BIOLOGY OF THE EMBRYONIC CORNEAL EPITHELIUM

这项资助的长期目标是验证角膜上皮细胞具有专门的细胞-细胞和细胞-基质结构域的假设，这些结构域负责稳定性、分化和通讯。在完整的胚胎角膜上皮组织中有一个特殊的基底-外侧结构域，包含许多肌动蛋白相关蛋白和信号蛋白。胞内蛋白在该区域的分布依赖于丝状肌动蛋白。下一组合乎逻辑的实验涉及进一步表征这种细胞基质附着复合物和细胞外基质引发的导致组织活力的信号转导途径。这些特定目标将通过分离无基板的整个禽类胚胎上皮和细胞外基质（ECM）分子培养来解决，在缺乏或存在阻断特定蛋白质合成的特异性抑制剂或寡核苷酸的情况下。具体目的：1 .验证酵素是ECM刺激的肌动蛋白束组装的成核位点的假设。肌动蛋白相关蛋白，zyxin与肌动蛋白成核蛋白同源，它从细胞核穿梭到成纤维细胞的病灶接触处。Zyxin在角膜上皮层中具有极化分布，依赖于肌动蛋白和ECM。确定酶合蛋白和肌动蛋白重组的序列，然后用反义寡核苷酸减少内源性酶合蛋白，以确定酶合蛋白是否作为ECM刺激的肌动蛋白束组装的成核位点。用免疫共沉淀法鉴定Zyxin结合蛋白，然后进行western blot分析。届时将确定Zyxin的周转率和稳定性。2。验证RhoGTP调节肌动蛋白重组和细胞质收缩的假说。Rho是一种小的信号蛋白，它在无活性形式（GDP）和活性形式（GTP）之间转换。一种参与改变Rho活性状态的蛋白已从胚胎角膜上皮（p190RhoGAP）中分离出来。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。验证上皮细胞-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保持细胞-细胞的附着。目前的项目将确定应力诱导信号转导通路是否在没有基底层的分离上皮中变得活跃。进一步的实验将确定外源性ECM是否可以拯救没有基底层的上皮细胞或用信号转导抑制剂治疗的上皮细胞。总之，在我们明确定义的角膜上皮模型中，这些实验将重点关注ECM介导的信号转导如何刺激肌动蛋白束的形成，并使细胞免于程序性细胞死亡。

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.