The Role of the Cytoskeleton in the Modulation of the Connexin36 Nexus in N2a Cells

We tested the hypothesis that the interaction of Cx36 with the cytoskeleton is necessary to achieve synaptic plasticity in neurons. In living cells, BioID and FRAP technology was used to demonstrate binding of Cx36 to actin and tubulin. Wild-type and mutant proteins, together with pharmacological blockers or TAT peptides were used to characterize the interaction. Major results of this study are that rCx36 interacts primarily with the neuronal βIII-tubulin isoform to regulate the trafficking and aggregation of connexons at the GJP. Amino acid Lys279 in the CTB domain is critical for this interaction. A potential actin-binding site in the CLB domain of rCx36 was identified, with actin mainly serving as an anchor to stabilize connexons. During a simulation of plasticity in vitro, GJPs became more stable; we attribute this to interactions with the cytoskeleton and associating proteins. We conclude that cytoskeletal-dependent interaction is required to modulate the strength of Cx36 synapses.