The affect of [beta]‐tubulin and actin on gap junction channel mobility in transformed and non‐transformed NRK cells

Gap junctions are aggregates of intracellular channels (plaques) that allow the direct exchange of small molecules and ions (≤ 1kD). We are utilizing a normal rat kidney cell line (NRK) containing a temperature‐sensitive viral oncogene (v‐src), pp60 src, to study the mobility of gap junction channels within plaques in transformed (v‐src active) and non‐transformed NRK cells (v‐src inactive). The mechanisms that underlie channel mobility or immobility are unknown. Others have shown Cx43 to associate with β‐tubulin and actin (Giepmans 2004). We asked whether these cytoskeletal elements influence channel mobility. Immunofluorescence was performed to identify the localization of Cx43, β‐tubulin and actin in transformed and non‐transformed NRK cells before and after disruption with nocodazole and cytochalasin B. Fluorescence recovery after photobleaching (FRAP) was used to visualize and quantify the mobility of EGFP tagged Cx43 gap junction channels in treated/non‐treated NRK cells. Disruption of the actin and microtubule networks was confirmed by immunofluorescence. Preliminary data suggest that disrupting microfilaments with cytochalasin B, or microtubules with nocodazole does not significantly affect fluorescence recovery t 1/2 . However, the microtubule data indicate additional experiments are needed to determine if the observed subtle changes of mobility are reflecting a biological response.