Equipment supplement

装备补充

• 批准号: 10614791
• 负责人: KATHLEEN M TRYBUS
• 金额: $ 1.9万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 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/10614791
• 关键词: Actins; Adaptor Signaling Protein; Affect; Amyotrophic Lateral Sclerosis; Animals; Axonal Transport; Binding Proteins; Biochemical; Biological; Biological Models; Biological Process; Biophysics; Cell Death; Cell Polarity; Cell physiology; Cells; Collaborations; Complex; Coupling; Cytokinesis; Data; Daughter; Defect; Dependence; Dynein ATPase; Equipment; Failure; Fission Yeast; Generations; Genetic Materials; Geometry; Goals; Huntington Disease; Huntington protein; 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; Proteins; Role; Scaffolding Protein; Speed; Techniques; Vesicle; anterograde transport; base; biophysical properties; biophysical techniques; cell motility; cell type; constriction; developmental disease; driving force; dynactin; insight; reconstitution; retrograde transport; scaffold; single molecule; trafficking; tumorigenesis

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体外重组的多组分，以洞悉复杂 货物运输和细胞因子等生物过程。在重组中使用的表达蛋白质将 在生化上表征，单分子和生化/生物物理技术将评估运动 功能。细胞质动力蛋白1和kiness在微管上驱动长距离运动，这是必需的 细胞极性和功能。动力蛋白移至极地MT的负末端，并驱动逆行运输，而 1、2和3级的动力学动力运动，向相反的末端，并驱动直流运输。生物学 这些电动机的货物包括结合膜的蔬菜，细胞器和mRNA。贩运的缺陷 有助于发育和神经退行性疾病（例如亨廷顿和肌萎缩症 硬化）。细胞货物和纯化的细胞器的双向运动由相对的电动机驱动 许多生物和细胞类型的方向性。 Dynein需要Dynactin和一个激活适配器才能完整 运动活动，这些适配器正在作为造成动力蛋白和动力蛋白电动机的支架。一个 主要目标是建立我们的体外重构复合物，该复合物含有动力蛋白 - 二奈乳蛋白，衔接蛋白 Bicauaudal D，MRNA结合蛋白平均值和mRNA货物，通过添加驱动蛋白1。初步的 数据表明，这种复合物概括了细胞中看到的双向运动。我们将使用生物物理和 单分子技术（TIRF和ISCAT显微镜），以确定步进模式和力 这些复合物的依赖性以了解电动机如何协调和/或竞争以实现这一目标 运动。我们将确定是否将动力蛋白与不同类别的运输驱动蛋白（驱动蛋白1，驱动蛋白2，， 或驱动蛋白3）影响结果，以及微管相关蛋白（地图）如何调节这些转运 复合物。为了概括发现，我们将根据脚手架重建一个动力蛋白 - 基因蛋白-1复合物 蛋白质亨廷顿蛋白，因为它在亨廷顿氏病中起因作用。第二个目标是进一步 涉及细胞因子的裂变酵母肌醇的生化/生物物理表征。主要的驱动力 因为细胞因子是肌球蛋白和肌动蛋白之间的相互作用，它可以为收缩环的收缩限制。 该过程在动物细胞中的复杂性已导致使用裂变酵母作为偏爱的模型系统。到 提案在裂变酵母中的细胞因子的一种更详细的分子机制，必须深入 主要收缩组件的表征。在这里，我们将使用生化/生物物理技术来 表征涉及细胞因子（MyO2和MyP2）的两个II类肌球蛋白，并确定轻链 磷酸化调节其速度和力输出。最后，我们将通过合作追求如何跟踪 几何形状影响载肌蛋白和肌动蛋白-1的货物运输（脂质体）在悬浮的肌动蛋白和 微管轨道，这与运动的主动性和终止均相关。