Crumbs3 and Epithelial Polarity

描述（由申请人提供）：肾上皮细胞的极化对于肾脏的正常发育和功能至关重要。我们实验室和其他人最近的工作指出了Crumbs蛋白家族在上皮细胞极性的发育和维持中的重要作用。我们的工作集中在Crumbs 3，这是肾上皮细胞中的主要形式。Crumbs 3是一种小的顶端跨膜蛋白，以两种不同的剪接异构体存在。一种亚型Crumbs 3a使用进化上保守的羧基末端尾来组织两种重要的极性复合物，Crumbs/帕尔斯/PATJ复合物和PAR 3/PAR 6/aPKC复合物。Crumbs 3表达的丧失或Crumbs 3的过表达可导致细胞极性的丧失和紧密连接的形成。认为Crumbs 3作为跨膜锚发挥作用，其定位信号传导分子如aPKC，以通过与位于基底外侧膜上的激酶的相互磷酸化步骤来限定顶端结构域。然而，一个根本的问题是Crumbs 3最初是如何定位的，以指导顶膜的形成。我们在三维培养中的最新研究表明，Crumbs 3在细胞动力学过程中的细胞分裂中很早就启动了顶端膜和管腔的形成。我们假设Crumbs 3在细胞动力学过程中靶向中间体的能力是初始顶膜和管腔形成的关键步骤。这一假设将在第一个特定的目标，我们将检查是否在胞质分裂过程中调节膜运输到中间体的因素也调节初始顶端膜的形成。在第二个具体的目标，我们将检查Crumbs 3的表达和运输的调节建立极化上皮细胞和上皮细胞进行间质转化。最后一个具体目标将通过在Crumbs 3小鼠中产生GFP敲除来检查这些体内过程。这将使我们能够确定我们在组织培养模型中产生的假设是否在肾脏发育、正常肾功能和肾损伤恢复期间成立。 公共卫生相关性：这项工作的目标是了解导致极化上皮细胞和肾小管形成的步骤。该提案的重点是一种名为Crumbs的蛋白质，它在这一过程中至关重要。正确的管形成是重要的许多疾病，包括多囊肾病和肾衰竭的恢复。我们的数据将描述Crumbs蛋白启动上皮极化的基本步骤。

DESCRIPTION (provided by applicant): The polarization of renal epithelia is crucial for proper kidney development and function. Recent work from our laboratory and others has pointed to an important role for the Crumbs family of proteins in development and maintenance of epithelial cell polarity. Our work has focused on Crumbs3 which is the predominant form in renal epithelia. Crumbs3 is a small apical transmembrane protein that exists as two different splice isoforms. One isoform, Crumbs3a, uses an evolutionarily conserved carboxy-terminal tail to organize two important polarity complexes, the Crumbs/PALS/PATJ complex and the PAR3/PAR6/aPKC complex. Loss of Crumbs3 expression or overexpression of Crumbs3 can lead to a loss of cell polarity and tight junction formation. It is felt that Crumbs3 functions as a transmembrane anchor that localizes signaling molecules such as aPKC to define the apical domain via reciprocal phosphorylation steps with kinases localized on the basolateral membrane. However a fundamental question is how is Crumbs3 initially localized to instruct creation of the apical membrane. Our newest studies in three-dimensional culture demonstrate that Crumbs3 initiates apical membrane and lumen formation very early in cell division during the cytokinetic process. We hypothesize that the ability of Crumbs3 to target to the mid-body during the cytokinetic process is a crucial step in initial apical membrane and lumen formation. This hypothesis will be tested in the first specific aim where we will examine if factors that regulate membrane trafficking to the midbody during cytokinesis also regulate initial apical membrane formation. In the second specific aim, we will examine the regulation of Crumbs3 expression and trafficking in established polarized epithelia and in epithelia undergoing mesenchymal transition. The last specific aim will examine these processes in vivo by generating a GFP knock in Crumbs3 mouse. This will allow us to determine if our hypothesis generated in tissue culture models holds during kidney development, normal kidney function and during renal recovery from injury. PUBLIC HEALTH RELEVANCE: The goal of this work is to understand the steps that lead to polarized epithelia and kidney tube formation. The proposal focuses on a protein called Crumbs that is crucial in this process. Proper tube formation is important in many diseases including polycystic kidney disease and in recovery from kidney failure. Our data will delineate the basic steps utilized by the Crumbs protein to initiate epithelial polarization.