FUNCTION OF CX43-INTERACTING PROTEINS IN NEURAL CREST MIGRATION

描述（由申请人提供）：神经嵴迁移的适当协调是正常心血管发育的绝对要求-尤其是流出道形态发生。间隙连接蛋白Cx43在心脏神经嵴细胞的定向迁移中起关键作用。Cx43影响神经嵴运动的分子机制尚不清楚。驱动细胞迁移的力量主要是由肌动蛋白细胞骨架的动力学产生的。我们假设介导连接蛋白和肌动蛋白之间联系的蛋白质是细胞运动调节因子的候选人。已知介导连接蛋白和肌动蛋白之间相互作用的分子是ZO1 -一种与果蝇肿瘤抑制蛋白盘大相关的PDZ蛋白。Gourdie实验室已经证明，Cx43-ZO1相互作用对心肌细胞间间隙连接接触程度的发育重塑至关重要。此外，我们的数据显示，在“抓伤”迁移实验中，ZO1- Cx43相互作用的抑制会减少体外神经嵴的生长，并降低培养单层中成纤维细胞和上皮细胞的运动能力。我们将测试ZO1-Cx43相互作用是参与调节细胞-细胞接触模式和神经嵴细胞迁移机制的一个组成部分的假设，通过确定：是否已知的刺激神经嵴细胞运动的因素，影响Cx43， ZO-1和其他连接蛋白相互作用蛋白之间的相互作用；2. 抑制ZO1-Cx43相互作用是否足以破坏体外神经嵴细胞迁移速率和方向性的调节；和3。如果ZO1-Cx43相互作用是神经嵴细胞在体内定向迁移的必要条件。这项研究将为胚胎神经嵴迁移的分子调控和人类出生缺陷的起源提供新的见解。

DESCRIPTION (provided by applicant): Proper coordination of neural crest migration is an absolute requirement for normal cardiovascular development - most particularly outflow tract morphogenesis. The gap junction protein Cx43 has a key function in this directed migration of cardiac neural crest cells. The molecular mechanism by which Cx43 affects neural crest motility is unknown. The forces driving cell migration are generated largely by the dynamics of the actin cytoskeleton. We hypothesize that proteins mediating linkage between connexins and actin are candidates for regulators of cell motility. One molecule known to mediate interaction between connexins and actin is ZO1 - a PDZ protein related to the Drosophila tumor suppressor protein discs large. The Gourdie lab has shown that Cx43-ZO1 interaction is critical to developmental remodeling of the extent of gap junctional contact between myocardial cells. Furthermore, we have data showing that inhibition of ZO1- Cx43 interaction decreases neural crest outgrowth in vitro and reduces motility of fibroblasts and epithelial cells in cultured monolayers in a "scratch wound" migration assay. We will test the hypothesis that ZO1-Cx43 interaction is an integral part of a mechanism involved in regulation of cell-cell contact pattern and migration of neural crest cells by determining: 1. Whether factors known to stimulate neural crest cell motility, affect interactions between Cx43, ZO-1 and other connexin interacting proteins in vitro; 2. Whether inhibition of ZO1-Cx43 interaction is sufficient to disrupt regulation of the rate and directionality of neural crest cell migration in vitro; and 3. if ZO1-Cx43 interaction is necessary for the directed migration of neural crest cells in vivo. This study will provide new insight into molecular regulation of neural crest migration in the embryo and origins of birth defects in humans.