Figure 1.
(A) Representative confocal images show complete nuclear translocation of YFP-GR after 3 hours of 1 µM dexamethasone treatment. (B) A representative CCD image of single molecules of YFP-GR after background subtraction shows two discernible Gaussian peaks of YFP fluorescence. (C) Regime for single molecule kinetics; images are taken with a time lag of 6.25 ms or 12.5 ms in 300 series of 8 per cell. In background-subtracted images, single molecules of YFP fluorescence are easily discernible. (D). PICS analysis of single molecule displacements, shown for dexamethasone-bound YFP-GR at time delay of 6.25 ms. The cumulative probability distribution as a function of the squared distance l (black line) is best fitted with a 2-population model (red dashed line), while a 1-population model gives a suboptimal fit (blue line) (n = 20 cells). (E) FRAP procedure of dexamethasone-bound YFP-GR. At t = 0 s a 100 ms bleach pulse is applied to a strip spanning the nucleus. Subsequently, FRAP recovery curves of 30 cells are recorded, combined and adjusted to baseline fluorescence (black line). Subsequently, Monte Carlo simulations are generated using a 3-population model and fitted to the combined FRAP curve. The top 10 fits are combined (red line) and show a good fit of the experimental data with small residuals (blue line).
Figure 2.
SMM and FRAP analyses provide a consistent model of the intranuclear mobility of the GR.
(A) A two-population fit of SMM analysis for dexamethasone-bound YFP-GR identifies two fractions of approximately equal size. (B) Both fractions show a linear increase in mean squared displacement (MSD) over time, but with a 40-fold difference in MSD. Diffusion coefficients (Dfast and Dslow) are calculated from a linear fit of the experimental data (dashed lines; D = slope/4). The Dfast of 1.31 µm2/s fits to diffusing molecules, while the Dslow of only 0.03 µm2/s best fits to the slow movement of chromatin and the molecules bound to it. (C) A 3-population Monte Carlo simulation of the FRAP curve for dexamethasone-bound YFP-GR shows that half of the nuclear population is diffusing, while the remainder is subdivided into two bound fractions that differ in their immobilization times. The fraction size of the diffusing fraction is similar in size as that obtained from SMM analysis. (D) Both bound fractions are only transiently immobilized, with a 3-fold difference in duration. (A and B) Data represented as best fit ± SEM (of 3 separate PICS analyses). (C and D) Data represented as average of top 10% best fits ± SEM.
Table 1.
Summary of all SMM and FRAP analyses of YFP-GR, YFP-MR and YFP-GR deletion mutants.
Figure 3.
Ligand structure determines the nuclear mobility of the GR.
A range of natural and synthetic agonists (black bars) and an antagonists (red bar) were tested for their effect on the intranuclear mobility of the GR by both SMM (A) and FRAP (B–C) analysis. Multiple structural elements of the steroids are associated with a reduced mobility of the receptor. Altered mobility can be reflected in all aspects of mobility: a larger bound fraction (SMM; white bars and FRAP; white and light grey bars combined) a lower diffusion coefficient (in µm2/s, written in its corresponding bar in A) and longer immobilization times (C). (D and E) A mutation of phenylalanine 623 to alanine (F623A) prevents interactions of the 9-fluoro group of steroids within the ligand binding pocket of the GR. F623A YFP-GR still translocates completely to the nucleus after 3 hours of 1 µM prednisolone or Δ-fludrocortisone treatment (D). SMM analyses of nuclear F623A YFP-GR kinetics shows that the mobility of F623A YFP-GR is highly similar after either Δ-fludrocortisone or prednisolone treatment (black bars for the diffusing fraction, with their corresponding diffusion coefficient (in µm2/s) written within their corresponding bar; (E)). SMM: n = 20, FRAP: n = 30. Data represented as total fit ± SEM (of 3 separate PICS analyses) for SMM and as average of top 10% fits ± SEM for FRAP. Δ-flu; Δ-fludrocortisone, dex; dexamethasone, Predn; prednisolone, csol; cortisol, cort; corticosterone. The data for GR-dexamethasone is the same as in Figure 2.
Figure 4.
Ligand structure determines the nuclear mobility of the MR.
A range of natural and synthetic agonists (black bars) and antagonists (red bars) were tested for their effect on the intranuclear mobility of the MR by both SMM (A) and FRAP (B–C) analysis. The MR and GR share several agonists, but the binding and functional characteristics differ. Indeed, a weak agonist for the GR, corticosterone (cort), which gave a very mobile GR, instead leads to a low mobility for the MR. A large bound fraction (SMM; white bars and FRAP; white and light grey bars combined) a low diffusion coefficient (in µm2/s, written within its corresponding bar in A) and long immobilization times (C). Of all functional steroid side groups, only the 18-keto (18 = O) group appears to affect the mobility of the MR. SMM: n = 20, FRAP: n = 30. Data represented as total fit ± SEM (of 3 separate PICS analyses) for SMM and as average of top 10% fits ± SEM for FRAP. Aldo; aldosterone, csol; cortisol, DOC; deoxycorticosterone, dex; dexamethasone, epler; eplerenone, spiro; spironolactone.
Figure 5.
Loss of either the DNA-binding or the ligand-binding domain results in a high GR mobility.
(A) Schematic representation of three functional YFP-GR deletion mutants tested. (B and C) Fraction distributions as analyzed by SMM (B) and FRAP (C). Diffusion coefficients are written within the corresponding bars in B (in µm2/s). (D) Immobilization times of both bound fractions in FRAP. While loss of the AF-1 domain hardly affects GR's nuclear mobility, deletion of the DBD and especially the LBD leads to a very mobile receptor with reduced frequency and average duration of DNA-binding and a higher diffusion coefficient. SMM: n = 20, FRAP: n = 30. Data represented as total fit ± SEM (of 3 separate PICS analyses) for B and as average of top 10% fits ± SEM for C and D. The data for wild type GR is the same as in Figure 3.
Figure 6.
A similar pattern of YFP-GR's nuclear mobility in stably transfected Hep3B cells.
SMM analysis of YFP-GR's nuclear mobility after treatment (3–6 h with 1 µM) with either dexamethasone or corticosterone was performed in Hep3B cells stably transfected with YFP-GR. These experiments were performed to check for effects of differences in cellular context and a lower level of YFP-GR expression on the mobility patterns. Both the size of the diffusing fraction (filled bars) and the diffusion coefficients (written in their corresponding bars in µm/s2) were highly similar between COS-1 and Hep3B cells. COS-1 data is the same as in Figure 2. All groups: n = 20. Data is represented as total fit ± SEM (of 3 separate PICS analyses).