Molecular Analysis of Interphase Centrosomal Structures

Centrosomes are the main microtubule-organizing centers in animal cells, contributing to mitotic spindle assembly and cell division, but also cell motility, polarity, intracellular transport and the positioning of organelles. Within the centrosome, microtubule nucleation and anchoring are mediated by the so-called pericentriolar material (PCM), recruited by a pair of centrioles. Previous work in the model organisms C. elegans and Drosophila has helped define the basic molecular mechanisms underlying centriole assembly, as well as the assembly and function of the mitotic PCM. In contrast, interphase PCM assembly remains poorly understood, both in the context of canonical, centriole-organized centrosomes, as well as the acentriolar PCM assemblies found on centriolar satellites or redistributed to other cellular locations in differentiated cells. The focus of this standalone project is the characterization of interphase centrosome structures, both canonical and non-canonical, taking advantage of the tools and assays available in C. elegans and Drosophila. The project is divided into two aims, a molecular characterization of the assembly of interphase centrosomes and centrosome-related structures in early embryos and later developmental stages of C. elegans (Aim 1) and of the assembly and function of centriolar satellites in C. elegans and Drosophila, building on the identification of orthologs of the core structural component PCM1 in those species (Aim 2). Aim 1 involves the application of in vivo imaging, proximity-dependent interaction mapping and targeted degradation to examine interphase centrosomes, comparing the underlying molecular mechanisms with those driving mitotic PCM assembly. Aim 2 centers on the characterization of PCM1 orthologs, seeking to identify conserved features underlying PCM1 function across species. It further includes a characterization of the in vivo and in vitro dynamics of PCM1 and PCM1-containing satellites, seeking to evaluate an alternative hypothesis for satellite assembly based on the principles of liquid-liquid phase separation. The power of invertebrate experimental models has hitherto not been extensively applied to interphase centrosomes. Given the evolutionary conservation of basic cellular structures, any findings will advance our understanding of a biomedically important cellular organelle.