Control of Epithelial Polarity

描述（由申请人提供）：许多内脏器官，如肾脏，由中空的小管和球体组成，内衬一层极化的上皮细胞。这些细胞具有面向中央腔的顶端质膜域和面向下面的基底膜的基底外侧质膜。这两个质膜域具有完全不同的蛋白质和脂质组成，并且蛋白质和脂质向这些不同的膜表面的运输是至关重要的。我们已经发现，信号脂质磷脂酰肌醇3，4，5-三磷酸（PIP 3）仅在基底外侧质膜上发现，并且是该表面的身份和形成的关键决定因素。相反，磷脂酰肌醇4，5-二磷酸（PIP 2）集中在顶端质膜，在那里它是这个表面的主要决定因素。我们将使用活细胞成像来检验PIP 2和PIP 3控制上皮极性发展的假设。我们将检验PIP 3是由磷脂酰肌醇3-激酶的一种特异性亚型在基底外侧质膜合成的假设。我们将测试脂质磷酸酶PTEN和SHIP 1/2在防止PIP 3在顶端质膜积累中的各自作用。我们将测试的假设，gp 135/podocalyxin和蛋白质与它相互作用直接参与形成的顶端表面。Gp 135是一种带负电荷的跨膜唾液粘蛋白，其通过其C末端的PDZ基序与支架蛋白NHERF 1结合。我们将gp 135错误定位到基底外侧表面，看看NHERF 1和其他蛋白质是否也跟随。质膜上的许多PIP 2由磷脂酰肌醇4-磷酸5-激酶（PI 5 K）合成。亚型PI 5 K1 β存在于顶端质膜并与NHERF 1相互作用。我们将测试PI 5 K1 β（以及α和γ亚型）在顶端质膜形成中的参与。我们还将测试是否通过与NHERF的相互作用将PTEN募集到顶端质膜。总之，这些实验将帮助我们了解顶端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.