Internalized gap junctions are degraded by autophagy

Abstract

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously—and effectively after treatment with natural inflammatory mediators—internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.

Keywords: :

• BECN1/(Atg6)
• LC3/(Atg8)
• annular gap junctions
• autophagy
• cell-cell junctions
• connexin43
• connexosome
• gap junctions
• p62/sequestosome1
• protein degradation

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank T. Yoshimori (National Institute of Genetics, Shizuoka, Japan) for providing fluorescent LC3 constructs; J.-P. Denizot (Unité de Neurosciences Intégratives et Computationnelles, CNRS, Gif sur Yvette, France) for the preparation of ultra-thin sections; D. Segretain (Institut National de la Santé et de la Recherche Médicale U895, Université Paris Descartes, 75006 Paris, France) for ultrastructural examination of our Cx43-GFP expressing HeLa cell samples; and L. Traub (University of Pittsburgh, Pittsburg, PA, USA), L. Cassimeris (Lehigh University), and Falk-lab members for critical advice. This work was supported by NIHs NIGMS (grant GM55725 to M.M.F.) and Lehigh University.

M.M.F. and A.M.G. designed the research; A.M.G., J.T.F, R.M.K. and M.M.F. performed the experiments; A.M.G., J.T.F., R.M.K., J.Y.M. and M.M.F. analyzed the data; M.W.D. provided essential research tools; A.M.G. and M.M.F. wrote the manuscript.

Supplemental Materials

Supplemental materials can be found at: www.landesbioscience.com/journals/autophagy/article/19390