ZP Domain Proteins and Epithelial Integrity

描述（由申请人提供）：ZP结构域蛋白和上皮完整性意义：上皮细胞是产生外（顶端）和内（基底）室的细胞片。ZP结构域蛋白存在于几乎所有上皮细胞的顶端细胞外基质（ECM）中，并且存在于从水母到人类的细胞中，但没有共同的活性被分配给它们。许多人类ZP结构域蛋白在疾病中突变，包括几乎普遍存在的hensin（癌症）、内耳tectorins（耳聋）和肾尿调蛋白（肾病）。利用C.在elegans amphid（一种由神经元和神经胶质组成的感觉上皮）中，我们鉴定了两种防止上皮细胞连接破裂所需的ZP结构域蛋白，DYF-7和FBN-1。这些结果表明ZP结构域蛋白的新作用，即维持上皮完整性，并提高了ZP结构域蛋白疾病可能被治疗的可能性。 细胞连接障碍创新性：除了我们的创新假设，C。作为上皮细胞模型的秀丽隐杆线虫在技术上是创新的。它允许基因操作的连接组件与单细胞分辨率;和，不像其他上皮细胞，破裂的感觉上皮细胞是不致命的，所以无效突变体是可行的。假设：我们推测DYF-7通过形成附着于上皮细胞顶端表面的丝状网状结构来防止细胞连接破裂。初步数据：ZP结构域蛋白DYF-7的缺失导致两栖类感觉上皮破裂。DYF-7精确定位于上皮细胞连接处附近的细胞外“帽”。DYF-7是所有与神经胶质形成连接的神经元所必需的，而不是其他神经元。异位DYF-7定位于所有上皮细胞连接附近。DYF-7在体外表达自发地组装成束的细丝，类似于在胚胎两栖类上皮表面通过电子显微镜（EM）观察到的细丝。ZP结构域蛋白FBN-1或推定的ZP结构域结合蛋白DEX-1的缺失模拟DYF-7的缺失。目的：首先，我们将在dyf-7突变体中用单细胞分辨率标记、耗尽和过表达上皮成分，以检验细胞连接破裂的假设，并确定其机制。第二，我们将测试的假设，DYF-7组装一个丝状的网状物连接到顶端表面，使用EM DYF-7细丝在体外和体内，DYF-7分泌和定位在上皮细胞培养模型中的分析，并在体内破坏其定位突变体的遗传筛选。第三，我们将阐明我们通过筛选DYF-7上游或下游的因子以及通过使用遗传学、生物化学和显微镜来测定DYF-7、FBN-1和DEX-1之间的相互作用而确定的新的上皮完整性途径。

DESCRIPTION (provided by applicant): ZP domain proteins and epithelial integrity Significance: Epithelia are sheets of cells that create outer (apical) and inner (basal) compartments. ZP domain proteins are found in the apical extracellular matrix (ECM) of nearly all epithelia, and are present from jellyfish to humans, but no shared activity has been assigned to them. Many human ZP domain proteins are mutated in disease, including the nearly-ubiquitous hensin (cancer), inner ear tectorins (deafness), and renal uromodulin (nephropathy). Using the C. elegans amphid, a sensory epithelium composed of neurons and glia, we identified two ZP domain proteins, DYF-7 and FBN-1, required to prevent rupture of epithelial cell junctions. These results suggest a new role for ZP domain proteins, namely maintenance of epithelial integrity, and raise the possibility that ZP domain protein diseases might be treated as cell junction disorders. Innovativeness: In addition to our innovative hypothesis, the use of the C. elegans amphid as a model epithelium is technically innovative. It allows genetic manipulation of junction components with single-cell resolution; and, unlike other epithelia, rupture of sensory epithelia is not lethal, so null mutants are viable. Hypothesis: We hypothesize that DYF-7 prevents cell junctions from rupture by forming a filamentous meshwork attached to the apical surfaces of epithelia. Preliminary data: Loss of the ZP domain protein DYF-7 causes rupture of the amphid sensory epithelium. DYF-7 localizes with exquisite precision to extracellular "caps" adjacent to epithelial cell junctions. DYF-7 is required by all neurons that form junctions with glia, but not by others. Ectopic DYF-7 localizes adjacent to cell junctions in all epithelia. DYF-7 expressed in vitro spontaneously assembles bundled filaments, similar to filaments seen by electron microscopy (EM) at the surface of the embryonic amphid epithelium. Loss of the ZP domain protein FBN-1 or the putative ZP-domain-binding protein DEX-1 mimic loss of DYF-7. Aims: First, we will label, deplete, and overexpress epithelial components with single-cell resolution in a dyf-7 mutant to test the hypothesis that cell junctions rupture, and determine the mechanism. Second, we will test the hypothesis that DYF-7 assembles a filamentous meshwork attached to apical surfaces, using EM of DYF-7 filaments in vitro and in vivo, analysis of DYF-7 secretion and localization in an epithelial cell culture model, and a genetic screen for mutants that disrupt its localization in vivo. Third, we will elucidate the new epithelial integrity pathway we identified by screening for factors upstream or downstream of DYF-7 and by using genetics, biochemistry, and microscopy to assay interactions between DYF-7, FBN-1, and DEX-1.