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First published online 8 August 2006
doi: 10.1242/jcs.03144

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ATP, phosphorylation and transcription regulate the mobility of plant splicing factors

Department of Biology and Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA

View larger version (51K): [in a new window]   Fig. 1. FRAP analyses of GFP-SR45. (A-D) Nuclei of root epidermal cells expressing GFP-SR45 or GFP alone were imaged before bleaching. A nucleoplasmic area (indicated by a circle) or a speckle (indicated by an arrow) were bleached and, following bleaching, images were taken for 60 seconds during recovery. Confocal images of a (A) nucleoplasmic area and (B) speckles before and after bleaching. (C) Images of fixed GFP-SR45, indicating no recovery of fluorescence. (D) Images of GFP alone; bleaching of the indicated area does not result in a well-defined bleached zone, indicating the extremely fast mobility of GFP alone. Time is given in seconds (A-C) or milliseconds (D). Bars, 5 µm. The entire dataset of images of A-D is animated in Movie 1, supplementary material. (E) Recovery kinetics of nucleoplasmic areas, speckles and fixed cells of GFP-SR45, and GFP alone. Data were normalized as described in Materials and Methods. Error bars are ± s.e.m.

View larger version (49K): [in a new window]   Fig. 2. FRAP and FLIP analyses reveal that the mobility of SR45 and SR1/SRp34 depends upon ATP. (A-D,F-I,K-L) A speckle (arrowhead) or a defined nucleoplasmic area (circle) in control or ATP-depleted cells was imaged before bleaching and during recovery for the time indicated. (A-D,F-I) Subsets of confocal images of a speckle (arrows in F) in ATP-depleted cells (A) SR45 and (F) SR1/SRp34, and control cells (B) SR45 and (G) SR1/SRp34. The entire set of time-lapse images of SR45 is animated in Movie 2, supplementary material. Subsets of confocal images of a nucleoplasmic area (circle) in ATP-depleted cells (C) SR45 and (H) SR1/SRp34, and control cells (D) SR45 and (I) SR1/SRp34. The entire time-lapse series of images for SR45 is animated in Movie 3, supplementary material. Bars, 5 µm. (E,J) Quantification of recovery kinetics of a speckle or a nucleoplasmic area in ATP-depleted cells of (E) SR45 and (J) SR1/SRp34. Curves shown are averages of at least ten nuclei in three different experiments. Error bars are ± s.e.m. (K,L) Subset of images of FLIP analyses of SR45. In each panel two adjacent nuclei of GFP-SR45 cells, either (K) untreated or (L) treated with NaN3 and 2-deoxyglucose are shown. A defined area (circle) in the lower nucleus of each panel was repeatedly bleached and photographed at the indicated times. The upper nucleus was not bleached and served as a reference for the normalization of fluorescence intensities. Very little loss of fluorescence in the unbleached zone of ATP-depleted nuclei indicates that the movement of GFP-SR45 is dependent upon ATP. The entire dataset of K and L is animated in Movie 4, supplementary material. (M) Quantitative FLIP analyses of the control and ATP-depleted GFP-SR45. Data presented are representative of five nuclei.

View larger version (68K): [in a new window]   Fig. 3. FRAP analyses of SR45 and SR1/SRp34 movement in cells treated with inhibitors of transcription or protein kinases. (A-C,E-G) Subsets of confocal images of epidermal nuclei treated with actinomycin D (A) SR45 and (E) SR1/SRp34, staurosporine (B) SR45 and (F) SR1/SRp34), and control (C) SR45 and (G) SR1/SRp34. A defined area or speckle (marked by a circle or arrow, respectively) was imaged before and after bleaching. The entire data set of A-C was animated and is presented in Movie 5, supplementary material. (D,H) Recovery kinetics of SR45 (D) and SR1/SRp34 (H) in nuclei not treated (control) or treated with actinomycin D or staurosporine. Curves are averages of at least ten nuclei of two experiments. Error bars are ± s.e.m. (I) Diffusion coefficients of SR45 and SR1/SRp34 in control and treated cells. Values are the mean ± s.e.m. of at least ten cells.

View larger version (49K): [in a new window]   Fig. 4. Localization of full-length SR45 and deletion mutants of SR45 fused to GFP. (A) Schematic diagram of the GFP-tagged SR45 deletion mutants. RS1 and RS2, arginine-serine-rich domain 1 and domain 2, respectively; RRM, RNA-recognition motif. Asterisks on full-length GFP model indicate location of predicted nuclear localization signals (NLS). Table on the right side summarizes the subcellular and subnuclear localization of GFP-tagged full length and deletion mutants of SR45. N, Nuclear; Cyt, Cytoplasmic; Nucleo, Nucleoplasmic; Spec, Speckle. (B) Arabidopsis mesophyll protoplasts were transiently transfected with vectors expressing the indicated GFP-tagged SR45 mutant and examined by confocal microscopy. Shown are single confocal optical sections. Fourth column of panels displays the expression of GFP-fusions in several protoplasts in a single-view field at low magnification (40x); bar, 100 µm. First, second and third column of panels show fluorescence images, chlorophyll autofluorescence images and their respective merged images (as indicated above each panel); bars, 10 µm. As expected, full-length SR45 localized in a speckled pattern in the nucleus. The rest of the mutants displayed a range of distributions, summarized in A.

View larger version (51K): [in a new window]   Fig. 5. Contribution of various domains to SR45 mobility and subcellular localization under control conditions, and after treatment with various inhibitors. (A-E) Optical sections from representative FRAP images of protoplasts expressing a deletion mutant. Circles indicate regions that were bleached (A) RS1; (B) RRM; (C) RS2; (D) RS1+RRM; (E) RRM+RS2. Bars, 10 µm. Quantitative FRAP analyses of the SR45-deletion mutants are shown to the right of each set of images. Error bars are ± s.e.m. of at least ten cells. Inset in C shows final recovery levels. Curves in red lines give the theoretical curves a protein of the molecular mass equivalent to the GFP-fusion protein would follow in the absence of any binding (see Materials and Methods). Curves in black give the best-fit curves to the experimental data of an exponential equation determined by the least-square method.