Mechanism and function of microvillar myosin-1

描述(申请人提供)：这项提案侧重于发展我们对1类肌球蛋白的理解：普遍表达的单体、膜结合、基于肌动蛋白的马达，参与不同的细胞功能，包括细胞器运输， 转录、宿主防御、细胞运动和机械感觉。我们对肌球蛋白-1的研究集中在“刷状边缘”，这是一个紧密排列的微绒毛阵列，从许多运输上皮细胞的顶端表面延伸出来。这个细胞器是许多肌球蛋白超家族成员的家园，其中最丰富的是肌球蛋白-1a(Myo1a)，它是8种脊椎动物1类肌球蛋白之一。我们的实验室利用细胞生物学和生物物理方法的独特组合发现：(I)Myo1a有助于膜-细胞骨架黏附，这对维持正常的刷状边缘结构至关重要，(Ii)Myo1a推动宿主防御机制中丰富的膜泡从微绒毛尖端释放到肠腔。最近的研究也强调了Myo1a功能的生理意义，研究表明这一马达的突变与人类结直肠肿瘤的形成有关。虽然我们在阐明Myo1a的生物学作用方面已经取得了实质性的进展，但控制该蛋白和其他肌球蛋白-1功能的分子特性、相互作用和事件仍然缺乏特征。这项提议的目的是加深我们对使Myo1a能够促进刷子边界功能的基本生化和生物物理特性的理解。为此，目标1将在体外检测单个Myo1a分子与活细胞质膜和支撑双层相互作用的单一性质，目标2将检测与支撑双层结合的Myo1a的力产生潜力，目标3将研究力感知在Myo1a动力学和功能调节中的作用。由于刷毛边缘形成和维持的缺陷是许多对人类健康构成重大威胁的疾病的核心，深入了解支配Myo1a行为的分子机制将提供信息，最终可能用于旨在修复畸形或受损刷子边缘的治疗方法的开发。

DESCRIPTION (provided by applicant): This proposal is focused on developing our understanding of class 1 myosins: ubiquitously expressed monomeric, membrane binding, actin-based motors that participate in diverse cellular functions, including organelle trafficking, transcription, host defense, cell motility, and mechano-sensation. Our studies of myosin-1 are centered on the 'brush border', a tightly packed array of microvilli that extends from the apical surface of many transporting epithelial cells types. This organelle is home to a number of myosin superfamily members, with the most abundant being myosin-1a (Myo1a), one of eight vertebrate class 1 myosins. Our laboratory has leveraged a unique combination of cell biological and biophysical approaches to discover that: (i) Myo1a contributes to membrane-cytoskeleton adhesion, which is critical for maintaining normal brush border structure, and (ii) Myo1a powers the release of membrane vesicles enriched in host defense machinery from microvillar tips into the intestinal lumen. The physiological significance of Myo1a function is also underscored by recent studies showing that mutations in this motor are linked to colorectal tumor formation in humans. While we have made substantial progress toward elucidating the biological roles of Myo1a, the molecular properties, interactions, and events that govern the function of this and other myosins-1 remain poorly characterized. The goal of this proposal is to develop our understanding of the fundamental biochemical and biophysical properties that enable Myo1a to contribute to brush border function. To this end, Aim 1 will examine the unitary properties of single Myo1a molecules interacting with the plasma membrane of live cells and supported bilayers in vitro, Aim 2 will examine the force generating potential of Myo1a bound to supported bilayers, and Aim 3 will investigate the role of force sensing in the regulation of Myo1a dynamics and function. Because defects in brush border formation and maintenance are at the core of numerous diseases that pose significant threats to human health, developing insight on the molecular mechanisms that govern Myo1a behavior will provide information that may ultimately be used in the development of therapeutics aimed at repairing malformed or damaged brush borders.