ZP Domain Proteins and Epithelial Integrity

ZP 结构域蛋白和上皮完整性

• 批准号: 8994290
• 负责人: Maxwell Heiman
• 金额: $ 33.63万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8994290
• 关键词: Affect; Animals; Apical; Autoantigens; Biochemistry; Biological; Biological Assay; Blood Vessels; Body Surface; Caenorhabditis elegans; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Crohn' s disease; DMBT1 gene; Data; Defect; Disease; Dysplasia; Electron Microscopy; Embryo; Endoglin; Endothelium; Enhancers; Epithelial; Epithelial Cell Junction; Epithelial Cells; Epithelium; Extracellular Matrix; Filament; Genetic; Genetic Screening; Goals; Human; In Vitro; Infection; Inherited; Intercellular Junctions; Jellyfish; Kidney; Kidney Diseases; Label; Labyrinth; Light; Maintenance; Malignant Neoplasms; Mechanics; Medical; Microscopy; Modeling; Molecular; Mutate; Mutation; Neuroglia; Neurons; Organ of Corti; Pancreas; Pathway interactions; Polymers; Pre-Eclampsia; Prevalence; Protein Binding Domain; Proteins; Resolution; Role; Rupture; Sense Organs; Sensory; Source; Structure; Supporting Cell; Surface; Taste Buds; Tertiary Protein Structure; Testing; Tumor Suppressor Proteins; UMOD gene; deafness; extracellular; genetic manipulation; hensin; in vivo; innovation; mutant; neurotensin mimic 1; novel; overexpression; prevent; protein function; public health relevance; receptor; resilience; screening

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.

描述(由申请人提供)：ZP结构域蛋白和上皮完整性意义：上皮是一层细胞，它产生外部(顶端)和内部(底部)间隔。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，从水母到人类都存在，但它们没有共同的活性。许多人类ZP结构域蛋白在疾病中发生突变，包括几乎无处不在的Hensin(癌症)、内耳tectorins(耳聋)和肾脏尿调蛋白(肾病)。利用线虫，一种由神经元和胶质细胞组成的感觉上皮，我们鉴定了两个ZP结构域蛋白DYF-7和FBN-1，它们是防止上皮细胞连接断裂所必需的。这些结果提示ZP结构域蛋白具有新的作用，即维持上皮完整性，并增加了ZP结构域蛋白病可能被作为 细胞连接紊乱。创新性：除了我们的创新假设之外，使用线虫作为模型上皮在技术上也是创新的。它允许以单细胞分辨率对连接部分进行遗传操作；而且，与其他上皮细胞不同，感觉上皮细胞的破裂不是致命的，因此零突变是可行的。假设：我们假设DYF-7通过形成附着在上皮细胞顶端表面的丝状网络来防止细胞连接断裂。初步数据：ZP结构域蛋白DYF-7的缺失会导致双眼感觉上皮破裂。DYF-7精确定位于上皮细胞连接附近的细胞外“帽子”。所有与神经胶质细胞形成连接的神经元都需要DYF-7，但其他神经元并不需要。异位DYF-7定位于所有上皮细胞连接附近。在体外表达的DYF-7自发地组装成束状细丝，类似于电子显微镜(EM)所见的胚胎两端上皮表面的细丝。ZP结构域蛋白FBN-1或可能的ZP结构域结合蛋白DEX-1的丢失与DYF-7的丢失类似。目的：首先，我们将在Dyf-7突变体中标记、耗尽和过度表达单细胞分辨率的上皮成分，以检验细胞连接断裂的假设，并确定其机制。其次，我们将使用DYF-7细丝在体外和体内的EM，分析DYF-7在上皮细胞培养模型中的分泌和定位，以及对扰乱其体内定位的突变体的遗传筛选，来验证DYF-7组装附着在顶端表面的丝状网络的假设。第三，我们将阐明我们通过筛选DYF-7上游或下游的因素以及通过遗传学、生物化学和显微镜来分析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。