Journal of Cell Science 4070 (Jordan et al., 1999;Falk, 2000) although Cx43-ZO-1 interaction might be reduced in those fusion proteins (Gaietta et al., 2002). More recently, by using dual immunofluorescence and immunoprecipitation analyses, we and others have reported that ZO-1 can participate in the internalization of gap junction plaques in different cell types, e.g. cardiomyocytes (Barker et al., 2002), astrocytes and Sertoli cells . In addition, accumulating evidence suggest that gap junction plaque endocytosis depends on the presence of additional Cx-protein partners. The nonreceptor tyrosine kinase Src is a well-known partner of Cx43, and can reduce the interaction of Cx43 with ZO-1 (Toyofuku et al., 2001). However, previously developed experimental approaches were not sufficient to elucidate the precise intermolecular interactions that occurs between these proteins during internalization of gap junction plaques and formation of annular gap junctions. This is partly due to the fact that biochemical techniques based on cell membrane fractionation do not allow the examination of sequential and molecular events that drive these processes.In this study, we have attempted to dissect the molecular interactions that occur between Cx43 and two of its binding partners, ZO-1 and Src, during the endocytic internalization of gap junctions. We also analyzed the effect of γ-hexachlorocyclohexane (HCH), a non-genomic carcinogen that is known to be a potent inducer of Cx43 internalization, on these molecular events. Our data reveal that a specific interaction between Cx43 and the activated form of the nonreceptor tyrosine kinase Src, concomitantly with a disruption of interaction of ZO-1 with Cx43 -specifically on one side of the gap junction plaque -are among the first events of endocytic internalization of gap junction plaques and formation of annular gap junctions. The current results further support the hypothesis that this mechanistic process may be abnormally accelerated in response to carcinogen exposure.
Results
Accelerated internalization of Cx43-GFP gap junction plaques in response to HCH exposureFluorescence deconvolution microscopy performed on Cx43-GFPtransfected 42GPA9 Sertoli cells allowed to observe many steps of Cx trafficking: cytoplasmic accumulation within the Golgi region (Fig. 1A, upper left panel) labeled using antibody CTR433, gap junction plaque between two adjacent cells at the plasma membrane visualized by the occludin signal (Fig. 1A, upper right panel), cytoplasmic spots of various sizes and shapes representing accumulation of early endosomes as depicted with Rab5 (Fig. 1A, left lower panel), and degradative elements revealed by using the lysosomal marker Lamp2 (Fig. 1A, right lower panel). Colocalization of Cx43-GFP in these different cellular compartments was evidenced by yellow fluorescence (Fig. 1A, insets). After 1-hour exposure to HCH that is known to induce Cx43-based gap junction internalization (Defamie et al., 2001), gap junction plaques had disappeared and only large green fluoresc...
