Role of FAK in Migrating Cells During Sea Urchin Embryogenesis

许多细胞运动依赖于细胞外基质和黏着斑蛋白复合物之间的动态相互作用，黏着斑蛋白复合物将细胞内细胞骨架锚定到细胞外基质上。 研究黏着斑激酶（FAK）在半透明海胆胚胎形态发生中的调控作用。 (1)最初的目标是确定FAK在哪些细胞和细胞运动类型中被发现，共聚焦显微镜将允许胚胎的三维重建。 初步研究表明，FAK存在于囊胚胚胎的增厚的植物板、在囊胚腔中迁移的初级间充质细胞、负责原肠与口板融合的次级间充质细胞以及延伸出pluteus臂的增厚的动物极中。 (2)第二个目标是通过显微注射只包含靶向结构域的FAK构建体到胚胎中来确定FAK的功能作用。 假设该结构域将与内源性FAK竞争，并通过减慢或中止细胞的正常运动而导致异常原肠胚形成。 (3)海胆胚胎至少有三种FAK亚型。目的是确定这些FAK亚型是否在海胆胚胎中差异表达以及何时表达。 这一信息可能是重要的，因为FAK的多种异构体也已从非洲爪蟾和小鼠胚胎中分离出来，但各种异构体的功能尚不清楚。 (4)最后一个目标是确定黏着斑复合物在胚胎中的动态变化。将FAK-绿色荧光蛋白构建体的mRNA和cDNA注射到卵中，以观察胚胎运动细胞中局部粘附形成的动力学。 这些观测将通过共焦显微镜在真实的时间内完成。 重要性：虽然黏着斑在培养的胚胎成纤维细胞迁移过程中的作用正在被广泛研究，但关于黏着斑在胚胎发生过程中的作用的报道很少，在活胚胎中也没有报道。 该提案引入了新技术来解决胚胎中细胞运动的基本机制，这些机制尚未在这样一个动态和复杂的系统中研究。

Many cell movements depend on dynamic interactions between theextracellular matrix and focal adhesion protein complexes, which anchorthe intracellular cytoskeleton to the extracellular matrix. The proposalis to study the regulatory role of focal adhesion kinase or FAK in themorphogenesis of the translucent sea urchin embryo. (1) The initial goal is to determine in which cells and types of cellmovements FAK found. Confocal microscopy will allow three-dimensionreconstruction of the embryos. Preliminary studies show that FAK is foundin the thickening vegetal plate of blastula embryos, in primary mesenchymecells migrating in the blastocoel, in secondary mesenchyme cellsresponsible for fusion of the archenteron with the oral plate, and in thethickening animal pole from which the pluteus arms extend. (2) The second goal will be to determine the functional role of FAK bymicroinjecting into embryos a construct of FAK that contains only thetargeting domain. The hypothesis is that this domain will compete withendogenous FAK and result in abnormal gastrulation by slowing or abortingthe normal movements of cells. (3) Sea urchin embryos have at least three isoforms of FAK. The goal is todetermine whether these isoforms of FAK are differentially expressed insea urchin embryos and when each is expressed. This information could besignificant since multiple isoforms of FAK have also been isolated fromXenopus and mouse embryos, but the function of the various isoforms is notknown. (4) The last goal is to determine how focal adhesion complexes dynamicallychange in embryos. mRNAs and cDNAs of FAK-green fluorescent proteinconstructs will be injected into eggs to watch the dynamics of focaladhesion formation in motile cells of the embryos. These observationswill be done with confocal microscopy in real time. Significance: Although the roles of focal adhesions during the migrationof embryonic fibroblast cells in culture are being extensively studied,little has been published on the roles of focal adhesions duringembryogenesis and none in living embryos. This proposal introduces newtechnology to address fundamental mechanisms of cell movements in embryosthat have not been studied in such a dynamic and complex system.