Figure 1. AR-V7 exhibits fast nuclear import kinetics independently of microtubules or the importin-α/β pathway, unlike AR-fl or AR-v567.

(A) Experimental design. Plasmids encoding GFP-tagged AR-fl, AR-v567, or AR-V7 were microinjected into the nuclei of the AR-null PC3 cells. As soon as GFP-tagged proteins were detected in the cytoplasm (~45 min post micro-injection), nuclear translocation kinetics was monitored by live-cell time-lapse confocal microscopy at 5 min intervals for a total of 90 min. (B) Representative time-lapse images showing subcellular localization of each GFP-tagged AR protein. Scale bar, 10 µm. (C) Quantitation of % nuclear GFP-AR protein in single cells (n=3–10 cells/condition/time point). (D) Effect of MT and importin-β inhibitors on AR-fl, AR-v567, and AR-V7 nuclear localization. M12 prostate cancer cells stably expressing GFP-tagged AR-fl or AR-v567 or AR-V7 were treated as indicated and subjected to live-cell time-lapse imaging. R1881: synthetic androgen; DTX: docetaxel, MT-stabilizing drug; IPZ: importazole, importin-β inhibitor. Representative images are shown. Arrows point to cells with cytoplasmic GFP-AR-fl or GFP-AR-v567. Scale bar, 10 µm. (E) Schematic overview of Fluorescence Recovery After Photobleaching (FRAP) assay and its quantitative output. (F) Effect of MT and importin-β inhibitors on AR-fl, AR-v567, and AR-V7 nuclear translocation kinetics following FRAP. T1/2 times in s are shown for each respective protein (n=4–12 cells/condition). Data represent mean ± SEM, p value (*p<0.05, **p<0.01, ****P<0.0001) was obtained using unpaired two-tailed t-test. Experiments were repeated at least twice.

Figure 1—figure supplement 1. AR-V7 exhibits fast nuclear import kinetics independently of microtubules, actin, or the importin-α/β pathway.

(A) Plasmid encoding GFP-tagged AR-V7 was microinjected into nuclei of PC3 cells and as soon as GFP was detected in the cytoplasm (~45 min post micro-injection) the kinetics of GFP-AR-V7 nuclear import were monitored by live-cell time-lapse confocal microscopy at 5 min intervals for a total of 180 min. Representative time-lapse images are shown at the indicated time points. Solid arrow: cell with both cytoplasmic and nuclear AR-V7 at time 0; Arrowhead: cells with cytoplasmic only AR-V7 at time 0; Dashed Arrow: cell with primarily nuclear AR-V7 first detected at +50 min after the start of imaging. Enhanced AR-V7 nuclear translocation is observed over time for all cells. Notice that there are cells with already extensive nuclear accumulation of AR-V7 at 0 min, suggesting very fast nuclear import kinetics from the time of microinjection (–45 min). Scale bar, 10 µm. (B–D) Corresponds to Figure 1D with additional time points. Briefly, M12 prostate cancer cells stably expressing GFP-tagged (B) AR-fl, (C) AR-v567, or (D) AR-V7 were treated as indicated and subjected to live-cell time-lapse imaging. R1881: synthetic androgen used to stimulate AR-fl nuclear translocation; DTX: docetaxel, MT-stabilizing drug; IPZ: importazole, importin-β inhibitor. Representative images are shown. Scale bar, 10 µm. (E) Table with T1/2 (half-time recovery) values for each variant (related to Figure 1F). (F) PC3 cells were treated with 1 μg/ml cytochalasin D (Cyto D) or vehicle control (VC) for 1 hr at 37°C following plasmid micro-injection into the nuclei of PC3 cells. Cells were then treated with 10 nM R1881 for 4 hr and subjected to point-scanning confocal microscopy. Representative images showing are shown. Scale bar, 10 µm. Experiments were repeated at least twice.

Figure 1—figure supplement 2. Dominant negative IPO11 does not abrogate the nuclear import of AR-fl or AR-V7.

(A) Plasmid encoding mCherry tagged AR-fl and/or a dominant negative mutant IPO11 was microinjected into PC3 cells.; 45 mins post microinjection, representative images were captured by live -cell confocal microscopy.