P-granules in C. elegans. Therefore, the work is significant in the fields of condensate biology and germline development as further studies focussing on the oosome can elucidate not only the molecular principles underlying oosome assembly but also address the plasticity in assembly of homologous MLOs across evolution.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this study, Kharel et al use endogenous antibodies to characterize the dynamics of germ granule components throughout development in the wasp Nasonia vitripennis. The authors observe several key differences in Nasonia germ granules as compared to the more well-studied germ granules of Drosophila. Kharel et al describe a novel isoform of the conserved granule component Vasa, which in Nasonia contains an FG domain. The authors use super-resolution microscopy to describe a core/shell architecture for the oosome, the single large germ granule that forms in the oocyte cytoplasm. Additionally, the authors find that a subset of Nasonia nurse cells have comparatively higher levels of perinuclear nuage which they hypothesize is related to high levels of DNA double strand breaks. The authors propose that these observed differences in conserved germ granule components may support the unique demands of germ granules in Nasonia.

    Major comments

    1. In many instances the authors make strong statements that are not directly supported by the presented data. For example, in the Abstract (line 62) the authors write "Our results point to the high degree of plasticity in the assembly of membraneless organelles, which adapt to specific developmental needs of different organisms, and suggest that novel molecular features of conserved proteins result in the unique architecture of the oosome in the wasp." Indeed, the authors have described several differences between germ granules in Drosophila versus Nasonia, but they have not presented data indicating that these differences are functionally relevant. They have also not shown that these differences result in the unique architecture of the oosome. The authors may of course speculate about the functional significance of their observations in the Discussion (emphasizing that certain statements are speculative), but should tone down or limit such statements in the Abstract and Results.

    A few more examples of over interpreted data:

    a. In line 111 of the Results the authors write: "Our data suggest that the Nv-Tud shell provides mechanical stability to contain a less dense oosome core during its migration in the early embryo." The authors have only observed an enrichment of Nv-Tud at the periphery of the oosome, which is not quantified. The authors have not performed experiments to test whether this enrichment is required for oosome integrity, or whether the core of the oosome is less dense.

    b. In line 113 of the Results, the authors state: "Nasonia egg chambers have distinct subset of nurse cells in anterior that show evidence of double-strand DNA breaks and assemble higher amounts of perinuclear nuage than their posterior counterparts, indicating a higher demand for anterior nurse cells to silence transposable elements." Indeed, a subset of nurse cells have strikingly higher levels of nuage, and a subset have significantly higher levels of gH2Av staining. However, a link between high levels of nuage and a need to silence transposable elements seems speculative.

    c. In line 421, the authors summarize their conclusions: "the assembly of the oosome...relies on the combination of highly conserved components...as well as a suite of novel features, including a novel Nv-Vas isoform, an unusual shell of Nv-Tud protein demarcating the edges of the oosome, and unusual distribution of nuage in the ovary." As the data presented in this study is largely descriptive, the authors have not directly tested whether any of these features are required for oosome assembly.

    1. Throughout the study, the authors often show single western blots or representative images. To determine reproducibility, the authors should quantify their data whenever possible or indicate the number of independent experiments used to generate a figure. Sample size should be included in the Legends.

    Some examples:

    a. In Figure 1a, the authors show that the short Nv-Vas isoform is decreased in the embryo. How many times was this western performed, and is the short isoform always similarly decreased in the embryo?

    b. In Figure 1d, the authors use RT-PCR to show that multiple vas RNA isoforms are present in ovaries, but only the long isoform is detected in embryos. How many times was the RT-PCR performed and how reproducible was this result? Also, there appears to be a third major RNA species present in ovaries, do the authors think this is relevant?

    c. In Figure 2d, Nv-Osk forms granules when expressed in S2 cells. What fraction of S2 cells expressing Nv-Osk had granules?

    d. In Figures 4 and 6, the authors should quantify core versus shell enrichment for Nv-Tud and other germ granule components (see Major comment 3 below).

    1. A main finding of this study is that Nv-Tud forms a fibrillar shell by concentrating at the periphery of the oosome. The authors propose that this shell "fulfills the role of a membrane" (line 371) to protect the integrity of the oosome. This model is based on a handful of images, some of which are not entirely convincing. For example, Nv-Tud does not appear to form a shell in the middle image of Figure 4b. In order to strengthen their model, the authors should quantify the enrichment of different germ granule components in the core versus shell of Nasonia oosomes. Optionally, the authors could directly test a role for Tud in oosome integrity by observing the fate of the oosome core components following tud mutation or RNAi.

    Minor comments

    1. It might be helpful to add the protein structure for Drosophila Vasa for comparison in Figure 1c. Similarly, Drosophila Oskar could be added to Figure 2b.
    2. The authors mis-reference Extended Data Fig 3 as Extended Data Fig 2 (starting in line 198).
    3. In the Figures throughout, it would be helpful to label each panel with the antibodies used. Relatedly, in both the Figures and text the authors should always clarify whether they used antibodies recognizing the long Nv-Vas isoform or the entire Nv-Vas. Also make sure to include MWs for all blots.
    4. For Extended Data Fig. 3d, I'm not sure that migration of an in vitro transcribed Osk "confirms" that the NTD is responsible for the higher molecular weight of Nasonia Osk. Is this experiment is needed?
    5. In Figure 2e, the authors don't observe recruitment of Nv-Vas to Nv-Osk granules in S2 cells, leading to the proposal that "contrary to Drosophila, Nv-Osk does not directly recruit or associate with Nv-Vas in Nasonia" (line 231). It's possible that Nv-Osk is necessary but not sufficient to recruit Nv-Vas, and the authors might consider directly testing this by RNAi depletion of Osk in Nasonia.
    6. The authors should mention and discuss the high level of gH2Av staining in the oocyte nucleus (Figure 3b). Has this been reported before?
    7. The authors find strong gH2Av staining in anterior nurse cells, leading them to write in line 277: "indicating that the same population of nurse cells that assemble high amounts of nuage, shows high level of DSBs." While it is likely that this is the same population, without co-staining of germ granules and gH2Av in the same egg chmaber the authors cannot conclude that this is the same population of cells.
    8. In Figure 3a, the authors note that Nv-Osk is produced in the cytoplasm of nurse cells, where it assembles into granules. It might be worthwhile to use osk RNAi as a control to make sure that the granular Osk signal in their IF is specific.
    9. Unless I've missed it, the authors never reference Extended data Figures 6a and b in the text.
    10. The authors write in line 362: "The spherical shape of these granule, point to their liquid characteristics and their formation inside the oosome core via liquid-liquid phase separation mechanism." Spherical shape alone is not sufficient to conclude liquid-like character or assembly via LLPS.

    Referees cross-commenting

    I might clarify that analyzing maternal RNA localization should be optional (Reviewer 3 Major Comment 5).

    Significance

    In this study Kharel et al use endogenous antibodies to observe the dynamics of conserved germ granule components in Nasonia. This approach allowed the authors to uncover several key differences between germ granules in Nasonia versus Drosophila. While these differences have the potential to be of interest to the specialized germ cell community, the data presented in this study are largely descriptive and not quantified. Therefore, the functional relevance of these differences remains uncertain.

    The finding that a subset of nurse cells have high levels of nuage is quite striking. Furthermore, the authors write in line 405: "The occurrence of DSBs in a distinct population of nurse cells in any organisms has not been reported before to our knowledge." Therefore, this population of nurse cells may be unique to Nasonia and would be of interest to be explored further in future studies. The authors could use granule mutants to test their hypothesis that high levels of nuage are required to silence transposable elements.

    A main goal of this study is to compare germ cell assembly between Nasonia and Drosophila, and thereby identify how unique features of Nasonia contribute to germ granule dynamics and function. Indeed, the oosome is quite unique as an enormous, solitary granule (though perhaps reminiscent of a Balbiani body?), and how it maintains integrity is an open question. The authors propose that Nv-Tud acts as a shell to stabilize the oosome, which would be remarkable for such a large germ granule. This finding could be of interest to a broader field of condensate researchers. Therefore, future studies should directly test whether Nv-Tud is required for oosome integrity. Finally, it would be helpful to the reader to more fully discuss direct comparisons between Nasonia and Drosophila germ granule components. For example, the authors should comment on what is known about isoforms of Drosophila Vasa, and whether Drosophila Vasa may have the potential to be alternatively spliced.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript entitled "Dynamic protein assembly and architecture of the large solitary membraneless organelle during germline development in the wasp Nasonia vitripennis" by Kharel et al. aims to examine structural features of the wasp oosome. Specifically, the authors look at the expression of the Vasa and Oskar protein isoforms, their accumulation in the oosome and nuage during oogenesis and early embryogenesis and aim to understand possible functional roles of the structure of these organelles in female germline development.

    However, several conclusions in this paper are not fully supported by the data and some of the experiments need additional experimentation and controls. Below I am listing my concerns (listed in the order they appear in the manuscript):

    Major concerns:

    Lane 141: Extended Data Fig. 1b: Control demonstrating that CIP treatment worked. Lack of change could be due to the enzyme not working.

    Lane 149: To demonstrate a differential splicing pattern the authors need to show PCR using primers spanning the intron that is retained or spliced out. These PCR products should also be quantified using qRT-PCR. The authors should explain the multitude of bands on the PCR gel - the gel presented by the authors is not convincing and shows issues with annealing efficiency (non-specificity) of primers.

    Lane 168-169: "we detected Nv-Vas outside the oosome, distributed in the embryos' cytoplasm (Fig. 1g)." The authors should show a control of the embryo/oocyte stained the same way but without the primary antibody to evaluate background fluorescence in the staining. The images should be quantified and imaged/displayed using the same imaging and normalization parameters as the one shown in Fig. 1g.

    Lane 171: Data showing the result of this mass spec experiment is not shown.3

    Lane 181: Extended Data Fig. 2: This graph is hard to interpret. A control is missing that shows how a curve of the structured protein or an FG-repeat containing fragment of the nucleoporin would look like. As it stands now, it is just a curve that cannot be interpreted by someone who has never used CD spectroscopy to study IDRs.

    Lane 202-203: "The Nv-osk mRNA 3' RACE mapping was consistent with the previously identified 3'-end of Nv-osk mRNA indicating that Nv-Oskis not extended at its C-terminus." Data for this is not shown in this manuscript.

    Lane 204: "However, 5'-end mapping revealed that Nv-osk RNA starts at more than 800 bps upstream of previously predicted Nv-osk transcription start site (Extended Data Fig. 2c)." The authors show a schematic which is not data. Instead, they should show raw dat.

    Lane 213-215: "in addition to major 51 kD band, a less intense ~100 kD band is detected, suggesting the formation of Osk dimers (Extended Data Fig. 3e) consistent with previous finding that LOTUS domain of Nv-Osk dimerizes" The authors are (presumably) running denaturing PAGE gels and they add beta-mercaptoethanol to the samples before they load them on the gel. Therefore, dimerization should not be detected on the gel. The band the authors see must be a contaminant.

    Lane 231: "that, contrary to Drosophila, Nv-Osk does not directly recruit or associate with Nv-Vas in Nasonia (Fig. 2e)." The authors cannot make this conclusion. It is possible that Osk and Vasa interact directly but that one of the proteins requires a post-translational modification for this interaction and this modification does not happen in S2 cells on Nasonia proteins.

    Lane 237: Lack of these specific amino acids in Nasonia proteins does not support the argument that Nasonia Osk and Vasa do not interact. Perhaps changes in amino acids in Vasa are compensated by changes in amino acids in Oskar.

    Lane 243: "co-expressed in S2 cells, fail to form granules (Fig. 2e). Overall, our data suggest that while Nv- Osk has the intrinsic ability to condense into spherical granules..." If the expression of Vasa is not high enough, then Vasa will not phase separate in S2 cells. It is possible that both proteins phase separate but at different critical concentrations, which would explain the lack of granule formation of Vasa in S2 cells.

    Lane 286: "transposon-related gene in Nasonia ovaries, we found no evidence that transposable elements are selectively upregulated in anterior nurse cells (Extended Data Fig. 5b), suggesting that high assembly of anterior nuage is needed to effectively silence transposable elements despite the prevalence of DSBs in anterior" This is an overstatement and incorrect interpretation. Since the staining is not mutually exclusive, the authors can conclude that there is no correlation between dsDNA breaks, nuage and transposon expression and that therefore nuage is not required for the regulation of transposon expression or dsDNA formation. Regardless, the data is correlative and existence of direct connection has not been tested. Lane 293: Control of IF staining without the primary antibody is missing to evaluate background fluorescence in the staining. The images should be quantified and imaged/displayed using the same imaging and normalization parameters. Also, the authors should do a western on oocytes that do not yet form germplasm to demonstrate Oskar protein expression in early oocytes.

    Lane 311: There is no data demonstrating that the oosome has migrated - just two images of an oosome in embryos of different ages. The developmental changes (progression) of the embryo are also not evident. The data currently presented are not evidence of migration. The authors should avoid interpretations connected with migration using the data correctly presented.

    Lane 314: the evidence that Tudor makes a shell is weak and only displayed in an image co-stained for Tud and Osk in Figure 4b. Co-staining of Tud and Vasa in the same panel does not display the shell convincingly. More data is needed to show a shell.

    Lane 314: There is no data showing that the shell is fibrillar.

    Lane 341-345: The authors overinterpret the data. Germ plasm is a cytoplasm and everything in a cell moves through the cytoplasm. This is not in itself evidence of a liquid nature of the oosome.

    Lane 363: Round granule shape is consistent with LLPS but is not evidence for it. The authors should fix their statement.

    Minor concerns:

    Lane 95: Statement" In particular, we provide evidence for the presence of a novel N-terminal segment of Nasonia Oskar (Nv-Osk) adjacent to the conserved LOTUS domain that is absent in other insect Osk proteins." Is not true as written. The Drosophila melanogaster (D mel) Oskar has a N-terminal extension which forms the Long Oskar isoforms. In fact, what the authors report here is that Nosonia Oskar protein is a lot more similar to the D mel Oskar than previously reported by the authors, and that both organisms express long and short Oskar isoforms that with very similar protein structures. The authors should correct their statement.

    Lane 134: Where is this data shown (Subsequently, we were able to confirm Nv-Vas identity of both IP-ed proteins with mass spectrometry.)- no mass spec data is reported in this manuscript

    Lane 171: " ass spectrometry..." is missing an M.

    Lane 200: Extended Data Fig 2b is referencing the wrong figure panel.

    In general, western blots are missing molecular weight standards.

    Lane 265: Tudor is not a component of nuage in D mel.

    Significance

    Overall, I found this manuscript interesting. It provided new insights in the expression of Vasa and Oskar and as well as new models of how these two proteins are regulated and how they accumulate in the oosome. Some of the features, like the newly identified N-terminal extension of Nasonia Oskar protein, appear to be shared with those of Drosophila melanogaster Oskar. This is an important finding because it indicates that mechanism by which Osk functions in the female germline might be conserved in insects.

  5. Version published to 10.1101/2023.12.13.571440v1 on bioRxiv