Specific inhibition of myosin-Va and - Vb

描述(由申请人提供)： 肌球蛋白对细胞内运输是必不可少的，编码肌球蛋白的基因突变已被证明会导致几种遗传性疾病。由于几个原因，对非传统肌球蛋白特定细胞功能的表征一直落后于它们的鉴定。首先，在相关肌球蛋白家族中存在大量冗余。一些研究小组已经通过表达肌球蛋白的货物结合域或尾部结构域来确定特定的功能；然而，这种方法无法检测到依赖于运动域先前定位的任何功能，并且可以从高水平的表达中产生非特异性的影响。其次，对突变体进行了研究。虽然基因消融的方法非常强大，但零突变体通常只阐明了特定肌球蛋白在发育过程中必不可少的初始过程，排除了对后来的生理功能的识别。本研究的最终目的是了解肌球蛋白-Va和-Vb在多种细胞功能中的作用。为此，开发了一种化学遗传策略，通过使用定点突变扩大肌球蛋白的ATP结合位点来实现选择性、特异性和时间控制。这使肌球蛋白对特定的ADP类似物的抑制反应敏感，同时仍允许它水解胞内的ATP。然后，类似物的急性应用导致肌动蛋白与肌动蛋白紧密结合，阻止了敏化突变肌球蛋白的运动。本申请中提出的实验将通过在HeLa、PC 12、MDCK和B16细胞系以及转基因和敲入突变小鼠的原代细胞中进行特异性抑制，来检验肌球蛋白-Va和肌球蛋白-Vb在基于微管的运动的启动、维持、拮抗或终止中发挥作用的假设。

DESCRIPTION (provided by applicant): Myosins are essential for intracellular transport, and mutations in the genes encoding them have been shown to cause several inherited diseases. The characterization of the specific cellular functions of unconventional myosins has lagged behind their identification for several reasons. First, there is a great deal of redundancy within families of related myosins. Several groups have identified specific functions by expressing the cargo-binding, or tail, domains of myosins; however, this approach fails to detect any functions dependent upon prior positioning by the motor domain and can yield nonspecific effects from high levels of expression. Second, the study of mutants has been employed. Although genetic ablation approaches are very powerful, null mutants often only illuminate the initial process for which a given myosin is essential during development, precluding the identification of later physiological functions. The ultimate goal of this research is to understand the roles of myosin-Va and -Vb in multiple cellular functions. To this end, a chemical-genetic strategy has been developed that achieves selectivity, specificity, and temporal control by enlarging the ATP-binding site of the myosin using site-directed mutagenesis. This sensitizes the myosin to inhibition by a specific ADP analog while still allowing it to hydrolyze cellular ATP. The acute application of the analog then causes tight binding to actin, arresting the movement of the sensitized mutant myosin. The experiments proposed in this application will test the hypothesis that myosin-Va and myosin-Vb function in initiation, maintenance, antagonism, or termination of microtubule-based motility, using specific inhibition in HeLa, PC 12, MDCK, and B16 cell lines as well as primary cells from transgenic and knock-in mutant mice.