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.

Previous work performed on the nematode worm C. elegans and the fruit fly Drosophila has helped define the fundamental molecular mechanisms underlying centriole assembly, as well as those underlying the assembly and function of mitotic centrosomes. The goal of this project was to apply the powerful tools and assays available in these model organisms to study the still rather poorly understood mechanisms underlying interphase centrosome assembly, both in the context of canonical, centriole-organized centrosomes and the acentriolar assemblies found on centriolar satellites. Work carried out in the lab over the past four years has largely followed the lines of investigation laid out in the original proposal. One notable early outcome was the development of a novel tool to identify protein proximity interactors in C. elegans in a developmental stage and tissue/cell-specific manner. Since its publication in 2022 we have distributed this tool to numerous labs worldwide. The same tool should be readily translatable to other experimental models. Indeed, we have employed the same approach to study centriolar satellites in Drosophila. Work on centrosomes has since progressed from a purely molecular description to a characterization of their material properties, which are central to their function as microtubule-organizing centers. Using a combination of quantitative microscopy-based approaches we found that, contrary to predictions of a hardening of the underlying centrosome scaffold, centrosomes become increasingly soft and thus more deformable as cells progress from interphase to mitosis. Theoretical modeling reveals that centrosome softening reduces variations in spindle length and chromosome positioning, suggesting it may mitigate the potentially damaging impact of spindle forces on chromosome segregation. A certain degree of elastic give may also be essential to expand the mitotic centrosome and develop its full microtubule nucleation potential. As for centriolar satellites, defining aspects of their function conserved between vertebrates and Drosophila led us to uncover a novel role for satellites, not in intracellular transport as previously thought, but in the coordinate synthesis of centrosomal and ciliary proteins, a paradigm shift that not only significantly advances our understanding of these cellular structures but, given that similar principles likely apply to components of other cellular structures, may also shed light on cellular compartmentalization more generally.