Mechanism of Cranial Neural Crest Cell Migration

描述(申请人提供)：脑神经脊细胞的迁移是由调节基因表达、细胞增殖、细胞信号和细胞黏附的蛋白质控制的。在这些蛋白质中，ADAM细胞表面金属蛋白水解酶可以调节细胞信号和细胞黏附。它们通过从细胞表面切割信号和黏附分子来实现这一点。在理解ADAM蛋白如何总体上控制细胞迁移，特别是CnC迁移方面，存在着根本的差距。我们的长期目标是了解在发育中的胚胎中数控系统的迁移是如何被控制的。本应用程序的目的是确定ADAM金属蛋白水解酶如何控制NC迁移。这一建议的中心假设是，基于广泛的初步数据，亚当金属蛋白水解酶控制细胞迁移既通过从质膜上脱落细胞黏附分子，也通过它们的细胞质结构域在细胞核内的独立活动来控制细胞迁移。目的1：研究钙粘连蛋白11的ADAM裂解是如何控制细胞迁移的。我们发现，在CNC迁移过程中，Cad-11被ADAM13切割，包含粘连位点的Cad-11胞外片段被脱落，并可以挽救缺乏ADAM13或过表达Cad-11的胚胎的迁移。我们的假设是，该片段通过受体与细胞表面结合，促进细胞迁移。我们建议确定Cad-11的胞外片段如何促进迁移。我们将确定，1)不切割Cad-11对CNC的规范和迁移有什么后果，2)Cad-11胞外片段是否刺激细胞定向迁移和/或干扰接触介导的抑制，以及3)这一过程中涉及的信号通路是什么。目的2：确定ADAM胞质结构域控制细胞迁移的机制。我们已经证明了ADAM13胞质结构域(C13)被切割并移位到细胞核中。我们发现，这对体内的基因表达和NC迁移至关重要。我们还发现了一个受C13调控的基因Calpain 8，它对数控系统的迁移是必不可少的。我们的假设是，ADAM13细胞质结构域通过改变转录因子的功能来促进NC迁移来控制基因的表达。我们将确定1)C13中负责控制基因表达和细胞迁移的氨基酸序列(S)，以及2)C13调节Calpain 8表达的机制。这种方法是创新的，因为它首次在任何系统中表明，亚当细胞质结构域在细胞核中发挥功能，调节特定的基因表达和细胞迁移。这项拟议的研究具有重要意义，因为它有望通过识别ADAM细胞质结构域中控制转录因子(例如FoxD3)活性的进化保守基序来垂直推进该领域。此外，Cad-11胞外结构域已被证明可以增加癌细胞的侵袭性，因此我们的结果将有助于阐明该结构域的作用机制。

DESCRIPTION (provided by applicant): The migration of cranial neural crest (CNC) cells is controlled by proteins that regulate gene expression, cell proliferation, cell signaling and cell adhesion. Among these proteins the ADAM cell surface metalloproteases can regulate both cell signaling and cell adhesion. They do so by cleaving signaling and adhesion molecules from the cell surface. There is a fundamental gap in understanding how ADAM proteins control cell migration in general and CNC migration in particular. Our long-term goal is to understand how CNC migration is controlled in a developing embryo. The objective of this application is to determine how ADAM metalloproteases control CNC migration. The central hypothesis of this proposal, based on extensive preliminary data, is that ADAM metalloproteases control cell migration both by shedding cell adhesion molecules from the plasma membrane and by the independent activity of their cytoplasmic domains inside of the nucleus. Aim 1: To identify how ADAM cleavage of Cadherin-11 controls CNC migration. We have shown that Cad-11 is cleaved by ADAM13 during CNC migration and that the Cad-11 extracellular fragment containing the adhesive site is shed and can rescue migration in embryos lacking ADAM13 or overexpressing Cad-11. Our hypothesis is that this fragment binds to the surface of CNC via a receptor and promotes migration. We propose to determine how the extracellular fragment of Cad-11 promotes migration. We will determine, 1) what are the consequences of not cleaving Cad-11 on CNC specification and migration, 2) if the Cad-11 extracellular fragment stimulates directed cell migration and/or interfere with contact mediated inhibition, and 3) What are the signaling pathways involved in this process. Aim 2: To identify the mechanism by which the ADAM cytoplasmic domain controls CNC migration. We have shown that the ADAM13 cytoplasmic domain (C13) is cleaved and translocates into the nucleus. We showed that this is critical for gene expression and CNC migration in vivo. We also showed that one gene regulated by C13, Calpain8 is essential for CNC migration. Our hypothesis is that the ADAM13 cytoplasmic domain controls gene expression by modifying the function of a transcription factor to promote CNC migration. We will determine 1) the amino acid sequence(s) in C13 that are responsible for the control of gene expression and cell migration, and 2) the mechanism by which C13 regulates the expression of Calpain8. The approach is innovative, because it shows for the first time in any system that ADAM cytoplasmic domains function in the nucleus to regulate specific gene expression and cell migration. The proposed research is significant, because it is expected to vertically advance the field by identifying evolutionary conserved motifs in ADAM cytoplasmic domain that control the activity of transcription factors (e.g. FoxD3). In addition, the Cad-11 extracellular domain has been shown to increase cancer cell invasion, thus our results will shed the light on the mechanism by which this domain operates.