Actin Cytoskeleton Rest Stops Regulate Anterograde Traffic of Connexin 43 Vesicles to the Plasma Membrane

• Published in: Circulation research 2012-03, Vol.110 (7), p.978-989
• Main Authors: Smyth, James W, Vogan, Jacob M, Buch, Pranali J, Zhang, Shan-Shan, Fong, Tina S, Hong, Ting-Ting, Shaw, Robin M
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - metabolism; Actins - metabolism; Animals; Biological and medical sciences; Biological Transport - physiology; Cell Line; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cells, Cultured; Connexin 43 - metabolism; Cytoplasmic Vesicles - metabolism; Cytoplasmic Vesicles - ultrastructure; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Fundamental and applied biological sciences. Psychology; Gap Junctions - metabolism; Gap Junctions - ultrastructure; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; Humans; Keratinocytes - cytology; Keratinocytes - metabolism; Keratinocytes - ultrastructure; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Models, Animal; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - ultrastructure; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.111.257964

RATIONALE:The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown. OBJECTIVE:We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking. METHODS AND RESULTS:High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 μm/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P