Function of Myosin VI

细胞内的运输过程依赖于载体蛋白，如分子运动肌凝蛋白VI，这是一种具有独特能力的肌凝蛋白运动蛋白，可以将货物运送到肌动蛋白丝的负端。尽管肌球蛋白VI与分泌和内吞运输途径都有关系，但对其在这些途径中具体作用的活细胞研究一直缺乏。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optinurin在分泌途径中的特定作用。在组成分泌过程中，在内质网（ER）合成的蛋白质被运送到高尔基复合体进行加工，然后到质膜合并或细胞外释放。基于小干扰rna的myosin VI敲低导致er到高尔基转运延迟，提示myosin VI在早期分泌途径中具有意想不到的功能。肌凝蛋白VI或视神经蛋白的消耗不会影响离开反式高尔基网络的囊泡数量，这表明这些蛋白质在反式高尔基囊泡形成中不起作用。然而，肌球蛋白VI和视神经蛋白与质膜上的分泌囊泡共定位。此外，活细胞全内反射荧光（TIRF）显微镜显示，肌球蛋白VI或视神经蛋白耗竭减少了质膜上囊泡融合事件的总数，增加了不完全融合事件的比例和该区域内停泊的囊泡数量。这些结果表明，肌球蛋白VI和优神经蛋白在调节分泌囊泡和质膜之间形成的融合孔中发挥了新的作用。为了补充这些关于肌球蛋白VI在分泌中的作用的研究，我们使用光漂白后活细胞荧光恢复（FRAP）来比较肌球蛋白VI在网格蛋白包被的囊泡和内吞摄取途径的早期核内体上的周转率。这些数据通过展示野生型肌凝蛋白VI与人工二聚化的肌凝蛋白VI、肌凝蛋白VI的耳聋突变体（D179Y）和肌凝蛋白VI结合伙伴Dab2之间的周转差异，为肌凝蛋白VI在内噬途径中的动力学以及运动蛋白及其结合伙伴在特定细胞内结构上周转的一般性质提供了新的见解。总的来说，对肌球蛋白VI分泌和内吞途径的检查增强了我们对细胞基本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变性。

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.