Mictotubules and actin differentially influence remodeling of connexin43 gap junctions

Abstract

Regulation of Cx43 gap junction (GJ) size and organization is poorly understood. Previously we showed that fusion of GFP to the C‐terminus of Cx43 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 polymer. 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. Supported by NIH grants HL07260, K12GM081265, HL56728, HL082802.