Microtubule Nucleation: Structure & Mechanism

微管成核：结构

• 批准号: 8027741
• 负责人: DAVID A. AGARD
• 金额: $ 43.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8027741
• 关键词: Animals; Antibodies; Baculoviruses; Bardet-Biedl Syndrome; Behavior; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Cell Cycle; Cell Polarity; Cells; Centrioles; Centrosome; Cilia; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Cytokinesis; Cytoskeleton; Data; Defect; Dependency; Development; Disease; Disulfides; Drosophila genus; Electron Microscopy; Electrons; Embryo; Embryonic Development; Engineering; Face; Filament; Freezing; Fungal Genome; GTP Binding; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Hydrolysis; Image; In Situ; In Vitro; Interphase; Kinetics; Laboratories; Life; Mass Spectrum Analysis; Measures; Methodology; Methods; Microscopic; Microtubule Triplet; Microtubules; Mining; Mitotic spindle; Modeling; Molecular; Molecular Conformation; Movement; Mutagenesis; Mutate; Nephronophthisis; Nucleotides; Oral; Organelles; Phosphorylation; Play; Positioning Attribute; Post-Translational Protein Processing; Preparation; Protein Binding; Protein Conformation; Protein Kinase; Proteins; Regulation; Resolution; Role; Saccharomyces cerevisiae; Site; Small Interfering RNA; Sodium Chloride; Structure; Sucrose; Surveys; Syndrome; Testing; Triplet Multiple Birth; Tubulin; Vesicle; Width; Work; Yeasts; adaptive optics; analog; base; cancer cell; cell type; digital; electron crystallography; electron tomography; genetic regulatory protein; genome wide association study; human disease; improved; in vitro Assay; in vitro testing; in vivo; innovation; light microscopy; monomer; new technology; optical imaging; particle; polymerization; protein complex; public health relevance; reconstitution; reconstruction; research study; spindle pole body; success; tomography; trafficking

DESCRIPTION (provided by applicant): The centrosome is of fundamental importance to animal cells in that it is the principal nucleator of the microtubule (MT) cytoskeleton and harbors the centrioles, specialized structures that are required for cilia formation and contribute to spindle-pole positioning. Through these principal roles, the centrosome is important in embryonic development and human disease. During interphase, MTs are required for vesicle trafficking and cell polarity. In most cell types, many of the MTs that form the mitotic spindle originate at the centrosome. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. The primary aims of this proposal are to determine the molecular mechanism of MT nucleation, and to understand how the nucleating machinery is assembled and regulated. Moreover we will begin to explore the mechanism by which microtubule doublets and triplets are formed at the centriole by focusing on the newly discovered ?- and ?-tubulins and their as yet unknown, binding partners. Our laboratory is uniquely poised to utilize a hierarchical array of structural approaches (x-ray crystallography, electron microscopic (EM) single particle reconstruction, and EM Tomography, small-angle x-ray scattering (SAXS)) to determine the structures of ?-, ?- and ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. By combining structural studies, MT assembly experiments and kinetic modeling, our previous work is leading to a paradigm shift in understanding the underlying principles of MT formation, suggesting a new role for GTP, a different model for MT assembly and an unexpected mode of regulation of nucleation in the ?-tubulin complexes. The proposed experiments will continue and expand upon these results, providing a detailed understanding of the physical origins of MT assembly, and the cellular machinery that dictates MT formation. In vitro predictions of modes of action or regulation will be assayed in vivo using mutagenesis and siRNA and live imaging. Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between 12-tubulin, ?-, ?- and ?-tubulin complexes, regulatory proteins and the centrosomal matrix. Public Health Relevance: The normal function of centrosomes and centrioles is directly relevant to human disease, and the proposed work is aimed at understanding the molecular mechanisms of these cellular components. Abnormal centrosome number and behavior is a common hallmark of cancer cells, and centriole defects are involved in many human ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel syndrome, and Oral-Facial-Digital syndrome.

描述（由申请人提供）：中心体对动物细胞具有至关重要的重要性，因为它是微管（MT）细胞骨架的主要核定器，并具有中心元素，这是纤毛形成所需的专业结构，并促进了螺旋杆杆的定位。通过这些主要角色，中心体在胚胎发育和人类疾病中很重要。在相间期间，囊泡运输和细胞极性需要MT。在大多数细胞类型中，形成有丝分裂纺锤体的许多MT源于中心体。中心体还具有超出MT成核的重要作用，包括细胞因子中的功能，在细胞周期中的进展以及隔离或定位一些蛋白质以控制它们何时何地活性。该提案的主要目的是确定MT成核的分子机制，并了解如何组装和调节成核机制。此外，我们将开始探索通过专注于新发现的？ - ？ - 微管蛋白及其尚不清楚的绑定伙伴，在中心位置形成微管双线和三倍的机制。我们的实验室可以独特地准备利用一系列结构方法（X射线晶体学，电子显微镜（EM）单个粒子重建和EM层析成像，小角度X射线散射（SAXS））来确定？ - ，？ - ，？ - 和？ - 和 - tubulin complect的结构，并了解其在体外的机制，并在体系中的作用，以确定其机制，以确定其机构的作用，以确定其机制，并确定其机制的作用，并确定其机构的机制，并确定其机制的机制，并且可以实现其机制，并且可以实现其机制，并且可以实现其机制，并且可以实现其机制，并且可以实现其机制，并且可以确定这些机制，并且可以实现其机制，并确定其机制和动作的作用。建模。通过结合结构研究，MT组装实验和动力学建模，我们以前的工作正在导致理解MT形成的基本原理的范式转移，这表明GTP具有新的作用，MT组装的不同模型以及一种意外的 - tububulin comploce中的成核调节模式。提出的实验将继续并扩展这些结果，从而对MT组装的物理起源以及决定MT形成的细胞机制提供了详细的理解。将使用诱变，siRNA和活成像在体内测定对作用或调节模式的体外预测。从原子到整个细胞器的跨度尺寸尺寸，我们的长期目标是综合12-微管蛋白之间的所有相关结构和功能相互作用的原子分辨率图片，？？ - ，？ - ？ - - 微管蛋白复合物，调节蛋白和中心体基质。公共卫生相关性：中心体和中心元素的正常功能与人类疾病直接相关，拟议的工作旨在了解这些细胞成分的分子机制。异常的中心体数量和行为是癌细胞的常见标志，中心缺陷与许多人类睫状性疾病有关，包括肾噬菌体，Bardet-Biedl综合征，Meckel综合征和口腔 - 偏种二牙综合征。