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多组分的体外重组，这将提供对复杂结构的洞察 生物过程，如货物运输和细胞质分裂。重组中使用的表达蛋白将 生物化学特征，单分子和生化/生物物理技术将评估马达 功能。胞浆动力蛋白-1和运动蛋白驱动微管上的长距离运动，这是 细胞的极性和功能。动力蛋白移动到极地MT的负端，并驱动逆行运输，而 1类、2类和3类运动蛋白的力量运动到相反的正端，并驱动顺行运输。生物学的 这些发动机的货物包括膜结合的囊泡、细胞器和信使核糖核酸。贩卖罪中的瑕疵 导致发育和神经退行性疾病(例如亨廷顿氏症和肌萎缩侧索硬化症 硬化症)。细胞货物和纯化细胞器的双向运动是由相反的马达驱动的。 在许多生物体和细胞类型中具有方向性。动力蛋白需要动力肌动蛋白和激活的适配器才能完全 这些适配器正在成为连接动力蛋白和动力蛋白发动机的支架。一个 我们的主要目标是建立在我们的体外重组复合体的基础上，其中包含连接蛋白dynein-dynactin 双尾D，信使核糖核酸结合蛋白平等化，和信使核糖核酸货物通过添加激动素-1。初步 数据显示，这个复合体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)确定步进模式和力 对这些复合体的依赖来理解发动机如何协调和/或竞争来实现这一点 动议。我们将确定dynein是否与不同类别的运动蛋白(kinesin-1，kinesin-2， 或Kinesin-3)影响转归，以及微管相关蛋白(MAP)如何调节这些转运 复合体。为了推广这些发现，我们将在支架的基础上重建动力蛋白-激动素-1复合体 亨廷顿蛋白，因为它在亨廷顿病中起着致病作用。第二个目标是推动我们的 参与胞质分裂的分裂酵母肌球蛋白的生化/生物物理特性。主要驱动力 因为胞质分裂是肌球蛋白和肌动蛋白之间的相互作用，促使收缩环收缩。这个 在动物细胞中这一过程的复杂性导致了分裂酵母作为一种受欢迎的模型系统的使用。至 提出更详细的分裂酵母胞质分裂的分子机制 主要收缩成分的表征。在这里，我们将使用生化/生物物理技术 鉴定参与胞质分裂的两个II类肌球蛋白(Myo2和Myp2)，并确定轻链是如何 磷酸化调节它们的速度和力量输出。最后，我们将通过协作探讨如何跟踪 几何形状影响结合了myoVa和kinesin-1的货物(脂质体)在悬浮肌动蛋白和肌动蛋白上的转运 微管轨迹，它与运动的启动和终止有关。