Function of Myosin VI

肌球蛋白 VI 的功能

• 批准号: 8344850
• 负责人: James Sellers
• 金额: $ 11.95万
• 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/8344850
• 关键词: Actins; Affect; Binding; Biological Assay; Biomechanics; Carrier Proteins; Cell membrane; Cell secretion; Cells; Clathrin-Coated Vesicles; Complement; Data; Disease; Docking; Early Endosome; Endoplasmic Reticulum; Etiology; Event; Exocytosis; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Goals; Golgi Apparatus; Hearing; Hypertrophic Cardiomyopathy; Investigation; Kinetics; Left; Life; Maintenance; Minus End of the Actin Filament; Molecular Motors; Motor; Mus; Mutation; Myosin ATPase; Nature; Nerve Degeneration; Pathway interactions; Process; Protein Secretion; Proteins; Role; Secretory Vesicles; Small Interfering RNA; Staging; Structure; Subcellular structure; Transport Process; Vesicle; base; deafness; extracellular; insight; mutant; myosin VI; novel; operation; stem; trafficking; trans-Golgi Network; uptake

Transport processes within the cell rely on carrier proteins such as the molecular motor myosin VI, a myosin motor protein with the unique ability to carry cargo towards the minus end of actin filaments. Although myosin VI has been implicated in both the secretory and endocytic trafficking pathways, live cell investigation of the specifics of its roles in these pathways has been lacking. As such, we have used a unique, live-cell secretion assay to investigate the specific roles of myosin VI and its binding partner optineurin in the secretory pathway. During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number of vesicles leaving the trans-Golgi network, indicating that these proteins do not function in trans-Golgi vesicle formation. However, myosin VI and optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence (TIRF) microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and optineurin in regulating the fusion pores that are formed between secretory vesicles and the plasma membrane during the final stages of secretion. To complement these studies on the role of myosin VI in secretion, we used live cell fluorescence recovery after photobleaching (FRAP) to compare the turnover rates of myosin VI on the clathrin-coated vesicles and early endosomes of the endocytic uptake pathway. These data offer novel insight into the kinetics of myosin VI in the endocytic pathway and the general nature of the turnover of a motor protein and its binding partners on specific intracellular structures, by demonstrating differences in turnover between wildtype myosin VI and an artificially dimerized myosin VI construct, a deafness mutant of myosin VI (D179Y), and the myosin VI binding partner Dab2. Overall, this examination of myosin VI in the secretory and endocytic pathways enhances our understanding of the basic, biomechanical operations of the cell and offers unique insights into the etiology of diseases stemming from mutations in myosin VI, such as hypertrophic cardiomyopathy and neurodegeneration.

细胞内的运输过程依赖于载体蛋白，如分子马达肌球蛋白VI，这是一种肌球蛋白马达蛋白，具有独特的能力将货物运送到肌动蛋白细丝的负端。虽然肌球蛋白VI参与了内分泌和胞内转运途径，但对其在这些途径中的具体作用尚缺乏活细胞研究。因此，我们使用了一种独特的活细胞分泌实验来研究肌球蛋白VI及其结合伙伴视神经磷酸酶在分泌途径中的特定作用。在结构性分泌过程中，在内质网(ER)合成的蛋白质被运输到高尔基复合体进行加工，然后被输送到质膜进行并入或细胞外释放。基于小干扰RNA的肌球蛋白VI的敲除导致内质网到高尔基体的转运延迟，这表明肌球蛋白VI在早期分泌途径中具有意想不到的功能。肌球蛋白VI或视神经磷酸酶的耗尽不影响离开反高尔基体网的小泡的数量，表明这些蛋白在反高尔基体囊泡的形成中不起作用。然而，肌球蛋白VI和视神经磷酸酶在质膜上与分泌小泡共存。此外，活细胞全内反射荧光(TIRF)显微镜显示，肌球蛋白VI或视神经磷酸酶耗竭减少了质膜上小泡融合事件的总数，增加了该区域不完全融合事件的比例和停靠的小泡数量。这些结果表明，在分泌的最后阶段，肌球蛋白VI和视神经磷酸酶在调节分泌囊泡和质膜之间形成的融合孔方面发挥了新的作用。为了补充这些关于肌球蛋白VI在分泌中的作用的研究，我们使用光漂白后的活细胞荧光恢复(FRAP)来比较肌球蛋白VI在内吞摄取途径的网状蛋白包裹的小泡和早期内小体上的周转率。这些数据通过展示野生型肌球蛋白VI与人工二聚肌球蛋白VI构建物、肌球蛋白VI耳聋突变体(D179Y)和肌球蛋白VI结合伙伴DAB2之间的周转差异，为肌球蛋白VI在内吞途径中的动力学以及运动蛋白及其在特定细胞内结构上的结合伙伴的一般性质提供了新的见解。总体而言，对肌球蛋白VI在分泌和内吞途径中的这种检查增强了我们对细胞基本的生物力学操作的理解，并为肌球蛋白VI突变引起的疾病的病因学提供了独特的见解，如肥厚性心肌病和神经退行性变。