STRUCTURE AND FUNCTION OF NONMUSCLE MYOSINS

非肌肉肌球蛋白的结构和功能

• 批准号: 2576745
• 负责人: J 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/2576745
• 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 V: We have shown by immunolocalization that myosin V is concentrated on melanosomes (MS), the specialized pigment-producing organelle of melanocytes (MC). A working model for melanosome movement within mammalian MCs was proposed in which the movement is microtubule- based centrally and actomyosin V-based peripherally (i.e. within MC dendrites and dendritic tips). The shape and pigment distribution of wild type MCs and MCs devoid of myosin V were compared both in vitro (using primary MC cultures) and in situ. In both cases, the principal defect in mutant MCs was found to be in the transport of MSs to the periphery of the cell rather than in cell shape, as was previously thought. Taken together, our studies indicate that the absence of a myosin V-dependent MS transport system, which functions to move MSs from the cell body to dendritic tips, is responsible for the coat color defect in myosin V- mice. Immunolocalizations of myosin V in nerve growth cones implicate this motor in organelle motility within neurons as well. Myosin XI: Dictyostelium myoJ, a class XI unconventional myosin, was shown by immunofluorescence microscopy to be concentrated on the membranes of the contractile vacuole (CV) complex. Analyses of myoJ- cells created by targeted gene disruption revealed, however, that myoJ is not required for normal CV functioning. MyoJ- cells are also normal as regards chemotactic aggregation and endocytosis. Biochemical studies indicate that myoJ uses calmodulin as a light chain. Myosin I: The light chain (LC) of Acanthamoeba myosin IC (AMIC) was cloned by RT-PCR and subsequent screening of a cDNA library. A full- length cDNA for the AMIC heavy chain was constructed and coexpressed with the LC as a fully-active enzyme in bacculovirus-infected cells. Several proteins that bind to the SH3 domain of Dictyostelium myosin IC have been identified. Myosin II: The flexibilities and bend angles of minifilaments assembled using wild-type and mutant Acanthamoeba myosin II rod domains were determined by electric birefringence. The results confirm that the flexibility of the rod resides entirely within the hinge domain, and indicate that while hinge residues other than the proline confer significant flexibility, the proline is required for the sharp bend to occur. The thermal unfolding of these molecules has also been studied. ER Dynamics: A chimeric GFP molecule was designed to ensure retention in the endoplasmic reticulum (ER)and used to show loss of ER membrane continuity within starfish eggs upon fertilization.

肌球蛋白V：我们已经通过免疫定位表明肌球蛋白V是 聚集在黑色素体（MS）上，是专门的色素产生的 黑素细胞的细胞器（MC）。 黑色素体运动的工作模型 在哺乳动物MC中提出了运动是微管的运动 基于中央和肌动蛋白V基于外周（即MC 树突和树突尖）。 形状和色素分布 在体外比较了没有肌球蛋白V的野生型MC和MC （使用原发性MC培养）和原位。 在这两种情况下，校长 发现突变MC中的缺陷是在MSS运输到该突变的缺陷 像以前一样 想法。 综上所述，我们的研究表明缺乏 肌球蛋白V依赖性MS传输系统，该系统的功能可以从 细胞体至树突状尖端，负责外套颜色缺陷 在肌球蛋白V鼠中。 神经生长锥中肌球蛋白V的免疫定位 这也暗示了该电动机在神经元内的细胞器运动中。 肌球蛋白XI：XI类非常规肌球蛋白的Dictyostelium myoj是 通过免疫荧光显微镜表明 收缩液泡（CV）复合物的膜。 分析myoj- 然而，由靶向基因破坏创建的细胞表明，Myoj 正常的CV功能不需要。 myoj-细胞也正常 关于趋化性聚集和内吞作用。 生化研究 表明Myoj使用钙调蛋白作为轻链。 肌球蛋白I：Acanthamoeba肌球蛋白IC（AMIC）的轻链（LC）是 由RT-PCR克隆，随后筛选cDNA库。 一个完整的 构建友好重链的长度cDNA，并与 LC作为受环病毒感染细胞中的完全活跃酶。 一些 与肌球蛋白IC的SH3结构域结合的蛋白质已经 确定。 肌球蛋白II：组装的微型图的灵活性和弯曲角度 使用野生型和突变的acanthamoeba肌球蛋白II杆域是 由电力双折射确定。 结果证实了 杆的柔韧性完全驻留在铰链域内，并且 表明虽然铰链残留物以外的脯氨酸会议 明显的灵活性，尖锐的弯曲需要 发生。 还研究了这些分子的热展开。 ER动力学：嵌合GFP分子旨在确保保留 在内质网（ER）中，用于显示ER膜的损失 受精后，海星卵内的连续性。