Crumbs3 and Epithelial Polarity

描述(由申请人提供)：肾上皮细胞的极化对肾脏的正常发育和功能至关重要。我们实验室和其他实验室最近的工作表明，Crumbs蛋白家族在上皮细胞极性的发育和维持中发挥了重要作用。我们的工作主要集中在肾上皮细胞中的主要形式Crums3上。Crums3是一种小的顶端跨膜蛋白，以两种不同的剪接异构体存在。Crums3a是一种异构体，它使用进化上保守的羧基末端尾巴来组织两个重要的极性复合体，Crumbs/PALS/PATJ复合体和PAR3/PAR6/aPKC复合体。Crums3表达缺失或过度表达可导致细胞极性丧失和紧密连接的形成。据认为，Crums3作为一个跨膜锚定，通过与基侧膜上的激酶相互作用的磷酸化步骤，定位aPKC等信号分子来定义顶端结构域。然而，一个基本的问题是，Crums3最初是如何定位的，以指导根尖膜的形成。我们在三维培养中的最新研究表明，在细胞动力学过程中，Crums3在细胞分裂的非常早期就启动了顶膜和管腔的形成。我们推测，在细胞运动过程中，Crums3靶向中体的能力是根尖膜和管腔形成的关键步骤。这一假设将在第一个特定目标中得到验证，我们将检查在细胞质分裂过程中调节膜向中体运输的因素是否也调节最初的顶膜形成。在第二个特定目标中，我们将研究Crums3在已建立的极化上皮和经历间充质转化的上皮中的表达和运输的调节。最后一个具体目标是通过在Crums3小鼠中产生GFP敲击来检查体内的这些过程。这将使我们能够确定我们在组织培养模型中产生的假设在肾脏发育、正常肾功能和肾脏损伤恢复期间是否成立。 公共卫生相关性：这项工作的目标是了解导致极化上皮和肾管形成的步骤。该提案的重点是一种名为面包屑的蛋白质，它在这一过程中至关重要。在包括多囊肾病在内的许多疾病中，正确的输卵管形成对于肾功能衰竭的恢复都是重要的。我们的数据将描绘出面包屑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.