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. 2022 Jul 18;11:e73396. doi: 10.7554/eLife.73396

Figure 2. Inactivation of Ran-GTP-mediated nuclear transport affects differentially the subcellular localization of AR-variants.

(A, B) Plasmids encoding GFP-tagged AR-fl, AR-v567, or AR-V7 were co-microinjected with plasmid encoding the catalytic mutant mCherry-tagged RanQ69L, into the nuclei of the AR-null PC3 cells. Cells expressing both tagged proteins were subjected to live-cell time-lapse imaging. Cells were treated with R1881 (10 nM) to induce AR-fl nuclear import and was kept present with the variants so that all conditions were the same. Expression of mCherry-tagged RanQ69L affected differentially the subcellular localization of each GFP-tagged AR proteins. Solid arrow: cell with both cytoplasmic and nuclear AR proteins; arrowheads: cytoplasmic AR proteins; dashed arrow: nuclear AR proteins. % Nuclear AR across conditions is graphically displayed in B (n>10 per condition). (C–F) AR-V7 nuclear import is impaired upon mutation of the dimerization box domain (D-box). PC3 cells stably expressing ARE-mCherry reporter were transfected with GFP-AR-V7 or GFP-AR-V7 D-box mutant (A596T and S597T). Representative images are shown (inset displays higher magnification of the indicated cell) and quantitative results are graphically displayed in (D) (n>10 cells per condition). (E, F) C4-2 cells stably expressing inducible GFP-AR-V7 or GFP-AR-V7 D-Box mutant were used to quantify subcellular AR-V7 localization following doxycycline induction. Representative images are shown (inset displays higher magnification of the indicated cell) and quantitative results are graphically displayed (F). ( n> 200 cells per condition). Data represent mean ± SEM, p value (**p<0.01, ***p<0.001, ****p<0.0001) was obtained using unpaired two-tailed t-test. Scale bar, 10 μm. Experiments were repeated at least twice.

Figure 2.

Figure 2—figure supplement 1. AR-V7 nuclear import requires active transport via the nuclear pore complex is dependent on Ran-GTP activity and is impaired upon mutation of the dimerization box domain (D-box).

Figure 2—figure supplement 1.

(A) Wheat germ agglutinin (WGA) blocks AR-V7 nuclear import: we incubated cells with WGA, an inhibitor of nucleoporin-mediated nuclear transport and monitored GFP-AR-V7 localization by live-cell imaging. WGA kept AR-V7 in the cytoplasm in the presence of doxycycline suggesting that AR-V7 nuclear import requires active transport via the NPC. Representative confocal microscopy images were shown. Red: WGA labeling of membranes; Green: GFP-AR-V7. Nuc: nuclear AR-V; Cyto: cytoplasmic AR-V7. Scale bars represent 10 µm. (B) (related to Figure 2B) HEK293T cells were transfected with plasmids encoding GFP-tagged AR-fl, AR-v567 and AR-V7 in the presence of the catalytic mutant Ran-GTP (mCherry-tagged RanQ69L). Nuclear accumulation of each AR variant was calculated. Representative confocal microscopy images (63× magnification) for each condition are shown. Solid arrow: cell with both cytoplasmic and nuclear proteins; arrowheads: cells with cytoplasmic protein; dashed arrow: cell with nuclear protein. Scale bars represent 10 µm. (C) Graphic display of % Nuclear AR across 30 individual cells per condition. Data represent mean ± SEM with n>10, p-value (**p<0.01, ****p<0.0001) was obtained using unpaired two-tailed t-test. (D) C4-2 cells with endogenous AR-fl stably expressing inducible GFP-AR-V7 were transfected with the catalytic mutant mCherry-tagged RanQ69L. Representative confocal microscopy images are shown. Cytoplasmic enrichment for AR-V7 is shown when the mutant Ran is co-expressed in the same cell. (E). 22RV1 cells with endogenous AR-fl and AR-V7 were transfected with the catalytic mutant mCherry-tagged RanQ69L. AR-V7 was detected by immunostaining with the RevMab antibody. Downregulation of endogenous AR-V7 protein was observed in cells co-expressing RanQ69L. Solid arrow: cell with cytoplasmic and nuclear AR-V7; dashed arrow: non-transfected cells with endogenous nuclear AR-V7 Dotted ellipses: cells transfected with mutant Ran where AR-V7 appears absent or downregulated. Scale bar, 10 µm. (F). PC3 cells were transfected with GFP-AR-fl or GFP-AR-fl-D-box mutant (A596T and S597T) and they were treated for 4 hr with 10 nM DHT. There was no effect of the D-box mutations on AR-fl nuclear import. Experiments were repeated at least twice.