MECHANISMS THAT MODULATE GAP JUNCTION SIZE, DISTRIBUTION AND TURNOVER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景细胞间的缝隙连接偶联模式被认为对许多类型的组织的正常功能是重要的。目前，人们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)结构来解决这个问题。表达外源导入野生型Cx43的HeLa细胞形成小的点状缝隙连接。相比之下，表达Cx43-GFP的细胞形成了大的片状缝隙连接。然而，通过用腺病毒载体共表达未标记的野生型Cx43，Cx43-GFP对缝隙连接大小的影响被挽救。Cx43的GFP标签已被证明可以取消ZO-1结合(Giepmann等人，2001)。这些结果表明，与Cx43的C-末端融合的GFP标签通过掩盖Cx43的C-末端氨基酸而改变间隙连接大小，这些氨基酸组成了ZO-1结合位点。我们目前正在使用直接针对Cx43和ZO-1之间相互作用的缺失和显性负向构造来验证这一假说。目标为了进一步解决ZO-1相互作用的丧失是否导致观察到的Cx43-GFP间隙连接大小的增加，我们将通过使用更小的表位标签来扩展我们的研究，该标签可以在分子内引入，适合活细胞和高分辨率成像。达到这一目的的最佳候选者是四半胱氨酸基序(在Tsien&Ellisman实验室开发)，它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记的前景(未发表的数据)。此外，MUSC的Hunter博士进行的体外研究将探讨微管与Cx43的C末端膜旁结构域结合的功能。具体地说，我们将使用珠结合实验来确定Cx43的微管蛋白结合域是否优先与微管末端相互作用。这些体外研究将补充吉普曼博士目前在NCMIR的工作，该工作侧重于真核细胞中Cx43与微管之间的相互作用。材料与方法标记和未标记的Cx43的周转率将使用生化(在MUSC)和荧光显微镜(在NCMIR和MUSC)脉冲追逐分析来确定。在用[35S]蛋氨酸进行代谢标记和不同时期的冷追逐后，表达重组连接蛋白的细胞将被分成洗涤剂可溶和不可溶的池，这将使连接连接蛋白(不溶池)的周转率与总细胞连接蛋白(可溶池)的周转率有所区别。或者，将使用Gaietta等人的技术来测量缝隙连接的周转率。(2002)，其中用荧光化合物(闪光)脉冲标记先前存在的四半胱氨酸标记的连接蛋白，在没有标记的情况下对细胞进行不同时期的追逐，然后用第二种荧光化合物(ReAsH)标记在追逐期间新合成的连接蛋白。测定由不同重组连接蛋白组成的缝隙连接的相对周转率(例如，四半胱氨酸融合到Cx43-GFP或野生型Cx43的C末端，以及内部标记的Cx43带有或不带有ZO-1结合所必需的残基)，结合上述病毒救援实验，将有助于阐明调控Cx43缝隙连接的大小、分布和周转的机制。在微管微球结合实验中，含有Cx43微管蛋白结合域和四半胱氨酸基序的纯化多肽将被结合到闪光偶联的微球上。为了确定Cx43是否优先结合到微管末端，将微珠与极性标记的荧光微管混合，并通过荧光显微镜分析微珠结合的位置。

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. BackgroundThe 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.