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结构域蛋白质和上皮完整性意义：上皮是产生外部（根尖）和内部（基础）隔室的细胞片。 ZP结构域蛋白几乎在几乎所有上皮的顶端细胞外基质（ECM）中都发现，并且从水母到人类存在，但没有分配给它们的共同活性。许多人类ZP结构域蛋白在疾病中被突变，包括几乎血液的Hensin（癌症），内耳Tecorins（耳聋）和肾尿素蛋白（肾病）。使用秀丽隐杆线虫两栖动物，一种由神经元和神经胶质组成的感觉上皮，我们确定了两个ZP结构域蛋白Dyf-7和FBN-1，以防止上皮细胞连接破裂。这些结果表明，ZP结构域蛋白质的新作用，即维持上皮完整性，并提高了ZP结构域蛋白质疾病的可能性。 细胞连接疾病。创新性：除了我们的创新假设外，秀丽隐杆线虫的使用在技术上是创新的。它允许对具有单细胞分辨率的连接组件进行遗传操纵；而且，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通过形成附着在上皮素的顶端表面的丝状网状工程来防止细胞连接破裂。初步数据：ZP结构蛋白蛋白Dyf-7的丢失会导致两栖感觉上皮的破裂。 Dyf-7以精确的精度定位，可与上皮细胞连接处相邻的细胞外“帽”。 DYF-7均由与GLIA结合的所有神经元所必需，但并非由其他神经胶质。异位DYF-7在所有上皮中的细胞连接处邻近。 DYF-7在体外自发地组装捆绑的细丝，类似于胚胎两栖上皮表面的电子显微镜（EM）所见的细丝。 ZP结构蛋白FBN-1或推定的ZP结合蛋白DEX-1模仿DYF-7的丧失。目的：首先，我们将在Dyf-7突变体中标记，耗尽和过表达单细胞分辨率，以测试细胞连接破裂的假设并确定机制。其次，我们将检验以下假设：dyf-7在体外和体内使用dyf-7丝的EM组装了附着在顶部表面上的丝状网状工作，对上皮细胞培养模型中的dyf-7分泌和定位的分析，以及对突变体的遗传筛选中的遗传筛选，这些突变体破坏了其本地化的位置。第三，我们将通过筛选DYF-7上游或下游的因子以及使用遗传学，生物化学和显微镜来测定DYF-7，FBN-1和DEX-1之间的相互作用来阐明我们确定的新上皮完整性途径。