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Jaya Aseervatham, Helen Yanran Wang, Cheryl Mitchell, Bi-Chang Chen, Ya-Ping Lin, Eric Betzig, John O'Brien; Cx36 coupling and structural plasticity is regulated by an association with the actin cytoskeleton. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3096.
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© ARVO (1962-2015); The Authors (2016-present)
Neuronal gap junction channels composed of Cx36 play an important role in neuronal network dynamics and show striking functional plasticity. We hypothesized that actin cytoskeleton may influence Cx36 function.
HaloTag open reading frame was inserted into an internal site in the C-terminus of perch Cx36 (Cx36-Halo). A truncated mutant lacking the last 7 aa was also prepared (Cx36-Halo-S298ter). They were transfected in Hela cells. Live cells were labeled with Tetramethylrhodamine or Oregon Green-conjugated HaloTag ligand and imaged live using Bessel Beam Plane Illumination, or after fixation and immunostaining using confocal microscopy. Functional coupling was assessed by scrape-loading using Neurobiotin and treatments to modulate PKA and PP2A activity (20 min: PKA activator 10 µM Sp-8-cpt-cAMPS, inhibitor 10 µM Rp-8-cpt-cAMPS, PP2A inhibitor 0.5 nM Microcystin LR) or actin dynamics (1 hr: 0.2 µM Cytochalasin D or Lantrunculin A). Hela cells without Cx36 were used as control.
Live imaging revealed that Cx36-Halo gap junctions change shape in minutes and extend dynamic finger-like processes called filadendrites. Fixed imaging showed that Cx36 gap junctions were always associated with actin bundles and fine filaments, but only occasionally with tubulin. Filadendrites co-localized with actin filaments. Disrupting actin with Cytochalasin D decreased gap junction size (p<0.05) and filadendrite presence (p<0.001), while preventing actin polymerization with Lantrunculin A reduced filadendrites (p<0.001) without reducing gap junction size (p=0.38). Truncation of the tip of the C-terminus reduced filadendrite presence (p<0.05), implying an interaction between the C-terminus tip and actin. Truncation mutant and Latrunculin A disrupted regulation of coupling by PKA in the same manner, causing PKA to increase coupling. Addition of microcystin in combination with PKA activator increased coupling, implying that PP2A suppresses coupling in Hela cells.
The results indicate that dynamic remodeling of the actin cytoskeleton is required for extension of mobile filadendrites from gap junction plaques. The results also suggest that the dynamically remodeling pool of actin is associated with the signaling complexes that control coupling through phosphorylation and dephosphorylation of Cx36, and that the Cx36 C-terminus tip is a focal point to integrate these signaling mechanisms.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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