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: 8169609
• 负责人: ROBERT G GOURDIE
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169609
• 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; Growth; Institution; Label; Life; Link; Mediating; Microfilaments; Microtubules; Molecular; Morphology; Movement; N-Cadherin; Proteins; Regulation; Research; Research Personnel; Resistance; Resources; Site; Source; Triton; United States National Institutes of Health; base; cell fixing; cellular imaging; polymerization

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 CX43间隙连接（GJ）的规模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，但是在细胞细胞接触部位控制钙粘着蛋白和连接蛋白之间相互作用的分子机制在很大程度上不确定。 ZO-1是一种肌动蛋白结合的maguk蛋白，它位于间隙连接和基于钙粘蛋白的连接处，因此是介导cadherins and Connexins在连接界面处的相互作用的好候选者。活细胞成像证实，尽管YFP-ZO-1的分布沿边缘分布得比N-钙粘蛋白-YFP更加漫射，但在GJ斑块边缘中，N-钙粘蛋白-YFP和YFP-ZO-1与CX43主要分布。边缘局部n-钙粘蛋白-YFP和YFP-ZO-1的运动通常与斑块的手指样突起相吻合，斑块似乎在张力下，就好像与细胞骨架动力学有关。与此相一致，细胞切拉斯蛋白治疗抑制了N-钙粘蛋白-YFP点的迁移率和强迫斑块成静态的，圆形的形态。在没有PDZ介导的CX43相互作用的情况下，ZO-1继续靶向斑块边缘，但假定与N-钙粘着蛋白相似的点状分布。此外，改变了斑块边缘的突出活性。 N-钙粘着蛋白，ZO-1和肌动蛋白丝在CX43斑块周围的广泛共定位通过固定细胞中的共聚焦显微镜证实。这些观察结果支持以下假设：N-钙粘着蛋白粘连靶向ZO-1至CX43斑块在ZO-1调节与肌动蛋白细胞骨架的链接的专用界面上。 以前，我们表明GFP与阻断ZO-1结合的CX43的C端融合会导致形成异常大的GJ。 CX43 GJ对Triton洗涤剂提取具有抗性，但CX43-GFP GJ在很大程度上是Triton可溶性的。有趣的是，Triton-Insoluble CX43-GFP主要定位于牙菌斑的边缘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稳定。荧光标记显示肌动蛋白丝与Triton-Insoluble CX43-GFP的相互作用最少。相反，由天然CX43组成的斑块与肌动蛋白丝广泛共定位。但是，与天然CX43 GJ相比，CX43-GFP斑块似乎获得了更多的微管触点。活细胞成像显示，GJ含有CX43-GFP的混合物，而天然CX43的组合比仅由CX43-GFP组成的斑块更具动态性。抑制肌动蛋白聚合或与肌动蛋白结合蛋白ZO-1相互作用抑制了混合CX43 GJ的动力学。这些结果表明，CX43 C末端元件（包括PDZ结合结构域）确定了GJ边缘处的细胞骨架相互作用，ZO-1介导的肌动蛋白连接促进了主动GJ重塑，而微管接触的肌肉连接则促进了GJ的稳定性和生长。