FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles

Abstract

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B1) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)-containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.

Figure 1. Dynamics of VDRB1 in living cells

(A) Transiently transfected COS-1 cells expressing VDRB1–EGFP or EGFP in Phenol Red-free medium were visualized by CLSM in the absence or presence of serum (FBS, containing ligand, see the Materials and methods section) as indicated. (B) Quantitative data (n=5) for F_n/c (=F_n/F_c, the ratio of the specific nucleoplasmic fluorescence F_n to the cytoplasmic fluorescence F_c) and F_sp/c (=F_sp/F_c, the ratio of the specific fluorescence associated with the nuclear speckles F_sp compared with F_c) in the absence (1.5 h) or presence of serum for VDRB1–EGFP- or EGFP-expressing cells. Significant differences (*P<0.05) were observed between untreated (−FBS) and serum treated (+FBS) cells (Student's t test). (C) Long-term imaging of VDRB1–EGFP-expressing cells as above in the absence of serum. Scale bar, 20 μm. (D) Quantitative data from (C) showing the dynamics of VDRB1–EGFP nucleoplasmic and nuclear speckle (three separate structures) localization.

Figure 2. FRAP analysis of the import of VDRB1–EGFP from the cytoplasm in transiently transfected untreated COS-1 cells and COS-1 cells treated with serum

(A) Transiently transfected COS-1 cells expressing VDRB1–EGFP or EGFP were bleached in the nucleus for 10 s (area indicated by the white box), and nuclear import was visualized at 10 s intervals over 400 s by CLSM to assess the return of fluorescence. Movie representations of these experiments [Fig2i.avi and Fig2ii.avi for VDRB1 in the presence or absence (1–2 h) of serum respectively; and Fig2iii.avi and Fig2iv.avi for EGFP in the presence or absence of serum respectively] are available at http://www.med.monash.edu.au/biochem/research/projects/nuclearsig/realtimemoviesVDRB1.html. Scale bar, 20 μm. (B) Quantitative results from (A) for fluorescence recovery, fitted according to the formulas F_n/c=(F_n/cmax)(1–e^−kt) or F_sp/c=(F_sp/cmax)(1–e^−kt), where k is the rate constant and t is time in seconds (30 s). The t_1/2 is indicated. (C) Pooled data (n=5 cells) for the fractional recovery of nuclear fluorescence after photobleaching (left panel) and t_1/2 (right panel) were determined as in (B), where significant differences (*P<0.05) were observed between the values for the return of nuclear VDRB1 for serum-treated cells (+) compared with untreated cells (−).

Figure 3. FRAP analysis of the export of VDRB1–EGFP from the nucleus in transiently transfected COS-1 cells treated with serum

(A) Transiently transfected COS-1 cells expressing VDRB1–EGFP or EGFP were bleached in the cytoplasm for 20 s (area indicated by white box), and export from the nucleus visualized at 10 s intervals over 400 s by CLSM. Movie representations of these experiments (Fig3i.avi and Fig3ii.avi for VDRB1 and EGFP respectively) are available at http://www.med.monash.edu.au/biochem/research/projects/nuclearsig/realtimemoviesVDRB1.html. Scale bar, 20 μm. (B) Quantitative analysis from (A) for specific nuclear and cytoplasmic fluorescence fitted exponentially [29]. (C) Pooled data of the extent of nuclear retention (left panel) and fractional recovery of cytoplasmic fluorescence (right panel) after cytoplasmic bleaching, and determined as in (B), where significant differences (*P<0.05) were observed between the values for VDRB1 and EGFP.