Mechanisms controlling the inactivation of microtubule organizing center function at the centrosome

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Project Summary/Abstract The centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Upon cell differentiation, MTOC activity at the centrosome is often maintained in an inactive state as MTOC function is reassigned to non-centrosomal sites to accommodate different cell functions. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers and has been linked to invasive cell behavior, little is known about how the centrosome is inactivated as an MTOC either during mitotic exit or maintained in an inactive state in differentiated cells. We are using C. elegans as a model to understand these fundamental knowledge gaps in MTOC biology in a live organism. Our analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of distinct protein territories organized into an inner and outer sphere that are removed from the centrosome at different rates and using different behaviors. We found that phosphatases oppose the addition of PCM by mitotic kinases, ultimately catalyzing the dissolution of inner sphere PCM proteins at the end of mitosis. The nature of the PCM appears to change such that the remaining aging PCM outer sphere is ruptured into sub-PCM ‘packets’ by microtubule based cortical pulling forces. Thus, the centrosome appears to be inactivated as an MTOC by a two-step mechanism beginning with PCM dissolution, followed by mechanically controlled rupture. In the proposed research, we will uncover the mechanisms underlying this two-step model for the inactivation of MTOC function at the centrosome. We will determine the mechanisms underlying PCM dissolution, identifying the pertinent phosphatases, their targets at the centrosome, and the role of the removal of these targets in disassembly (Aim 1). We will then specifically uncover the protein-protein interactions underlying the PCM outer sphere and packets (Aim 2). Finally, we will probe the mechanisms underlying the inactivation of MTOC function in differentiated cells and test the role of centrosome inactivation in cell differentiation (Aim 3). Proper microtubule organization is essential for normal development and cell function and hyperactive MTOC function at the centrosome is a hallmark of some cancers. Thus, the molecules uncovered in these studies could provide potential therapeutic targets as well as shed light on this important, but understudied topic in cell and developmental biology.