FUNCTION OF CX43-INTERACTING PROTEINS IN NEURAL CREST MIGRATION

描述(由申请人提供)：适当协调神经脊迁移是正常心血管发育的绝对要求--尤其是流出道的形态发生。缝隙连接蛋白Cx43在心脏神经脊细胞的定向迁移中起着关键作用。Cx43影响神经脊运动的分子机制尚不清楚。推动细胞迁移的力量很大程度上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的候选蛋白。已知的一个介导连接蛋白和肌动蛋白相互作用的分子是ZO1--一种与果蝇肿瘤抑制蛋白大圆盘相关的PDZ蛋白。古尔迪实验室已经证明，Cx43-ZO1的相互作用对于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ZO1-Cx43相互作用减少了体外培养的神经脊的生长，并在“划痕”迁移实验中降低了培养单层成纤维细胞和上皮细胞的运动性。我们将验证这一假设，即ZO1-Cx43相互作用是参与调节神经脊细胞的细胞-细胞接触模式和迁移的机制的组成部分，通过确定：1.已知的刺激神经脊细胞运动的因素是否影响Cx43、ZO-1和其他连接蛋白相互作用蛋白在体外的相互作用；2.抑制ZO1-Cx43相互作用是否足以扰乱神经脊细胞在体外迁移的速度和方向的调节；以及3.ZO1-Cx43相互作用是否对神经脊细胞在体内的定向迁移是必要的。这项研究将为人类胚胎中神经脊迁移的分子调控和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.