Control of Epithelial Polarity

描述（由申请人提供）：许多内部器官，如肾脏，由中空的小管和球体组成，内衬一层极化上皮细胞。这些细胞有一个面向中央管腔的顶端质膜域和一个面向下层基膜的基底外侧质膜。这两种质膜结构域具有完全不同的蛋白质和脂质组成，因此将蛋白质和脂质运送到这些不同的膜表面是至关重要的。我们发现信号脂质磷脂酰肌醇3,4,5-三磷酸（PIP3）仅存在于基侧质膜上，并且是该表面的身份和形成的关键决定因素。相比之下，磷脂酰肌醇4,5-二磷酸（PIP2）集中在顶质膜上，在那里它是该表面的主要决定因素。我们将使用活细胞成像来验证PIP2和PIP3控制上皮极性发展的假设。我们将验证PIP3是由磷脂酰肌醇3-激酶的特定异构体在基侧质膜上合成的假设。我们将测试脂质磷酸酶PTEN和SHIP1/2在阻止PIP3积聚在根尖质膜中的各自作用。我们将验证gp135/podocalyxin及其相互作用的蛋白质直接参与根尖表面形成的假设。Gp135是一种带负电荷的跨膜唾液粘液蛋白，通过其c端PDZ基序与支架蛋白NHERF1结合。我们将把gp135错定位到基底外侧表面，看看NHERF1和其他蛋白是否会跟随。质膜上的PIP2大部分是由磷脂酰肌醇4-磷酸5激酶（PI5K）合成的。PI5K1beta亚型存在于根尖质膜上，并与NHERF1相互作用。我们将测试pi5k1β（以及α和γ亚型）在顶端质膜形成中的作用。我们还将测试PTEN是否通过与NHERF的相互作用被募集到根尖质膜上。这些实验将有助于我们了解根尖质膜和管腔形成的分子机制。

DESCRIPTION (provided by applicant): Many internal organs, such as the kidney, consist of hollow tubules and spheres lined by a layer of polarized epithelial cells. These cells have an apical plasma membrane domain facing the central lumen and a basolateral plasma membrane facing the underlying basement membrane. These two plasma membrane domains have completely different protein and lipid compositions, and trafficking of proteins and lipids to these distinct membrane surfaces is vital. We have found that the signaling lipid phosphatidyl inositol 3,4,5- trisphosphate (PIP3) is found only at the basolateral plasma membrane and is a key determinant of the identity and formation of this surface. In contrast phosphatidyl inositol 4,5-bisphosphate (PIP2) is concentrated at the apical plasma membrane, where it is a main determinant of this surface. We will test the hypothesis that PIP2 and PIP3 control the development of epithelial polarity, using live cell imaging. We will test the hypothesis that PIP3 is synthesized at the basolateral plasma membrane by a specific isoform(s) of phosphatidyl inositiol 3- kinase. We will test the respective roles of the lipid phosphatases PTEN and SHIP1/2 in preventing PIP3 from accumulating at the apical plasma membrane. We will test the hypothesis that gp135/podocalyxin and proteins with which it interacts are directly involved in formation of the apical surface. Gp135 is a negatively charged, transmembrane sialomucin, which binds via a PDZ motif at its C-terminus to the scaffolding protein NHERF1. We will mislocalize gp135 to the basolateral surface and see if NHERF1 and other proteins follow. Much of the PIP2 at the plasma membrane is synthesized by phosphatidyl inositol 4- phosphate 5-kinase (PI5K). The isoform PI5K1beta is found at the apical plasma membrane and interacts with NHERF1. We will test the involvement of PI5K1beta (as well as the alpha and gamma isoforms) in formation of the apical plasma membrane. We will also test if PTEN is recruited to the apical plasma membrane by its interaction with NHERF. Together, these experiments will help us understand the molecular mechanism of apical plasma membrane and lumen formation.