Role of myosin-1 in membrane and actin dynamics

描述(由申请人提供)： 本研究的目的是了解肌球蛋白I在膜收缩和肌动蛋白逆行流动中的作用。肌球蛋白是人类中最大的非传统肌球蛋白之一，但对其在脊椎动物细胞中的活性、分布或调节知之甚少。1)研究转移性哺乳动物细胞系MTLn3细胞在板脂伸缩过程中myo1c的分布。这将通过免疫荧光显微镜和瞬时表达EGFP-肌球蛋白融合蛋白来完成。2)利用小干扰RNA(SiRNA)技术研究肌球蛋白I基因敲除对细胞伸展、膜回缩和肌动蛋白逆行流动的影响。对照细胞和肌球蛋白-I基因敲除细胞中的逆行流动将通过表面附着的乳胶珠的运动性来评估。3)确定myo1c的机械活性与膜动力学的关系。我们将表达改变了马达特性的myo1c突变体，并将检测这些突变体对膜动力学的影响。

DESCRIPTION (provided by applicant): The objectives of the proposed research are to understand the roles of myosin I in membrane retraction and retrograde actin flow. Myosin Is represent one of the largest classes of unconventional myosins in humans, yet little is known about their activity, distribution, or regulation in vertebrate cells. Specific aims include the following: 1) To characterize the distribution of myo1c during lamellipodial extension and retraction in MTLn3 cells, a metastatic mammalian cell line. This will be accomplished by immunofluorescence microscopy and transient expression of eGFP-myosin fusion proteins. 2) To determine the effect of myosin-I "knockdown" on cell spreading, membrane retraction, and retrograde actin flow using small interfering RNA (siRNA). Retrograde flow in control and myosin-I knockdown cells will be assessed by motility of surface-attached latex beads. 3) To determine the relationship between the mechanical activity of myo1c and membrane dynamics. We will express myo1c mutants that have altered motor properties, and we will examine the effect of these mutants on membrane dynamics.