Figure 1.
FRAP Analysis of Splicing Proteins in Different Subnuclear Compartments
FRAP experiments were performed on HeLa cells expressing GFP-tagged splicing proteins, as indicated.
(A) Images of representative cells. The circles illustrate bleach regions localized in the nucleoplasm (arrows, green circles), nuclear speckles (arrowheads, red circles), or CBs (cb, blue circle). Bar indicates 5 μm.
(B) FRAP recovery curves of indicated GFP-tagged splicing proteins in the nucleoplasm (green curves), nuclear speckles (red curves), and CBs (blue curve). The fluorescence intensity I was monitored over time (I was corrected for the background intensity and the amount of total fluorescence lost during the bleach and imaging). Each recovery curve corresponds to a pool of three independent experiments, with ten different cells analyzed per experiment. The recovery for the immobile protein GFP-coilin-PABPN1 [60] is shown for comparison (black curve).
(C) Experimental values obtained for the diffusion coefficient (D), apparent immobile fraction (I.F.), recovery time at 50% of initial fluorescence (t50%), and recovery time at 90% of initial fluorescence (t90%) in the speckles, CBs, and nucleoplasm.
Figure 2.
The Transcriptional Inhibitor DRB Induces a Reversible Accumulation of GFP-U2AF65 in Enlarged Nuclear Speckles
Steady-state distribution of GFP-U2AF65 in the nucleus of a HeLa cell after addition (A,B) or removal (D,E) of DRB.
(A,B) Depict the same cell imaged immediately after addition of DRB (A) and 35 min later (B).
(D,E) Depict the same cell imaged immediately after removal of DRB (D) and 35 min later (E). The arrows point to a nuclear speckle, the arrowheads to the nucleoplasm. Bar indicates 5 μm.
(G,H) Plot of the ratio between fluorescence intensities in the nuclear speckles (n = 6 speckles) and in the nucleoplasm over time after addition (G) or removal (H) of DRB. Error bars represent standard deviations.
(C,F) Depict the threshold segmentations of images in (A,B) and (D,E), revealing the outline of nuclear speckles in the presence (red outlines) and absence (white outlines) of DRB. The nuclear boundary is outlined in yellow.
(I) Quantification of the projected areas in (C,F) reveal an approximate 2-fold increase in speckles size (n = 25 speckles, 4 cells), when transcription is inhibited by DRB.
Figure 3.
Splicing Proteins Are More Mobile in Cells Treated with DRB
FRAP (A,B) and FLIP (D–F) experiments were performed on cells mock-treated (−DRB) or treated (+DRB) with DRB for 30 min. The GFP-tagged splicing proteins are indicated on each graph. For FRAP experiments, the bleached region was localized on a nuclear speckle. For FLIP experiments, the bleach region corresponded to half of the cell nucleus (dashed outline in (C), and the fluorescence decay was analyzed over a nuclear speckle. Each recovery or decay curve corresponds to a pool of three independent experiments, with ten different cells analyzed per experiment. Error bars represent standard deviations. The differences observed in the FLIP curves between DRB treated and untreated cells are statistically significant (p < 0.0001 for GFP-U2AF65 and GFP-U2AF35, and p < 0.005 for SF1).
Figure 4.
A Deletion Variant of Snurportin 1 Affects the Subnuclear Distribution of snRNPs
HeLa cells were transfected with either wt snurportin1 (wt SPN1) (A–D) or the deletion variants SPN1ΔN (E–H), and SPN1ΔC (I–L) fused to GFP. The cells were labeled with mAb Y12 directed against Sm proteins.
(A,E,I) Depict the superimposition of red (Y12 antibody staining) and green (GFP-tagged protein) images.
(B,C,F,G,J,K) Depict the same cell imaged for GFP-tagged protein and Y12 staining, as indicated; arrows point to CBs and arrowheads to nuclear speckles. Bar indicates 10 μm. (D,H,L) Show the western blot analysis of each fusion protein. Molecular weight markers (kDa) are indicated on the left.
Figure 5.
Expression of SPN1ΔN Induces Disassembly of Cajal Bodies and Enlargement of Speckles
HeLa cells were transfected with either wt snurportin1 (wt SPN1) (A,C,E,G) or the deletion variant SPN1ΔN (B,D,F,H). Cells were imaged for green, red, and blue fluorescence.
(A) Cell cotransfected with GFP-SmE (green) and CFP-wt SPN1 (blue). Arrows point to CBs.
(B) Cell cotransfected with GFP-SmE (green) and CFP-SPN1ΔN (blue). Superimposition of green and blue shows a perfect colocalization of SmE and SPN1ΔN in round and enlarged speckles.
(C) Cell transfected with CFP-wtSPN1 (blue) and immunolabeled with the antibody 4G3 directed against the U2 snRNP B′′ protein (red). Arrows point to CBs and arrowheads to nuclear speckles.
(D) Cell transfected with CFP-SPN1ΔN (blue) and immunolabeled with the antibody 4G3 (red). Superimposition of red and blue images shows colocalization of snRNPs and SPN1ΔN in enlarged speckles (arrowheads).
(E) Cell transfected with GFP-wtSPN1, double-labeled with antibodies directed against coilin (blue) and fibrillarin (red); arrows point to CBs and arrowheads to nucleoli.
(F) Cell transfected with GFP-SPN1ΔN, double-labeled with antibodies directed against coilin (blue) and fibrillarin (red). Note that coilin relocalized from CBs to the nucleolus.
(G) Cell transfected with GFP-wtSPN1, double-labeled with antibodies directed against coilin (blue) and SMN protein (red). Although SMN is detected both in the cytoplasm and in nuclear foci, the cytoplasmic staining is not well-visualized because this image was focused on CBs (arrows), which were in a confocal plane distinct from the cytoplasm. Arrowheads indicate additional minor coilin bodies that are apparently devoid of SMN.
(H) Cell transfected with GFP-SPN1ΔN, double-labeled with antibodies directed against coilin (blue) and SMN (red); arrowheads point to nucleoli that now accumulate coilin and arrows point to nuclear SMN foci. Bar indicates 10 μm.
Figure 6.
Expression of SPN1ΔN Affects the Mobility of Splicing Proteins in Living Cells
FLIP experiments were performed on cells expressing GFP-U2AF65 (A) and GFP-SmE (B) together with the wt (+wt SPN1, red curves) or the dominant-negative mutant (+SPN1ΔN, green curves) forms of snurportin1. The fluorescence decay was analyzed over nuclear speckles. The results obtained for GFP-U2AF65 and GFP-SmE in cells that were not transfected with any SPN1 construct are also shown (black curves). Each decay curve corresponds to a pool of three independent experiments, with ten different cells analyzed per experiment.
Figure 7.
Modeling Splicing Protein Kinetics in the Cell Nucleus
(A) The scheme illustrates the steady-state distribution of splicing proteins when splicing is either active (Splicing ON) or inactive (Splicing OFF). Splicing factors are represented in green and nuclear binding sites in grey. The lower row depicts positions inside the whole nucleus, and the upper row shows a zoom of the area delimited by the dashed square. The large grey circle in the upper row represents a nuclear speckle, and grey dots in the nucleoplasm represent intron-containing nascent transcripts. Note the larger number of splicing factors inside the nuclear speckle when splicing is inhibited. The parameters used for binding reactions were = 3.28 s−1, koff,nuc = 10 s−1,
= 0.045 s−1, and koff,spk = 0.066 s−1 (Splicing ON). To model splicing inhibition, we assumed a decrease in available nucleoplasmic binding sites; accordingly, the pseudo on-rate in the nucleoplasm was decreased by a factor of approximately 30,000,
= 10−4 s−1.
(B) Color-code representation of the concentration of splicing factors throughout the nucleoplasm and within nuclear speckles (pink squares define dimensions and number). The simulation space corresponding to the nucleus was divided into squares (25 × 25 pixels), and the number of splicing proteins inside each square was computed (Max is the maximum value obtained in the simulations). After splicing inhibition, the number of splicing proteins at nuclear speckles is higher. Consequently, there is an increase in the local concentration (the pale pink squares become dark red) as well as in the total area occupied by highly concentrated splicing proteins (pink/red squares); this closely mimics the enlarged nuclear speckles observed in HeLa cells treated with DRB (Figure 2B and 2D) or expressing SPN1ΔN (Figure 4G).
Figure 8.
Influence of and
on the Relative Concentration of Splicing Proteins in Nuclear Speckles
The number of splicing proteins in a speckle and in an equivalent nucleoplasmic area was counted and their ratio calculated.
(A) The graph plots the ratio as a function of . The ratio increases with decreasing
values, but stabilizes below a threshold of approximately 10−1.5 s−1.
(B) The graph plots the ratio as a function of . This ratio increases unbounded with increasing
values.
Figure 9.
(A) Schematic illustration of a simulated FLIP sequence. The green dots represent the positions of unbleached molecules, while the black dots represent the bleached ones. The area that was repeatedly bleached corresponds to the first and the fourth quadrant of the circle that defines the nucleus. The diffusion model was run for an initial 100 s to achieve a steady state, and then a cycle of repetitive bleaching events was started. Fluorescence was monitored in the unbleached portion of the circle, both in a nuclear speckle and in a nucleoplasmic region of the same size, and at the same distance from the bleached region.
(B) FLIP decay curves were generated by counting the number of fluorescent molecules inside the monitored regions at defined time intervals. For normalization, these values are divided by the number of fluorescent molecules in those regions immediately before bleaching. Normalized FLIP curves are then fitted by an exponential function of the form: f(t) = exp(−Kt), where K is the rate of fluorescence decay. Only FLIP decay curves in the nuclear speckles are depicted. The model parameters were as follows: = 0.045 s−1 , koff,spk = 0.066 s−1, koff,nuc = 10 s−1, and
= 3.28 s−1 (blue line) or 0.03 s−1 (red line). The decay is faster for the lower
.
(C) Plot of FLIP decay rates (K) as a function of . The decay is faster for lower
values and stabilizes below approximately 10−1.5 s−1, which implies at least a 100-fold reduction in the concentration of nucleoplasmic binding sites.
(D) The effect of increasing . FLIP decay curves in the speckles was obtained with the following parameters:
= 0.045 s−1 (blue line) or 0.5 s−1 (red line), koff,spk = 0.066 s−1, koff,nuc = 10 s−1, and
= 3.28 s−1. The decay is slower for the higher
.
(E) Plot of FLIP decay rates (K) as a function of . This decay is increasingly slower for the higher values of
.