Myosin-1 interactions with membranes

描述（由申请人提供）：本提案中详细的研究旨在了解肌球蛋白-1与膜和膜组分在分子水平上的相互作用，并阐明这些相互作用在膜组织和动力学中的作用。肌球蛋白-1家族成员（人类中表达的8种不同基因）是单头运动蛋白，在膜动力学和其他细胞过程中起重要作用。肌球蛋白-1异构体的一个关键特性似乎是它们能够直接将肌动蛋白细胞骨架与细胞膜连接起来。几个肌球蛋白-1同工型已被证明直接与膜结合，而一个肌球蛋白-1同工型已被证明通过假定的尾部结构域的pleckstrin同源结构域与磷酸肌肽头基紧密结合。鉴于肌凝蛋白-1家族在驱动膜动力学中的重要性，值得注意的是，大多数肌凝蛋白-1的膜相互作用尚未被探索。因此，本研究的重点是表征肌球蛋白-1-膜的相互作用。我们将使用各种生化和生物物理技术来检查膜结合的特异性、亲和力和动力学。具体目的如下：目的1：肌球蛋白-1家族成员与膜和磷酸肌苷的结合；目的1.1：磷酸肌苷结合的表征；目的1.2：磷酸肌苷特异性；目的1.3：结合动力学；目的2：膜与肌动球蛋白系统的相互作用；目的2.1膜表面肌动蛋白运动的重构；目的2.2肌球蛋白介导的PIP2聚类；目的2.3膜中肌球蛋白-1与PIP2结合的动力学。

DESCRIPTION (provided by applicant): The studies detailed in this proposal seek to understand myosin-1 interactions with membranes and membrane components at the molecular level and to elucidate the role of these interactions in membrane organization and dynamics. Myosin-1 family members (eight different genes expressed in humans) are single-headed motor proteins that play essential roles in membrane dynamics and other cellular processes. A key property of myosin-1 isoforms appears to be their ability to directly link the actin cytoskeleton to cell membranes. Several myosin-1 isoforms have been shown to bind directly to membranes, and one isoform, myo1c, has been shown to bind tightly to phosphoinositide headgroups via a putative pleckstrin homology domain in its tail domain. Given the importance on the myosin-1 family in driving membrane dynamics, it is remarkable that the membrane interactions of most of the myosin-1 have been unexplored. Therefore, this proposal is focused on characterizing the myosin-1-membrane interactions. We will use a variety of biochemical and biophysical techniques to examine membrane binding specificities, affinities, and dynamics. The specific aims are as follows: Aim 1: Binding of myosin-1 family members to membranes and phosphoinositides Subaim 1.1: Characterization of phosphoinositide binding Subaim 1.2: Phosphoinositide specificity Subaim 1.3: Kinetics of binding Aim 2: Interplay between membranes and the actomyosin system Subaim 2.1 Reconstitution of actin motility on membrane surfaces Subaim 2.2 Myosin-mediated PIP2 clustering Subaim 2.3 Dynamics of myosin-1 binding to PIP2 in a membrane. PUBLIC HEALTH RELEVANCE: Myosin-1 family members are important for numerous basic cellular processes. In humans, myosin-1 mutations have been linked to deafness, while myosin-1 deletions in mice result in increased susceptibility to infection, kidney inflammation, and defects in the gut endothelium. Understanding the specificity of and roles for myosin-1 interactions with lipid components of the cell membrane will provide insight into these normal and diseased physiological states.