Microtubule Nucleation and its Regulation

中心体的基本功能是成核和稳定起隔离作用的微管。 染色体(纺锤体微管)或定位纺锤体(纺锤体微管)。微管 成核是细胞周期中的一个关键事件，细胞调节中心体的成核能力以 当需要更多的微管时，在有丝分裂开始时增加。这个项目的重点是成核。 采用体外和体内实验相结合的方法。 γ-微管蛋白是微管成核过程中一种保守的必需成分。与另外两种保守的蛋白质 它形成了伽马-微管蛋白小复合体。小复合体的多聚体可以形成一个被认为是模板的环 微管二聚体将α-β微管蛋白二聚体组装成原丝环，从而形成新生微管 (2)。但是，控制小复合环的形成，激活环的成核能力 复杂性，捕获和稳定α-β微管蛋白二聚体的机制，以及反馈 控制成核能力的机制仍不是很清楚。 前两个目的是针对y-微管蛋白环复合体的激活。Y-6.5A细丝结构 微管蛋白复合体显示，γ-微管蛋白以13个环的形式存在。然而，它们的位置太远，无法 模板：在微管中发现的13根原丝(1)。这一结果提出了目前的假设：y- 微管蛋白小复合体可以通过结构转变被激活以形成成核模板，该结构转化 定位y-微管蛋白，以匹配形成 微管。这种转变既需要关闭γ-微管蛋白小分子复合体，又需要变构激活。 (1)(未公布数据)。我们将使用遗传、生化和细胞生物学方法来执行功能 γ-微管蛋白复合体的体内外分析。我们的工作直接补充了结构和 阿加德实验室采用的生物物理方法。我们将一起详细了解 微管成核的物理和生物学基础。 在第三个目标中，我们从成核机制后退一步，检查总体上的控制 中心体的成核能力。中心体的成核能力扩大了几倍 有丝分裂准备(3)。这种扩张被称为中心体成熟。对于中心体来说 在高等真核生物中，一种模型开始出现，即中央周围三极细胞的组装和成熟 材料涉及由有丝分裂酶PIk1的蛋白磷酸化驱动的核心蛋白的富集化 和极光-A(4)。然而，中央周围物质在超微结构和成分上的复杂性 阻碍了进步。所涉及的全部蛋白质是未知的，上游信号 触发磷酸化的因素尚不清楚，而磷酸化对成熟的影响仍有待研究。 被发现。在酵母中，成核能力受细胞周期事件和细胞倍性的调节。值得注意的是扩张 当有丝分裂检查点被激活时，纺锤体的极体发生。这提供了一种非常简单的方法 通过实验控制膨胀，从而研究其要求(5-7)。在酵母中，所有的结构 SPB中的蛋白质是已知的，它们的许多磷酸化位点已经确定(8)。在……里面 此外，我们通过基因筛查确定了三种参与扩增过程的蛋白质(9)。在……里面 第三个目标，膨胀和周转的动力学，这三种蛋白质在膨胀过程中的作用 伽马-微管蛋白复合体的磷酸化调节作用将作为中心体的模型进行检验。 成熟。这项工作将补充Winey实验室项目中的目标，该项目将确定 中心体组装中核心蛋白的磷酸化。

The fundamental function of centrosomes is to nucleate and stabilize microtubules that serve to segregate chromosomes (the spindle microtubules) or position the spindle (the aster microtubules). Microtubule nucleation is a critical event in the cell cycle and cells regulate the nucleation capacity of the centrosome to increase at the start of mitosis when more microtubules are required. This project focuses on the nucleation process with a combination of both in vitro and in vivo experimental approaches. Gamma-tubulin is a conserved essential element of microtubule nucleation. With two other conserved proteins it forms the gamma-tubulin small complex. Multimers of the small complex can form a ring that is thought to template the assembly of alpha-beta tubulin dimers into a ring of protofilaments and thereby form a nascent microtubule (2). However, the control of small complex ring formation, the activation of the nucleation capacity of the ring complex, the mechanism by which alpha-beta tubulin dimers are captured and stabilized, and the feedback mechanisms that control the nucleation capacity are still not well understood. The first two aims address the activation of the y-tubulin ring complex. The 6.5 A structure of filaments of the y- tubulin complex showed the y-tubulins are held in a ring of 13. However they are positioned too far apart to template the 13 protofilaments found in microtubules (1). This result suggested the current hypothesis: the y- tubulin small complex could be activated to form a template for nucleation by a structural transformation that positions the y-tubulins to match the orientation and geometry of the alpha-beta tubulin dimers that form the microtubule. This transformation requires both closure of the gamma-tubulin small complex and allosteric activation (1) (unpublished data). We will use genetic, biochemical and cell biological approaches to perform functional analyses of the y-tubulin complex in vitro and in vivo. Our work is directly complementary to structural and biophysical approaches taken by the Agard lab. Together, we will provide a detailed understanding of the physical and biological basis of microtubule nucleation. In the third aim we step back from the mechanism of nucleation and examine the control of the overall nucleation capacity of the centrosome. The nucleation capacity of the centrosome expands several fold in preparation for mitosis (3). This expansion has been termed centrosome maturation. For the centrosome of higher eukaryotes, a model is beginning to emerge that the assembly and maturation of the pericentriolar material involves the enrichment of core proteins driven by protein phosphorylation by the mitotic kinases PIkl and Aurora-A (4). However the complexity of the pericentriolar material both in ultrastructure and composition has hampered progress. The full complement of proteins that are involved is not known, the upstream signals that trigger phosphorylation are not known, and the consequences of phosphorylation on maturation remains to be discovered. In yeast nucleation capacity is regulated by cell cycle events and cell ploidy. Notably expansion of the spindle pole body occurs upon activation of the mitotic checkpoint. This provides a very simple method to experimentally control expansion and thereby study its requirements (5-7). In yeast, all the structural proteins in the SPB are known and many of their sites of phosphorylation have been determined (8). In addition we have identified three proteins involved in the expansion process through a genetic screen (9). In the third aim, the kinetics of expansion and turnover, the role of these three proteins in the expansion process and the role of phospho-regulation of the gamma-tubulin complex will be examined as a model for centrosome maturation. This work will complement the aim in the Winey lab project that will determine the role of the phosphorylation of core proteins in centrosome assembly.