Regulation of Brush Border Structure by Myosin-1a

描述(申请人提供)：我们的长期目标是阐明刷状边缘(BB)细胞骨架及其相关的运动蛋白在肠上皮细胞(肠细胞)的正常生理和病理生理学中的功能。作为自然界观察到的最有序的F-肌动蛋白集合体之一，BB是肠道吸收营养物质的主要部位。BBS由长度和直径几乎相同的微绒毛(MV)紧密排列组成；与每一MV相关的质膜通过由肌球蛋白-1a(Myo1a)组成的螺旋式桥接阵列连接到支持肌动蛋白束上，肌球蛋白-1a(Myo1a)是一种沿着肌动蛋白细丝产生力和运动的马达蛋白。我们最近对Myo1a基因敲除小鼠的分析表明，该马达在稳定顶膜形态方面发挥了关键作用。缺乏Myo1a的BBS表现出明显的BB膜囊泡和突出，这些缺陷与吸收不良肠病(如乳糜泻和细菌性胃肠炎)的症状惊人地相似。这项建议的目的是通过确定如何利用Myo1a的运动活性来调节BB膜结构来研究这些表型背后的分子机制。我们的中心假设是，Myo1a通过产生有助于膜-细胞骨架黏附的机械力来稳定BB膜。我们将通过实施一种多方面的方法来检验这一假设，包括基于光学捕获的生物物理分析。具体地说，我们将：(1)确定分离的BBS所展示的ATP诱导的膜脱落是否代表Myo1a力产生的表现，(2)在重建的MV运动分析中直接测量Myo1a与天然BB组分相互作用时产生的力，(3)测量由Myo1a提供的总BB膜黏附的比例，以及(4)确定Myo1a的生物物理性质如何控制其在膜-细胞骨架黏附中的作用。完整的BBS对粘膜的正常吸收功能是必不可少的；MV丢失是许多对人类健康构成重大威胁的G1疾病的常见症状。通过阐明控制MV稳定性的因素，这项工作将加深我们对正常肠细胞功能的理解，并有助于阐明在肠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.