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

我们的总体方法是专注于多组分体外重建，这将为复杂的 生物过程，如货物运输和胞质分裂。在重组中使用的表达蛋白将 生物化学特征，单分子和生物化学/生物物理技术将评估运动 功能细胞质动力蛋白-1和驱动蛋白驱动微管长距离运动，这是细胞生长所必需的。 细胞极性和功能。动力蛋白移动到极地MT的负端并驱动逆行运输， 1、2和3类驱动蛋白驱动运动到相反的正端并驱动顺行运输。生物 这些马达的货物包括膜结合囊泡、细胞器和mRNA。贩运缺陷 导致发育和神经退行性疾病（例如亨廷顿病和肌萎缩侧索硬化症） 硬化症）。细胞货物以及纯化的细胞器的双向运动由相反的马达驱动。 方向性在许多生物体和细胞类型。动力蛋白需要动力肌动蛋白和激活衔接子来完成 这些适配器正在成为连接动力蛋白和驱动蛋白马达的支架。一 我们的主要目标是建立在我们的体外重组复合物含有动力蛋白-动力肌动蛋白，衔接蛋白 Bicaudal D，mRNA结合蛋白平等主义者，以及通过添加驱动蛋白-1的mRNA货物。初步 数据显示，该复合体再现了细胞中所见的双向运动。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 这些复合体的依赖性，以了解如何协调和/或竞争，以实现这一点 议案我们将确定是否将动力蛋白与不同类型的转运驱动蛋白（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（MAPs）如何调节这些转运 配合物为了概括研究结果，我们将在支架的基础上重建动力蛋白-驱动蛋白-1复合物 亨廷顿蛋白，因为它在亨廷顿病中起着致病作用。第二个目标是进一步加强我们的 涉及胞质分裂的裂殖酵母肌球蛋白的生物化学/生物物理学表征。的一大动力 胞质分裂是肌球蛋白和肌动蛋白之间的相互作用，为收缩环的收缩提供动力。的 动物细胞中该过程的复杂性导致使用分裂酵母作为有利的模型系统。到 提出一个更详细的分子机制，胞质分裂酵母，这是必要的，有一个深入的 主要收缩成分的表征。在这里，我们将使用生物化学/生物物理技术， 表征参与胞质分裂的两种II类肌球蛋白（Myo 2和Myp 2），并确定轻链 磷酸化调节它们的速度和力量输出。最后，我们将通过合作来追求如何跟踪 几何形状影响具有结合的myoVa和驱动蛋白-1的货物（脂质体）在悬浮的肌动蛋白上的运输， 微管轨道，这是有关的启动和终止的运动。

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.