MECHANISMS THAT MODULATE GAP JUNCTION SIZE, DISTRIBUTION AND TURNOVER

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。细胞间缝隙连接偶联的模式被认为对于许多类型组织的正常功能是重要的。目前，对控制缝隙连接大小和分布的分子机制知之甚少。我们通过在连接蛋白缺陷的HeLa细胞中表达连接蛋白43（Cx43）构建体来解决这个问题。HeLa细胞表达外源性引入的野生型Cx43形成小的点状间隙连接。相比之下，表达Cx43-GFP的细胞形成大的片状间隙连接。然而，Cx43-GFP对间隙连接大小的影响通过使用腺病毒载体共表达未标记的野生型Cx43来挽救。Cx43的GFP标记已显示消除ZO-1结合（Giepmans等人，2001）这些结果表明，与Cx43的C末端融合的GFP标签通过掩蔽Cx43的C末端氨基酸（包含闭合小带-1（ZO-1）结合位点）来改变间隙连接的大小。我们目前正在使用直接靶向Cx43和ZO-1之间相互作用的缺失和显性阴性构建体来测试这一假设。为了进一步解决ZO-1相互作用的丧失是否是观察到的Cx43-GFP间隙连接大小增加的原因，我们将通过使用更小的表位标签来扩展我们的研究，该表位标签可以分子内引入并且适合于活细胞和高分辨率成像。用于此目的的最佳候选者是四半胱氨酸基序（在Tsien和Ellisman实验室开发），其以高亲和力结合荧光双砷化合物，并已显示出作为全功能分子内标签的前景（未发表的数据）。此外，MUSC的Hunter博士进行的体外研究将解决微管与Cx43的C-末端质膜结构域结合的功能。具体来说，我们将确定是否Cx43的微管蛋白结合域的相互作用优先与微管的末端使用珠结合试验。这些体外研究将补充目前的工作，由博士Giepmans在NCMIR的重点是Cx43和微管之间的相互作用，在真核cells.Materials & MethodsThe周转率的标记和未标记的Cx43将使用生化（在MUSC）和荧光显微镜（在NCMIR和MUSC）脉冲追踪测定。在用[35 S]甲硫氨酸进行代谢标记和不同时间段的冷追踪后，表达重组连接蛋白的细胞将被分级分离成去污剂可溶性和不溶性池，这将允许将连接连接蛋白（不溶性池）的转换率与总细胞连接蛋白（可溶性池）的转换率区分开。或者，间隙连接的转换率将使用Gaietta等人（2002）的技术测量，其中预先存在的四半胱氨酸标记的连接蛋白用荧光化合物（FlAsH）脉冲标记，细胞在没有标记的情况下被追踪不同的时间段，然后在追踪期间新合成的连接蛋白用第二荧光化合物（ReAsH）标记。由不同重组连接蛋白组成的间隙连接的相对转换率的测定（例如，融合到Cx43-GFP或野生型Cx43的C末端的四半胱氨酸，以及具有或不具有ZO-1结合所必需的残基的内部标记的Cx43），与上述病毒拯救实验相结合，将有助于阐明调节大小的机制，Cx43间隙连接的分布和周转。对于微管珠结合测定，将包含Cx43的微管蛋白结合结构域和四半胱氨酸基序的纯化多肽结合至FlAsH缀合的珠。为了确定Cx43是否优先结合微管末端，将珠子与极性标记的荧光微管混合，并通过荧光显微镜测定珠子结合的位置。

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. The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in connexin-deficient HeLa cells. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. However, the effect of Cx43-GFP on gap junction size was rescued by co-expression of untagged wild-type Cx43 using an adenoviral vector. GFP-tagging of Cx43 has been shown to abolish ZO-1 binding (Giepmans et al., 2001) These results suggest that the GFP tag, which is fused to the C-terminus of Cx43, alters gap junction size by masking the C-terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.GoalTo further address whether the loss of ZO-1 interaction is responsible for the observed increase in Cx43-GFP gap junction size, we will extend our studies by using a smaller epitope tag that can be introduced intramolecularly and that is suitable for live cell and high-resolution imaging. The best candidate for this purpose is the tetracysteine motif (developed in the Tsien & Ellisman labs), which binds fluorescent biarsenic compounds with high affinity, and has shown promise as a fully functional intramolecular tag (unpublished data). In addition, in vitro studies performed by Dr. Hunter at MUSC will address the function of microtubule binding to the C-terminal juxtamembrane domain of Cx43. Specifically, we will determine whether the tubulin binding domain of Cx43 interacts preferentially with the ends of microtubules using a bead binding assay. These in vitro studies will complement current work by Dr. Giepmans at NCMIR which focuses on the interaction between Cx43 and microtubules in eukaryotic cells.Materials & MethodsThe turnover rates of tagged and untagged Cx43 will be determined using both biochemical (at MUSC) and fluorescence microscopy (at NCMIR and MUSC) pulse-chase assays. Subsequent to metabolic labeling with [35S]methionine and various periods of cold chase, cells expressing recombinant connexins will be fractionated into detergent soluble and insoluble pools, which will allow the turnover rate of junctional connexin (insoluble pool) to be differentiated from that of total cellular connexin (soluble pool). Alternatively, the turnover rate of gap junctions will be measured using the technique of Gaietta et al. (2002), in which pre-existing tetracysteine-tagged connexins are pulse-labeled with a fluorescent compound (FlAsH), cells are chased for various periods without label, and then connexins newly synthesized during the chase period are labeled with a second fluorescent compound (ReAsH). Determination of the relative turnover rates of gap junctions composed of different recombinant connexins (e.g. tetracysteine fused to the C-terminus of Cx43-GFP or wild-type Cx43, and internally tagged Cx43 with or without the residues essential for ZO-1 binding), combined with the viral rescue experiments described above, will help to elucidate the mechanisms that regulate the size, distribution and turnover of Cx43 gap junctions. For the microtubule bead binding assay, purified polypeptide comprising the tubulin binding domain of Cx43 and a tetracysteine motif will be bound to FlAsH-conjugated beads. To determine if Cx43 binds preferentially to microtubule ends, beads will be mixed with polarity-marked fluorescent microtubules and the location of bead binding will be assayed by fluorescence microscopy.