BIOLOGY OF THE EMBRYONIC CORNEAL EPITHELIUM

这笔赠款的长期目标是测试角膜上皮细胞具有特殊的细胞-细胞和细胞-基质结构域的假设，这些结构域负责稳定、分化和通讯。在完整的胚胎角膜上皮组织中，含有许多肌动蛋白相关蛋白和信号蛋白的特殊的基底区-侧区已经被定义。该结构域中的细胞内蛋白分布依赖于丝状肌动蛋白。下一个符合逻辑的实验包括进一步描述这种细胞基质附着复合体以及由细胞外基质启动的导致组织存活的信号转导通路。这些特定的目标将通过分离整个没有基膜的禽类胚胎上皮细胞来实现，并在没有或存在阻止特定蛋白质合成的特定抑制剂或寡核苷酸的情况下与细胞外基质(ECM)分子一起培养。具体目标：1.验证齐辛是ECM刺激的肌动蛋白束组装的成核部位的假设。肌动蛋白相关蛋白，凝集素是肌动蛋白核蛋白的同源蛋白，它从细胞核穿梭到成纤维细胞的焦点接触。凝集素在角膜上皮片中呈极化分布，这种分布依赖于肌动蛋白和细胞外基质。确定凝乳素和肌动蛋白重组的顺序，然后用反义寡核苷酸还原内源性凝乳素，以确定凝乳素是否作为ECM刺激的肌动蛋白束组装的成核部位。用免疫共沉淀法鉴定凝血酶结合蛋白，然后进行蛋白质印迹分析。智信S的离职率和稳定性将得到确定。2.验证RhoGTP调控肌动蛋白重组和细胞质回缩的假设。Rho是一种小的信号蛋白，在非活性形式(GDP)和活性形式(GTP)之间洗牌。从胚胎角膜上皮中分离到一种参与改变Rho活性状态的蛋白质(P190RhoGAP)。目前的项目将确定该蛋白(P190RhoGAP)和其他相关下游信号蛋白(ROCK、Dia、MAP激酶和P13激酶)在细胞外基质刺激的肌动蛋白束形成中的作用。3.检验上皮细胞-细胞和细胞-基质相互作用抑制细胞凋亡的假设。该模型在分离细胞-基质附着复合体的同时，维持细胞-细胞的附着。目前的项目将确定在分离的没有基底膜的上皮细胞中，压力诱导信号转导通路是否变得活跃。进一步的实验将确定外源ECM是否可以挽救分离的没有基底膜的上皮细胞，或者是否可以用信号转导抑制剂治疗。总之，在我们定义良好的角膜上皮模型中，这些实验将集中于ECM介导的信号转导如何刺激肌动蛋白束的形成，并将细胞从程序性细胞死亡中拯救出来。

The long-term objective of this grant is to test the hypothesis that corneal epithelial cells have specialized cell-cell and cell-matrix domains that are responsible for stability, differentiation and communication. A specialized basal-lateral domain in the intact embryonic corneal epithelial tissue that contains many actin associated and signaling proteins has been defined. The intracellular protein distribution in this domain is filamentous-actin dependent. The next logical set of experiments involves further characterization of this cell matrix attachment complex and the signal transduction pathways initiated by extracellular matrix that lead to tissue viability. These specific aims will be addressed with whole avian embryonic epithelia isolated without basal lamina and cultured with extracellular matrix (ECM) molecules in the absence or presence of specific inhibitors or oligonucleotides that block synthesis of specific proteins. Specific aims: I. Test the hypothesis that zyxin is a nucleating site for ECM stimulated actin bundle assembly. The actin associated protein, zyxin is homologous to an actin nucleating protein and it shuttles from the nucleus to fibroblast focal contacts. Zyxin has a polarized distribution in corneal epithelial sheets that is actin and ECM dependent. The sequence of zyxin and actin reassembly will be determined, then endogenous zyxin will be decreased with antisense oligonucleotides to establish if zyxin acts as a nucleation site for ECM stimulated actin bundle assembly. Zyxin binding proteins will be identified with co-immunoprecipitation, followed by western blot analysis. Zyxin s turnover rate and stability will be determined. II. Test the hypothesis that RhoGTP regulates actin reorganization and cytoplasmic retraction. Rho is a small signaling protein that shuffles between an inactive form (GDP) to an active form (GTP). One of the proteins that participates in changing the active state of Rho has been isolated from the embryonic corneal epithelium (p190RhoGAP). The current project will determine the role of this protein (p190RhoGAP) and other related downstream signaling proteins (ROCK, Dia, MAP kinase and P13 kinase) in extracellular matrix stimulated actin bundle formation. III. Test the hypothesis that epithelial cell-cell and cell-matrix interactions suppresses apoptosis. This model maintains cell-cell attachments while separating the cell-matrix attachment complex. The current project will determine if the stress-inducing signal transduction pathways become active in epithelia isolated without the basal lamina. Further experiments will determine if exogenous ECM can rescue epithelia isolated without the basal lamina or treated with signal transduction inhibitors. In summary, these experiments in our well defined corneal epithelial model will focus on how ECM mediated signal transduction stimulates actin bundle formation and also save the cells from programmed cell death.