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 edges?the site of GJ growth?suggesting 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.
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