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

肌球蛋白I，ARP2/3复合物和肌动蛋白动力学：融合蛋白包含肌球蛋白IB（MYOB）和IC（MYOC）结合116KDA蛋白（P116）的SH3结构域，再加上通过Microsequenc鉴定的七个其他成员ARP2/3 Complects Capz Alpha，Capz Alpha和Microsequency鉴定出的九个其他蛋白质。使用野生型和突变细胞提取物进行的免疫沉淀表明，Myob和Myoc与P116，ARP2/3和体内的CAPZ一起存在于复合体中，其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与capfold i，Capz i，Capz and arp2/arp2/3相结合。 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迁移的细胞和背，杯状，大型细胞延伸（冠）。 P116基因敲除细胞在这些大型细胞结构的形成中表现出严重的缺陷，以及液相内吞作用率的伴随降低（WT的约50％）。 Myob/Myoc双突变体在冠状形成中也表现出明显的缺陷（但差异很有趣），并部分错误地定位了ARP3。这些结果确定了将主要肌动蛋白丝核定器和钢丝末端插头连接到无处不在的钢丝末端指导的电动机的复杂，表明这种复合物在生理上很重要，并表明先前报道的肌球蛋白I突变体表型至少部分归因于ARP2/3和Capz.myososs.myosos vyics dynamisics的缺陷，以表明肌动症的功能。小鼠黑素细胞中的黑素体，并与微管电动机合作，以驱动哺乳动物黑色素细胞的黑素体特征的周围积累。负责与黑色素体相互作用的肌球蛋白V的结构域已被映射，现在被用来搜索黑色素体表面上的肌球蛋白V受体。 -ARP2/3复合物，肌球蛋白I，肌动蛋白动力学，肌球蛋白V，黑色素体，黑色素细胞，鼠标，dictyostelium