[
International Worm Meeting,
2021]
RNA interference is a widely conserved mechanism of gene regulation and silencing wherein small RNAs, 18-30 nucleotides in length, target and downregulate deleterious transcripts. In C. elegans germ cells, RNA silencing is coordinated through perinuclear nuage containing at least four compartments: P granules, Z granules, Mutator foci, and SIMR foci. Multiple of these compartments are phase-separated condensates and maintain distinct structures within the surrounding bulk cytoplasmic phase, similar to the immiscibility of oil droplets in water. Fluorescent widefield microscopy reveals that these compartments are closely adjacent to one another, yet also appear as separate and distinct puncta. It remains unclear 1) how these compartments are organized on the nanomolecular scale, 2) what constitutes the interface between compartments, and 3) how these compartments exchange small RNAs, proteins, and transcripts to facilitate RNA silencing. Our work aims to address the unknowns of nuage organization and interaction to model RNA silencing through phase-separated condensates. We use ectopic protein expression, aliphatic alcohol, and heat stress to probe the interactions and characteristics of P granules and Mutator foci, and have discovered that these condensates remain separate yet adjacent in ectopic environments, respond differentially to perturbation of hydrophobic interactions and, after disruption, can re-establish adjacency in a dynamic manner. The re-establishment of granule interaction may indicate the separate and adjacent nature of these condensates is essential for proper siRNA routing and silencing. Using 3D-STORM imaging, we reveal the nanomolecular scale of Mutator foci and capture a preliminary view of protein density and distribution within the condensate. To image multiple condensates in high resolution we use 3D-Structured Illumination Microscopy and find previously uncharacterized P granule "pocket" formations that house Z granules, Mutator foci and SIMR foci, alluding to a more interesting granule organization than previously suspected. Ultimately this work on understanding RNA silencing through C. elegans germ granules may also provide fundamental biological insight to how phase-separated condensates coordinate cellular processes.