Regulation of Brush Border Structure by Myosin-1a

描述（由申请人提供）：我们的长期目标是阐明刷状缘（BB）细胞骨架及其相关运动蛋白在肠上皮细胞（肠上皮细胞）正常生理学和病理生理学中的功能。作为自然界中观察到的最高度有序的F-肌动蛋白组装体之一，BB作为肠道中营养吸收的主要部位发挥作用。BB由长度和直径几乎相同的紧密排列的微绒毛（MV）阵列组成;与每个MV相关的质膜通过由肌球蛋白-1a（Myo 1a）组成的螺旋排列的桥连接到支持肌动蛋白束，肌球蛋白-1a（Myo 1a）是一种产生力和运动的马达蛋白，其定向沿着肌动蛋白丝。我们最近对Myo 1a基因敲除小鼠的分析表明，这种马达在稳定顶端膜形态方面起着关键作用。缺乏Myo 1a的BB表现出显著的BB膜囊泡形成和疝形成，这些缺陷与吸收不良肠病（如乳糜泻和细菌性胃肠炎）中观察到的症状惊人相似。该提案的目标是通过确定Myo 1a的运动活性如何被利用来调节BB膜结构来研究这些表型的分子机制。我们的中心假设是Myo 1a通过产生有助于膜-细胞骨架粘附的机械力来稳定BB膜。我们将通过实施多方面的方法来测试这一假设，包括基于光学捕获的生物物理测定。具体而言，我们将：（1）确定由分离的BB证明的ATP诱导的膜脱落是否代表Myo 1a力产生的表现，（2）在重构的MV运动性测定中直接测量Myo 1a和天然BB组分之间相互作用期间产生的力，（3）测量由Myo 1a提供的总BB膜粘附的分数，以及（4）确定Myo 1a的生物物理特性如何支配其在膜-细胞骨架粘附中的作用。完整的BB对于粘膜的正常吸收功能是必不可少的; MV损失是对人类健康构成重大威胁的许多GI疾病中的常见症状。通过阐明控制MV稳定性的因素，这项工作将发展我们对正常肠上皮细胞功能的理解，并有助于阐明在肠上皮细胞功能障碍的情况下可能受到干扰的途径和过程。

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the function of the brush border (BB) cytoskeleton and its associated motor proteins in the normal physiology and pathophysiology of the intestinal epithelial cell (enterocyte). As one of the most highly-ordered F-actin assemblies observed in nature, the BB functions as the primary site of nutrient absorption in the intestinal tract. BBs are composed of tightly packed arrays of microvilli (MV) that are nearly identical in length and diameter; the plasma membrane associated with each MV is linked to a supporting actin bundle by a spiraling array of bridges composed of myosin-1a (Myo1a), a motor protein that generates force and motion directed along actin filaments. Our recent analyses of Myo1a knockout mice indicate that this motor plays a critical role in stabilizing apical membrane morphology. BBs lacking Myo1a exhibit significant vesiculation and herniation of the BB membrane, defects that are strikingly similar to symptoms observed in malabsorption enteropathies such as Celiac disease and bacterial gastroenteritis. The goal of this proposal is to investigate the molecular mechanism underlying these phenotypes by determining how the motor activity of Myo1a is exploited to regulate BB membrane structure. Our central hypothesis is that Myo1a stabilizes the BB membrane by generating mechanical forces that contribute to membrane-cytoskeleton adhesion. We will test this hypothesis by implementing a multifaceted approach, including biophysical assays based on optical trapping. Specifically, we will: (1) determine if ATP-induced membrane shedding demonstrated by isolated BBs represents a manifestation of Myo1a force generation, (2) directly measure the forces generated during interactions between Myo1 a and native BB components in a reconstituted MV motility assay, (3) measure the fraction of total BB membrane adhesion provided by Myo1a, and (4) determine how the biophysical properties of Myo1a govern its role in membrane-cytoskeleton adhesion. Intact BBs are essential for the normal absorptive function of the mucosa; MV loss is a common symptom in a number of Gl diseases that pose significant threats to human health. By elucidating factors that control MV stability, this work will develop our understanding of normal enterocyte function and help illuminate pathways and processes that may be perturbed in cases of enterocyte dysfunction.