Molecular Mechanisms Controlling Centriole Duplication

Abstract

Centrioles are barrel shaped, non-membrane bound organelles that typically exist in pairs where the younger ‘daughter’ centriole emanates orthogonally off of the proximal end of the older ‘mother’ centriole. The mother-daughter centriole pair and their surrounding proteins constitute the centrosome, which is the primary regulator of chromosome separation and cell division in animal cells. The centrosome controls these processes by acting as the microtubule organizing center (MTOC) of the cell – it nucleates microtubules during mitosis to form the mitotic spindle required to promote chromosome segregation. In order for proper chromosome segregation to occur, each cell needs to contain only two centrosomes entering mitosis – one at each pole to achieve spindle bipolarity. To achieve this, each centrosome must duplicate itself throughout the cell cycle. This duplication event is orchestrated by the centrioles – a single daughter centriole is built off of each mother centriole as the cell cycle progresses. The formation of excess daughter centrioles around a mother is a mechanism of centriole amplification, which is a driver of aneuploidy and tumor formation. Because of this, the process of centriole duplication is tightly controlled on a molecular level.The process of centriole duplication is coordinated in part by two conserved proteins: the Ser/Thr kinase Plk4 and its multifunctional regulator, Asterless. Previous work has shown that an excess of Asl protein levels, Plk4 protein levels and Plk4 catalytic activity can contribute to centriole amplification. Thus, it is crucial to obtain a complete understanding of how Plk4 and Asl coordinate their functions to ensure centrioles duplicate properly. It is known that Asl can regulate Plk4 by targeting it to the centriole as well as control its stability in a cell-cycle dependent manner. However, we still have an incomplete understanding of exactly how these two proteins promote the formation of a single daughter centriole during each cell cycle. Here we identify multiple new regulatory mechanisms that Plk4 and Asl utilize in order to control centriole assembly. First, we identify Asl as a multifunctional phosphorylation-dependent regulator of Plk4 catalytic activity. When dephosphorylated, Asl can bind Plk4 and stimulate its activity. Asl itself then becomes phosphorylated, and functions as a Plk4 inhibitor, invoking a negative feedback mechanism to thought prevent Plk4 from inducing centriole amplification through its catalytic activity. In the following chapter, we identify the two phospho-residues in Asl necessary for Plk4 inhibition, while also determining that this inhibition is dependent on the interaction between Plk4 and Asl. Importantly, we describe a role for this negative feedback mechanism in limiting centriole assembly: Phospho-Asl localizes to puncta around the mother centriole where they keep Plk4 inhibited. The phosphatase PP2A is then recruited to the centriole to dephosphorylate Asl at only one of the puncta, where Plk4 can then be activated to promote centriole assembly. Lastly, we reveal data that will be important for future studies on this topic, such as the mechanism of PP2A recruitment to the centriole, dynamics of Plk4 activation and inhibition, and the possible existence of an unidentified cellular Plk4 inhibitor.