Unconventional Myosin - Organelle Interactions

小鼠“稀释”非常规肌球蛋白（Myo 5a）基因座提供了一个出色的遗传系统，以研究分子马达货物的相互作用和非常规肌球蛋白家族密切相关的成员的重叠功能。 “稀释”系统的几个方面为回答这些问题提供了强有力的方法：在“稀释”基因座和“灰色”、“铅灰色”和“稀释抑制”基因座上多个突变等位基因的可用性;美世最近对“稀释”家族的第二个哺乳动物成员myr 6（Myo 5 b）的表征;操纵小鼠基因组的能力;以及原代黑素细胞培养系统的可用性，该系统提供了对“稀释”肌球蛋白功能的视觉测定。这些研究将通过提供对马达-货物相互作用的见解和测试非传统肌球蛋白家族中的功能冗余来影响细胞器运输领域。这些研究有以下目标：1、利用细胞生物学、生物化学和遗传学方法，在原代黑素细胞和黑色素瘤细胞系中确定“稀释”的功能，并鉴定与“稀释”相互作用的蛋白质。2、利用具有“稀释”肌球蛋白部分的嵌合cDNA构建体，鉴定“稀释”肌球蛋白尾部赋予货物结合特异性的区域。（肌球蛋白-Va）和它的近亲，myr 6（肌球蛋白-Vb），在培养的细胞和转基因小鼠。

The murine "dilute" unconventional myosin (Myo5a) locus provides an outstanding genetic system to study molecular motor-cargo interactions and overlapping functions of closely related members of unconventional myosin families. Several aspects of the "dilute" system contribute to a powerful approach to answering these questions: the availability of multiple mutant alleles at the "dilute" locus and the "ashen," "leaden," and "dilute suppressor" loci; the recent characterization by Mercer of a second mammalian member of the "dilute" family, myr 6 (Myo5b); the ability to manipulate the genome of the mouse; and the availability of a primary melanocyte cell culture system that provides a visual assay for "dilute" myosin function. The studies will impact the organelle transport field by providing insights into motor-cargo interactions and testing for functional redundancy within an unconventional myosin family.The studies have the following objectives:1, Determine the functions of "dilute" and identify proteins that interact with "dilute" in both primary melanocyte and melanoma cell lines, using cell biological, biochemical, and genetic methods.2, Identify regions of the "dilute" myosin tail that confer cargo-binding specificity using chimeric cDNA constructs with portions of the "dilute" myosin (myosin-Va) and its close relative, myr 6 (myosin-Vb), in both cultured cells and in transgenic mice.