Gap junctions create direct cell-cell communication in most cell types. These membrane specializations contain one plasma membrane from two apposing cells and tens to thousands of dodecameric connexin channels spanning the two membranes. These form discrete and recognizable cellular structures during quiescent (non-mitotic) phases. Cells dynamically modulate gap junctional communication by regulating the synthesis, transport, gating and turnover of these connexin channels. Current textbook descriptions depicting gap junctions as static, stand-alone structures now are replaced with a new paradigm of connexins, hemichannels, channels and gap junctions as very mobile, dynamic and interactive assemblies. Protein phosphorylation is an important regulatory mechanism by which proteins can control cellular function and/or localization in a process newly termed "spatial cell biology". The gap junction protein, connexin43, has a highly regulated life cycle during which several, hierarchical phosphorylation events occur at several specific serine residues in its C-terminus. Different phosphorylation events occur during all stages of the cell cycle and can change which proteins interact with connexin43, the kinetics and/or localization of connexin43 trafficking, assembly, gating, and turnover in a cell cycle stage specific manner that affects important biological processes such as cell migration and proliferation. This project is focused on imaging the elegant interplay between connexin43 phosphorylation, its cellular localization and the cell cycle. The three specific aims of this proposed research are: (1) determine whether certain kinases form complexes with connexin43 at particular stage(s) of its life cycle;(2) to correlate the phosphorylation of specific serine residues with their cellular location singly and in tandem;and (3) to elucidate how specific phosphorylation events are linked to cellular localization during the cell cycle. This proposal focuses on the identification and characterization of connexin trafficking structures using live cell imaging, correlative light and electron tomography with protein tags or probes to produce 3D reconstructions of selectively labeled connexins in cells. Methods for developing and applying multiple probes for correlated light and electron microscopy are essential to the success in imaging trafficking intermediates. In combination with biochemical and inhibitor analyses of wild type and mutant Cx43 proteins, the overall goal is to study these phospho-forms at electron tomographic resolution (~40-60 E) in 3D to determine their morphologies and locations within the context of other cellular components. From these studies, we will gain a mechanistic understanding through advanced imaging how connexin phosphorylation in controls gap junction communication-dependent functions in quiescent cells and during the cell cycle. The importance of this research is driven by the fact that changes in connexin localization and gap junctional communication are part the exquisite control of cellular proliferation, migration and with a loss of growth control during carcinogenesis.

Public Health Relevance

Direct cell-cell communication as mediated by gap junctions has been shown repeatedly to be a necessary component of homeostasis and is highly regulated during the cell cycle, developmental processes and cell proliferation. Connexin diseases result when gap junction proteins mis-traffic or mis-function and loss of gap junction intercellular communication is concomitant with carcinogenesis. We investigate the connexin43 trafficking process using an imaging based approach examining the hierarchy of connexin43 phosphorylation events and where within the cell cycle, connexin43-kinase(s) interactions occurs.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microscopic Imaging Study Section (MI)
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Nie, Zhongzhen
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University of California San Diego
Schools of Medicine
La Jolla
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Cone, Angela C; Cavin, Gabriel; Ambrosi, Cinzia et al. (2014) Protein kinase C?-mediated phosphorylation of Connexin43 gap junction channels causes movement within gap junctions followed by vesicle internalization and protein degradation. J Biol Chem 289:8781-98
Wang, Junjie; Ambrosi, Cinzia; Qiu, Feng et al. (2014) The membrane protein Pannexin1 forms two open-channel conformations depending on the mode of activation. Sci Signal 7:ra69
Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D et al. (2013) Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix. PLoS One 8:e70916
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Ambrosi, Cinzia; Gassmann, Oliver; Pranskevich, Jennifer N et al. (2010) Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. J Biol Chem 285:24420-31
Ambrosi, Cinzia; Boassa, Daniela; Pranskevich, Jennifer et al. (2010) Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability. Biophys J 98:1809-19
Boassa, Daniela; Solan, Joell L; Papas, Adrian et al. (2010) Trafficking and recycling of the connexin43 gap junction protein during mitosis. Traffic 11:1471-86
Gassmann, Oliver; Kreir, Mohamed; Ambrosi, Cinzia et al. (2009) The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes. J Struct Biol 168:168-76
Sosinsky, Gina E; Crum, John; Jones, Ying Z et al. (2008) The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications. J Struct Biol 161:359-71
Boassa, Daniela; Qiu, Feng; Dahl, Gerhard et al. (2008) Trafficking dynamics of glycosylated pannexin 1 proteins. Cell Commun Adhes 15:119-32

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