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、Arp 2/3复合物和肌动蛋白动力学：含有网骨藻肌球蛋白IB（myoB）和IC（myoC）的SH 3结构域的融合蛋白结合116 kDa蛋白（p116），以及通过微测序鉴定为七元Arp 2/3复合物的其他九种蛋白，CapZ α和CapZ β。使用野生型和突变体细胞提取物进行的免疫沉淀表明，myoB和myoC存在于与p116，Arp 2/3和CapZ在体内的复合物中，它们的SH 3结构域是这种相互作用所需的，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Arp 2/3复合物。p116的克隆揭示了一个富含亮氨酸的重复结构域，一个WASP样WH 2/酸性结构域，和富含脯氨酸的序列，我们显示含有SH 3结构域结合位点，并表明p116是Acan 125的Dictyosteoblastoma同系物。p116与Arp 3、肌动蛋白、冠蛋白、myoB和myoC在动态肌动蛋白丰富的细胞延伸中广泛共定位，特别是迁移细胞和背侧、杯状、巨胞饮细胞延伸（冠）的前缘。p116基因敲除的细胞在这些巨胞饮结构的形成中表现出严重的缺陷，并伴随着液相内吞率的降低（约为WT的50%）。MyoB/myoC双突变体也表现出显着的缺陷冠形成（但有趣的差异）和部分错误定位Arp 3。这些结果鉴定了一种将主要肌动蛋白丝成核剂和有刺末端封端剂连接到普遍存在的有刺末端定向马达的复合物，表明该复合物在生理学上是重要的，并表明先前报道的肌球蛋白I突变表型至少部分是由于Arp 2/3和CapZ的功能缺陷。先前的研究表明，肌球蛋白V定位于小鼠黑素细胞中的黑素体，并且它与微管马达合作以驱动哺乳动物黑素细胞特征的黑素体的外周积累。负责与黑素体相互作用的肌球蛋白V的结构域已经被绘制出来，现在正被用来寻找黑素体表面的肌球蛋白V受体。- Arp 2/3复合物，肌球蛋白I，肌动蛋白动力学，肌球蛋白V，黑素体，黑素细胞，小鼠，网骨藻