Study of Cell Adhesion Through an Analysis of Myosin 7

Myosin VII （M7）是一种基于肌动蛋白的运动蛋白，已知在人类、小鼠和斑马鱼的听力和平衡中起重要作用。简单真核生物Dictyostelium表达M7同源物（DdM7），零突变分析表明，它在吞噬和细胞迁移所需的细胞底物粘附中起关键作用，这是肌球蛋白的一种新功能。来自哺乳动物系统的新证据与M7直接参与粘附一致，表明这类肌凝蛋白的作用在整个进化过程中一直保持不变。我们对Ddm7的分析表明，这种肌球蛋白在将受体组织成高亲和力复合物（粘附复合物）方面发挥作用，该复合物能够与表面结合，而不参与蛋白质向质膜的运输。此外，我们发现Ddm7的尾部区域对于这种粘附复合物的形成至关重要。我们的工作目标是通过回答以下主要问题来确定基于DdM7的粘附的分子基础：1)连接DdM7与粘附复合物的蛋白质是什么？生化和遗传方法将用于鉴定与DdM7结合并参与细胞粘附的蛋白质；2)连接蛋白如何与DdM7相互作用形成粘附复合物。互补分析将用于确定连接肌球蛋白和粘附复合物所需的DdM7分子区域。考虑到M7功能在整个进化过程中的保守性，在Dictyostelium等遗传易感系统中对M7功能的详细分子分析将提供一个独特的机会，以深入了解基于M7的粘附如何在听觉和前庭系统的特化细胞中起作用，以及M7突变（如在Usher综合征IB型中发生）如何导致耳聋。

Myosin VII (M7) is an actin-based motor protein known to play an essential role in hearing and balance in humans, mice and zebrafish. The simple eukaryote Dictyostelium expresses a M7 homologue (DdM7) and null mutant analysis has revealed that it plays a critical role in cell substrate adhesion required for phagocytosis and cell migration, a novel function for a myosin. Emerging evidence from mammalian systems is consistent with M7's direct involve involvement in adhesion, indicating that the role of this class of myosins has been conserved throughout evolution. Our analysis of Ddm7 suggests that this myosin plays a role in organizing receptors into a high affinity complex (the adhesion complex) that is capable of binding to surfaces and is not involved in the transport of proteins to the plasma membrane. Furthermore, we have found that the tail region of Ddm7 is essential for the formation of this adhesion complex. The goal of our work is to identify the molecular basis of DdM7-based adhesion by answering the following major questions: 1) What are the proteins that link DdM7 to the adhesion complex? Both biochemical and genetic approaches will be used to identify proteins that bind to DdM7 and participate in cell adhesion; and 2) How do the linker proteins interact with DdM7 to make an adhesion complex. A complementation analysis will be employed to identify regions of the DdM7 molecule required for linking this myosin to the adhesion complex. Given the conservation of M7 function throughout evolution, the detailed molecular analysis of M7 function in a genetically tractable system such as Dictyostelium shall offer a unique opportunity to gain insight into how M7-based adhesion functions in the specialized cells of the auditory and vestibular system and how mutations in M7, such as occur in Usher's syndrome type IB, might lead to deafness.