STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

Summary

Principal Investigator: Gina E Sosinsky
Abstract: DESCRIPTION (provided by applicant): 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: Project Narrative 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.
Funding Period: 2005-02-01 - 2014-12-31
more information: NIH RePORT

Top Publications

  1. ncbi Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel
    Derek L Beahm
    Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA
    J Biol Chem 281:7994-8009. 2006
  2. pmc Trafficking and recycling of the connexin43 gap junction protein during mitosis
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA
    Traffic 11:1471-86. 2010
  3. pmc Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels
    Atsunori Oshima
    Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo ku, Kyoto 606 8502, Japan
    J Mol Biol 405:724-35. 2011
  4. pmc Pannexin channels are not gap junction hemichannels
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA
    Channels (Austin) 5:193-7. 2011
  5. pmc Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly
    Yong Chun Yu
    Institute of Neurobiology, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China
    Nature 486:113-7. 2012
  6. pmc Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging
    Mark H Ellisman
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Methods Cell Biol 111:139-55. 2012
  7. pmc Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation
    Elena Dolmatova
    Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
    Am J Physiol Heart Circ Physiol 303:H1208-18. 2012
  8. pmc Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
    Jeffrey D Martell
    Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
    Nat Biotechnol 30:1143-8. 2012
  9. pmc Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, California, United States of America
    PLoS ONE 8:e70916. 2013
  10. pmc Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093 0608, USA
    J Biol Chem 285:24420-31. 2010

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Detail Information

Publications21

  1. ncbi Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel
    Derek L Beahm
    Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA
    J Biol Chem 281:7994-8009. 2006
    ..This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo...
  2. pmc Trafficking and recycling of the connexin43 gap junction protein during mitosis
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA
    Traffic 11:1471-86. 2010
    ..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...
  3. pmc Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels
    Atsunori Oshima
    Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo ku, Kyoto 606 8502, Japan
    J Mol Biol 405:724-35. 2011
    ..Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration...
  4. pmc Pannexin channels are not gap junction hemichannels
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA
    Channels (Austin) 5:193-7. 2011
    ..Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature...
  5. pmc Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly
    Yong Chun Yu
    Institute of Neurobiology, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China
    Nature 486:113-7. 2012
    ..These results suggest a strong link between lineage-dependent transient electrical coupling and the assembly of precise excitatory neuron microcircuits in the neocortex...
  6. pmc Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging
    Mark H Ellisman
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Methods Cell Biol 111:139-55. 2012
    ..Here we review and discuss the current generation of genetic labels for direct protein identification by CLEM, addressing their relative strengths and weaknesses and in what experiments they would be most useful...
  7. pmc Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation
    Elena Dolmatova
    Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
    Am J Physiol Heart Circ Physiol 303:H1208-18. 2012
    ..ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury...
  8. pmc Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
    Jeffrey D Martell
    Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
    Nat Biotechnol 30:1143-8. 2012
    ..Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen...
  9. pmc Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, California, United States of America
    PLoS ONE 8:e70916. 2013
    ..Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure. ..
  10. pmc Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093 0608, USA
    J Biol Chem 285:24420-31. 2010
    ..We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other...
  11. pmc Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, California, USA
    Biophys J 98:1809-19. 2010
    ..In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating...
  12. pmc The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes
    Oliver Gassmann
    Institute for Organic and Biomolecular Chemistry, University of Gottingen, Germany
    J Struct Biol 168:168-76. 2009
    ..The conductance levels of Cx26M34A were found at around 45 and 70 pS...
  13. ncbi Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels
    Jinshu Yu
    BioTechnological Center, University of Technology Dresden, Tatzberg 47 51, 01307 Dresden, Germany
    J Biol Chem 282:8895-904. 2007
    ..5 degrees rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure...
  14. ncbi Markers for correlated light and electron microscopy
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research and Center for Research in Biological Systems, University of California, San Diego, La Jolla, California 92093, USA
    Methods Cell Biol 79:575-91. 2007
  15. pmc The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Biochem J 408:375-85. 2007
    ..These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43...
  16. ncbi Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, Department of Neurosciences, University of California, San Diego, La Jolla, California 92093 0608, USA
    J Biol Chem 282:31733-43. 2007
    ..We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues...
  17. pmc The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, 1070 Basic Science Building MC 0608, 9500 Gilman Drive, La Jolla, CA 92093 0608, USA
    J Struct Biol 161:359-71. 2008
    ....
  18. pmc Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy
    Jason Lanman
    Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Struct Biol 161:439-46. 2008
    ..Two features of the mitochondrial modification are approximately 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle...
  19. pmc Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves
    Guy A Perkins
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093 0608, USA
    J Struct Biol 161:469-80. 2008
    ..Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication...
  20. pmc Trafficking dynamics of glycosylated pannexin 1 proteins
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, California 92093 0608, USA
    Cell Commun Adhes 15:119-32. 2008
    ..Therefore, Panx1 has kinetics and dynamics that make it unique to serve distinct functions separate from connexin-based channels...
  21. pmc Protein kinase Cδ-mediated phosphorylation of Connexin43 gap junction channels causes movement within gap junctions followed by vesicle internalization and protein degradation
    Angela C Cone
    From the National Center for Microscopy and Imaging Research
    J Biol Chem 289:8781-98. 2014
    ....

Research Grants30