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.

Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission

“…Such fissions may be critical to gap junction protein degradation but the cellular need for this process can at this point, only be speculated and remains to be elucidated. Both lysosomal and more recently autophagolysosomal degradation of the annular gap junction vesicle have been demonstrated (Bejarano et al, 2012;Fong et al, 2012;Lichtenstein et al, 2011;Murray et al, 1981). In addition, other mechanisms of internalization may exist for removing gap junction proteins from the cell surface that do not result in annular vesicle formation, possibly involving lipid rafts (Kirkham and Parton, 2005), and caveolin (Kirkham and Parton, 2005).…”

Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus

“…Consequently, removal from the plasma membrane is directionally regulated as internalization of the channels within one of the coupled cells forms a double membrane macrostructure called an annular gap junction or connexosome (10); clathrin and other endocytic adaptors have been shown to be involved in this process (11)(12)(13)(14). Degradation of connexins involves both the lysosomal (endosome or autophagy) and proteasomal pathways (15)(16)(17). Implicated in the regulation of these processes are post-translational modifications (18).…”

Low pH enhances connexin32 degradation in the pancreatic acinar cell

“…Cx32 degradation is complex, with established roles for proteasomal and lysosomal pathways (20,23,45). The autophagic pathway has been implicated in the degradation of some connexins (2,6,26) but has not been studied for Cx32.…”