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复合物和肌动蛋白动力学：融合蛋白含有肌动蛋白IB（Myob）和IC（Myoc）结合116 kDa蛋白（P116），再加上九个其他蛋白质，以及被鉴定为7-MEMBER ARP2/COMPLECT和Alpa-Beta beta subunits的九个其他蛋白质。 免疫沉淀反应表明，这些成分存在于体内复合物中，肌球蛋白的SH3结构域在这种复合物中的存在所必需，并且P116充当复杂组装，结合肌球蛋白I，限制肌球蛋白I，上限蛋白和ARP2/3复合物的脚手架。 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经历趋化性迁移的细胞以及背，杯状，大细胞延伸的细胞。缺乏P116的细胞在这些大型细胞结构的形成，流体相位吞作用速率的伴随降低以及化学术聚集效率显着降低时，表现出明显的缺陷。 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肌动蛋白的大会状态。我们已经净化了Acan 125至同质性。它与封盖蛋白相称，表明它们的相互作用很强。 包含垂直蛋白样和酸性序列的ACAN 125片段刺激ARP2/3依赖性肌动蛋白成核。 肌球蛋白V，RAB 27A和黑色素体动力学：在先前的研究中，我们已经表明，野生型小鼠黑素细胞的黑色素体特征的周围积累是由涉及快速，远距离，双向，双向，微管依赖性运动与肌动蛋白V依赖性捕获和梅兰运动相关的合作的合作过程驱动的。现在，我们已经通过证明稀释度（肌球蛋白v NULL）黑素细胞的恢复，通过重新插入肌球蛋白V的恢复需要远程，双向，微管依赖性运动来验证该模型。 肌球蛋白V影响黑色素体位置的能力绝对需要在其中存在替代剪接的，27个残基，黑色素特异性外显子。 我们目前正在寻找与此重要序列相互作用的蛋白质。 最近，灰座基因座的产物被证明是一种新颖的Rab Rab 27a。 我们发现，与稀释的黑素细胞相关的灰色黑素细胞的表型，例如灰小鼠的外套颜色缺陷。 这一事实与数据一起表明该GTPase和肌球蛋白V在野生型黑素细胞中的周围黑素体上进行了共定位，这表明RAB 27A在富含肌动蛋白的外周中启用了肌球蛋白V依赖性捕获。关于灰黑素细胞的救援实验的结果支持了这一结论，并与其他数据一起表明肌球蛋白V和RAB 27A在黑色素体表面上处于复合体。