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: 7722440
• 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: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722440
• 关键词: Actin-Binding Protein; Actins; Adhesions; Binding; C-terminal; Cadherins; Cell Adhesion; Cells; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Connexin 43; Connexins; Cytochalasins; Cytoskeleton; Depressed mood; Detergents; Elements; Fingers; Fluorescence; Funding; Gap Junctions; Grant; Green Fluorescent Proteins; Growth; Institution; Label; Life; Link; Localized; Mediating; Microfilaments; Microtubules; Molecular; Morphology; Movement; N-Cadherin; Proteins; Regulation; Research; Research Personnel; Resistance; Resources; Site; Source; Thinking; Triton; United States National Institutes of Health; base; cell fixing; cellular imaging; polymerization; 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. Regulation of Cx43 gap junction (GJ) size and organization is poorly understood. Cadherin-mediated cell adhesion is thought to be required for gap junction formation, yet the molecular mechanisms that govern interplay between cadherins and connexins at sites of cell-cell contact are largely undefined. ZO-1, an actin-binding MAGUK protein, localizes to both gap junctions and cadherin-based junctions, and thus is a good candidate to mediate interactions between cadherins and connexins at junctional interfaces. Live cell imaging confirmed that both N-cadherin-YFP and YFP-ZO-1 colocalize with Cx43 predominately at GJ plaque edges, although YFP-ZO-1 was distributed more diffusely along edges than N-cadherin-YFP. Movement of edge-localized N-cadherin-YFP and YFP-ZO-1 often coincided with finger-like protrusions of plaque that appeared to be under tension, as if linked to cytoskeletal dynamics. Consistent with this, cytochalasin treatment depressed the mobility of N-cadherin-YFP punctae and forced plaques into a static, rounded morphology. In the absence of PDZ-mediated interaction with Cx43, ZO-1 continued to target to plaque edges but assumed a punctate distribution similar to N-cadherin; moreover, the protrusive activity at plaque edges was altered. Extensive colocalization of N-cadherin, ZO-1 and actin filaments at the periphery of Cx43 plaques was confirmed by confocal microscopy in fixed cells. These observations support the hypothesis that N-cadherin adhesions target ZO-1 to Cx43 plaques at specialized interfaces where ZO-1 modulates linkages to the actin cytoskeleton. Previously we showed that fusion of GFP to the C-terminus of Cx43, which blocks ZO-1 binding, leads to the formation of aberrantly large GJs. Cx43 GJs are resistant to Triton detergent extraction, yet Cx43-GFP GJs are largely Triton-soluble. Interestingly, Triton-insoluble Cx43-GFP localizes predominately to plaque edgesthe site of GJ growthsuggesting that GJ edges are stabilized by cytoskeletal interactions that influence GJ size. Fluorescence labeling revealed minimal interaction of actin filaments with Triton-insoluble Cx43-GFP. In contrast, plaques composed of native Cx43 were extensively colocalized with actin filaments. However, Cx43-GFP plaques appear to acquire more microtubule contacts than native Cx43 GJs. Live cell imaging showed GJs containing a mix of Cx43-GFP and native Cx43 are more dynamic than plaques comprised solely of Cx43-GFP. Inhibition of either actin polymerization or Cx43 interaction with the actin binding protein ZO-1 suppressed the dynamics of mixed Cx43 GJs. These results suggest that Cx43 C-terminal elements, including the PDZ binding domain, determine cytoskeletal interactions at GJ edges, with ZO-1-mediated actin connections promoting active GJ remodeling, whereas microtubule contacts confer GJ stability and growth.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Cx43间隙连接（GJ）的大小和组织的调节知之甚少。钙粘蛋白介导的细胞粘附被认为是间隙连接形成所必需的，然而在细胞-细胞接触位点处钙粘蛋白和连接蛋白之间相互作用的分子机制在很大程度上是不确定的。ZO-1是一种肌动蛋白结合MAGUK蛋白，定位于间隙连接和钙粘蛋白连接，因此是介导钙粘蛋白和连接蛋白在连接界面相互作用的良好候选者。活细胞成像证实，N-钙粘蛋白-YFP和YFP-ZO-1都与Cx43共定位，主要在GJ斑块边缘，尽管YFP-ZO-1比N-钙粘蛋白-YFP沿着边缘分布更弥散。边缘定位的N-cadherin-YFP和YFP-ZO-1的运动通常与似乎处于张力下的斑块的指状突起一致，好像与细胞骨架动力学有关。与此相一致，细胞松弛素治疗抑制了N-钙粘蛋白-YFP斑点的移动性，并迫使斑块成为静态的圆形形态。在没有PDZ介导的与Cx43的相互作用的情况下，ZO-1继续靶向斑块边缘，但呈现类似于N-钙粘蛋白的点状分布;此外，斑块边缘的肿胀活性发生改变。在固定的细胞中，通过共聚焦显微镜证实了N-钙粘蛋白，ZO-1和肌动蛋白丝在Cx43斑块周围的广泛共定位。这些观察结果支持了这样的假设，即N-钙粘蛋白粘附靶向ZO-1到Cx43斑块在专门的接口，其中ZO-1调节连接到肌动蛋白细胞骨架。 先前我们表明，GFP融合到Cx43的C-末端，阻断ZO-1结合，导致异常大的GJ的形成。Cx43 GJ对Triton去污剂提取具有抗性，但Cx43-GFP GJ在很大程度上是Triton可溶的。有趣的是，Triton不溶性Cx43-GFP主要定位于斑块边缘GJ生长部位这表明GJ边缘通过影响GJ大小的细胞骨架相互作用而稳定。荧光标记显示肌动蛋白丝与Triton不溶性Cx43-GFP的最小相互作用。与此相反，斑块组成的天然Cx43广泛共定位与肌动蛋白丝。然而，Cx43-GFP斑块似乎比天然Cx43 GJ获得更多的微管接触。活细胞成像显示，含有Cx43-GFP和天然Cx43的混合物的GJ比仅由Cx43-GFP组成的斑块更具动态性。抑制肌动蛋白聚合或Cx43与肌动蛋白结合蛋白ZO-1的相互作用抑制了混合Cx43 GJs的动力学。这些结果表明，Cx43 C-末端元件，包括PDZ结合域，确定在GJ边缘的细胞骨架相互作用，ZO-1介导的肌动蛋白连接促进主动GJ重塑，而微管接触赋予GJ稳定性和生长。