Internalization of gap junctions as a regulatory mechanism of direct GJIC

Summary

Principal Investigator: Matthias M Falk
Abstract: DESCRIPTION (provided by applicant): Communication between adjacent cells through gap junctions (GJs) occurs in nearly every tissue and is fundamental to coordinated cell behavior during development, differentiation, and tissue maintenance. In addition, a growing number of publications indicate that connexins and GJs also play important, yet underestimated functions in physical cell-cell coupling, and in cellular guiding during migration. Cx43-based gap junctional intercellular communication (GJIC) is of particular interest because it is regulated by both physiological and pathological stimuli. While the modulation of GJIC is well documented, surprisingly little is known about how up- or down-regulation of GJIC is actually achieved. GJ channels are known to open and close (gate) in response to physiological parameters;however GJ-based intercellular coupling could also be regulated through altering the number of GJ channels in the plasma membrane (PM). Ultra-structural analyses identified cytoplasmically located, spherical GJ vesicles in situ, leading to the hypothesis that cytoplasmic GJ vesicles (termed annular gap junctions, AGJs) represent internalized GJs. To test this hypothesis, we investigated the spatiotemporal process of GJ internalization in primary vasculature endothelial cells (PAECs), and in cells that transiently or stably expressed fluorescent protein-tagged Cx43. Our studies demonstrate that rapid and efficient internalization of GJ plaques occurs naturally, for example in response to inflammatory mediators such as thrombin and endothelin. Further studies revealed that entire, or large portions of GJ plaques internalize in a ZO-1-mediated, clathrin-dependent process that forms intracellular double-membrane GJ vesicles. These vesicles are then fragmented into smaller vesicles that can be degraded by lysosomal pathways. Based on these results, we hypothesize that GJ internalization may be used widely to efficiently modulate GJIC and cell-cell coupling. To test this hypothesis, we will address three specific questions: (1) Is GJ internalization commonly used to down-regulate intercellular communication and physical cell-cell coupling? (2) How do clathrin and clathrin-associated proteins internalize double-membrane spanning GJs? And (3) which cellular pathway/s degrade/s internalized GJs, and can internalized GJs re-insert into the PM to restore/up-regulate GJIC and physical cell-cell coupling? We will use a comprehensive, integrated experimental approach that includes the expression of fluorescence-tagged Cx43, molecular biology, biochemistry, immunological and functional assays, and temporal high-resolution light and electron microscopic analyses. The physiological role of GJ internalization will be investigated in two endothelial cell systems in which cellular uncoupling/re-coupling occurs naturally: (a) inflammatory mediator (thrombin, endothelin), and (b) VEGF-mediated stimulation. These studies will contribute to our understanding which mechanisms cells use to maintain and modulate intercellular signaling and cell-cell contacts, and will add novel insights into the many evolving functions of endocytic and autophagosomal protein degradation pathways. PUBLIC HEALTH RELEVANCE: Connexin (Cx) family members (of which there are at least 20 in humans) are the protein constituents of gap junction (GJ) channels that provide direct cell-to-cell communication. Cxs are expressed in many different tissues, with Cx43 predominantly found in most tissues. Human mutations in several Cxs (including Cx26, Cx30, Cx30.3, Cx31, Cx32, Cx43, Cx46, and Cx50) are implicated in a number of diseases including neuropathies, deafness, cataracts, skin disorders, and cranio-facial developmental defects, demonstrating the fundamental need of gap junctional intercellular communication (GJIC) at all stages of life, including development, differentiation, and tissue maintenance. Cx43-based GJIC is of particular interest because it is regulated by both physiological and patho-physiological stimuli. While the modulation of GJIC is well documented, surprisingly little is known about how up- or down-regulation of cell-cell communication is actually achieved. Indeed, cells not only need to be able to precisely up or down-regulate the amount of GJIC, but also to internalize all GJs (physically uncouple from their neighbors) if they need to become migratory (development, tissue differentiation, wound healing, etc.) or need to uncouple for other reasons (mitosis, apoptosis, etc.). An understanding of these processes will contribute to our knowledge of how cells maintain and modulate intercellular signaling and cell-cell contacts, and may in future allow us to prevent unwanted GJ internalization/degradation and human diseases that are linked to the mis-regulation of GJIC.
Funding Period: 1998-02-01 - 2014-12-31
more information: NIH RePORT

Top Publications

  1. ncbi Phylogenetic analysis of three complete gap junction gene families reveals lineage-specific duplications and highly supported gene classes
    Stephen D Eastman
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca B 217, Bethlehem, PA 18015, USA
    Genomics 87:265-74. 2006
  2. pmc Degradation of connexins and gap junctions
    Matthias M Falk
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, D 218, Bethlehem, PA 18015, USA Electronic address
    FEBS Lett 588:1221-9. 2014
  3. pmc Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis
    John T Fong
    Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015
    Mol Biol Cell 24:2834-48. 2013
  4. pmc Proteins and mechanisms regulating gap-junction assembly, internalization, and degradation
    Anastasia F Thévenin
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, USA
    Physiology (Bethesda) 28:93-116. 2013
  5. pmc Nanoporosity significantly enhances the biological performance of engineered glass tissue scaffolds
    Shaojie Wang
    Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015, USA
    Tissue Eng Part A 19:1632-40. 2013
  6. pmc Degradation of endocytosed gap junctions by autophagosomal and endo-/lysosomal pathways: a perspective
    Matthias M Falk
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, D 218, Bethlehem, PA 18015, USA
    J Membr Biol 245:465-76. 2012
  7. pmc Internalized gap junctions are degraded by autophagy
    John T Fong
    Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA
    Autophagy 8:794-811. 2012
  8. ncbi Evaluation of 3D nano-macro porous bioactive glass scaffold for hard tissue engineering
    S Wang
    Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA, USA
    J Mater Sci Mater Med 22:1195-203. 2011
  9. pmc Adherens junctions remain dynamic
    Matthias M Falk
    Department of Biology, Lehigh University, Bethlehem, PA, USA
    BMC Biol 8:34. 2010
  10. pmc Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes
    Susan M Baker
    Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA
    BMC Cell Biol 11:15. 2010

Research Grants

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
  2. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013

Detail Information

Publications16

  1. ncbi Phylogenetic analysis of three complete gap junction gene families reveals lineage-specific duplications and highly supported gene classes
    Stephen D Eastman
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca B 217, Bethlehem, PA 18015, USA
    Genomics 87:265-74. 2006
    ..The identification of a third complete connexin gene family provides novel insight into the evolution of connexins, and sheds light into the phenotypic evolution of intercellular communication via gap junctions...
  2. pmc Degradation of connexins and gap junctions
    Matthias M Falk
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, D 218, Bethlehem, PA 18015, USA Electronic address
    FEBS Lett 588:1221-9. 2014
    ..In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting for degradation. ..
  3. pmc Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis
    John T Fong
    Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015
    Mol Biol Cell 24:2834-48. 2013
    ..Our analyses provide a mechanistic model for clathrin's efficient internalization of large plasma membrane structures, such as GJs. ..
  4. pmc Proteins and mechanisms regulating gap-junction assembly, internalization, and degradation
    Anastasia F Thévenin
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, USA
    Physiology (Bethesda) 28:93-116. 2013
    ..We also look closely at the atomic resolution structure of a GJ channel, since the structure harbors vital cues relevant to GJ biosynthesis and turnover...
  5. pmc Nanoporosity significantly enhances the biological performance of engineered glass tissue scaffolds
    Shaojie Wang
    Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015, USA
    Tissue Eng Part A 19:1632-40. 2013
    ....
  6. pmc Degradation of endocytosed gap junctions by autophagosomal and endo-/lysosomal pathways: a perspective
    Matthias M Falk
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, D 218, Bethlehem, PA 18015, USA
    J Membr Biol 245:465-76. 2012
    ..Furthermore, we highlight and discuss structural criteria that seem required for an alternate degradation via the endo-/lysosomal pathway...
  7. pmc Internalized gap junctions are degraded by autophagy
    John T Fong
    Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA
    Autophagy 8:794-811. 2012
    ..Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general...
  8. ncbi Evaluation of 3D nano-macro porous bioactive glass scaffold for hard tissue engineering
    S Wang
    Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA, USA
    J Mater Sci Mater Med 22:1195-203. 2011
    ..Thus, the new nano-macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration...
  9. pmc Adherens junctions remain dynamic
    Matthias M Falk
    Department of Biology, Lehigh University, Bethlehem, PA, USA
    BMC Biol 8:34. 2010
    ....
  10. pmc Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes
    Susan M Baker
    Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA
    BMC Cell Biol 11:15. 2010
    ..Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered...
  11. pmc Gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles
    Matthias M Falk
    Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA
    Mol Biol Cell 20:3342-52. 2009
    ..Our observations may have implications for the process of endocytic vesicle budding in general...
  12. ncbi Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen
    Jerome Gilleron
    INSERM U 895, Team 5 Physiopathologic control of germ cell proliferation genomic and non genomic mechanisms, Universite Paris Descartes, 45 rue des Saint Peres, 75006, Paris, France
    J Cell Sci 121:4069-78. 2008
    ....
  13. pmc Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation
    Susan M Baker
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, Bethlehem, PA 18015, USA
    FEBS Lett 582:4039-46. 2008
    ..These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response...
  14. pmc Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery
    Anna M Gumpert
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA
    FEBS Lett 582:2887-92. 2008
    ..To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells...
  15. pmc Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels
    M Kathryn Iovine
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA
    FEBS Lett 582:165-70. 2008
    ..Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro...
  16. pmc EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368
    John T Fong
    Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, Bethlehem, PA 18015, USA
    FEBS Lett 588:836-44. 2014
    ..Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation. ..

Research Grants30

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
    ..Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program. ..
  2. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
    ....