Development of Polarity in Hepatic Cells

肝细胞极性的发展

• 批准号: 7110343
• 负责人: Ann L Hubbard
• 金额: $ 41.79万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110343
• 关键词: RNA interference; biological signal transduction; cell adhesion molecules; cell growth regulation; cell population study; cellular polarity; hepatectomy; histogenesis; immunofluorescence technique; immunoprecipitation; laboratory rat; liver cells; liver regeneration; membrane activity; membrane biogenesis; membrane proteins; phosphoprotein phosphatase; protein kinase C; tight junctions

DESCRIPTION (provided by applicant): This project studies proteins ("complex 1") that participate in specifying polarity. A membrane protein, junctional adhesion molecule (JAM-1), PAR3, PAR6 (two PDZ proteins), and atypical protein kinase C (aPKC) form complex 1. The complex plays a role in assembly of tight junctions (TJ), even though it is absent from the functional structure. The hypothesis to be tested is that JAM-1 is a membrane anchor that recruits PAR3, a scaffold to which a Cdc42-GTP/PAR6 full length/aPKC sub-complex docks, thus locating the complex apical of "primordial" adherens junctions. Activated aPKC then phosphorylates proteins that help form the final TJ. Our newly- identified PAR6A-short, which cannot bind aPKC or Cdc42 but retains a semi-PDZ domain, regulates TJ formation and/or stabilizes mature TJs. In vitro models are WlF-B cells, which develop a polarized hepatic phenotype, and intestinal Caco-2 cells, which express proteins in a tetracycline-inducible manner. In vivo experiments extend in vitro results to the regenerating liver, where TJ formation is crucial to recovery of organ function. Approaches are molecular biology, morphology (eg, live cell imaging), quantitative biochemistry (eg, fractionation, in vitro binding, protein identification) and use of recombinant adenoviruses to alter/knock-down complex 1 proteins. Aim #1 tests JAM's role. In #1A, constructs mutated in the cytoplasmic or ectoplasmic (adhesive) domain of JAM are expressed in WlF-B cells to determine if/when these domains function in progression to an hepatic phenotype. Live-cell imaging of GFP-reporters provides insight into the dynamics of the process. In #1B, mutants expressed in hepatic Fao cells are used in a novel bead assay to identify the proteins JAM recruits. In #1C, mutant JAM or RNAi is expressed in livers of 2/3 hepatectomized rats to determine the effects on TJ formation. Aim #2 tests the roles of PAR6 and PAR6A-short. In #2A, PAR6A-short is induced in tet-off Caco-2 cells at various stages of maturation to distinguish a role in formation versus maintenance of TJs. A second approach is use of RNAi to silence all PAR6 or just PAR6A-short in WIF-B cells. In #2B, in vitro assays test candidates reported to bind full-length PAR6 for binding to PAR6A-short. In #2C, PAR6A-short or RNAi is expressed in vivo to determine the effects on liver regeneration. Aim #3 uses pharmacological approaches to test the roles of aPKC and protein phosphatase 2A in the development of WlF-B polarity.

描述（由申请人提供）：该项目研究参与极性的蛋白质（“复杂1”）。一种膜蛋白，连接粘附分子（JAM-1），PAR3，PAR6（两个PDZ蛋白）和非典型蛋白激酶C（APKC）形成复合物1。即使缺乏功能结构，该复合物在紧密连接（TJ）的组装中起作用。要测试的假设是JAM-1是招募PAR3的膜锚，它是CDC42-GTP/PAR6全长/APKC子复合码头的脚手架，因此位于“原始”粘附剂连接的复杂顶点。然后，激活的APKC磷酸化蛋白质，有助于形成最终的TJ。我们新鉴定的Par6a-short无法结合APKC或CDC42，但保留了半PDZ结构域，可以调节TJ形成和/或稳定成熟的TJ。体外模型是WLF-B细胞，它们会形成偏振肝表型和肠Caco-2细胞，它们以四环素诱导的方式表达蛋白质。体内实验将体外结果扩展到再生肝脏，其中TJ形成对于器官功能的恢复至关重要。方法是分子生物学，形态学（例如，活细胞成像），定量生物化学（例如，分级，体外结合，蛋白质鉴定）以及使用重组腺病毒来改变/敲除染色体复合物1蛋白。 AIM＃1测试JAM的角色。在＃1a中，在WLF-B细胞中表达果酱的细胞质或胞质（粘合剂）结构域中突变的构建体，以确定这些结构域是否在进展为肝表型中起作用。 GFP重新携带者的活细胞成像提供了对过程动力学的见解。在＃1b中，在新型珠测定中使用了在肝粮农组织细胞中表达的突变体，以鉴定蛋白质果酱的新兵。在＃1C中，突变果酱或RNAi在2/3肝切除大鼠的肝脏中表达，以确定对TJ形成的影响。 AIM＃2测试PAR6和PAR6A-SHORT的角色。在＃2a中，在成熟的各个阶段，在Tet-Off Caco-2细胞中诱导PAR6A-Short，以区分地层与维持TJ的作用。第二种方法是使用RNAi沉默所有PAR6或仅在WIF-B细胞中的Par6a-short。在＃2B中，体外测定测试候选者据报道结合了全长PAR6，以结合与PAR6A-SHORT结合。在＃2C中，在体内表达par6a-short或RNAi，以确定对肝脏再生的影响。 AIM＃3使用药理学方法来测试APKC和蛋白质磷酸酶2a在WLF-B极性发展中的作用。