Investigating Tight Junction-Mediated Spatial Organization in Polarized Epithelia.

 描述（由申请方提供）：上皮细胞的不对称性对于后生动物的正常器官功能和阻碍异常上皮-间质转化是必要的。上皮细胞膜分为两个域，顶端和基底侧，这是由紧密连接（TJ）划分。这些电子致密的结构环绕着细胞，并连接着相对细胞的顶点。TJ被认为通过限制结构域特异性组分（如顶端的、高度糖基化的粘蛋白）的侧向扩散来维持细胞极性。然而，TJ复合物作为屏障的调节和机制仍然不清楚。该提案将确定TJ蛋白的物理约束如何驱动维持细胞极性所必需的空间组织。跨膜TJ蛋白，例如密蛋白-1，通过胞质TJ蛋白，例如ZO-1连接到肌动蛋白细胞骨架。最近的工作表明，肌动蛋白网络和蛋白质的大小是膜不对称的关键决定因素，因此，我们建议，TJ的机械调节。我们将研究力对TJ的能力，以防止易位的结构域特异性磷脂酰肌醇磷酸使用图案化的悬臂梁和定制的原子力显微镜与侧视光学成像系统的影响。我们还将测试TJ是否基于大小依赖的分选机制分离蛋白质。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.