Proteome-scale Isolation and Tagging of P Granules Uncover Unique Solid-like Condensates to Ensure Spermatogenesis
Abstract
P granules are membrane-less organelles essential for germ cell fate specification, post-transcriptional regulation, and genome stability. However, the molecular organization and developmental plasticity of P granules remain incompletely understood. Here, we isolated P granules from enriched C. elegans germ cells and identified 256 associated proteins by mass spectrometry. Using CRISPR/Cas9 together with programmatically generated primer sets, we established a proteome-scale collection of endogenously tagged strains for visualizing P-granule proteins. The corresponding fluorescence imaging data, together with genome-wide primer sets for constructing endogenously tagged C. elegans strains, were integrated into an annotated, searchable online database (https://scgerm-atlas.sjtu.edu.cn/website/#/P-granules). Using this resource, we systematically visualized P-granule protein dynamics and mapped their spatiotemporal expression trajectories during gametogenesis. Following the remodeling of perinuclear P granules during spermatogenesis, a distinct set of male-specific germ granules emerged, that likely forms diverse solid-like condensates to meet the multifaceted physiological demands of spermatogenesis. Together, this work uncovers a developmental transition in germ granule composition and highlight their critical role in male fertility and germline integrity, laying the groundwork for future studies on germ granules in development and disease.
References
Eddy, E.M. (1975). Germ plasm and the differentiation of the germ cell line. Int Rev Cytol 43, 229–280. doi:10.1016/s0074-7696(08)60070-4
Seydoux, G. (2018). The P Granules of C. elegans: A Genetic Model for the Study of RNA-Protein Condensates. J Mol Biol 430, 4702–4710. doi:10.1016/j.jmb.2018.08.007
Hird, S.N., Paulsen, J.E., and Strome, S. (1996). Segregation of germ granules in living Caenorhabditis elegans embryos: cell-type-specific mechanisms for cytoplasmic localisation. Development 122, 1303–1312. doi:10.1242/dev.122.4.1303
Updike, D.L., Hachey, S.J., Kreher, J., and Strome, S. (2011). P granules extend the nuclear pore complex environment in the C. elegans germ line. J Cell Biol 192, 939–948. doi:10.1083/jcb.201010104
Ouyang, J.P.T., Folkmann, A., Bernard, L., Lee, C.Y., Seroussi, U., Charlesworth, A.G., Claycomb, J.M., and Seydoux, G. (2019). P Granules Protect RNA Interference Genes from Silencing by piRNAs. Dev Cell 50, 716–728.e716. doi:10.1016/j.devcel.2019.07.026
Phillips, C.M., and Updike, D.L. (2022). Germ granules and gene regulation in the Caenorhabditis elegans germline. Genetics 220. doi:10.1093/genetics/iyab195
Updike, D., and Strome, S. (2010). P granule assembly and function in Caenorhabditis elegans germ cells. J Androl 31, 53–60. doi:10.2164/jandrol.109.008292
Strome, S., and Wood, W.B. (1982). Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans. Proc Natl Acad Sci U S A 79, 1558–1562. doi:10.1073/pnas.79.5.1558
Gruidl, M.E., Smith, P.A., Kuznicki, K.A., McCrone, J.S., Kirchner, J., Roussell, D.L., Strome, S., and Bennett, K.L. (1996). Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci U S A 93, 13837–13842. doi:10.1073/pnas.93.24.13837
Kawasaki, I., Shim, Y.H., Kirchner, J., Kaminker, J., Wood, W.B., and Strome, S. (1998). PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans. Cell 94, 635–645. doi:10.1016/s0092-8674(00)81605-0
Spike, C.A., Bader, J., Reinke, V., and Strome, S. (2008). DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells. Development 135, 983–993. doi:10.1242/dev.015552
Chen, J.X., Cipriani, P.G., Mecenas, D., Polanowska, J., Piano, F., Gunsalus, K.C., and Selbach, M. (2016). In Vivo Interaction Proteomics in Caenorhabditis elegans Embryos Provides New Insights into P Granule Dynamics. Mol Cell Proteomics 15, 1642–1657. doi:10.1074/mcp.M115.053975
Branon, T.C., Bosch, J.A., Sanchez, A.D., Udeshi, N.D., Svinkina, T., Carr, S.A., Feldman, J.L., Perrimon, N., and Ting, A.Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36, 880–887. doi:10.1038/nbt.4201
Price, I.F., Hertz, H.L., Pastore, B., Wagner, J., and Tang, W. (2021). Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans. Elife 10. doi:10.7554/eLife.72276
Zhao, C., Cai, S., Shi, R., Li, X., Deng, B., Li, R., Yang, S., Huang, J., Liang, Y., Lu, P., et al. (2024). HERD-1 mediates multiphase condensate immiscibility to regulate small RNA-driven transgenerational epigenetic inheritance. Nat Cell Biol 26, 1958–1970. doi:10.1038/s41556-024-01514-8
Hubstenberger, A., Courel, M., Benard, M., Souquere, S., Ernoult-Lange, M., Chouaib, R., Yi, Z., Morlot, J.B., Munier, A., Fradet, M., et al. (2017). P-Body Purification Reveals the Condensation of Repressed mRNA Regulons. Mol Cell 68, 144–157.e145. doi:10.1016/j.molcel.2017.09.003
Wheeler, J.R., Jain, S., Khong, A., and Parker, R. (2017). Isolation of yeast and mammalian stress granule cores. Methods 126, 12–17. doi:10.1016/j.ymeth.2017.04.020
Pepling, M.E., Wilhelm, J.E., O'Hara, A.L., Gephardt, G.W., and Spradling, A.C. (2007). Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body. Proc Natl Acad Sci U S A 104, 187–192. doi:10.1073/pnas.0609923104
Cox, R.T., and Spradling, A.C. (2003). A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130, 1579–1590. doi:10.1242/dev.00365
Schreier, J., Dietz, S., Boermel, M., Oorschot, V., Seisrup, A.S., de Jesus Domingues, A.M., Bronkhorst, A.W., Nguyen, D.A.H., Phillis, S., Gleason, E.J., et al. (2022). Membrane-associated cytoplasmic granules carrying the Argonaute protein WAGO-3 enable paternal epigenetic inheritance in Caenorhabditis elegans. Nat Cell Biol 24, 217–229. doi:10.1038/s41556-021-00827-2
Batista, P.J., Ruby, J.G., Claycomb, J.M., Chiang, R., Fahlgren, N., Kasschau, K.D., Chaves, D.A., Gu, W., Vasale, J.J., Duan, S., et al. (2008). PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. Mol Cell 31, 67–78. doi:10.1016/j.molcel.2008.06.002
Gu, W., Shirayama, M., Conte, D., Jr., Vasale, J., Batista, P.J., Claycomb, J.M., Moresco, J.J., Youngman, E.M., Keys, J., Stoltz, M.J., et al. (2009). Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol Cell 36, 231–244. doi:10.1016/j.molcel.2009.09.020
Spieth, J., Brooke, G., Kuersten, S., Lea, K., and Blumenthal, T. (1993). Operons in C. elegans: polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73, 521–532. doi:10.1016/0092-8674(93)90139-h
Evans, D., Zorio, D., MacMorris, M., Winter, C.E., Lea, K., and Blumenthal, T. (1997). Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis. Proc Natl Acad Sci U S A 94, 9751–9756. doi:10.1073/pnas.94.18.9751
Kuhn, S.Z. and J.R. (2013). Cell isolation and culture. In WormBook, Hobert, O., ed. doi:10.1895/wormbook.1.157.1, http://www.wormbook.org
Hamburger, A.W., Dunn, F.E., and White, C.P. (1985). Percoll density gradient separation of cells from human malignant effusions. Br J Cancer 51, 253–258. doi:10.1038/bjc.1985.36
Chodosh, L.A. (2001). UV crosslinking of proteins to nucleic acids. Curr Protoc Mol Biol Chapter 12, Unit 12.5. doi:10.1002/0471142727.mb1205s36
Aebersold, R., and Mann, M. (2003). Mass spectrometry-based proteomics. Nature 422, 198–207. doi:10.1038/nature01511
Maduro, M.F. (2015). 20 Years of unc-119 as a transgene marker. Worm 4, e1046031. doi:10.1080/21624054.2015.1046031
Kim, H.M., and Colaiacovo, M.P. (2019). CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans. Curr Protoc Mol Biol 129, e106. doi:10.1002/cpmb.106
Tzur, Y.B., Winter, E., Gao, J., Hashimshony, T., Yanai, I., and Colaiacovo, M.P. (2018). Spatiotemporal Gene Expression Analysis of the Caenorhabditis elegans Germline Uncovers a Syncytial Expression Switch. Genetics 210, 587–605. doi:10.1534/genetics.118.301315
Alberti, S., Gladfelter, A., and Mittag, T. (2019). Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 176, 419–434. doi:10.1016/j.cell.2018.12.035
Martin, E.W., and Holehouse, A.S. (2020). Intrinsically disordered protein regions and phase separation: sequence determinants of assembly or lack thereof. Emerg Top Life Sci 4, 307–329. doi:10.1042/ETLS20190164
Youn, J.Y., Dyakov, B.J.A., Zhang, J., Knight, J.D.R., Vernon, R.M., Forman-Kay, J.D., and Gingras, A.C. (2019). Properties of Stress Granule and P-Body Proteomes. Mol Cell 76, 286–294. doi:10.1016/j.molcel.2019.09.014
Seydoux, G., and Braun, R.E. (2006). Pathway to totipotency: lessons from germ cells. Cell 127, 891–904. doi:10.1016/j.cell.2006.11.016
Brangwynne, C.P., Eckmann, C.R., Courson, D.S., Rybarska, A., Hoege, C., Gharakhani, J., Julicher, F., and Hyman, A.A. (2009). Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732. doi:10.1126/science.1172046
Wang, J., and Zhang, H. (2019). Phase separation in membrane biology: interplay between membrane-bound organelles and membraneless condensates. Dev Cell 50, 421–435. doi:10.1016/j.devcel.2019.07.011
Zhang, Y., Yan, L., Zhou, Z., Yang, P., Tian, E., Zhang, K., Zhao, Y., Li, Z., Song, H., Han, J., et al. (2009). SEPA-1 mediates the specific recognition and degradation of PGL granules in C. elegans. Nat Cell Biol 11, 1340–1346. doi:10.1038/ncb1977
Tian, E., Wang, J., Zhang, Y., Yan, L., Zhang, K., and Zhang, H. (2010). Epigenetic regulation of PGL granule assembly and autophagy in C. elegans. Development 137, 999–1007. doi:10.1242/dev.045906
Brangwynne, C.P., Mitchison, T.J., and Hyman, A.A. (2011). Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc Natl Acad Sci U S A 108, 4334–4339. doi:10.1073/pnas.1017150108
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