STRUCTURE AND FUNCTION OF UNCONVENTIONAL MYOSINS

非常规肌球蛋白的结构和功能

• 批准号: 6290376
• 负责人: JOHN A HAMMER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290376
• 关键词: Acanthamoeba; Dictyostelium; birefringences; cell motility; chimeric proteins; endoplasmic reticulum; fertilization; immunologic assay /test; intracellular transport; melanosomes; molecular cloning; myosins; phagocytosis; protein folding; protein isoforms; protein structure function; protoplasm motility; site directed mutagenesis; tissue /cell culture

Myosin I, the Arp2/3 Complex, and Actin Dynamics: Fusion proteins containing the SH3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116kDa protein (p116), plus nine other proteins identified by microsequencing as the seven-member Arp2/3 complex, CapZ alpha and CapZ beta. Immunoprecipitations performed using wild type and mutant cell extracts indicate that myoB and myoC are present in a complex with p116, Arp2/3, and CapZ in vivo, that their SH3 domains are required for this interaction, and, together with other experiments, that p116 acts as a scaffold, binding myosin I, CapZ, and the Arp2/3 complex at independent sites. Cloning of p116 reveals a leucine-rich- repeat domain, a WASP-like WH2/acidic domain, and proline-rich sequences which we show contain the SH3 domain binding site, and indicates that p116 is a Dictyostelium homolog of Acan 125. p116 colocalizes extensively with Arp3, actin, coronin, myoB and myoC in dynamic actin-rich cellular extensions, especially the leading edge of migrating cells and dorsal, cup-like, macropinocytic extensions (crowns). p116 knockout cells exhibit a profound defect in the formation of these macropinocytic structures, and a concomitant reduction in the rate of fluid phase endocytosis (~ 50% of WT). MyoB/myoC double mutants also exhibit a striking defect in crown formation (but with interesting differences) and partially mislocalize Arp3. These results identify a complex linking the major actin filament nucleator and barbed-end capper to a ubiquitous barbed-end-directed motor, indicate that this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes are due at least in part to defects in the function of Arp2/3 and CapZ.Myosin V and Melanosome Dynamics: Previous studies have shown that myosin V localizes to melanosomes in mouse melanocytes, and that it cooperates with microtubule motors to drive the peripheral accumulation of melanosomes characteristic of mammalian melanocytes. The domain of myosin V that is responsible for the interaction with the melanosome has been mapped and is now being used to search for the myosin V receptor on the melanosome surface. - Arp2/3 complex, myosin I, actin dynamics, myosin V, melanosomes, melanocytes, mouse, Dictyostelium

Myosin I， Arp2/3复合物和肌动蛋白动力学：包含Dictyostelium Myosin IB （myoB）和IC (myoC) SH3结构域的融合蛋白结合116kDa蛋白（p116），以及通过微测序鉴定为七成员Arp2/3复合物CapZ α和CapZ β的其他九种蛋白。使用野生型和突变型细胞提取物进行的免疫沉淀表明，myoB和myoC存在于体内与p116、Arp2/3和CapZ的复合物中，它们的SH3结构域是这种相互作用所必需的，并且，与其他实验一起，p116作为支架，在独立的位点结合肌球蛋白I、CapZ和Arp2/3复合物。p116的克隆揭示了富含亮氨酸的重复结构域、类似wasp的WH2/酸性结构域和富含脯氨酸的序列，这些序列包含SH3结构域结合位点，表明p116是Acan 125的盘基骨属同源物。p116与Arp3、肌动蛋白、冠状蛋白、myoB和myoC广泛共定位于动态富肌动蛋白的细胞延伸，特别是迁移细胞的前缘和背侧、杯状、巨红细胞延伸（冠）。p116敲除细胞在这些巨噬细胞结构的形成中表现出深刻的缺陷，并伴随着液相内吞率的降低（约占WT的50%）。MyoB/myoC双突变体在冠形成方面也表现出显著的缺陷（但存在有趣的差异），并部分错定位Arp3。这些结果确定了一个将主要肌动蛋白丝成核器和带刺末端盖帽器连接到普遍存在的带刺末端定向马达的复合体，表明该复合体在生理上是重要的，并表明先前报道的肌球蛋白I突变表型至少部分归因于Arp2/3和CapZ的功能缺陷。Myosin V和黑素小体动力学：先前的研究表明，Myosin V定位于小鼠黑素细胞中的黑素小体，并与微管马达协同驱动哺乳动物黑素细胞特征的黑素小体的外周积累。肌凝蛋白V负责与黑素小体相互作用的结构域已经被绘制出来，现在正被用来寻找黑素小体表面的肌凝蛋白V受体。- Arp2/3复合体，肌凝蛋白I，肌动蛋白动力学，肌凝蛋白V，黑素小体，黑素细胞，小鼠，盘状骨