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，一种具有独特能力的肌球蛋白马达蛋白，将货物运送到肌动蛋白丝的负端。 虽然肌球蛋白VI已被牵连在分泌和内吞运输途径，活细胞的调查，其在这些途径中的作用的细节一直缺乏。 因此，我们使用了一个独特的，活细胞分泌试验，以研究肌球蛋白VI及其结合伙伴视神经磷酸酶的分泌途径中的具体作用。 在组成性分泌期间，在内质网（ER）处合成的蛋白质被转运到高尔基复合体进行加工，然后转运到质膜进行掺入或细胞外释放。小干扰RNA为基础的敲低肌球蛋白VI导致ER到高尔基体的运输延迟，这表明肌球蛋白VI在早期分泌途径中的意想不到的功能。耗尽的肌球蛋白VI或视神经磷酸酶不影响离开反式高尔基体网络的囊泡的数量，这表明这些蛋白质不起作用的反式高尔基体囊泡的形成。然而，肌球蛋白VI和视神经磷酸酶共定位与分泌囊泡在质膜上。此外，活细胞全内反射荧光（TIRF）显微镜表明，肌球蛋白VI或视神经磷酸酶耗尽减少了质膜囊泡融合事件的总数，并增加了不完全融合事件的比例和该区域停靠囊泡的数量。这些结果表明，肌球蛋白VI和optineurin在调节的融合孔之间形成的分泌囊泡和质膜在分泌的最后阶段，一个新的作用。为了补充这些研究的作用，肌球蛋白VI的分泌，我们使用活细胞荧光恢复后光漂白（FRAP）比较周转率的网格蛋白包被的囊泡和早期内吞摄取途径的肌球蛋白VI。这些数据提供了新的见解肌球蛋白VI的动力学的内吞途径和电机蛋白及其结合伙伴的营业额的一般性质的特定的细胞内结构，通过展示野生型肌球蛋白VI和人工二聚肌球蛋白VI的构建，耳聋突变体的肌球蛋白VI（D179Y），和肌球蛋白VI的结合伙伴Dab2之间的营业额的差异。总的来说，这种肌球蛋白VI的分泌和内吞途径的检查增强了我们对细胞的基本生物力学操作的理解，并对肌球蛋白VI突变引起的疾病（如肥厚性心肌病和神经变性）的病因学提供了独特的见解。