Development of Polarity in Hepatic Cells

描述(申请人提供)：本项目研究参与指定极性的蛋白质(“复合体1”)。膜蛋白、连接黏附分子(JAM-1)、PAR3、PAR6(两个PDZ蛋白)和非典型蛋白激酶C(APKC)形成复合体1。该复合体在紧密连接(TJ)的组装中发挥作用，尽管它不存在于功能结构中。要检验的假设是，JAM-1是一个膜锚定，它招募了PAR3，PAR3是一种支架，CDC42-GTP/PAR6全长/aPKC亚复合体与其对接，从而定位“原始”连接的复合体顶端。激活的aPKC然后使有助于形成最终TJ的蛋白质磷酸化。我们新发现的PAR6A-Short不能与PKC或CDC42结合，但保留了一个半PDZ结构域，调节TJ的形成和/或稳定成熟的TJ。体外模型是WLF-B细胞和肠道Caco-2细胞，WLF-B细胞形成极化的肝脏表型，肠道Caco-2细胞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，在那里TJ的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(如活细胞成像)、定量生物化学(如分离、体外结合、蛋白质鉴定)和使用重组腺病毒改变/敲除复合体1蛋白。Aim#1测试Jam的角色。在#1a中，在WLF-B细胞中表达在JAM的胞浆或胞外(粘附性)结构域中突变的结构，以确定这些结构域是否/何时在进展为肝脏表型的过程中发挥作用。绿色荧光蛋白-记者的活细胞成像提供了对这一过程的动态的洞察。在#1B中，在肝脏FAO细胞中表达的突变体被用于一种新的珠状试验来鉴定果酱新兵的蛋白质。在#1C中，突变的JAM或RNAi在2/3肝切除大鼠的肝脏中表达，以确定其对TJ形成的影响。目的#2测试PAR6和PAR6A-Short的作用。在#2a中，在不同成熟阶段的tet-off Caco-2细胞中诱导PAR6A-Short，以区分在TJ形成和维持中的作用。第二种方法是使用RNAi沉默WIF-B细胞中的所有PAR6或仅PAR6A-Short。在#2B中，据报道，体外测试候选结合全长PAR6以结合PAR6A-Short。在#2C中，PAR6A-Short或RNAi在体内表达，以确定对肝再生的影响。目的#3利用药理学方法检测蛋白激酶C和蛋白磷酸酶2A在WLF-B极性形成中的作用。

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.