STRUCTURE AND FUNCTION OF UNCONVENTIONAL MYOSINS

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一个116 kDa蛋白（p116），加上其他九个被鉴定为七成员Arp2/复合物的蛋白，以及封盖蛋白的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，肌凝蛋白的SH3结构域是其在该复合体中存在所必需的，p116作为复合体组装的支架，在独立的位点结合肌凝蛋白I、capping蛋白和Arp2/3复合体。p116的克隆揭示了一个富含亮氨酸的重复结构域，一个类似于verprolin的序列，后面是一个酸性区域，以及富含脯氨酸的序列，我们发现这些序列包含SH3结构域结合位点，这表明p116是Acan 125的盘基骨门同源物。p116与Arp2/3复合物、myoB和myoC一起定位于动态的富含肌动蛋白的细胞延伸，包括进行趋化迁移的细胞的前缘，以及背侧、杯状、巨噬细胞的延伸。缺乏p116的细胞在这些巨噬细胞结构的形成中表现出明显的缺陷，伴随的是液相内吞作用速率的降低，以及化学聚集效率的显著降低。这些结果确定了一个将肌动蛋白丝组装的成核（Arp2/3）和终止（盖帽蛋白）的关键参与者与普遍存在的刺端定向马达连接起来的复合体，表明负责该复合体形成的蛋白质（p116/Acan 125）在生理上是重要的，并表明先前报道的盘状骨中肌球蛋白I突变表型至少部分归因于肌动蛋白组装状态的缺陷。我们已经将Acan 125纯化到均匀。它与盖帽蛋白广泛结合，表明它们的相互作用相当强。Acan 125的一个片段含有类似于verprolin和酸性序列，刺激arp2 /3依赖性肌动蛋白成核。肌凝蛋白V、Rab 27a和黑素小体动力学：在之前的研究中，我们已经表明野生型小鼠黑素细胞特征的黑素小体的外周积累是由一个合作过程驱动的，该过程涉及快速、远距离、双向、微管依赖的运动，以及肌动蛋白丰富的外周中肌凝蛋白V依赖的捕获和黑素小体的局部运动。我们现在已经验证了这个模型，证明通过重新引入肌球蛋白V来恢复稀黑素细胞（肌球蛋白V缺失）中的黑素小体位置需要存在长距离、双向、微管依赖的运动。肌凝蛋白V影响黑素小体位置的能力绝对需要在其中存在一个选择性剪接的27个残基的黑素小体特异性外显子。我们目前正在寻找与这个重要序列相互作用的蛋白质。灰色位点的产物最近被证明是一种新的兔，兔27a。我们发现淡色黑素细胞的表型，如淡色小鼠的毛色缺陷，与淡色黑素细胞的表型是难以区分的。这一事实，再加上数据显示GTPase和肌凝蛋白V在野生型黑素细胞的外周黑素体上共定位，表明rab 27a能够在富含肌动蛋白的外周细胞中实现肌凝蛋白V依赖性捕获。对苍白黑色素细胞的修复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位于黑色素小体表面的复合体中。

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.