Editor's evaluation: AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer (2021)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions. Editor's evaluation This work performs a careful study of AR-V7, a splice variant of androgen receptor (AR) that lacks the androgen-binding domain, is constitutively active, and is typically expressed as prostate cancers become resistant to anti-androgen therapies. Clinically, there is intense interest in overcoming anti-androgen resistance, and part of this includes understanding differences between AR-V7 and AR, to be able to therapeutically target AR-V7. This manuscript provides a robust analysis of the regulation of nuclear import and the chromatin-binding features of AR-V7 versus AR. The work reveals that AR-V7 exhibits fast nuclear import kinetics in an NLS- and importin-α/β- independent manner, dependent on the dimerization (D-box) domain mediates AR-V7 nuclear import, revealing a new function for this domain versus its role in the full-length AR. The work also shows that AR-V7 employs an unconventional mode of transcription characterized by high intranuclear mobility, with transient and unstable chromatin interactions, likely reflecting a "Hit-and-Run" mechanism. This greatly enlarges the mechanistic understanding of AR-V7 function, and may help with developing new therapeutic agents. https://doi.org/10.7554/eLife.73396.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Androgen receptor (AR) remains a critical therapeutic target in the treatment of metastatic castration-resistant prostate cancer (CRPC), due to overactive AR signaling (Feldman and Feldman, 2001). Next-generation AR inhibitors targeting either androgen biosynthesis (abiraterone acetate) or AR ligand binding (enzalutamide) have shown improved clinical outcomes including survival. However, these new therapies are not curative due to the development of resistance (Watson et al., 2015). Expression of active AR splice variants (AR-Vs) which re-activate AR transcriptional program in CRPC (Antonarakis et al., 2016; Maughan and Antonarakis, 2015) is one of the key drivers in disease progression and is believed to be one mechanism of resistance to abiraterone and enzalutamide. Structurally, the majority of AR-Vs lack the ligand-binding domain (LBD), which is the target of most AR-targeted therapies, and are constitutively active in the nucleus driving AR-signaling (Uo et al., 2018; Watson et al., 2015). Among more than 20 alternatively spliced AR variants identified to date, AR-V7, which arises from cryptic exon inclusion, is the most prevalent variant in CRPC followed by the exon-skipping AR-v567. Expression of AR-V7 has been clinically associated with adverse patient outcomes including increase rates of metastases and inferior survival rates, and resistance to current standard of care treatment with abiraterone, enzalutamide, and taxane chemotherapy (Antonarakis et al., 2014; Antonarakis et al., 2017; Guo et al., 2009; Hörnberg et al., 2011; Hu et al., 2009; Maughan and Antonarakis, 2015; Robinson et al., 2015 #80; Rizzo et al., 2021; Robinson et al., 2015; Tagawa et al., 2019). Together these data suggest that AR-V7 is a driver of CRPC progression and a desirable therapeutic target. However, the exact mechanism(s) underlying AR-V7 oncogenic functions are not well understood. Chromatin immunoprecipitation (ChIP), transcriptomic, and epigenetic studies have identified AR-V7 cistromes and target genes, both distinct and shared with AR-fl, as well as splicing factors that drive AR-V7 production, in an effort to elucidate potentially unique to AR-V7 regulatory mechanisms (Cao et al., 2014; Hu et al., 2009; Li et al., 2013; Melnyk et al., 2020; Xu et al., 2015). Previously we showed that AR-fl binds microtubules (MTs) via the hinge domain and uses them as tracks for fast nuclear import (Thadani-Mulero et al., 2014; Zhu et al., 2010). Taxanes stabilize MTs and inhibit AR signaling by impairing AR-fl nuclear import and subsequent activation of target genes (Antonarakis et al., 2017; Darshan et al., 2011; Thadani-Mulero et al., 2014; Zhu et al., 2010). Similar to AR-fl, the hinge-containing AR-v567 binds MTs and is sensitive to taxane treatment. In contrast, the hinge-less AR-V7 does not bind MTs, conferring taxane resistance in xenografts and patients with CRPC (Thadani-Mulero et al., 2014). In this study, we set out to investigate the mechanisms mediating AR-V7 nuclear import and its subnuclear biophysical properties in association with chromatin to identify unique, targetable biological features. Results AR-V7 exhibits fast nuclear import kinetics in a MT and importin-α/β independent pathway Nuclear translocation is a pre-requisite for the transcriptional activity of AR-fl and all other nuclear hormone receptors. Nuclear import is mediated by a conserved bipartite nuclear localization signal (NLS) motif, which in AR-fl and AR-v567 is comprised of parts of exons 3 and 4. AR-V7 lacks exon 4, which is the second half of the canonical bipartite NLS, and although its cryptic exon 3 has been implicated in NLS reconstitution (Chan et al., 2012; Guo et al., 2009; Hu et al., 2009), the canonical NLS motif of AR-V7 is compromised. Yet, AR-V7 is constitutively localized to the nucleus in both cell lines and clinical samples (Sun et al., 2010; Watson et al., 2010), indicating efficient nuclear import. To measure basal nuclear import kinetics of the AR-Vs (AR-V7, AR-v567, and AR-fl), plasmids encoding each GFP-tagged AR were microinjected into the nuclei of AR-null PC3 cells and nuclear translocation kinetics was monitored by live-cell time-lapse confocal microscopy (Figure 1A-B). For each protein, we calculated the extent and rate of nuclear import by quantifying the % nuclear GFP-AR protein in single cells over time (Figure 1C). AR-fl remained largely in the cytoplasm under basal condition, exhibiting ~20% nuclear accumulation which remained steady over the duration of the experiment, indicating very low basal nuclear import kinetics. AR-v567 reached a maximum of ~50% by 90 min. In contrast, AR-V7 exhibited fast nuclear import kinetics, reaching ~50% nuclear accumulation within the first 25 min and ~75% nuclear accumulation by 90 min (Figure 1B-C and Figure 1—figure supplement 1A). These data suggested that AR-V7 exhibited the highest basal nuclear import rates, likely via a more efficient nuclear import mechanism. It is established that canonical AR-fl utilizes the classical importin-α/β nuclear import mechanism where the importin-α binds to the NLS of AR protein followed by importin-β binding, forming a trimeric (cargo-NLS/importin-α/importin-β) complex in the cytoplasm which enters the nucleus through the nuclear pore complex (NPC) using the Ran-GTP (Pemberton and Paschal, 2005). To identify the mechanisms mediating AR-V7 nuclear import, we examined the involvement of the MT-transport system and the importin-α/β-Ran-GTP pathway (Darshan et al., 2011; Jenster et al., 1993; Kaku et al., 2008; Thadani-Mulero et al., 2014; Zhou et al., 1994; Zhu et al., 2010). We analyzed the translocation kinetics of each variant by live-cell time-lapse imaging using chemical probes that disrupt MTs (docetaxel [DTX]) or importin-β (Importazole [IPZ]) (Figure 1D and Figure 1—figure supplement 1B–1D). In agreement with our published data, AR-fl readily translocated to the nucleus upon addition of the synthetic AR ligand R1881, while perturbation of MTs with DTX, abrogated this effect (Thadani-Mulero et al., 2014). IPZ inhibited the R1881-induced AR-fl nuclear translocation, confirming the role of importin-β in the canonical AR-fl nuclear import pathway (Kaku et al., 2008), and that AR-v567 shares the same pathway of nuclear import with AR-fl (Figure 1D and Figure 1—figure supplement 1C). In contrast, neither DTX nor IPZ had an effect on AR-V7 nuclear localization, indicating that nuclear import of AR-V7 is both MT and importin-α/β independent (Figure 1D and Figure 1—figure supplement 1D). Figure 1 with 2 supplements see all Download asset Open asset 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. To quantify nuclear translocation kinetics of AR proteins in response to treatment, we performed fluorescence recovery after photobleaching (FRAP) analysis in the M12 human metastatic prostate cancer cells stably expressing each GFP-tagged AR protein (Thadani-Mulero et al., 2014). The half-time of recovery (T½; defined as the time required for fluorescence intensity to reach 50% of its pre-bleach intensity) was then calculated and used as a readout of nuclear import dynamics (Figure 1E). Treatment with R1881 accelerated AR-fl nuclear import by decreasing the T½ from 23 to 11 s while addition of DTX or IPZ significantly attenuated T½ to 23 and 19 s, respectively (Figure 1F and Figure 1—figure supplement 1E). The nuclear recovery of AR-v567, which retains the MT-binding hinge region, was also significantly impaired by DTX or IPZ treatment (T½ 23 and 22 s, respectively). In contrast, AR-V7 nuclear import kinetics was much faster than those of unliganded AR-fl (T½ 11 vs. 23 s) and was not affected by DTX or IPZ. To determine the involvement of the actin cytoskeleton in AR-V7 nuclear import, we treated PC3 cells microinjected with GFP-tagged AR plasmids with the actin-depolymerizing agent cytochalasin D (Cyto D) and identified that there was no effect on the nuclear import of AR-V7 nor in that of AR-fl or AR-V567 (Figure 1—figure supplement 1F). AR-V7 nuclear import requires active transport via the NPC and is partially dependent on Ran-GTP activity Most nuclear import pathways involve the small GTPase Ran, which catalyzes the release of cargo protein from importin in the nucleus. As AR-V7 does not use importin-α/β pathway for nuclear import, we set out to determine whether it requires active transport via interaction with the nucleoporins, NPC components that mediate transport of proteins larger than 40 kDa. Thus, we incubated cells with wheat germ agglutinin (WGA), a well-established inhibitor of nucleoporin-mediated nuclear transport (Finlay et al., 1987; Whitehurst et al., 2002; Yoneda et al., 1987) and identified that WGA resulted in cytoplasmic sequestration of GFP-AR-V7 (Figure 2—figure supplement 1A), suggesting that interaction with nucleoporins is required for AR-V7 nuclear import. Next, to investigate whether AR-V7 depends on Ran-GTP for nuclear import, we quantified the nuclear fraction of GFP-tagged AR-fl, AR-v567, or AR-V7 proteins in the presence of the catalytic Ran-GTP mutant (mCherry-fused Ran Q69L; GTP hydrolysis deficient mutant). Our data identified that AR-fl and AR-v567 nuclear import was inhibited in the presence of the catalytic Ran-GTP mutant (Figure 2A); while AR-V7 nuclear import was partially inhibited, as evidenced by AR-V7 localization in both nucleus and cytoplasm (Figure 2A; solid arrows). Quantitation of the percent nuclear signal of each AR protein identified significant decrease in nuclear localization for all proteins in the presence of the catalytic mutant RanQ69L (Figure 2B). Similar results were observed when HEK293T cells were transiently co-transfected with GFP-AR-fl, GFP-AR-v567, or GFP-AR-V7 and mCherry-tagged Ran Q69L (Figure 2—figure supplement 1B and C). In addition, to confirm these results in cells expressing endogenous AR-fl, we generated C4-2 cells stably expressing inducible GFP-AR-V7 and examined its subcellular localization, following induction with doxycycline, in the presence of the catalytic mutant RanQ69L. We observed, using live-cell imaging, enhanced cytoplasmic localization of AR-V7 in cells co-expressing the Ran mutant, consistent with our earlier findings (Figure 2—figure supplement 1D). Interestingly, when the mutant Ran was introduced into the 22RV1 cells endogenously expressing both AR-fl and AR-V7, expression of AR-V7 appeared to be downregulated in the presence of the mutant Ran (Figure 2—figure supplement 1E). Figure 2 with 1 supplement see all Download asset Open asset 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. AR-V7 nuclear import is impaired upon mutation of the dimerization box domain (D-box) Androgen-regulated gene expression requires AR-fl receptor dimerization in the nucleus mediated by the zinc finger (D-box) domain, prior to DNA binding. AR-V7 transcriptional activity has been shown to depend on the D-box domain which mediates AR-V7 homodimerization in the nucleus (Centenera et al., 2008; Xu et al., 2015). To determine the potential impact of D-box domain on AR-V7 nuclear localization and transcriptional activity, we generated a construct encoding GFP-AR-V7 containing two functionally inactivating D-box mutations (A596T and S597T) and transiently transfected PC3 cells stably expressing the ARE-mCherry reporter (Azeem et al., 2017). Single-cell analysis showed nuclear GFP-AR-V7 was transcriptionally active as evidenced by concurrent mCherry-ARE expression. In contrast, the GFP-AR-V7-D-box mutant was both transcriptionally inactive and enriched in the cytoplasm compared to wild-type GFP-AR-V7 (29% in cytoplasmic AR-V7 vs. 71% in AR-V7-D-box mutant; p<0.0001) (Figure 2C-D). Interestingly, the same D-box mutations had no effect on AR-fl nuclear localization (Figure 2—figure supplement 1F) consistent with the role of D-box on AR-fl homodimerization in the nucleus. To expand these results to additional cell lines, we generated C4-2 cells harboring endogenous AR-fl, to stably express inducible GFP-AR-V7-D-Box mutant and quantified its subcellular localization by live-cell imaging. Our results indicated significantly enhanced cytoplasmic localization of the AR-V7-D-box mutant in comparison to its inducible wild-type counterpart (Figure 2E-F). Taken together, these results indicate that AR-V7 nuclear import partially requires an intact D-box domain, identifying a novel, variant-specific function for this conserved domain. AR variants drive ligand-independent fractional nuclear translocation of AR-fl with no evidence of heterodimerization In CRPC, AR-fl is often co-expressed with AR variants (AR-V) in patient tumors and it has been suggested that active AR signaling in castrate conditions is partially due to AR-V heterodimerization with AR-fl resulting in its nuclear translocation in the absence of ligand (Cao et al., 2014; Xu et al., 2015). To determine the effect of AR-V7 on AR-fl nuclear localization, we microinjected mCherry-AR-fl with GFP-AR-V7 in PC3 cells and quantified AR-fl nuclear accumulation across conditions (Figure 3A-B). Under basal conditions, we observed low AR-fl nuclear localization which was increased by twofold in the presence of AR-V7 in the absence of ligand (12% vs. 24%, p<0.001). Similar results were observed in PC3 cells and C4-2 cells following transient transfection with mCherry-AR-fl and GFP-AR-V7 (Figure 3—figure supplement 1A–1B). Interestingly, a similar pattern of enhanced AR-fl nuclear localization in the absence of ligand, was observed in the presence of GFP-AR-v567 (Figure 3—figure supplement 1C), suggesting that this interaction is not unique to AR-V7 and may apply to additional nuclear AR-variants. As AR-V7 is the most prevalent AR-V expressed in patient tumors, we sought to determine next whether enhanced nuclear localization of AR-fl in the presence of AR-V7 occurs in an androgen sensitive model of prostate cancer. To that end, we transfected LNCaP cells with mCherry-AR-fl and/or GFP-AR-V7. The percentage of nuclear AR-fl was numerically higher in the presence versus in the absence of AR-V7 co-expression (31% vs. 25%) (Figure 3C-D). Figure 3 with 1 supplement see all Download asset Open asset AR-V7 drives nuclear translocation of AR-fl in the absence of ligand. (A, B) Plasmids encoding mCherry-AR-fl or GFP-AR-V7 were micro-injected in PC3 cells. Representative microscopic images (scale bar, 10 μm) and % nuclear AR is shown. Data represent a box plot with n>10cells per condition, p value (***p<0.001) was obtained using unpaired two-tailed t-test. (C, D) LNCaP cells were transfected with mCherry-ARfl and/or GFP-AR-V7, and cells were treated 10 nM R1881 as indicated (scale bar, 5 μm). Data quantification is shown as a box plot and was obtained using unpaired t-test with n≥10 cells per condition. (E) PC3 cells were transfected with mCherry-AR-fl, or GFP-AR-V7, and cells were treated 10 nM R1881 or 50 μM Importazole, as indicated. Representative confocal microscopy images are shown with arrows pointing to nuclear AR-fl across conditions. Scale bar, 10 μm. Experiments were repeated at least twice. (F, G) C4-2 cells stably expressing inducible GFP-AR-V7 or GFP-AR-V7 D-Box mutant were induced by doxycycline and transfected with mCherry-AR-fl (scale bar, 5 μm). Representative images are shown, data quantification is shown as a box plot, and obtained using a Mann-Whitney test with n≥ 8 per condition. Mechanistically, we sought to determine whether AR-V7 might form a heterodimer with unliganded AR-fl in the cytoplasm driving the into the nucleus. Treatment with IPZ showed that when AR-V7 and AR-fl were co-expressed in the same IPZ inhibited the nuclear localization of AR-fl while it had no effect on AR-V7 (Figure consistent with our earlier results (Figure These results not cytoplasmic in agreement with published et al., Next, we sought to determine whether nuclear heterodimerization might the enhanced nuclear of AR-fl. As the D-box domain was shown to be partially in heterodimerization et al., we generated C4-2 cells stably expressing inducible AR-V7 or D-Box mutant, in addition to GFP-AR-V7 and transfected them with Our results show no in % nuclear AR-fl when co-expressed with either wild-type or D-box mutant AR-V7 (Figure Taken together, these results the presence of interaction between AR-fl and AR-V7 in the cytoplasm or nucleus as the underlying mechanism of enhanced unliganded nuclear AR-fl. To investigate whether AR-V7 transcriptional mediates the fractional AR-fl nuclear translocation, we introduced the domain mutation known to the transcriptional activity of canonical AR, into AR-V7 We that the mutation abrogated AR-V7 transcriptional activity, but not its nuclear localization, compared to AR-V7 (Figure This is evidenced by the decrease in ARE-mCherry reporter activity and the expression of the endogenous AR-V7 target the transcriptionally inactive mutant was co-expressed with AR-fl, we observed a similar increase in nuclear AR-fl in the presence of mutant or AR-V7 as compared to (31% vs. vs. p<0.0001) (Figure Figure Download asset Open asset mutation AR-V7 transcriptional activity (A, B) PC3 cells stably expressing ARE-mCherry reporter were transfected with the indicated plasmids for and the expression of protein with protein was analyzed by confocal imaging. Representative images and quantitative results are shown (n>10 cells per condition). Scale bar, 10 µm. (C) for the endogenous was quantified in PC3 cells after transfection of indicated Data with AR-fl are as a (D) PC3 cells were transfected with mCherry-AR-fl or GFP-AR-V7 or and by confocal microscopy (scale bar, 10 and % Nuclear AR protein was quantified cells per condition). Data represent mean ± SEM, p value was obtained using unpaired two-tailed t-test. Experiments were repeated at least twice. AR-V7 exhibits high subnuclear mobility kinetics and chromatin time It is well established that AR-fl is transcriptionally active and in the while AR-fl is and transcriptionally inactive et al., et al., 2005). AR-V7, on the other is transcriptionally active in the absence of the exact mechanism underlying its nuclear activity is not Thus, we the rate of AR-V7 with chromatin using and confocal live-cell imaging in cells transfected with GFP-tagged AR-V7 or AR-fl as a analysis of AR-fl identified fluorescence recovery of nuclear AR was significantly compared to unliganded AR s vs. 3 s, p<0.0001) (Figure suggesting chromatin time of AR, in agreement with on AR and other nuclear hormone including the and et al., et al., et al., In contrast, the fluorescence recovery of nuclear GFP-AR-V7 was very fast compared to AR-fl s and 8 s, p<0.0001) (Figure indicating that AR-V7 exhibits chromatin transcriptionally Figure 5 with 1 supplement see all Download asset Open asset AR-V7 exhibits high intranuclear mobility compared with liganded AR-fl. (A) was performed in PC3 cells transiently expressing GFP-AR-fl the absence or presence of 10 nM or GFP-AR-V7. was monitored at 1 s Representative images of cells at time are shown. (B) of proteins recovery after photobleaching at 1 s intervals are graphically (C) of half-time of recovery in for each condition, was performed in PC3 cells transiently expressing or protein. Cells were at 1 s intervals to fluorescence recovery of the proteins and nuclear of proteins Scale bar, 10 µm. Data represent mean ± SEM, p value was obtained using unpaired two-tailed t-test. Experiments were repeated at least twice. To the kinetics of subnuclear mobility of each protein, we generated or and followed them by live-cell imaging et al., 2015). of AR-fl in a small region, to a in the from to which was monitored by time-lapse imaging at 1 s intervals (Figure Our data identified that AR-fl remained within the of the fluorescence recovery by AR suggesting stable and chromatin binding (Figure the other AR-V7, in s the (Figure the nucleus in than This high intranuclear mobility of AR-V7 was by fluorescence recovery of (Figure These data reveal for the first time a between the chromatin rates and intranuclear mobility of AR-V7 and canonical AR-fl, To investigate potential nuclear of both proteins when co-expressed in the same we co-microinjected mCherry-AR-fl and GFP-AR-V7 in the nuclei of PC3 cells and monitored respective recovery after We observed a similar pattern of intranuclear kinetics for each protein, when co-expressed in the same as the kinetics observed when each protein was expressed with fast AR-V7 fluorescence recovery of AR-fl (Figure supplement DNA binding mutation AR-V7 and nuclear mobility kinetics To the between the high nuclear mobility of AR-V7 and its high transcriptional activity, we introduced the mutation into AR-V7 or AR-fl expression plasmids and introduced them into PC3 cells. We detected a significant increase in the mobility of the mutant compared to AR-fl, in the presence of ligand, with of s versus s, (Figure and Figure supplement was observed in the absence of ligand, suggesting that of the AR-fl is mediated by of AR-fl. we that the high intranuclear mobility of AR-V7 was accelerated by the mutation with recovery for at 2 s s for AR-V7, (Figure These data suggested that mediates the transient chromatin exhibited by AR-V7. Next, we examined the effect of the mutation on the transcriptional