Centrosomes play several fundamental roles in the cell including the nucleation and organization of microtubules for spindle assembly and molecular motor-driven processes. They also anchor important regulatory activities that control centrosome and spindle function and may contribute to tumorigenesis through the organization of dysfunctional spindles that fail to segregate chromosomes properly. The overall objective of our research is to understand the molecular basis of centrosome function. Our general strategy is to focus on the function of pericentrin, a centrosome protein I identified in six years ago, using a combination of molecular, biochemical and morphological strategies. Over the past budget period, we have made significant progress in understanding pericentrin function. We determined that pericentrin forms a complex with microtubule nucleating proteins including gamma tubulin and is in close proximity with gamma tubulin at the centrosome. We found that pericentrin interacts directly with cytoplasmic dynein light intermediate chain 1 and that dynein mediates assembly of pericentrin and gamma tubulin onto centrosomes. Moreover, pericentrin overexpression disrupts dynein localization, causes spindle defects and creates aneuploid cells. In collaboration with Dr. J. Scott (Vollum Inst.), we showed that pericentrin anchors kinase A to centrosomes and that this interaction is important for spindle function. Based on the observations outlined above, we have formulated a model in which pericentrin serves to specifically transport, anchor and organize important functional and regulatory activities at the centrosome. Over the next budget period we will continue to study the role of pericentrin and pericentrin- interacting proteins in centrosome and spindle function using both in vivo approaches and in vitro reconstitution. In the first aim of this proposal we will investigate the role of pericentrin in the assembly of microtubule nucleating complexes onto centrosomes. We will use cytoplasmic extracts prepared from Xenopus eggs to test the ability of protein complexes containing pericentrin to mediate the assembly of gamma tubulin complexes onto centrosomes in vitro. The second objective is to determine the significance of the interaction between pericentrin and dynein light intermediate chain 1. Specifically, we will use a dominant negative form of pericentrin to uncouple the pericentrin-dynein light intermediate chain interaction and examine the role of this interaction in centrosome assembly and spindle organization.
The third aim i s to identify and characterize other pericentrin-interacting proteins and to characterize a novel centriole protein which, like pericentrin, was identified using autoimmune sera.
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|Hehnly, Heidi; Doxsey, Stephen (2014) Rab11 endosomes contribute to mitotic spindle organization and orientation. Dev Cell 28:497-507|
|Chen, Chun-Ting; Hehnly, Heidi; Yu, Qing et al. (2014) A unique set of centrosome proteins requires pericentrin for spindle-pole localization and spindle orientation. Curr Biol 24:2327-2334|
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|Foraker, Amy B; Camus, Stéphane M; Evans, Timothy M et al. (2012) Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG. J Cell Biol 198:591-605|
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