The centriole engagement linker controls centrosome number and contributes to cytoskeletal patterning in large cytoplasms

Centrosomes are non-membrane bound organelles that are composed of microtubule- based centrioles and associated pericentriolar material (PCM) that allows for organization of the microtubule cytoskeleton during interphase. Proliferating cells typically begin the cell cycle with two centrioles, which duplicate concordantly with DNA replication during S-phase. During duplication, it is essential that each existing centriole nucleates formation of precisely one new centriole that remains orthogonally linked until disengagement in mitosis. In normal cycling cells, aberrant centriole duplication can give rise to defective signaling, genome instability and tumorigenesis. In contrast, control of centriole number in terminally differentiating cell types that have exited the canonical cell cycle is poorly understood. Differentiation programs that modify the cell cycle, such as an endocycle, are of particular interest due to the link between the cell cycle and centriole duplication. Thus, the fundamental questions of 1) how cycling cells control centriole number fidelity through the entire cell cycle and 2) terminally differentiating cells, such as those undergoing an endocycle, control their centriole number in the context of a modified cell cycle. To address these questions, we developed in-vitro assays to explore the physical properties underlying the fidelity of centriole duplication in cycling cells as well as in- cellulo approaches to investigate the control of centriole number during the endocycle with a new primary cell culture system. First, we used isolated centrioles with a xenopus extract system to demonstrate the high fidelity of engaged centrioles undergoing duplication. We find that the number of centrosomes in a continuous cytoplasm increases exponentially as a function of ~2n in tight coordination with nucleus-driven cell cycle signaling and that these centrosomes serve as organizing subunits in a large, shared cytoplasm. To understand how these findings applied to a developmentally relevant context, we generated a primary cell culture using trophoblast giant cells (TGCs). Like the Xenopus extract system, these cells have large cytoplasms and undergo multiple modified cell cycles without dividing. We demonstrate that unlike what was observed in the cycling xenopus extract, TGCs in the endocycle do not increase their centriole number exponentially and thus uncouple these processes during development. We also show that TGCs are able to disassemble the robust engagement linker in the absence of mitosis to give rise to supernumerary centrosomes that organize those cells' larger-than- normal cytoplasm, a behavior that has not previously been observed in an unperturbed cell. Finally, we again use isolated centrosomes to interrogate the molecular and structural underpinnings of this high-fidelity engagement link. We find that the connection between the duplicating mother and daughter centrioles is scaffolded by the PCM, has little identifiable ultrastructure, and it is extremely robust to physical and chemical challenges. Together, these results reveal that the engagement linker allows for centriole and centrosome number fidelity by maintaining enforcement of a previously described reduplication block via a physically resilient, PCM-based scaffolding that enables attachment of the daughter to the mother centriole

Keywords: Molecular and Cellular Physiology.
Advisor: Stearns Tim
Ferrell James Ellsworth
Lewis Richard (Richard Sheridan)
Straight Aaron