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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01544

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Protein 4.1R regulates interphase microtubule organization at the centrosome

¹ Departamento de Biología Molecular, Centro de Biología Molecular `Severo Ochoa' (UAM/CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain
² European Molecular Biology Laboratory (EMBL), Cell Biology and Cell Biophysics Programme, 69117 Heidelberg, Germany

View larger version (65K): [in a new window]   Fig. 1. Effect of exogenous 4.1R expression on interphase tubulin and actin cytoskeletons in COS-7 cells. (A) Schematic representation of the exon map for the 4.1R protein (top) and the cDNA constructs used in the transfection experiments shown in B,C (bottom). Exons are coded as follows: striped, alternative; white, constitutive; black, noncoding. The number of each exon is shown at the bottom of the most upstream scheme. Three translation-initiation sites at exons 2' (ATG-1), 4 (ATG-2) and 8 (ATG-3) are indicated, as is the stop codon (TGA) at exon 21. (B) Conventional fluorescence micrographs showing COS-7 cells transfected with the indicated 4.1R-encoding cDNAs and subjected to double-immunofluorescence analysis with anti-tag 9E10 (left), and anti-tubulin YL1/2 (right) antibodies. (C) Cells were transfected with 4.1R6016,18-encoding cDNA and triple stained with anti-tag 9E10 (top) and anti-tubulin YL1/2 (middle) antibodies, and with Alexa-Fluor-594/phalloidin (bottom). Projections of complete x-y optical section stacks were acquired by confocal microscopy. Arrows indicate transfected cells. Bar, 20 µm.

View larger version (75K): [in a new window]   Fig. 2. Effect of exogenous 4.1R expression on the distribution of the microtubule nucleating proteins -tubulin and pericentrin. Cells were triple labeled to detect exogenous 4.1R6016,18, tubulin and -tubulin (top) or exogenous 4.1R6016,18, tubulin and pericentrin (bottom). Arrows and asterisks indicate transfected and untransfected cells, respectively. Images are projections of x-y optical section stacks acquired by confocal microscopy. Bar, 20 µm.

View larger version (78K): [in a new window]   Fig. 3. Effects of exogenous 4.1R expression on centrosomal proteins of the dynein-dynactin complex and on centrosomal protein 4.1R. (A) Projection of optical section stacks obtained by confocal microscopy of cells triple labeled to detect exogenous 4.1R6016,18 (blue), p150Glued or dynein (green) and -tubulin or -tubulin (red). Arrows and asterisks indicate transfected and untransfected cells, respectively. The insets are enlargements showing the distribution of each protein at the centrosome in the transfected cells. Notice that the transfected cells have altered distributions of p150Glued and dynein. (B) Detergent lysates of cells transfected with empty plasmid (Ct) or with 4.1R6016,18/GFP-encoding cDNA (Tr) were sedimented into 5-20% sucrose gradients as described (Echeverri et al., 1996). Fractions were analysed by immunoblotting using antibodies to p150Glued (p150Glued), to dynein intermediate chain (dynein) and to the control proteins actin (actin) and -tubulin (-tubulin). Notice that, in fraction 1 isolated from transfected cells (F1, arrow), p150 and dynein intermediate chain proteins are present, whereas they are absent from fraction 1 in control cells. F1 from transfected cells (Tr) also contains endogenous 4.1R (Endogenous 4.1R) and exogenous 4.1R (Exogenous 4.1R). The sucrose gradient, the fractions collected and the position of sedimentation of the standard proteins, bovine serum albumin (4.4S), catalase (11.3S) and thyroglobulin (19S) are all indicated at the top. (C) Effect on endogenous centrosomal 4.1R. Cells were triple stained to detect endogenous and exogenous 4.1R (4.1R), tubulin (tubulin) and -tubulin (-tubulin). The images are projections of optical-section stacks acquired by confocal microscopy and insets are enlargements showing the distributions of each protein at the centrosome in a transfected cell. Notice that -tubulin is detected as a pair of foci, whereas centrosomal endogenous 4.1R is absent. By contrast, untransfected cells contain both centrosomal 4.1R and -tubulin (asterisks). Bar, 20 µm.

View larger version (69K): [in a new window]   Fig. 4. Effects of exogenous 4.1R expression on microtubule nucleation and retention at the centrosome. Cells were treated with nocodazole to promote microtubule disassembly and then washed to allow microtubule regrowth. At the indicated times, untransfected (A) and transfected (B) cells were fixed and triple labeled with the following antibodies: YL1/2 to detect microtubules (tubulin); GTU-88 to detect -tubulin (-tubulin); and 10b to detect endogenous and exogenous 4.1R (4.1R). Notice that, in cells with disorganized microtubules, -tubulin remains at the centrosome, whereas centrosomal 4.1R does not. The images of untransfected (A) and transfected (B) cells were taken from the same field. The images are projections of optical-section stacks acquired by confocal microscopy and insets are enlargements showing the distribution of each protein at the centrosome. Bar, 20 µm.

View larger version (52K): [in a new window]   Fig. 5. Endogenous 4.1R is present in isolated centrosomes. (A) Centrosomes isolated from Molt-4 T cells were subjected to double immunofluorescence with anti-4.1R (4.1R) and anti--tubulin (-tubulin) or anti--tubulin (-tubulin) antibodies. The samples were analysed by epifluorescence microscopy. (B) Western-blot analyses of isolated centrosomes were carried out with anti-4.1R (4.1R), anti--tubulin (-tubulin) and anti-actin (actin) antibodies. (C) In-vitro-assembled microtubule asters were triple stained with anti--tubulin (-tubulin), anti-4.1R (4.1R) and anti--tubulin (-tubulin) antibodies. The images are projections of optical-section stacks acquired by confocal microscopy. Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 6. Effect of recombinant protein GST-4.1R6016,18 on microtubule aster formation. The isolated centrosomes were preincubated with buffer (Ct), GST (GST), GST-4.1R8016 (GST-4.1R80) or GST-4.1R6016,18 (GST-4.1R60) for 45 minutes at 4°C and then tubulin was added to start in-vitro microtubule-aster formation. The microtubule asters stained with anti--tubulin antibody are shown (red). A representative aster (boxed field in the GST-4.1R60 panel) double stained with anti--tubulin (red) and anti-GST (green) antibodies is shown in the enlargements. The recombinant GST-4.1R60 protein is incorporated into the center of the asters and along the microtubules (merge). Four experiments were performed in duplicate. The results are shown in the histogram as the mean ± the standard deviation of the number of microtubule asters assembled in vitro. The images are projections of optical-section stacks acquired by confocal microscopy. Bar, 10 µm.

View larger version (38K): [in a new window]   Fig. 7. Protein 4.1R associates with - and -tubulin. (A) Coomassie-stained gels showing the purified (P) GST fusion proteins used in the binding assays and the eluates (E) from glutathione beads coupled with the indicated GST fusion proteins and incubated with COS-7 cell extracts. (B) Immunoblots of the eluates revealed with antibodies against - and -tubulin. Positions of molecular-weight markers are indicated on the left.

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