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Molecular Mechanisms of Motility Deduced from in Vitro Reconstituted Microtubule- and Actin-Based Motor Complexes

从体外重建的基于微管和肌动蛋白的运动复合体推导出运动的分子机制

基本信息

批准号：
10592401
负责人：
KATHLEEN M TRYBUS
金额：
$ 39万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10592401
关键词：
Actins Adaptor Signaling Protein Affect Amyotrophic Lateral Sclerosis Animals Axonal Transport Binding Binding Proteins Biochemical Biological Biological Models Biological Process Biophysics Cell Death Cell Polarity Cell physiology Cells Collaborations Complex Coupling Cytokinesis Cytoplasm Data Daughter Defect Dependence Dynein ATPase Failure Fission Yeast Generations Genetic Materials Geometry Goals Huntington Disease Huntington gene In Vitro Kinesin Light Liposomes Membrane Messenger RNA Microscopy Microtubule-Associated Proteins Microtubules Mitosis Modeling Molecular Molecular Motors Motion Motor Motor Activity Myosin ATPase Myosin Type II Neurodegenerative Disorders Organelles Organism Outcome Output Pattern Phosphorylation Play Ploidies Process Property Proteins Role Scaffolding Protein Speed Techniques Vesicle anterograde transport biophysical properties biophysical techniques cell motility cell type constriction developmental disease driving force dynactin insight reconstitution retrograde transport scaffold single molecule trafficking transmission process tumorigenesis

项目摘要

Our overall approach is to focus on multi-component in vitro reconstitutions that will provide insight into complex biological processes such as cargo transport and cytokinesis. Expressed proteins used in the reconstitutions will be biochemically characterized, and single-molecule and biochemical/biophysical techniques will assess motor function. Cytoplasmic dynein-1 and kinesins drive long-distance motion on microtubules, which is required for cell polarity and function. Dynein moves to the minus-end of the polar MT and drives retrograde transport, while kinesins of class 1, 2 and 3 power motion to the opposite plus-end and drive anterograde transport. The biological cargoes of these motors include membrane-bound vesicles, organelles and mRNA. Defects in trafficking contribute to developmental and neurodegenerative diseases (e.g. Huntington’s and amyotrophic lateral sclerosis). Bidirectional motion of cellular cargoes as well as purified organelles are driven by motors of opposite directionality in many organisms and cell types. Dynein requires both dynactin and an activating adaptor for full motor activity, and these adaptors are emerging as scaffolds for coupling both dynein and kinesin motors. A major goal is to build on our in vitro reconstituted complex containing dynein-dynactin, the adaptor protein Bicaudal D, the mRNA-binding protein Egalitarian, and mRNA cargo by the addition of kinesin-1. Preliminary data show that this complex recapitulates the bidirectional motion seen in the cell. We will use biophysical and single molecule techniques (TIRF and iSCAT microscopy) to determine the stepping patterns and force dependence of these complexes to understand how the motors co-ordinate and/or compete to achieve this motion. We will determine if coupling dynein with different classes of transporting kinesins (kinesin-1, kinesin-2, or kinesin-3) affects the outcome, and how microtubule-associated proteins (MAPs) regulate these transport complexes. To generalize findings, we will reconstitute a dynein-kinesin-1 complex based on the scaffolding protein huntingtin, because it plays a causative role in Huntington’s disease. A second goal is to further our biochemical/biophysical characterization of fission yeast myosins involved in cytokinesis. A major driving force for cytokinesis is the interaction between myosin and actin that powers constriction of the contractile ring. The complexity of this process in animal cells has led to the use of fission yeast as a favored model system. To propose a more detailed molecular mechanism for cytokinesis in fission yeast it is essential to have an in depth characterization of the principal contractile components. Here we will use biochemical/biophysical techniques to characterize the two class II myosins involved in cytokinesis (Myo2 and Myp2), and determine how light chain phosphorylation regulates their speed and force output. Lastly, we will pursue via collaboration how track geometry influences transport of cargo (liposomes) with bound myoVa and kinesin-1 on suspended actin and microtubule tracks, which is relevant to both the initiation and termination of motility.

我们的总体方法是专注于多组分体外重组，这将提供复杂的见解