Structure and function of Myosin VI

描述（由申请人提供）：VI类肌球蛋白可能是非常规肌球蛋白类别中最非常规的。与所有其他特征肌球蛋白相比，它们以相反的方向（肌动蛋白丝上的负末端方向）运输。肌球蛋白VI也有一个不寻常的和有争议的延长其短杠杆臂，以增加其步长。 它重新排列其转换器构象，以实现大冲程，并通过未知的机制进行二聚化。肌球蛋白VI参与许多细胞功能，但对于毛细胞的静纤毛的维持以及因此的突变是必需的 会导致耳聋。虽然它可以作为进行性肌球蛋白马达，在内吞作用中运输货物，但它也可以作为应变依赖性锚，参与组织结构如高尔基体。肌球蛋白VI在细胞中被发现为单体，但最佳地作为二聚体发挥功能。 我们已经提出，这表明肌球蛋白VII和X可能共有一种新的调节形式，即货物引发的二聚化。本研究的目的是：（1）进一步阐明肌球蛋白VI的杠杆臂延伸、负责二聚化的区域和肌球蛋白VI门控的机制。（2）开始探索肌球蛋白VIIa和X的结构方面，它们似乎在细胞中通过货物启动的二聚化来调节，就像肌球蛋白VI的情况一样。 使用生物化学和光学陷阱测定进一步表征导致耳聋的肌球蛋白VI突变，以及（4）使用细胞测定（Caco-2细胞）和突变体来确定肌球蛋白VI的锚定与运输作用。

DESCRIPTION (provided by applicant): Class VI myosins are perhaps the most unconventional of the unconventional myosin classes. They traffic in the reverse direction (minus end-directed on the actin filament) as compared to all other characterized myosins. Myosin VI also has an unusual and controversial extension of its short lever arm to increase its step size. It rearranges its converter conformation to achieve a large strok and dimerizes by an unknown mechanism. Myosin VI is involved in a number of cellular functions, but is essential for maintenance of the stereocilia of the hair cells and thus mutations in myosin VI can result in deafness. While it can function as a processive myosin motor, transporting cargoes in endocytosis, it also can function as a strain-dependent anchor that is involved in organizing structures such as the Golgi apparatus. Myosin VI is found as a monomer in cells, but functions optimally as a dimer. We have proposed that this is indicative of a novel form of regulation that may be shared by myosins VII and X~ namely, cargo-initiated dimerization. The aims of the study are: (1) further delineate the lever arm extension of myosin VI, the regions responsible for dimerization, and the mechanism of myosin VI gating~ (2) begin to probe structural aspects of myosins VIIa and X, which appear to be regulated in cells by cargo-initiated dimerization, as is the case for myosin VI~ (3) further characterize myosin VI mutations that result in deafness using both biochemical and optical trap assays~ and (4) use cellular assays (Caco-2 cells) and mutants to define anchoring vs. transport roles of myosin VI.