Compartmentalized localization of perinuclear proteins within germ granules in C. elegans

Biomolecular condensates are nonmembrane-enclosed organelles that play important roles in distinct biological processes. Germ granules are RNA-rich condensates that are often docked on the cytoplasmic surface of germline nuclei and exist in a variety of organisms. The C. elegans germ granule is a condensate that is subcompartmentalized into at least 6 distinct regions, including the P granule, Z granule, E granule, Mutator foci, SIMR foci and P-body. Although a number of perinuclear proteins have been found to be enriched in germ granules, their precise localization within the subcompartments of germ granules is still unclear. Here, we systematically labeled perinuclear localized proteins with fluorescent proteins via CRISPR/Cas9 technology and further explored the perinuclear localization of these proteins. Using this nematode strain library, we identified a series of proteins localized in Z or E granules. We found that proteins that participate in distinct piRNA processing steps were localized in particular subcompartments, including the P-, Z-, and E-granules. We further identified a novel subcompartment, the D granule, which was enriched between the P granule and the nuclear pore complex. Finally, we analyzed the perinuclear localization of these germ granule subcompartments in mip-1/eggd-1 mutants and found that the LOTUS domain protein MIP-1/EDDG-1 was required for the establishment of the multiphase architecture of germ granules. Overall, our work revealed germ granule architecture and redefined the localization of perinuclear proteins within germ granules. Additionally, the library of genetically modified nematode strains expressing peri-nuclear proteins labeled with fluorescent tags will facilitate research on germ granules in C. elegans.