Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen

Abstract

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...

The Blood-Testis Barrier and Its Implications for Male Contraception

“…7). This finding is consistent with an earlier report that Cx43-ZO-1-Src form a functional complex at the gap junction plaques in the cell epithelium of the 42GPA9 Sertoli cell line (Gilleron et al, 2008). Knockdown of Cx43 alone in Sertoli cells by RNAi did not interfere with Sertoli cell TJ permeability barrier function (Li et al, 2009b), consistent with a recent study reporting that the BTB remains intact in Sertoli cellspecific Cx43 knockout (KO) mice when examined by electron microscopy (Carette et al, 2010b), even though these mice were infertile.…”

Vesicular and conductive mechanisms of nucleotide release

“…Cx43 may form complexes with membrane receptors, cell signaling molecules, scaffolding proteins, cytoskeleton components, and other proteins independently of Cx43 hemichannel activity [134][135][136][137][138]. Deletion of genes encoding Cx32, Cx36, or Cx43 affected the transcription of unrelated genes [139,140].…”

Connexins, cell motility, and the cytoskeleton

“…The components described in this study include clathrin, the alternative clathrin-adaptor Dab2, the GTPase dynamin, the unconventional myosin-VI, and actin filaments [Piehl et al, 2007]. The mechanism by which Cx43 GJ plaques are internalized might involve Src-mediated dissociation of ZO-1 from one side of the GJ plaque [Gilleron et al, 2008].…”