Figure 3.

RGS14 is a nucleocytoplasmic shuttling protein in hippocampal neurons.A, RGS14 rapidly shuttles into and out of the nucleus in dividing cells directed by a nuclear localization sequence (NLS) and nuclear export sequence (NES). Studies validated that RGS14 followed these same cellular dynamics in primary hippocampal neurons. B, neurons were infected with AAV2/9-eYFP-RGS14 for 18 h and then imaged using live cell confocal microscopy. Leptomycin B (LMB) was added (final concentration 20 nM) and YFP signal was measured. At time zero, YFP-RGS14 is entirely cytosolic. After 20 min, YFP-RGS14 was detectable in the nucleus, at 40 min YFP-RGS14 was mostly nuclear, and by 80 to 120 min YFP-RGS14 was entirely nuclear. C, wild-type YFP-RGS14 does not colocalize with Hoechst in vehicle-treated conditions, but colocalizes with Hoechst entirely under LMB conditions. D, mutation of the NES (L503A/L504A, NESm) in RGS14 sequesters RGS14 entirely in the nucleus under vehicle-treated conditions. Approximately ten images were collected per condition and representative images are shown.