Mechanism and impact of de novo centriole assembly in mammalian cells

中心粒决定细胞的组织结构，在细胞分裂中起着重要作用。中心粒在S期复制，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极。虽然复制现有的中心粒对是中心粒生成的典型模式，但如果使用激光或显微手术移除现有的中心粒，新的中心粒可以形成“从头开始”。因此，细胞有能力形成新的中心粒，但对这一途径知之甚少，也缺乏合适的模型来研究其机制。 研究发现，新受精的小鼠胚胎缺乏中心粒，在囊胚期胚胎(~细胞期)出现协调的从头中心粒制造爆发。我们最近开发了一种方法来观察这波从头开始的中心粒生物发生，使用表达GFP标记的中心蛋白Centin-2(CETN2：：GFP)的转基因小鼠。此外，在这种情况下，我们拥有成像微管动力学和细胞分裂的尖端工具，使用反义方法和融合蛋白过度表达来操纵基因功能在我们实验室是司空见惯的。在这里，我们建议利用这些新的方法来研究中心粒组装的机制和影响。我们的三个目标是： 目的1.测定从头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，并检查每一步的细胞周期时相依赖性。这些实验将为理解从头中心粒组装的分子要求提供可靠的基础。 目的2.探讨从头中心粒组装的分子机制。一小部分因子已被确定为典型中心粒复制所必需的，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是一种新的特征蛋白，被发现在纤毛发生的背景下对于内质醇的放大是必不可少的。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个细胞分裂中抑制形成。 目的3.确定改变中心粒组装时间的影响。我们将分析失败的中心粒出现对细胞分裂、结构、极化和命运的影响。我们将使用不同的方法引入外源中心粒，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，并观察进入正常无中心体环境的中心体的命运。 这个项目将首次利用小鼠胚胎的易操纵性来研究从头中心粒形成的机制以及额外的中心体对分裂细胞的影响。

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.