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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 对 Cx43 间隙连接 (GJ) 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，但在细胞与细胞接触部位控制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尚不清楚。 ZO-1 是一种肌动蛋白结合 MAGUK 蛋白，定位于间隙连接和基于钙粘蛋白的连接，因此是介导连接界面处钙粘蛋白和连接蛋白之间相互作用的良好候选者。活细胞成像证实，N-cadherin-YFP 和 YFP-ZO-1 主要与 Cx43 共定位于 GJ 斑块边缘，尽管 YFP-ZO-1 沿边缘分布比 N-cadherin-YFP 更分散。边缘定位的 N-钙粘蛋白-YFP 和 YFP-ZO-1 的运动通常与斑块的手指状突起同时发生，这些突起似乎处于张力下，似乎与细胞骨架动力学有关。与此一致的是，细胞松弛素治疗抑制了 N-钙粘蛋白-YFP 点的移动性，并迫使斑块变成静态的圆形形态。在没有 PDZ 介导的与 Cx43 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，斑块边缘的突出活动也发生了改变。通过固定细胞中的共聚焦显微镜证实了 N-钙粘蛋白、ZO-1 和肌动蛋白丝在 Cx43 斑块外围的广泛共定位。这些观察结果支持这样的假设：N-钙粘蛋白粘附在专门的界面上将 ZO-1 靶向 Cx43 斑块，其中 ZO-1 调节与肌动蛋白细胞骨架的连接。 之前我们表明，GFP 与 Cx43 C 末端的融合会阻断 ZO-1 结合，从而导致异常大的 GJ 的形成。 Cx43 GJ 对 Triton 去污剂萃取具有抗性，但 Cx43-GFP GJ 在很大程度上可溶于 Triton。有趣的是，Triton 不溶性 Cx43-GFP 主要定位于 GJ 生长部位的斑块边缘，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 的相互作用最小。相比之下，由天然 Cx43 组成的斑块与肌动蛋白丝广泛共定位。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 混合物的 GJ 比仅由 Cx43-GFP 组成的斑块更具活力。抑制肌动蛋白聚合或抑制 Cx43 与肌动蛋白结合蛋白 ZO-1 的相互作用会抑制混合 Cx43 GJ 的动力学。这些结果表明，Cx43 C 末端元件（包括 PDZ 结合域）决定 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活跃的 GJ 重塑，而微管接触则赋予 GJ 稳定性和生长。