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。 对Cx43缝隙连接(GJ)的大小和组织的调控知之甚少。钙粘附素介导的细胞黏附被认为是缝隙连接形成所必需的，然而控制细胞间接触部位钙粘附素和连接蛋白相互作用的分子机制在很大程度上还不清楚。ZO-1是一种肌动蛋白结合的Maguk蛋白，定位于缝隙连接和基于钙粘连蛋白的连接，因此是介导连接界面上连接蛋白和钙粘连蛋白相互作用的一个很好的候选者。活细胞成像证实N-钙粘素-YFP和YFP-ZO-1都与Cx43主要分布在GJ斑块边缘，尽管YFP-ZO-1比N-钙粘素-YFP沿边缘分布更广泛。边缘定位的N-钙粘素-YFP和YFP-ZO-1的运动经常与似乎处于张力下的指状斑块突起相一致，似乎与细胞骨架动力学有关。与此一致的是，细胞松弛素治疗降低了N-钙粘素-YFP斑点的流动性，并迫使斑块进入静态的圆形形态。在没有PDZ介导的与Cx43相互作用的情况下，ZO-1仍然以斑块边缘为靶点，但呈类似N-钙粘素的点状分布；此外，斑块边缘的突起活动也发生了变化。共聚焦显微镜观察到固定细胞中N-钙粘蛋白、ZO-1和肌动蛋白细丝广泛共存于Cx43斑块的周边。这些观察结果支持这样一种假设，即N-钙粘附素粘附靶向ZO-1到Cx43的特殊界面上的斑块，其中ZO-1调节与肌动蛋白细胞骨架的连接。 先前，我们发现GFP与Cx43的C-末端融合，阻断了ZO-1的结合，导致了异常大的GJS的形成。Cx43-GFP GJS对Triton洗涤剂提取具有抗性，但Cx43-GFP GJS基本上可溶于Triton。有趣的是，不溶于Triton的Cx43-GFP主要定位于GJ生长部位的斑块边缘，这表明GJ边缘是通过影响GJ大小的细胞骨架相互作用而稳定的。荧光标记显示肌动蛋白细丝与Triton不溶性Cx43-GFP之间的相互作用很小。相反，由天然Cx43组成的斑块与肌动蛋白细丝广泛共存。然而，Cx43-GFP斑块似乎比天然Cx43 GJS获得了更多的微管接触。活细胞成像显示，含有Cx43-GFP和天然Cx43的GJS比仅由Cx43-GFP组成的斑块更具活力。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的C末端元件，包括PDZ结合域，决定了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了活动的GJ重塑，而微管接触则提供了GJ的稳定和生长。

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.