Cadherin endocytosis in the cranial neural crest

颅神经嵴中的钙粘蛋白内吞作用

• 批准号: 8805561
• 负责人: LISA A TANEYHILL
• 金额: $ 7.6万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-12 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8805561
• 关键词: Abbreviations; Address; Adherens Junction; Affect; Amino Acids; Binding; Biochemistry; Biological; Biological Assay; Biotinylation; Cadherins; Cell membrane; Cell surface; Cell-Cell Adhesion; Cells; Cellular biology; Cephalic; Chick Embryo; Cues; Cytoplasmic Tail; Data; Development; Disease; Dorsal; Down-Regulation; Embryo; Embryology; Embryonic Development; Endocytosis; Endosomes; Epithelial Cells; Event; Excision; Face; Gene Expression; Generations; Genes; Goals; Health; Heart Part; Human; Human body; Imagery; In Vitro; Inherited; Knowledge; Lead; Lysosomes; Malignant Neoplasms; Mediating; Mission; Modeling; Molecular; Molecular Biology; Mutation; N-Cadherin; Nerve; Neural Crest; Neural Crest Cell; Neural tube; Pathway interactions; Pigments; Play; Population; Process; Proteins; Proteolysis; Proteolytic Processing; Public Health; Publishing; Quality of life; Regulation; Research; Research Design; Role; Skin; Somites; Staging; Structure; Syndrome; System; Testing; Work; base; bone; cell behavior; cell motility; cell type; embryo tissue; epithelial to mesenchymal transition; gene repression; human disease; improved; in vitro Assay; in vitro Model; in vivo; in vivo Model; innovation; insight; malformation; migration; molecular marker; mutant; prevent; public health relevance; response; tissue repair; tool

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.

描述（由申请人提供）：神经嵴细胞（NCC）发育过程中发生的异常直接导致许多人类先天性和遗传性畸形、疾病和癌症。迁移前的 NCC 在胚胎背神经管中以贴壁上皮细胞的形式存在，但随后通过上皮间质转化 (EMT) 响应众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。尽管这些正常和异常的 EMT 之间存在一些相似之处，但其中最主要的是由细胞表面钙粘蛋白的丢失和粘附连接的分解以促进细胞运动而引发的级联事件。我们实验室的工作表明，迁移前颅内 NCC 中 Cadherin6B (Cad6B) 的丢失对于 NCC EMT 至关重要。体外钙粘蛋白的减少是通过蛋白水解和内吞作用在转录和翻译后介导的。我们之前的研究描述了 NCC EMT 期间 Cad6B 体内转录抑制和蛋白水解加工的类似机制。然而，我们新的初步数据现在表明，Cad6B 具有假定的内吞基序，并在体外和体内与内体和溶酶体标记物共定位。这些初步研究结果表明，Cad6B 也通过内吞作用进行翻译后调节，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 Cad6B 内吞作用在 NCC EMT 和迁移过程中发挥着关键作用，通过从迁移前的 NCC 质膜上去除 Cad6B 蛋白，从而促进粘附连接的拆除和 NCC 迁移。本申请的具体目标是 1) 定义假定的内吞基序中的突变如何影响 Cad6B 内化，2) 评估 Cad6B 内吞作用中断对 NCC EMT 和迁移的影响。在目标 1 中，我们将改变基序内的特定氨基酸，这些氨基酸可以调节 Cad6B 内吞作用并评估对 Cad6B 内化的影响（体外和体内）。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 Cad6B 内吞作用的破坏如何影响 NCC EMT 和迁移。拟议的研究具有创新性，因为它采用了结合胚胎学、生物化学以及细胞和分子生物学的多学科方法来检查生物学相关的 EMT 过程中钙粘蛋白调节的翻译后机制。这些研究意义重大，因为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。