MECHANISMS THAT MODULATE GAP JUNCTION SIZE, DISTRIBUTION AND TURNOVER

调节间隙连接尺寸、分布和周转的机制

• 批准号: 7358113
• 负责人: ROBERT G GOURDIE
• 金额: $ 0.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358113
• 关键词: MECHANISMS; MODULATE; GAP; JUNCTION; SIZE

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fluorescent biarsenic compounds with high affinity, and has shown promise as a fully functional intramolecular tag (unpublished data).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.

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。细胞间的间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，对控制间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失的HeLa细胞中表达连接蛋白43 （Cx43）构建物来解决这个问题。表达外源引入的野生型Cx43的HeLa细胞形成小的、点状的间隙连接。相比之下，表达Cx43-GFP的细胞形成了大的片状间隙连接。然而，通过使用腺病毒载体共表达未标记的野生型Cx43，可以挽救Cx43- gfp对间隙连接大小的影响。Cx43的GFP标签已被证明可以消除ZO-1结合（Giepmans等，2001）。这些结果表明，融合到Cx43的c端的GFP标签通过掩盖Cx43的c端氨基酸来改变间隙连接的大小，该氨基酸包含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性结构来验证这一假设。为了进一步确定ZO-1相互作用的缺失是否导致了观察到的Cx43-GFP缝隙连接大小的增加，我们将通过使用一种更小的表位标签来扩展我们的研究，这种标签可以在分子内引入，并且适用于活细胞和高分辨率成像。这方面的最佳候选是四胱氨酸基元（由Tsien和Ellisman实验室开发），它以高亲和力结合荧光双砷化合物，并显示出作为全功能分子内标签的前景（未发表的数据）。此外，由MUSC的Hunter博士进行的体外研究将解决微管与Cx43的c端近膜结构域结合的功能。具体来说，我们将使用头结合试验确定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Giepmans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）脉冲追踪测定。在用[35S]蛋氨酸进行代谢标记和不同时期的冷追赶后，表达重组连接蛋白的细胞将被分离成洗涤剂可溶性池和不溶池，这将使连接蛋白（不溶池）的周转率与总细胞连接蛋白（可溶性池）的周转率区分开来。或者，使用Gaietta等人（2002）的技术测量间隙连接的周转率，其中预先存在的四胱氨酸标记的连接蛋白用荧光化合物（FlAsH）进行脉冲标记，在不标记的情况下追赶细胞不同的时期，然后在追赶期间新合成的连接蛋白用第二种荧光化合物（ReAsH）进行标记。测定由不同重组连接蛋白（如四胱氨酸融合到Cx43- gfp或野生型Cx43的c端，以及内部标记带有或不带有ZO-1结合必需残基的Cx43）组成的缝隙连接的相对周转率，结合上述病毒拯救实验，将有助于阐明Cx43缝隙连接大小、分布和周转率的调控机制。对于微管珠结合试验，纯化的多肽包含Cx43的微管蛋白结合结构域和四胱氨酸基序将被结合到flash共轭珠上。为了确定Cx43是否优先与微管末端结合，将微管与极性标记的荧光微管混合，并通过荧光显微镜检测微管结合的位置。