Mechanism of centrosome maturation in vertebrates

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Project Summary/Abstract Faithfully segregating chromosomes during mitosis relies on a properly assembled spindle apparatus with centrosomes anchoring mitotic microtubules on both sides of it. The centrosome is a major microtubule- organizing center (MTOC) in animal cells. It consists of a pair of centrioles surrounded by the pericentriolar material (PCM). The PCM nucleates and anchors microtubules and thus dictates the MTOC activity of the centrosome. Centrosomes rapidly expand their PCM at the onset of mitosis. This process, termed centrosome maturation, is critical for spindle organization and chromosome segregation. However, there is a fundamental gap in understanding how the PCM is assembled and regulated at the onset of mitosis. In addition, while the framework of centrosome maturation has been elucidated at the molecular level, the fundamental principle of PCM assembly remains elusive at the organellar level—without an enclosing membrane, what keeps the crowded PCM proteins from dispersing? What glue holds this membraneless ensemble together as a micron- sized centrosome during mitosis? In vertebrates, centrosome maturation is driven in part by pericentrin (PCNT), a large PCM protein linked to human developmental disorders, including primordial dwarfism, microcephaly, and Down syndrome. PCNT acts as a scaffold in the recruitment of other PCM proteins during centrosome maturation. Our recent work reveals that PCNT is delivered co-translationally to mitotic centrosomes and this co-translational targeting of PCNT facilitates centrosome maturation. Our long-term goal is to understand how the centrosome is assembled and functions. The overall objective is to elucidate the role of PCNT in regulating centrosome maturation and PCM assembly. Based on our recent work and preliminary studies, we hypothesize that co-translational protein targeting promotes PCNT phase separation via proximity- driven condensation, a process that facilitates proper PCM assembly, and that PLK1 regulates the co- translational targeting process in addition to its role at the mitotic centrosome. We will test our hypothesis in three specific aims: (1) determine the contribution of PCNT condensation in centrosome maturation, (2) determine the relationship between co-translational targeting and phase separation of PCNT, and (3) determine the molecular mechanisms through which dynein and PLK1 regulate co-translational targeting of PCNT. The molecular mechanisms underlying centrosome assembly in vertebrate cells remain to be elucidated. Understanding these processes is the key to fully understanding how centrosome function is normally regulated and disrupted in human disease.