The molecular mechanisms that direct the centrosome, the primary microtubule-organizing center of the cell, to its two distinct and alternate fates: spindle formation or flagella nucleation, are not known. An improved understanding of the pathways involved and their regulation is essential to develop interventions to prevent disorders associated with disregulation of centrosome fate determination including cilia/flagella malformations as well as cancer development. The long-term goal of this work is to understand how dramatic alterations in microtubule architecture occurring during spermatogenesis are properly regulated. In order to accomplish this goal, experiments are designed to determine how the activity of Protein Phosphatase-1 (PP1) is coordinated between cell division and cilia/flagella formation. The central hypothesis to be tested is that dynamic regulation of PP1 acts as the molecular switch between these distinct activities of the centrosome through regulation of AurA and Nek2 kinases. The rationale for the proposed work is that dissection of the PP1 regulatory network will expand understanding of how the fate of the centrosome becomes determined to form a flagellum instead of proceeding through the cell cycle to nucleate the spindle. In order to achieve the experimental objectives, a bimodal approach will be employed by first determining the role of PP1 regulatory subunits in each pathway using a cultured cell model that recapitulates flagella formation followed by integration of this information using an animal model deficient in a component of the pathway. The central hypothesis will be tested with the following specific aims: (1) determine the mechanism by which modulation of PP1 activity controls centrosome separation, (2) determine the mechanism by which activating and inhibiting subunits modulate PP1 to control cilia length and number, and (3) determine the role of Nek2 and AurA in flagella formation in male germ cells. The contribution of this work is expected to be a detailed understanding of how centrosome fate determination, and therefore flagella biogenesis, is controlled by activating and inhibiting regulators of PP1 including the newly identified PP1 regulatory subunit PPP1R42. This innovative approach examines the pivotal role of PP1 regulatory subunits in controlling the molecular switch at the intersection of a signaling network that specifies flagella biogenesis versus spindle formation.