Mechanism and Coordination of Cytoplasmic Dynein Motility

细胞质动力蛋白运动的机制和协调

基本信息

批准号：
8865640
负责人：
Ahmet Yildiz
金额：
$ 28.32万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8865640
关键词：
ATP Hydrolysis ATP phosphohydrolase ATPase Domain Affect Affinity Alzheimer&apos s Disease Axonal Transport Behavior Binding Biochemical Biological Process Cell division Cell physiology Cells Cellular biology Chemicals Complex Development Disease Dynein ATPase Elements Engineering Eukaryotic Cell Event Fluorescence Polarization Generations Genetic Head Health Image In Vitro Investigation Kinesin Knock-out Lead Learning Measurement Measures Mechanics Methods Microtubules Mitosis Mitotic spindle Modeling Molecular Molecular Conformation Molecular Motors Motion Motor Movement Mutate Mutation Neurobiology Nucleotides Pathogenesis Peptides Play Positioning Attribute Property Proteins Recombinants Registries Regulation Relative (related person)Research Role Saccharomyces cerevisiae Site Structure Surface System Techniques Testing Vesicle Walking Work Yeasts base cell motility controlled release dimer imaging modality millisecond motor neuron degeneration mutant optical imaging optical traps premature prevent retrograde transport single molecule temporal measurement tool trafficking

项目摘要

DESCRIPTION (provided by applicant): Molecular motors drive key biological processes such as intracellular cargo transport and cell division. Two dimeric motors, kinesin and cytoplasmic dynein, can take many consecutive steps along microtubules to transport cargos over long distances. This continuous movement, termed processivity, requires coordination between the two motor domains to prevent premature release from the microtubule. Detailed structural and mechanistic models exist for kinesin, but the mechanism and coordination of dynein motility remains largely unknown. Dynein's unconventional structure and distinct origin suggest that it has different mechanistic features than other cytoskeletal motors. Dynein forms a large multisubunit complex, the core of which consists of a ring of AAA ATPase domains. Conformational changes driven by ATP hydrolysis within the ring underlie dynein force generation and motion. Recent structural and biochemical studies have identified the major conformational states of monomeric dynein constructs. However, studies of active dynein dimers are lacking. As a result, the molecular basis by which ATP driven structural changes lead to unidirectional motion of a dimer as a whole is unknown. In our preliminary work, we have used S. cerevisiae to express recombinant dynein motors and characterized dynein stepping behavior in vitro. In this proposal, using single-molecule imaging methods, we propose to dissect the coordination between the nucleotide and conformational states of the motor domains in native and engineered dynein constructs. We have three specific aims. First, using multicolor tracking methods, we will directly observe how the AAA ring domains coordinate their nucleotide cycles and move relative to each other. The specific roles of distinct AAA domains will be studied by selectively mutating out the ATPase sites in one ring. Second, we will investigate how ATP-driven conformational states of the motor domain drive the dynein powerstroke and alter microtubule-binding affinity. The ability to perform these measurements as dynein walks will allow us to demonstrate whether the mechanical cycle of one head is gated until the other head completes its forward step. Third, we will establish the structural basis of dynein's minus-end directionality. Together, our proposed research represents a focused investigation of the conformational and chemical states of dynein at a single-molecule level, as active dynein dimers move along surface-immobilized MTs. We hope to significantly advance understanding of dynein's fundamental mechanochemistry and learn how it achieves retrograde transport of intracellular cargos.