FUNCTION OF CX43 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-特别是流出道形态发生。差距连接蛋白Cx43在心脏神经嵴细胞的定向迁移中具有关键功能。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，蛋白质介导连接蛋白和肌动蛋白之间的联系是候选人的调节细胞运动。一种已知介导连接蛋白和肌动蛋白之间相互作用的分子是ZO 1-一种与果蝇肿瘤抑制蛋白盘相关的PDZ蛋白。Gourdie实验室已经表明，Cx43-ZO 1相互作用对心肌细胞之间缝隙连接接触程度的发育重塑至关重要。此外，我们有数据表明，抑制ZO 1- Cx43的相互作用，减少体外神经嵴的生长，并减少运动的成纤维细胞和上皮细胞在培养的单层在“划痕”迁移试验。我们将通过以下方法来检验这一假设，即ZO 1-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的机制的组成部分：1.体外实验中，刺激神经嵴细胞运动的因素是否影响Cx43、ZO-1与其他连接蛋白相互作用蛋白的相互作用; ZO 1-Cx43相互作用的抑制是否足以破坏体外神经嵴细胞迁移的速率和方向性的调节;和3.如果ZO 1-Cx43相互作用对于体内神经嵴细胞的定向迁移是必要的。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.