Microtubule Nucleation and its Regulation

微管成核及其调控

• 批准号: 8668221
• 负责人: Trisha N. Davis
• 金额: $ 32.52万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668221
• 关键词: Address; Affect; Alleles; Back; Behavior; Binding; Biochemical; Biological; Cell Cycle; Cell Cycle Stage; Cells; Centrosome; Chromosomes; Collection; Complement; Complex; Core Protein; Cytoplasm; Data; Defect; Elements; Ensure; Eukaryota; Event; Face; Feedback; Filament; Genes; Genetic; Genetic Screening; Geometry; In Vitro; Kinetics; Kinetochores; Linker-Scanning Mutagenesis; Mammalian Cell; Measures; Methods; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic Checkpoint; Modeling; Modification; Mutation; Nuclear; Phosphorylation; Phosphorylation Site; Phosphotransferases; Photobleaching; Ploidies; Positioning Attribute; Post-Translational Protein Processing; Preparation; Process; Property; Protein Binding; Proteins; Recovery; Regulation; Role; Set protein; Side; Signal Transduction; Structural Protein; Structure; System; Temperature; Testing; Tubulin; Work; Yeasts; base; beta Tubulin; biophysical techniques; cell cortex; dimer; gamma Tubulin; human STK6 protein; in vivo; insight; mutant; novel; protein degradation; receptor; reconstruction; spindle pole body; temperature sensitive mutant; tool

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.

中心体的基本功能是使微管成核和稳定， 染色体（纺锤体微管）或定位纺锤体（星形微管）。微管 成核是细胞周期中的关键事件，细胞调节中心体的成核能力， 在有丝分裂开始时需要更多的微管。本项目的重点是成核 方法与体外和体内实验方法的组合。 γ-微管蛋白是微管成核的保守的必需元件。与另外两种保守的蛋白质 它形成γ-微管蛋白小复合物。多聚体的小复合体可以形成一个环，被认为是模板 α-β微管蛋白二聚体组装成原丝环，从而形成新生微管 （二）、但是，控制小络合物环的形成，活化环的成核能力 复合物，α-β微管蛋白二聚体被捕获和稳定的机制，以及反馈 控制成核能力的机制仍然没有很好地理解。 前两个目标解决了γ-微管蛋白环复合物的激活。6.5A的y- 微管蛋白复合物显示Y-微管蛋白保持在13的环中。然而，它们的位置相距太远， 在微管中发现的13种原丝的模板（1）。这一结果表明了目前的假设：y- 微管蛋白小复合物可被激活以通过结构转变形成成核模板， 定位y-微管蛋白以匹配形成微管的α-β微管蛋白二聚体的取向和几何形状， 微管。这种转化需要γ-微管蛋白小复合物的闭合和变构激活 (1)（未公布数据）。我们将使用遗传学、生物化学和细胞生物学方法来进行功能性研究。 体外和体内的γ-微管蛋白复合物的分析。我们的工作是对结构和 Agard实验室采用的生物物理学方法。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，检查整体的控制。 中心体的成核能力。中心体的成核能力扩大了几倍， 有丝分裂的准备（3）。这种扩张被称为中心体成熟。对于 在高等真核生物中，一个模型开始出现，即近中心粒的组装和成熟 材料涉及由有丝分裂激酶PIK 1的蛋白磷酸化驱动的核心蛋白的富集 和Aurora-A（4）。然而，由于其超微结构和成分的复杂性， 阻碍了进展。涉及的蛋白质的完整补充是未知的，上游信号 引发磷酸化的原因尚不清楚，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调节。显著扩张 在有丝分裂检查点激活时发生纺锤体极体的分裂。这提供了一个非常简单的方法 通过实验控制膨胀，从而研究其要求（5-7）。在酵母中， SPB中的蛋白质是已知的，并且已经确定了它们的许多磷酸化位点（8）。在 此外，我们还通过遗传筛选鉴定了参与扩增过程的三种蛋白质（9）。在 第三个目标，扩张和周转的动力学，这三种蛋白质在扩张过程中的作用 和磷酸化调节的γ-微管蛋白复合物的作用将作为中心体的模型进行检查 成熟这项工作将补充Winey实验室项目的目标，该项目将确定 中心体组装中核心蛋白的磷酸化。