STRUCTURE AND FUNCTION OF UNCONVENTIONAL MYOSINS

非常规肌球蛋白的结构和功能

• 批准号: 6432642
• 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/6432642
• 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 116 kDa protein (p116), plus nine other proteins identified as the seven-member Arp2/complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that these components are present together in a complex in vivo, that the SH3 domain of the myosin is required for its presence in this complex, and that p116 acts as the scaffold for complex assembly, binding myosin I, capping protein, and the Arp2/3 complex at independent sites. Cloning of p116 reveals a leucine-rich-repeat domain, a verprolin-like sequence followed by an acidic region, 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 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase endocytosis, and a significant decrease in the efficiency of chemtactic aggregation. These results identify a complex that links key players in the nucleation (Arp2/3) and termination (capping protein) of actin filament assembly with a ubiquitous barbed-end-directed motor, indicate that the protein responsible for the formation of this complex (p116/Acan 125)is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium are due at least in part to defects in the assembly state of actin. We have purifed Acan 125 to homogeneity. It copurifies extensively with capping protein, indicating that their interaction is quite strong. A fragment of Acan 125 containing the verprolin-like and acidic sequences stimulates Arp2/3-dependent actin nucleation. Myosin V, Rab 27a, and Melanosome Dynamics: In previous studies we have shown that the peripheral accumulation of melanosomes characteristic of wild type mouse melanocytes is driven by a cooperative process involving rapid,long-range, bidirectional, microtubule-dependent movements coupled to the myosin V-dependent capture and local movement of melanosomes in the actin-rich periphery. We have now verified this model by demonstrating that the restoration of melanosome position in dilute (myosin V null) melanocytes by reintoduction of myosin V requires the presence of the long range, bidirectional, microtubule-dependent movements. The ability of myosin V to influence melanosome position absolutely requires the presence in it of an alternatively spliced, 27-residue, melanoctye-specific exon. We are currently searching for proteins that interact with this important sequence. The product of the ashen locus was recently shown to be a novel rab, rab 27a. We find that the phenotype of ashen melanocytes, like the coat color defect in ashen mice, is indistinquishable from that of dilute melanocytes. This fact, together with data showing that this GTPase and myosin V colocalize on peripheral melanosomes in wild type melanocytes, suggests that rab 27a enables myosin V-dependent capture in the actin-rich periphery. The results of rescue experiments on ashen melanocytes support this conclusion, and, together with other data, suggest that myosin V and rab 27a are in a complex on the melanosome surface.

肌球蛋白I，Arp2/3复合体，和肌动蛋白动力学：包含Dictyostelialmyosin IB(MYOB)和IC(MyoC)SH3结构域的融合蛋白结合一个116 kDa的蛋白(P116)，加上另外9个被鉴定为7个成员的Arp2/复合体的蛋白质，以及帽蛋白的α和β亚基。免疫沉淀反应表明，这些成分在体内共同存在于一个复合体中，肌球蛋白的SH3结构域是它存在于这个复合体中所必需的，p116作为复合体组装的支架，在独立的位置结合肌球蛋白I、封端蛋白和Arp2/3复合体。P116的克隆显示了一个富含亮氨酸的重复结构域，一个类似马鞭草的序列，后面跟着一个酸性区域，以及我们发现的富含Pro的序列，其中包含SH3结构域的结合位点，表明p116是Acan 125的Dictyostoma同源物。P116与Arp2/3复合体、MYOB和myoC一起定位在动态的富含肌动蛋白的细胞延伸中，包括经历趋化迁移的细胞的前沿，以及背部的杯状巨噬细胞延伸。缺乏p116的细胞在这些大胞质结构的形成中表现出显著的缺陷，伴随而来的是液相内吞速率的降低，以及化学聚集效率的显著降低。这些结果确定了一个复合体，该复合体将肌动蛋白细丝组装的成核(Arp2/3)和终止(封顶蛋白)的关键角色与普遍存在的带刺末端导向的运动联系在一起，表明负责形成该复合体的蛋白质(p116/Acan 125)具有重要的生理意义，并表明先前报道的Dictyostelial肌球蛋白I突变表型至少部分是由于肌动蛋白组装状态的缺陷造成的。我们已将Acan 125纯化至均一。它与包膜蛋白广泛交配，表明它们之间的相互作用相当强。Acan 125的一个片段包含类维拉普林和酸性序列，可刺激Arp2/3依赖的肌动蛋白成核。肌球蛋白V、Rab 27A和黑素小体动力学：在以前的研究中，我们已经证明，野生型小鼠黑素细胞特有的黑素小体的外围积累是由一个合作过程驱动的，该过程涉及快速、远程、双向、微管依赖的运动，以及依赖肌球蛋白V的捕获和富含肌动蛋白的外围黑素小体的局部移动。我们现在已经验证了这一模型，证明了通过重新诱导肌球蛋白V来恢复稀释型(肌球蛋白V缺失)黑素细胞中的黑素小体位置需要存在远程、双向、微管依赖的运动。肌球蛋白V影响黑素小体位置的能力绝对需要存在一个选择性剪接的、27个残基的黑色素特异性外显子。我们目前正在寻找与这一重要序列相互作用的蛋白质。灰白基因的产物最近被证明是一个新的Rab，Rab 27A。我们发现，灰白的黑素细胞的表型，就像灰白小鼠的毛色缺陷一样，与稀释的黑素细胞的表型是难以区分的。这一事实，再加上数据显示，该GTPase和肌球蛋白V共同定位在野生型黑素细胞的外周黑素小体上，表明Rab 27A能够在肌动蛋白丰富的外周捕获肌球蛋白V依赖的外周。对灰白黑素细胞的拯救实验结果支持这一结论，并与其他数据一起表明，肌球蛋白V和Rab 27A在黑素小体表面的复合体中。