ZP Domain Proteins and Epithelial Integrity

ZP 结构域蛋白和上皮完整性

• 批准号: 8612003
• 负责人: Maxwell Heiman
• 金额: $ 33.31万
• 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/8612003
• 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

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.

ZP结构域蛋白与上皮完整性 意义：上皮是一层细胞，形成外（顶端）和内（基底）室。ZP结构域 在几乎所有上皮细胞的顶端细胞外基质（ECM）中发现了蛋白质，并且存在于水母中 人类，但没有分配给他们的共享活动。许多人ZP结构域蛋白是突变的 在疾病中，包括几乎无处不在的hensin（癌症），内耳tectorins（耳聋）和肾 尿调素（肾病）。利用C. elegans amphid，由神经元和神经胶质组成的感觉上皮， 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破裂所必需的 交叉点这些结果表明ZP结构域蛋白的新作用，即维持上皮细胞的增殖。 完整性，并提高ZP结构域蛋白疾病可能作为细胞连接障碍治疗的可能性。 创新性：除了我们的创新假设，C。以秀丽隐杆线虫为模型 上皮细胞是技术上的创新。它允许基因操作的连接组件与单细胞 并且，与其他上皮不同，感觉上皮的破裂不是致命的，因此无效突变体是可行的。 假设：我们假设DYF-7通过形成一个丝状的连接来防止细胞连接的破裂。 附着在上皮细胞顶端表面的网状物。 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都需要，但其他神经元不需要。异位DYF-7定位于邻近 所有上皮细胞的细胞连接。在体外表达的DYF-7自发地组装成束的细丝，类似于 电子显微镜（EM）在胚胎两栖类上皮表面看到的细丝。失去ZP 结构域蛋白FBN-1或推定的ZP结构域结合蛋白DEX-1模拟DYF-7的缺失。 目的：首先，我们将标记，耗尽，并过表达上皮成分与单细胞分辨率在一个dyf-7 突变体，以测试假设，细胞连接破裂，并确定机制。第二，我们将测试 假设DYF-7组装了一个附着在根尖表面的丝状网络，使用DYF-7的EM 体外和体内的纤维，上皮细胞培养模型中DYF-7分泌和定位的分析， 以及对破坏其体内定位的突变体进行遗传筛选。第三，我们将阐明新的上皮细胞 我们通过筛选DYF-7上游或下游的因子，并使用 遗传学、生物化学和显微镜以测定DYF-7、FBN-1和DEX-1之间的相互作用。