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.

细胞内的转运过程依赖于载体蛋白，例如分子运动肌球蛋白VI，这是一种具有独特的肌球蛋白运动蛋白，具有将货物携带到肌动蛋白丝的负端的独特能力。 尽管肌球蛋白VI与分泌和内吞交易途径有关，但缺乏对其在这些途径中角色的细节的实时调查。 因此，我们使用了独特的现场活细胞分泌测定法来研究肌球蛋白VI及其结合伴侣Optineurin在分泌途径中的特定作用。 在本构分泌过程中，将内质网（ER）合成的蛋白质转运到高尔基体配合物进行加工，然后转移到质膜以掺入或细胞外释放。基于RNA的小型肌球蛋白VI敲低会导致ER到Golgi的运输延迟，这表明在早期分泌途径中，肌球蛋白VI的功能出乎意料。肌球蛋白VI或Optineurin的耗竭不会影响离开反式高尔基网络的囊泡数量，表明这些蛋白质在反式高尔基囊泡形成中不起作用。但是，肌球蛋白VI和Optineurin与质膜的分泌囊泡共定位。此外，活细胞总内反射荧光（TIRF）显微镜表明，肌球蛋白VI或Optineurin耗竭减少了质膜处的囊泡融合事件的总数，并增加了不完整的融合事件的比例和该区域中停泊的囊泡的数量。这些结果表明，在分泌的最后阶段，肌球蛋白VI和Optineurin在调节分泌囊泡和质膜之间形成的融合孔中的新作用。为了补充有关肌球蛋白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）比较了肌球蛋白VI在粘蛋白涂层囊泡和内粒细胞摄取途径的早期内体上的周转率。这些数据为内吞途径中肌球蛋白VI动力学的动力学提供了新颖的见解，以及运动蛋白及其在特定细胞内结构上的结合伴侣的一般性，通过证明野生型肌球蛋白VI的离职差异和人为型肌动蛋白VI结构，肌动蛋白的肌动蛋白和肌动蛋白差异（D199）（d179）（d179） DAB2。总体而言，这种对分泌和内吞途径中肌球蛋白VI的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（例如肥大性心肌病变和神经变性）提供了对疾病的病因的独特见解。