Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY Centrosomes, the major microtubule organizing centers in animal cells, are duplicated precisely once per cell cycle to catalyze microtubule generation for rapid bipolar spindle assembly and ensure accurate chromosome segregation. Centrosome amplification and fragmentation are frequently observed in cancer cells and are thought to contribute to their chromosome instability and aneuploidy. Centrosomes are comprised of a centriolar core that recruits pericentriolar material matrix to dock microtubule-nucleating g-tubulin-containing complexes (γTuCs). Given the central role of centrosomes in cell division and their frequent aberration in cancer, there has been significant interest in developing centrosome-targeting agents for cancer therapy. In this proposal, we elucidate distinct centrosomal microtubule generation mechanisms that contribute to spindle formation and address how the primacy of the centrosome as the dominant site for spindle microtubule generation is maintained. The dominance of centrosomes as mitotic microtubule nucleation centers, which reinforces bipolarity of the spindle, is ensured in part by the mitotic kinase PLK1, which independently controls expansion of the pericentriolar matrix and matrix-anchored microtubule nucleation during mitotic entry. PLK1 executes both tasks by phosphorylating distinct regions of the primary structural component of the pericentriolar matrix (CDK5RAP2 in humans, Cnn in Drosophila and SPD-5 in C. elegans). In Aim 1, we will pursue parallel biochemical and in vivo approaches in the C. elegans embryo to determine how PLK-1 phosphorylation remodels the pericentriolar matrix component SPD-5 to enable gTuC docking and activation. We will also determine how centrioles recruit and organize the mitotic pericentriolar matrix and dictate its size, inspired by preliminary data indicating that the mitotic pericentriolar matrix is not simply an expansion of interphase pericentriolar matrix, but is an independent entity. In addition to phosphoregulation enforcing dominance of centrosomes as microtubule-generating sites, the primacy of the centrosome is ensured by the ubiquitin ligase TRIM37, which functions as a “guardian of the centrosome”. TRIM37 prevents rogue collections of centrosomal proteins from forming ectopic microtubule- organizing centers. In Aim 2, we address how TRIM37 recognizes and suppresses ectopic centrosomal protein assemblies, by focusing on its signature TRAF domain that is unique in the TRIM superfamily, its potential ability to oligomerize, and its ubiquitin ligase activity. Centrosomes in human cells have at least two mechanisms for microtubule generation that independently support spindle assembly: the well-studied pericentriolar matrix- anchored mechanism and a pericentriolar matrix-independent centriole-anchored mechanism. In Aim 3, we investigate the assembly and function of a specific type of ectopic microtubule-organizing center formed in TRIM37 mutant cells that is independent of the pericentriolar matrix, concentrates core centriolar proteins, and robustly supports spindle formation in the absence of centrosomes. This effort should shed light on poorly understood pericentriolar matrix-independent mechanisms for spindle microtubule generation in human cells.
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