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Trafficking and recycling of the connexin43 gap junction protein during mitosis.

ABSTRACT: During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)-containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse-chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time-lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43-GFP-4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno-labeled endogenous Cx43-expressing cells. Photo-oxidation of ReAsH-labeled Cx43-GFP-4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse-chase labeling and single label time-lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit.

SUBMITTER: Boassa D

PROVIDER: S-EPMC3272544 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Trafficking and recycling of the connexin43 gap junction protein during mitosis.

Boassa Daniela D Solan Joell L JL Papas Adrian A Thornton Perry P Lampe Paul D PD Sosinsky Gina E GE

Traffic (Copenhagen, Denmark) 20100910 11

During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)-containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse-chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time-lapse imaging of gree ...[more]

PMID: 20716111

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Project description:Human immunodeficiency virus-1 (HIV-1) infection compromises the central nervous system (CNS) in a significant number of infected individuals, resulting in neurological dysfunction that ranges from minor cognitive deficits to frank dementia. While macrophages/microglia are the predominant CNS cells infected by HIV, our laboratory and others have shown that HIV-infected astrocytes, although present in relatively low numbers with minimal to undetectable viral replication, play key role in NeuroAIDS pathogenesis. Our laboratory has identified that HIV "hijacks" connexin (Cx) containing channels, such as gap junctions (GJs) and hemichannels (HCs), to spread toxicity and apoptosis to uninfected cells even in the absence of active viral replication. In this study, using a murine model with an astrocyte-directed deletion of Cx43 gene (hGFAP-cre Cx43fl/fl) and control Cx43fl/fl mice, we examined whether few HIV-infected human astrocytoma cells (U87-CD4-CCR5), microinjected into the mouse cortex, can spread toxicity and apoptosis through GJ-mediated mechanisms, into the mouse cells, which are resistant to HIV infection. In the control Cx43fl/fl mice, microinjection of HIV-infected U87-CD4-CCR5 cells led to apoptosis in 84.28 ± 6.38% of mouse brain cells around the site of microinjection, whereas hGFAP-cre Cx43fl/fl mice exhibited minimal apoptosis (2.78 ± 1.55%). However, simultaneous injection of GJ blocker, 18?-glycyrrhetinic acid, and Cx43 blocking peptide along with microinjection of HIV-infected cells prevented apoptosis in Cx43fl/fl mice, demonstrating the Cx43 is essential for HIV-induced bystander toxicity. In conclusion, our findings demonstrate that Cx43 expression, and formation of GJs is essential for bystander apoptosis during HIV infection. These findings reveal novel potential therapeutic targets to reduce astrocyte-mediated bystander toxicity in HIV-infected individuals because despite low to undetectable viral replication in the CNS, Cx channels hijacked by HIV amplify viral neuropathogenesis.

Project description:The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.

Dataset Information

Trafficking and recycling of the connexin43 gap junction protein during mitosis.

Publications

Trafficking and recycling of the connexin43 gap junction protein during mitosis.

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