Boermel, Mandy, Butter, Falk, Dietz, Sabrina, Schreier, Jan, Ketting, Rene, Seistrup, Ann-Sophie, Domingues, Antonio, Bronkhorst, Alfred, Oorschot, Viola, L'Hernault, Steven, Phillips, Carolyn, Nguyen, Dieu
[
International Worm Meeting,
2021]
Germ cells possess specialized perinuclear, phase-separated compartments, also named condensates. Amongst others, they contain the mRNA surveillance machinery responsible for transposon silencing and fertility. In the nematode Caenorhabditis elegans, three condensates, P granules, Z granules and Mutator foci, are home to RNA interference-related pathways, driven by a highly diversified Argonaute sub-clade (WAGO) that mediates gene silencing. Intriguingly, it has been shown that WAGO-mediated gene silencing can be inherited via both oocyte and sperm. Especially the inheritance via sperm is remarkable, since significant amounts of cellular material, including Argonaute proteins, are expelled from maturing spermatids into so-called residual bodies. How then does sperm-mediated inheritance of cytoplasmic RNAi work? We genetically identify WAGO-3 as a major Argonaute protein required for the paternal inheritance of endogenous small RNAs. Just like other Argonaute proteins, like WAGO-1 and ALG-3, WAGO-3 localizes to P granules in naive germ cells. During spermatogenesis, however, P granules disappear and WAGO-3, but not WAGO-1 and ALG-3, accumulates in a newly identified condensate, the PEI granule. In contrast to P granules, PEI granules remain stable during later stages of spermatogenesis. They are retained within maturing spermatids and selectively keep WAGO-3 from accumulating in the residual body. Using immunoprecipitation experiments followed by label-free quantitative mass spectrometry, we identified two uncharacterized proteins: PEI-1 and PEI-2. Both proteins are specifically expressed during spermatogenesis and we dissect their roles in PEI granule transport and function. Based on correlative light and electron microscopy (CLEM) and genetic studies, proper segregation of PEI granules in mature sperm is coupled, likely via S-palmitoylation, to the myosin-driven transport of membranous organelles. Our results identify a new sperm-specific condensate, which we call PEI granules. While not essential for spermatogenesis, PEI granules are required for paternal inheritance of small RNAs and we reveal a novel mechanism for the subcellular sorting of condensates through coupling to transport of membranous structures. pei-like genes are also found in human and often expressed in testis, suggesting that the here identified mechanism of subcellular transport of membraneless organelles may be more broadly conserved.
[
West Coast Worm Meeting,
2004]
The sense of touch is still the most enigmatic of the ways organisms learn about their environment. C. elegans is an outstanding organism for the study of sensory mechanotransduction since the response to gentle touch is behaviorally simple and mediated by a set of 6 touch receptor neurons. Genetic analyses have led to the formulation of a molecular model for touch transduction. Central to this model is an ion channel formed by at least four proteins assembled in an unknown stoichiometry: two pore-forming subunits, MEC-4 and MEC-10, and two accessory proteins, MEC-2 and MEC-6. When co-expressed in Xenopus oocytes, MEC-2 and MEC-6 dramatically and synergistically increase current generated by MEC-4/10 ion channels 1,2 . This is consistent with the observation that mutations in any one of the genes encoding these four proteins ruin touch sensation. Moreover, mechanoreceptor currents, recorded in vivo, require intact copies of all four genes (see poster this meeting). Whole-cell recordings in oocytes are unable, however, to distinguish between possible mechanisms for the action of MEC-2 and MEC-6. Each protein must either enhance the single channel conductance, open probability, or some combination of both parameters. Single channel recordings, by contrast, are ideal to differentiate between mechanisms. So far, we have eliminated increases in single channel conductance as an explanation for the effects of MEC-2 and MEC 6 on macroscopic current. We are currently investigating how each accessory protein modulates open probability. 1. Chelur, D. S., et al. (2002). Nature 420(6916): 669-73. 2. Goodman, M. B., et al. (2002). Nature 415(6875): 1039-42. Supported by an AHA Grant-in-Aid, an Alfred P. Sloan and Terman Fellowships to MBG.