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

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

项目成果

期刊论文数量(0)
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KATHLEEN M TRYBUS其他文献

KATHLEEN M TRYBUS的其他文献

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{{ truncateString('KATHLEEN M TRYBUS', 18)}}的其他基金

Equipment Supplement
装备补充
  • 批准号:
    10796279
  • 财政年份:
    2020
  • 资助金额:
    $ 39万
  • 项目类别:
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
  • 批准号:
    10133095
  • 财政年份:
    2020
  • 资助金额:
    $ 39万
  • 项目类别:
Equipment supplement
装备补充
  • 批准号:
    10614791
  • 财政年份:
    2020
  • 资助金额:
    $ 39万
  • 项目类别:
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
  • 批准号:
    10368927
  • 财政年份:
    2020
  • 资助金额:
    $ 39万
  • 项目类别:
Structure and function of the Plasmodium myosin XIV-actin glideosome.
疟原虫肌球蛋白 XIV-肌动蛋白滑胶体的结构和功能。
  • 批准号:
    10650841
  • 财政年份:
    2017
  • 资助金额:
    $ 39万
  • 项目类别:
Mutational Studies of Processive Myosin Motors
进行性肌球蛋白运动的突变研究
  • 批准号:
    7807806
  • 财政年份:
    2009
  • 资助金额:
    $ 39万
  • 项目类别:
MUTATIONAL STUDIES OF PROCESSIVE MYOSIN MOTORS
进行性肌球蛋白运动的突变研究
  • 批准号:
    7910491
  • 财政年份:
    2007
  • 资助金额:
    $ 39万
  • 项目类别:
Mutational Studies of Processive Myosin Motors
进行性肌球蛋白运动的突变研究
  • 批准号:
    9268016
  • 财政年份:
    2007
  • 资助金额:
    $ 39万
  • 项目类别:
Mutational studies of processive myosin motors
进行性肌球蛋白运动的突变研究
  • 批准号:
    8289420
  • 财政年份:
    2007
  • 资助金额:
    $ 39万
  • 项目类别:
Mutational studies of processive myosin motors
进行性肌球蛋白运动的突变研究
  • 批准号:
    8499349
  • 财政年份:
    2007
  • 资助金额:
    $ 39万
  • 项目类别:
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