Development of Polarity in Hepatic Cells

描述（由申请人提供）：本项目研究参与指定极性的蛋白质（“复合物1”）。膜蛋白、连接粘附分子（JAM-1）、PAR 3、PAR 6（两种PDZ蛋白）和非典型蛋白激酶C（aPKC）形成复合物1。该复合物在紧密连接（TJ）的组装中起作用，即使它不存在于功能结构中。待测试的假设是JAM-1是募集PAR 3的膜锚，PAR 3是Cdc 42-GTP/PAR 6全长/aPKC亚复合物对接的支架，从而定位“原始”粘附连接的复合物顶端。活化的aPKC然后磷酸化蛋白质，帮助形成最终的TJ。我们新鉴定的PAR 6A-短，其不能结合aPKC或Cdc 42但保留半PDZ结构域，调节TJ形成和/或稳定成熟TJ。体外模型是产生极化肝表型的WIF-B细胞和以四环素诱导方式表达蛋白质的肠Caco-2细胞。体内实验将体外结果扩展到再生肝脏，其中TJ形成对器官功能的恢复至关重要。方法包括分子生物学、形态学（例如，活细胞成像）、定量生物化学（例如，分级分离、体外结合、蛋白质鉴定）和使用重组腺病毒改变/敲低复合物1蛋白。目标#1测试JAM的作用。在#1A中，在JAM的细胞质或细胞外质（粘附）结构域中突变的构建体在WIF-B细胞中表达，以确定这些结构域是否/何时在进展为肝脏表型中发挥作用。GFP报告基因的活细胞成像提供了对该过程动态的洞察。在#1B中，在肝Fao细胞中表达的突变体用于新的珠测定以鉴定蛋白质JAM募集物。在#1C中，突变体JAM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。目标#2测试PAR 6和PAR 6A-short的作用。在#2A中，PAR 6A-短在不同成熟阶段的tet-off Caco-2细胞中被诱导，以区分TJ形成与维持中的作用。第二种方法是使用RNAi沉默WIF-B细胞中的所有PAR 6或仅PAR 6A-短。在#2B中，体外测定测试据报道结合全长PAR 6的候选物与短PAR 6A的结合。在#2C中，PAR 6A-短或RNAi在体内表达以确定对肝再生的影响。目的#3使用药理学方法来测试aPKC和蛋白磷酸酶2A在WIF-B极性发展中的作用。

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.