Role of FAK in Migrating Cells During Sea Urchin Embryogenesis

许多细胞的运动依赖于细胞外基质和粘着斑蛋白复合体之间的动态相互作用，粘着斑蛋白复合体将细胞内的细胞骨架锚定在细胞外基质上。本研究旨在研究粘着斑激酶(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在哪些细胞和类型的细胞运动中发现。共聚焦显微镜将允许对胚胎进行三维重建。初步研究表明，FAK存在于囊胚增厚的植物板、迁移于囊胚腔内的初级间充质细胞、负责原肠与口腔板融合的次生间充质细胞、以及增厚的动物极部。(2)第二个目标将是通过向胚胎中微量注射只包含靶向结构域的FAK结构来确定FAK的功能作用。假设该结构域将与内源性FAK竞争，通过减缓或中止细胞的正常运动而导致异常的原肠形成。(3)海胆胚胎至少有3种FAK亚型。我们的目标是确定FAK的这些亚型在海胆胚胎中是否有差异表达，以及每种亚型何时表达。这一信息可能具有重要意义，因为也从非洲爪哇和小鼠胚胎中分离出了多种亚型的FAK，但各种亚型的功能尚不清楚。(4)最后一个目标是确定焦点黏附复合体如何在胚胎中动态变化。FAK-绿色荧光蛋白结构的mRNAs和cDNAs将被注射到卵子中，以观察胚胎活动细胞中焦点黏附形成的动力学。这些观察将用共聚焦显微镜实时完成。意义：虽然在培养的胚胎成纤维细胞迁移过程中，粘着斑细胞的作用得到了广泛的研究，但很少有人发表关于粘着斑粘附子在胚胎发生过程中的作用，在活体胚胎中也没有发表。这项建议引入了新技术来解决胚胎中细胞运动的基本机制，这些机制还没有在这样一个动态和复杂的系统中研究过。

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.