Mechanism and impact of de novo centriole assembly in mammalian cells

中心粒决定细胞组织，在细胞分裂中起重要作用。中心粒在s期复制，因此在细胞分裂过程中，有一对中心粒可用于组织有丝分裂纺锤体的每一个极点。虽然现有中心粒对的复制是中心粒生成的典型模式，但如果使用激光或显微手术去除现有中心粒，新的中心粒可以形成“de-novo”。因此，细胞具有重新形成新中心粒的能力，但对这一途径知之甚少，而且检测其机制的合适模型也很少。***新受精的小鼠胚胎缺乏中心粒，在囊胚期胚胎（~64细胞期）发生协调爆发的新生中心粒制造。我们最近开发了一种方法来观察这种新生中心粒生物发生的浪潮，使用转基因小鼠表达GFP标记的中心粒蛋白centrin-2拷贝（CETN2::GFP）。此外，我们有先进的工具来成像微管动力学和细胞分裂在这种情况下，和操作基因功能使用反义方法和融合蛋白过表达是常规在我们的实验室。在这里，我们建议利用这些新方法来研究中心粒组装的机制和影响。我们的三个目标是：***目标1。确定新生中心粒组装的时空动力学。我们将使用CETN2::GFP胚胎来确定中心粒出现的精确动力学。我们将建立中心粒组装的结构时间过程，并检查每个步骤的细胞周期相依赖性。这些实验将为理解重新组装中心粒的分子要求提供坚实的基础。* * *的目标2。目的：探讨新生中心粒组装的分子机制。已经确定了一小部分因子对典型中心粒复制至关重要，如CPAP和SAS6。我们将确定这些因素在新生中心粒生物发生中的作用，以及DEUP1的作用，DEUP1是一种新发现的蛋白质，在纤毛发生的背景下对中心粒扩增至关重要。这些实验将建立从头形成的分子要求，并解决如何在最初的几次细胞分裂中限制形成。* * *的目标3。确定改变中心粒组装时间的影响。我们将分析中心粒出现失败对细胞分裂、结构、极化和命运的影响。我们将使用不同的方法介绍外源性中心粒，包括我们实验室最近开发的一种创新的微操作方法。我们将评估额外中心体的影响，并观察中心体引入正常无中心体环境后的命运。***该程序将首次利用小鼠胚胎的易感性来研究新生中心粒形成的机制和额外中心体对细胞分裂的影响。********

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.********