MECH & PATHWAYS INVOLVED IN CONNEXIN ASSEMBLY & GAP JUNCTION INTERNALIZATION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。所有高等多细胞生物都需要细胞间的通讯来维持正常的组织和器官功能。这个过程通常涉及离子、小代谢物和信使从一个细胞到邻近细胞的运输。一种细胞-细胞连接，即间隙连接，在相邻细胞之间形成，并在分子从一个细胞的细胞质到其相邻细胞的通道中发挥重要作用，具有离子、激素和稳态能力。间隙连接的功能包括协调心脏动作电位、同步神经元放电和开关、共享代谢物、胚胎发育、促进激素分泌、通过信号分子传递信号转导等。近年来，研究人员对间隙连接蛋白缺陷在遗传性疾病中的作用进行了深入的研究。间隙连接在信号转导途径中发挥积极作用，使用经典的信号分子，如cAMP和其他核苷酸、钙离子和肌醇三磷酸。这些小分子通过间隙连接从一个细胞移动到另一个细胞，然而，这些信号通路的细节及其作用正在深入研究中。间隙连接的组装和降解是一个动态过程，其半衰期通常为~3 ~ 6小时。已发表的研究表明，在细胞内和细胞表面有相当多的连接蛋白活性池参与连接蛋白的运输途径，然而，这些研究仅限于生化分析和共聚焦显微镜。我们的目标是表征选定的心脏连接蛋白（Cx43、Cx45和Cx37）在分泌途径和间隙连接内化过程中的运输、组装和分子相互作用。我们假设心脏连接蛋白的运输和组装存在多种途径，这些连接蛋白是由网格蛋白依赖和/或独立机制内化的。以前研究连接蛋白出口到质膜和内化降解的技术是有限的。我们计划使用新的和新的四胱氨酸（TC）标记连接蛋白作为荧光探针来检测心脏连接蛋白的生命周期。使用FlAsH/ReAsH进行tc标记和标记提供了几个优点，包括：1)相同的tc标记的连接蛋白可以是绿色或红色荧光，2)可以很容易地从gfp标记的连接蛋白中光学分离出ReAsH荧光，3)FlAsH和ReAsH结合是永久性的，允许脉冲标记的连接蛋白在分泌和内吞途径中被跟踪，4)通过光学显微镜观察到的相同的ReAsH标记的细胞可以用强交联试剂进行固定，用于高分辨率EM。5)即使预先用FlAsH标记细胞，也可以选择性地通过EM定位ReAsH； 6)与大得多的GFP （27 kD）标签相比，含有四胱氨酸的基序很小（2 kD），不太可能干扰连接蛋白功能。我们正在开发带有四系半胱氨酸（TC）基序标记的心脏连接蛋白，它将表现出野生型连接蛋白的特征，并允许对心脏连接蛋白的生命周期进行高分辨率的光和EM分析。间隙连接是动态的，其体内半衰期在肝细胞为5小时，在心脏为1.3小时，在培养心肌细胞为2小时。与其他多聚膜蛋白一样，间隙连接蛋白，即连接蛋白，是共翻译插入到内质网（ER）中的。Cx43和可能的Cx46在内质网中似乎不会寡聚成连接子，但最有可能在反式高尔基网络（TGN）中。然而，大量证据表明，至少Cx32和Cx26可以在内质网膜内寡聚。利用抗体显微注射研究和gfp标记的Cx43，我们最近表明，间隙连接内在化可以通过环形间隙连接的形成发生，其中整个间隙连接或连接的片段被移除到两个接触细胞中的一个。不排除间隙连接去除的另一种途径，即间隙连接分解成小聚集体，连接子随后内化到核内体中。尽管迄今为止存在的证据很少，但内化连接子可能会循环形成新的间隙连接通道。我们对tc标记的Cx43 （Cx43- tc）的初步研究已经对Cx43的转运和周转产生了令人兴奋的新见解。我们将tc标记的Cx37和Cx45与Cx43-TC并行进行表征，并将其与fp标记和野生型连接蛋白进行比较。为了检查Cx45-TC和Cx37-TC是否都具有功能，将野生型和表达Cx37-TC或Cx45-TC的HeLa细胞微注射路西法黄或神经生物素，并确定这些小分子通过间隙连接的扩散情况。我们目前拥有所有心脏连接蛋白的cDNA，这些连接蛋白在许多细胞系统中表达时已被fp标记和表征。我们目前正在生成tc标记的Cx37和Cx45，并预计这些tc标记的连接蛋白将表现出野生型连接蛋白的特征。有了Cx43-TC，我们已经开始按照描述进行tc标记的Cx45和Cx37的工程。简而言之，编码TC肽或该序列的连接子变异的DNA将在框架中融合到连接蛋白cDNA的3¿-末端，并测序以确保保真度。使用标准转染方案，将选定的细胞系转染或感染编码野生型、fp标记或tc标记心脏连接蛋白的cdna。我们开发了一种针对TC¿777¿标签的抗体，作为跟踪和验证TC结构的附加工具。我们有四种细胞系来检查心脏连接蛋白的运输和组装。1)小鼠HL-1细胞保留了成年心肌细胞表达&#61537；-心肌肌球蛋白重链和&#61537；-心肌肌动蛋白的表型特征，使其在培养中能够主动收缩。2)我们使用具有良好特征的人冠状动脉内皮细胞（HCAEC; Clonetics）来模拟主要血管的内皮内层。3)最后，我们使用了具有通讯能力的正常大鼠肾（NRK）细胞和通讯缺陷的人癌（HeLa）细胞系，这两种细胞系在之前的研究中已被常规使用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. All higher, multicellular organisms require intercellular communication for normal tissue and organ function. This process typically involves the transport of ions, small metabolites, and messengers from one cell to its neighbors. One kind of cell-cell junction, gap junctions, form between neighboring cells and serve an essential role in the passage of molecules from the cytoplasm of one cell to its neighbor in both ionic, hormonal and homeostatic capabilities. The functions of gap junctions include coordinating action potentials in the heart, synchronizing neuronal firing and switching, sharing of metabolites, embryonic development, facilitation of hormonal secretion, and transmission of signal transduction by passage of signal molecules. Recent intense investigations have been pursuing the effects that defects in gap junction proteins have in hereditary diseases. Gap junctions play an active role in signal transduction pathways using classic signaling molecules such cAMP and other nucleotides, calcium ions, and inositol triphosphate. These small molecules move through gap junctions from cell to cell, however, details of these signaling pathways and their effects are under intense investigation. The assembly and degradation of gap junctions are dynamic processes and their half-lives are typically ~3-6 hours. Published work has shown that there are considerable active pools of connexins within the cell and at the cell surface that are involved in the connexin trafficking pathways, however, these studies have been limited to biochemical analysis and confocal microscopy. Our goal is to characterize the transport, assembly and molecular interactions of selected heart connexins (Cx43, Cx45 and Cx37) along the secretory pathway and during gap junction internalization. We hypothesize that multiple pathways exist for the transport and assembly of heart connexins and these connexins are internalized by clathrin-dependent and/or independent-mechanisms. Previous technologies to investigate connexin export to the plasma membrane and internalization for degradation have been limited. We plan to use the new and novel tetracysteine (TC)-tagging of connexins as fluorescence probes for examining the lifecycle of heart connexins. TC-tagging and labeling with FlAsH/ReAsH provides several advantages which include: 1) the same TC-tagged connexin can be green or red fluorescence, 2) ReAsH fluorescence can easily be separated optically from GFP-tagged connexins developed for, 3) FlAsH and ReAsH binding is permanent allowing for pulse labeled connexins to be followed thought both the secretory and endocytic pathways, 4) the same ReAsH labeled cells that were observed by light microscopy can be fixed with strong cross-linking reagents for high resolution EM, 5) ReAsH can selectively be localized by EM even though the cells are pre-labeled with FlAsH, and 6) the tetracysteine containing motif is small (2 kD) and less likely to interfere with connexin function in comparison to the much larger GFP (27 kD) tag. We are developing heart connexins tagged with the tetracysteine (TC) motif will exhibit wild-type connexin characteristics and allow for high resolution light and EM analysis of the life-cycle of heart connexins. Gap junctions are dynamic with an in vivo half-life of ~5 hours in liver hepatocyte, 1.3 hours in the heart and 2 hours in cultured cardiac myocytes. Consistent with other polytopic membrane proteins, the gap junction proteins, connexins, are co-translationally inserted into the endoplasmic reticulum (ER). Cx43 and possibly Cx46 do not appear to oligomerize into connexons while in the ER but most likely in the trans Golgi Network (TGN). However, substantial evidence indicates that at least Cx32 and Cx26 can oligomerize within ER membrane. Using antibody microinjection studies and GFP-tagged Cx43, we have recently shown that gap junction internalization can occur via the formation of annular gap junctions where an entire, gap junction or a fragment of a junction is removed into one of the two contacting cells. An alternate pathway of gap junction removal has not been ruled-out where gap junctions disassemble into small aggregates and connexons subsequently internalize into endosomes. Although little evidence exists to date, it is plausible that internalized connexons may recycle to form new gap junction channels. Our initial study with TC-tagged Cx43 (Cx43-TC) has produced exciting new insights into Cx43 transport and turnover. We are characterize TC-tagged Cx37 and Cx45 in parallel with Cx43-TC and comparing these to their FP-tagged and wild-type connexin counterparts. To examine if both Cx45-TC and Cx37-TC are functional, wild-type and Cx37-TC or Cx45-TC expressing HeLa cells will be microinjected with Lucifer yellow or neurobiotin and spreading of these small molecules via gap junctions will be determined. We currently have at disposal the cDNA¿s for all the heart connexins and these connexins have been FP-tagged and characterized when expressed in a number of cell systems. We are currently generating TC-tagged Cx37 and Cx45 and anticipate that these TC-tagged connexins will exhibit wild type connexin characteristics. With Cx43-TC in hand we have begun engineering TC-tagged Cx45 and Cx37 as described. Briefly, DNA encoding the TC peptide or linker variations of this sequence will be fused in frame to the 3¿-end of the connexin cDNA and sequenced to ensure fidelity. Selected cell lines are either transfected or infected with cDNAs encoding wild-type, FP-tagged or TC-tagged heart connexins using standard transfection protocols. We developed an antibody against the TC ¿777¿ tag as an additional tool for tracking and verifying TC-constructs. We have four cell lines to examine the transport and assembly of heart connexins. 1) Mouse HL-1 cells retain phenotypic characteristics of adult cardiomyocytes expressing -cardiac myosin heavy chain and -cardiac actin, allowing them to actively contract in culture. 2) We are using well- characterized human coronary artery endothelial cells (HCAEC; Clonetics) which mimic the endothelial lining of major vessels. 3) Finally, we use communication-competent Normal Rat Kidney (NRK) cells and communication-deficient human carcinoma (HeLa) cell lines that have been routinely used for several previous studies.