Apical membrane modeling in tubulogenesis

肾小管发生中的顶膜建模

• 批准号: 9117605
• 负责人: VERENA GOBEL
• 金额: $ 33.06万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-11 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9117605
• 关键词: Actins; Animal Model; Apical; Biogenesis; Biological; Caenorhabditis elegans; Cell Polarity; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Clathrin; Clathrin-Coated Vesicles; Coated vesicle; Complex; Cues; Cytomegalovirus Infections; Cytoskeleton; Data; Defect; Development; Disease; Engineering; Enzymes; Epithelial; Epithelium; Extracellular Matrix; Genes; Genetic; Glycosphingolipids; Golgi Apparatus; Grant; Growth; Health; Human; In Vitro; Intestines; Label; Lateral; Life; Lipids; Malignant Neoplasms; Membrane; Membrane Microdomains; Mining; Morphogenesis; Nature; Nematoda; Organ; Organism; Organogenesis; Orphan; Pathway interactions; Phenotype; Plasma; Positioning Attribute; Process; RNA Interference; RNA interference screen; Recruitment Activity; Regulation; Resolution; Resources; Role; Route; Sorting - Cell Movement; Sphingolipids; Testing; Thinking; Tissues; Transcription Factor AP-1; Tube; Tubular formation; Vesicle; apical membrane; base; basolateral membrane; cellular microvillus; cofilin; design; genome-wide; human disease; in vivo; luminal membrane; membrane biogenesis; membrane model; mutant; novel; self organization; theories; tissue processing; trafficking; Actins; Animal Model; Apical; Biogenesis; Biological; Caenorhabditis elegans; Cell Polarity; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Clathrin; Clathrin-Coated Vesicles; Coated vesicle; Complex; Cues; Cytomegalovirus Infections; Cytoskeleton; Data; Defect; Development; Disease; Engineering; Enzymes; Epithelial; Epithelium; Extracellular Matrix; Genes; Genetic; Glycosphingolipids; Golgi Apparatus; Grant; Growth; Health; Human; In Vitro; Intestines; Label; Lateral; Life; Lipids; Malignant Neoplasms; Membrane; Membrane Microdomains; Mining; Morphogenesis; Nature; Nematoda; Organ; Organism; Organogenesis; Orphan; Pathway interactions; Phenotype; Plasma; Positioning Attribute; Process; RNA Interference; RNA interference screen; Recruitment Activity; Regulation; Resolution; Resources; Role; Route; Sorting - Cell Movement; Sphingolipids; Testing; Thinking; Tissues; Transcription Factor AP-1; Tube; Tubular formation; Vesicle; apical membrane; base; basolateral membrane; cellular microvillus; cofilin; design; genome-wide; human disease; in vivo; luminal membrane; membrane biogenesis; membrane model; mutant; novel; self organization; theories; tissue processing; trafficking

DESCRIPTION (provided by applicant): Generating and maintaining polarity is fundamental to multicellular life and a prerequisite for spatial diversity and morphogenesis. Epithelial membrane biogenesis requires distinct polarizing cues from: (a) outside the cell (e.g. the matrix), (b) insie the cell (e.g. through directional trafficking), and (c) the plasma membrane and its junctions (e.g by plasma-membrane associated polarity determinants such as the partitioning-defective PAR s). Many of these highly conserved polarity cues have been identified, but their integration during the complex process of tissue morphogenesis is not well understood. We have conducted genome-wide unbiased as well as targeted RNAi-based screens in the transparent roundworm C. elegans, engineered with fluorescently-labeled apical membranes, to explore polarized membranes biogenesis in simple multi- and unicellular tubes at single-cell resolution. Here, tubular organogenesis involves coincident apical domain and lumen morphogenesis, permitting polarity analysis in a 3D in vivo setting during morphogenesis. These screens identified, among other novel tube phenotypes, an intriguing intestinal polarity and multiple-ectopic-lumen phenotype, where all apical molecules tested, including the apical polarity complex component PAR-6 were found displaced to the basolateral membrane. Among other molecules, the loss of several unrelated lipid -biosynthetic enzymes, trafficking-related genes, as well as actin-modulating genes were found to cause this phenotype. Subsequent analyses identified membrane sphingolipids (SLs) as well as clathrin/AP-1 as trafficking molecules required for epical sorting and lumen positioning, a novel in vivo function for these proposed raft components and an unexpected trafficking route for this classical post-Golgi vesicle coat and its adaptor. Here, we will characterize this new vesicular sorting pathways for apical polarity and lumen morphogenesis by functionally, genetically, morphologically and biochemically characterizing some of the other molecules suggested by these screens are to participate in polarity regulation and lumen formation, and explore their relationship to SLs, clathrin/AP-1, each other and classical polarity determinants such as the PARs in this function. Specifically, we will assess the role of the actin cytoskeleton in apical sorting via three identified actin modulators (Aim1); analyze the relationship of PAR-6 and CDC-42 to SLs, clathrin/AP-1 and these actin modulators (Aim2); and delineate the components and routes of this new apical sorting pathway by integrating multiple trafficking-related genes also identified as involved in apical membrane biogenesis by our multi-and unicellular tubulogenesis screens (Aim3). Given the fundamental nature of polarity and the high structural and functional conservation of all molecules examined, the results of these studies should be directly relevant to human epithelial polarity and the related human diseases, two examples being cancer( associated with loss of polarity) and diseases of internal organs (all composed of, and functioning through, polarized tubular epithelia).

描述（由申请人提供）：产生和维持极性是多细胞寿命的基础，也是空间多样性和形态发生的先决条件。 Epithelial membrane biogenesis requires distinct polarizing cues from: (a) outside the cell (e.g. the matrix), (b) insie the cell (e.g. through directional trafficking), and (c) the plasma membrane and its junctions (e.g by plasma-membrane associated polarity determinants such as the partitioning-defective PAR s).这些高度保守的极性提示已被鉴定出来，但是在组织形态发生过程中它们在复杂的过程中的整合尚不清楚。我们已经在透明的round虫秀丽隐杆线虫中进行了全基因组无偏见以及靶向RNAi的筛选，该秀丽隐杆线虫采用荧光标记的顶端膜设计，以在单细胞分辨率下在简单的多细胞和单细胞管中探索极化的膜生物发生。在这里，管状器官发生涉及一致的顶端结构域和腔形态发生，可以在形态发生过程中3D体内环境中的极性分析。这些筛选在其他新型的管表型中鉴定出了一个有趣的肠极性和多切片 - 轻型表型，其中所有测试的根尖分子（包括顶极性复合物成分PAR-6）都被置于基底外侧膜上。在其他分子中，发现几种无关的脂质生物合成酶，与运输相关的基因以及肌动蛋白调节基因的丧失会导致这种表型。随后的分析鉴定出膜鞘脂（SLS）以及网格蛋白/AP-1是流量分类和管腔定位所需的运输分子，这是这些提出的筏的新型体内功能 这款古典后高尔基囊泡外套及其适配器的组件和意外的贩运途径。在这里，我们将通过功能，遗传，形态学和生化在遗传，形态和生化上表征这些新的囊泡分选途径，以表征这些筛选所建议的其他分子的表征是参与极性调节和腔体形成，并探索与SLS，clathrin/ap-1的相关性，每个性分子的范围，每个性分类的范围，这些功能均可确定这些功能。具体而言，我们将通过三个确定的肌动蛋白调节剂来评估肌动蛋白细胞骨架在顶端分选的作用（AIM1）；分析PAR-6和CDC-42与SLS，Clathrin/ap-1以及这些肌动蛋白调节剂的关系（AIM2）；并通过整合多个运输相关的基因来描述这种新的顶端分选途径的组件和途径，也通过我们的多细胞肾小管生成筛选也鉴定出与顶膜生物发生相关的基因（AIM3）。鉴于极性的基本性质以及所检查的所有分子的高结构和功能保护，这些研究的结果应与人类上皮极性和相关的人类疾病直接相关，两个例子是癌症（与极性丧失）和内部器官的疾病（所有人都可以通过极性小管的疾病）。