STRUCTURE AND FUNCTION OF UNCONVENTIONAL MYOSINS

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），加上被鉴定为七元Arp 2/3复合物的其他九种蛋白，以及加帽蛋白的α和β亚基。 免疫沉淀反应表明，这些组件一起存在于一个复杂的体内，肌球蛋白的SH 3域是需要其存在于这个复杂的，和p116作为支架复杂的组装，结合肌球蛋白I，加帽蛋白，和Arp 2/3复合物在独立的网站。 p116的克隆揭示了一个富含亮氨酸的重复结构域，一个verprolin样序列，然后是一个酸性区域，和脯氨酸丰富的序列，我们显示含有SH 3结构域结合位点，并表明p116是一个Dictyosteoblastoma同系物Acan 125。p116与Arp 2/3复合物、myoB和myoC一起沿着定位于动态的富含肌动蛋白的细胞延伸，包括经历趋化性迁移的细胞的前缘，以及背侧、杯状、巨胞饮细胞延伸。缺乏p116的细胞在这些巨胞饮结构的形成中表现出明显的缺陷，同时降低了液相内吞的速率，并显着降低了趋化聚集的效率。这些结果确定了一种复合物，该复合物将成核（Arp 2/3）和终止中的关键参与者联系起来（帽蛋白）的肌动蛋白丝装配与一个普遍存在的倒钩末端定向马达，表明蛋白质负责形成这种复合物（p116/Acan 125）在生理上是重要的，并建议，以前报道的肌球蛋白I突变体表型在网骨藻是由于至少部分的缺陷，在装配状态的肌动蛋白。我们已经将Acan 125纯化至均匀。它与加帽蛋白广泛共纯化，表明它们的相互作用相当强。 Acan 125的片段含有verprolin样和酸性序列刺激Arp 2/3依赖性肌动蛋白成核。 肌球蛋白V，Rab 27 a和黑素体动力学：在以前的研究中，我们已经表明，野生型小鼠黑素细胞的黑素体特征的外周积累是由一个合作过程驱动的，该过程涉及快速，长距离，双向，微管依赖性运动，与肌球蛋白V依赖性捕获和黑素体在肌动蛋白丰富的外周的局部运动相结合。我们现在已经证实了这个模型，证明恢复黑素小体的位置在稀释（肌球蛋白V空）黑素细胞的肌球蛋白V的reintroduction需要存在的长距离，双向，微管依赖的运动。 肌球蛋白V影响黑素体位置的能力绝对需要存在于其中的一个选择性剪接的27个残基的黑素辛基特异性外显子。 我们目前正在寻找与这一重要序列相互作用的蛋白质。 ashen基因座的产物最近被证明是一种新的rab，rab 27 a。 我们发现，灰色黑素细胞的表型，如灰色小鼠的毛色缺陷，是不可区分的，从稀释的黑素细胞。 这一事实，连同数据显示，这种GT酶和肌球蛋白V共定位于野生型黑素细胞的外周黑素体，表明rab 27 a使肌球蛋白V依赖性捕获肌动蛋白丰富的周边。对灰白色黑素细胞的拯救实验的结果支持这一结论，并且与其他数据一起，表明肌球蛋白V和rab 27 a在黑素体表面上处于复合物中。

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.