Investigating Tight Junction-Mediated Spatial Organization in Polarized Epithelia.

 描述(申请人提供)：上皮细胞的不对称性对于后生动物正常的器官功能和阻止异常的上皮-间充质转变是必要的。上皮细胞膜分为两个区域，顶端和基底外侧，这两个区域由紧密连接(TJ)划分。这些电子致密结构包围细胞，并将相对细胞的尖端连接起来。TJS被认为通过限制区域特定成分的横向扩散来维持细胞的极性，例如顶端高度糖基化的粘蛋白。然而，TJ复合体作为屏障的调节和机制仍不清楚。这一提议将决定对TJ蛋白的物理限制如何驱动维持细胞极性所必需的空间组织。跨膜TJ蛋白，如Claudin-1，通过胞内TJ蛋白，如ZO-1，与肌动蛋白细胞骨架相连。最近的工作表明，肌动蛋白网络和蛋白质大小是膜不对称的关键决定因素，因此，我们认为TJs是机械调节的。我们将使用有图案的悬臂和带有侧视光学成像系统的定制原子力显微镜来研究力对TJ防止结构域特定的磷脂酰肌醇磷酸盐移位的能力的影响。我们还将测试TJ是否基于大小相关的排序机制来分离蛋白质。这项工作将为TJ如何维持细胞极性以及TJ的失调如何导致疾病，特别是癌症提供新的见解。

 DESCRIPTION (provided by applicant): Asymmetry of epithelial cells is necessary for proper organ function in metazoans and for impeding aberrant epithelial-mesenchymal transition. Epithelial cell membranes are divided into two domains, apical and basolateral, which are demarcated by tight junctions (TJs). These electron dense structures encircle the cell and connect the apices of opposing cells. TJs are thought to maintain cell polarity by limiting the lateral diffusion of domain-specific components, such as the apical, heavily glycosylated mucins. However, the regulation and mechanism of the TJ complex as a barrier remains obscure. This proposal will determine how physical constraints on TJ proteins can drive spatial organization essential for maintaining cell polarity. Transmembrane TJ proteins, e.g. claudin-1, are linked to the actin cytoskeleton through cytosolic TJ proteins, e.g. ZO-1. Recent work has suggested that actin networks and protein size are critical determinants for membrane asymmetry, and as such, we propose that TJs are mechanically regulated. We will examine the effect of force on TJ's ability to prevent translocation of domain-specific phosphatidylinositol phosphates using a patterned cantilever and a custom-built atomic force microscope with a side-view optical imaging system. We will also test whether TJs segregate proteins based on a size-dependent sorting mechanism. This work will provide new insight into how TJs are able to maintain cell polarity and how dysregulation of TJs leads to disease, particularly cancer.