Mechanism and impact of de novo centriole assembly in mammalian cells

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Centrioles dictate cell organisation and are instrumental in cell division. Centrioles replicate in S-phase, such that one pair is available to organize each pole of the mitotic spindle during cell division. Whilst replication of the existing centriole pair is the canonical mode of centriole generation, new centrioles can form `de-novo' if the existing centrioles are removed using lasers or microsurgery. Thus, cells have the capability to form new centrioles de novo, but very little is known about this pathway, and appropriate models for examining its mechanism are scarce.***          Newly fertilized mouse embryos lack centrioles and a coordinated burst of de novo centriole manufacture occurs in blastocyst-stage embryos (~64 cell stage). We recently developed means for observing this wave of de novo centriole biogenesis using a transgenic mouse expressing a GFP-tagged copy of the centriole protein centrin-2 (CETN2::GFP). Moreover we have cutting edge tools for imaging microtubule dynamics and cell division in this setting, and manipulation of gene function using antisense approaches and fusion-protein overexpression are routine in our lab. Here we propose to exploit these novel approaches to examine the mechanism and impact of centriole assembly. Our three Aims are: ***Aim 1. To determine the spatiotemporal dynamics of de novo centriole assembly. We will use CETN2::GFP embryos to determine the precise dynamics of centriole emergence. We will establish the structural time-course of centriole assembly and examine the cell cycle phase-dependency of each step. These experiments will provide a robust basis for understanding the molecular requirements for de novo centriole assembly. ***Aim 2. To examine the molecular mechanism of de novo centriole assembly. A small cadre of factors have been identified as essential for canonical centriole replication, such as CPAP and SAS6. We will determine the role of these factors in de novo centriole biogenesis, as well as that of DEUP1, a newly characterised protein found to be essential for entriole amplification in the context of ciliogenesis. These experiments will establish the molecular requirements for de novo formation, and address how formation is restrained for the first several cell divisions. ***Aim 3. To determine the impact of altering the timing of centriole assembly. We will analyze the impact of failed centriole emergence upon cell division, structure, polarisation, and fate. We will introduce exogenous centrioles using different approaches, including an innovative micromanipulation approach developed recently in our lab. We will assess the impact of extra centrosomes, and observe the fate of centrosomes introduced into a normally centrosome-free environment. ***This program will for the first time exploit the tractability of the mouse embryo to examine the mechanism of de novo centriole formation and the impact of extra centrosomes upon dividing cells.********