DETERMINING THE STRUCTURE, FUNCTION AND REGULATION OF DYNEIN AND FLAGELLA

确定动力蛋白和鞭毛的结构、功能和调节

• 批准号: 8171279
• 负责人: DANIELA NICASTRO
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171279
• 关键词: Attenuated; Biological; Caliber; Cell Shape; Cell division; Cells; Chemicals; Chronic; Cilia; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Cytoskeleton; Defect; Development; Disease; Dynein ATPase; Environment; Enzymes; Event; Flagella; Foundations; Freezing; Funding; Future; Genetic; Goals; Grant; Human; Imaging Techniques; Imaging technology; In Situ; Institution; Knowledge; Link; Lung diseases; Male Sterility; Malignant Neoplasms; Mechanics; Mediating; Microtubule-Associated Proteins; Microtubules; Molecular; Motor; Obesity; Organ; Organelles; Polycystic Kidney Diseases; Primary Ciliary Dyskinesias; Process; Proteins; Protocols documentation; Regulation; Research; Research Personnel; Resources; Source; Specimen; Structure; Technology; Three-Dimensional Imaging; United States National Institutes of Health; Work; base; cell behavior; cell motility; driving force; electron tomography; human disease; image processing; innovation; insight; protein complex; research study; retrograde transport; therapeutic development; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dyneins are microtubule-based motor enzymes that convert chemical energy into mechanical work. The dynein motors occur either in the cytoplasm, where they mediate retrograde transport, or within the integral structure of cilia and flagella, where they generate the forces that drive these motile organelles. While progress has been made in understanding aspects of dynein's function, the complexity and size of this motor enzyme have made it difficult to elucidate its molecular mechanism. Electron tomography of rapidly frozen specimens has been shown to be an exciting new technique for imaging well-preserved biological structures in an unperturbed cellular environment. Axonemes are excellent specimens for cryo-electron tomography and for the study of dynein in situ, thanks to the small diameter and the highly ordered arrangement of the microtubules and associated protein complexes in this organelle. We are using the cutting-edge technology of cryo-electron tomography to study the three-dimensional ultrastructures of dynein and intact flagella both preserved in their native states. Our approaches use modern and innovative tools, including integrated genetic and structural approaches that allow us to overcome current limitations in imaging technology and to directly visualize gene products in cells. Our work is aimed at contributing fundamental knowledge to our understanding of the mechanisms underlying motor function and control on a molecular level and to our understanding of the functional organization of cells in general. A major benefit of the proposed experiments will be the development of new tools for 3D imaging and image processing, which in the future will provide insights into cellular events including those gone amiss as in major diseases. In humans the normal function of several organs requires the activity of cilia. Defects in the motility and assembly of cilia and flagella have been linked to important human diseases, such as primary ciliary dyskinesia (PCD), polycystic kidney disease (PKD), chronic respiratory disease, male sterility, and human obesity disorders (reviewed by Snell et al., 2004). In addition dynein-driven transport along the microtubule cytoskeleton has major impact on cell behavior and organization, including cell division, signaling and cell shape; defects in this organization are often hallmarks of cancer. We expect that the proposed project will lay the foundation for our long-term goal to understand the mechanisms of ciliary-linked disorders in humans, a prerequisite to the development of therapeutic protocols capable of attenuating these disease processes.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 动力蛋白是基于微管的运动酶，可将化学能转化为机械能。动力蛋白马达要么发生在细胞质中，在那里它们介导逆行运输，要么发生在纤毛和鞭毛的整体结构中，在那里它们产生驱动这些运动细胞器的力。虽然在理解动力蛋白功能方面取得了进展，但这种运动酶的复杂性和大小使得阐明其分子机制变得困难。快速冷冻标本的电子断层扫描已被证明是一种令人兴奋的新技术，可在未受干扰的细胞环境中对保存完好的生物结构进行成像。轴丝是冷冻电子断层扫描和原位动力蛋白研究的优秀标本，这要归功于该细胞器中微管和相关蛋白质复合物的小直径和高度有序的排列。我们正在利用冷冻电子断层扫描的尖端技术来研究保存在原始状态的动力蛋白和完整鞭毛的三维超微结构。我们的方法使用现代和创新的工具，包括集成的遗传和结构方法，使我们能够克服当前成像技术的限制并直接可视化细胞中的基因产物。我们的工作旨在为我们理解分子水平上运动功能和控制的机制以及我们对细胞功能组织的总体理解提供基础知识。所提出的实验的一个主要好处是开发用于 3D 成像和图像处理的新工具，这些工具在未来将提供对细胞事件的见解，包括那些在重大疾病中出现问题的事件。 在人类中，多个器官的正常功能需要纤毛的活动。纤毛和鞭毛的运动和组装缺陷与重要的人类疾病有关，例如原发性纤毛运动障碍（PCD）、多囊肾病（PKD）、慢性呼吸道疾病、男性不育症和人类肥胖症（由 Snell 等人综述）等，2004）。此外，动力蛋白驱动的沿微管细胞骨架的运输对细胞行为和组织有重大影响，包括细胞分裂、信号传导和细胞形状；该组织的缺陷通常是癌症的标志。我们期望拟议的项目将为我们了解人类纤毛相关疾病机制的长期目标奠定基础，这是开发能够减轻这些疾病过程的治疗方案的先决条件。