Cadherin endocytosis in the cranial neural crest

描述（由申请人提供）：神经嵴细胞（NCC）发育期间发生的异常是许多人类先天性和遗传性畸形、疾病和癌症的直接原因。迁移前NCC在胚胎背神经管中以贴壁上皮细胞的形式存在，但后来通过上皮-间充质转化（EMT）响应于许多内在和外在线索而变得能动。重要的是，这一事件在范围上类似于强调人类疾病和癌症的异常EMT。虽然这些正常和异常EMT之间存在几个相似之处，其中最主要的是由细胞表面钙粘蛋白蛋白的丢失和粘附连接的解体引发的级联事件，以促进细胞运动。我们实验室的工作表明，迁移前颅NCC中钙粘蛋白6 B（Cad 6 B）的丢失对NCC EMT至关重要。钙粘蛋白在体外的减少是通过蛋白质水解和内吞作用在转录和转录后介导的。我们先前的研究描述了在NCC EMT期间Cad 6 B在体内的转录抑制和蛋白水解加工的可比机制。然而，我们新的初步数据显示，Cad 6 B具有推定的内吞基序，并与内体和溶酶体的标记物在体外和体内共定位。这些初步的研究结果表明，Cad 6 B也通过内吞作用进行调节，据我们所知，这是体内EMT期间钙粘蛋白内吞作用的首次证明。基于这些数据，我们假设Cad 6 B内吞作用在NCC EMT和迁移过程中起着关键作用，通过从迁移前NCC质膜中去除Cad 6 B蛋白，从而促进粘附连接的拆除和NCC迁移。本申请的具体目的是1）定义推定的内吞基序中的突变如何影响Cad 6 B内化和2）评估Cad 6 B内吞作用中的破坏对NCC EMT和迁移的影响。在目标1中，我们将改变特定的氨基酸的基序，可以调节Cad 6 B的内吞作用，并评估对Cad 6 B的内化，在体外和体内的影响。在目标2中，我们将通过体内和体外细胞和分子测定来评估Cad 6 B内吞作用的破坏如何影响NCC EMT和迁移。拟议的研究是创新的，因为它需要一个多学科的方法，结合胚胎学，生物化学，细胞和分子生物学检查钙粘蛋白的翻译后调节机制，在生物学相关的EMT。这些研究具有重要意义，因为这些结果将增强我们对正常发育和异常EMT期间产生迁移细胞类型相关机制的理解，并可能导致人类疾病新疗法的开发。

DESCRIPTION (provided by applicant): Abnormalities that occur during neural crest cell (NCC) development are directly responsible for many human congenital and hereditary malformations, diseases and cancers. Premigratory NCCs exist as adherent epithelial cells in the embryonic dorsal neural tube but later become motile through an epithelial-to- mesenchymal transition (EMT) in response to numerous intrinsic and extrinsic cues. Importantly, this event is analogous in scope to abnormal EMTs that underscore human disease and cancer. Although several similarities exist between these normal and aberrant EMTs, the chief amongst these is the cascade of events initiated by loss of cell surface cadherin proteins and the disassembly of adherens junctions to facilitate cell motility. Work from our lab has shown that loss of Cadherin6B (Cad6B) in premigratory cranial NCCs is crucial for NCC EMT. Cadherin reduction in vitro is mediated both transcriptionally and post-translationally through proteolysis and endocytosis. Our prior studies described comparable mechanisms of transcriptional repression and proteolytic processing of Cad6B in vivo during NCC EMT. Our new preliminary data, however, now reveal that Cad6B possesses putative endocytic motifs and co-localizes with markers of endosomes and lysosomes, both in vitro and in vivo. These initial findings suggest that Cad6B is also regulated post-translationally via endocytosis and, to our knowledge, are the first demonstration of cadherin endocytosis during an in vivo EMT. Based on these data, we hypothesize that Cad6B endocytosis plays a critical function during NCC EMT and migration through removal of Cad6B protein from premigratory NCC plasma membranes, thereby promoting the dismantling of adherens junctions and NCC migration. The Specific Aims of this application are to 1) define how mutations in putative endocytic motifs affect Cad6B internalization and 2) assess effects of disruptions in Cad6B endocytosis on NCC EMT and migration. In Aim 1, we will alter specific amino acids within motifs that may regulate Cad6B endocytosis and assess effects on Cad6B internalization, both in vitro and in vivo. In Aim 2, we will evaluate how disruptions in Cad6B endocytosis impact NCC EMT and migration through in vivo and in vitro cell and molecular assays. The proposed research is innovative because it takes a multi-disciplinary approach that combines embryology, biochemistry, and cell and molecular biology to examine post- translational mechanisms of cadherin regulation during a biologically relevant EMT. These studies are significant because the results will enhance our understanding of mechanisms associated with generating migratory cell types during both normal developmental and aberrant EMTs, and could lead to the development of new treatments for human diseases.