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First published online 3 May 2005
doi: 10.1242/jcs.02345

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Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

¹ Molecular Mechanisms of Biological Regulation, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
² Department of Anatomy, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
³ Department of Internal Medicine, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
⁴ Department of Environmental Sciences, Faculty of Science, Osaka Women's University, 2-1 Daisen-cho, Sakai 590-0035, Japan
⁵ Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
⁶ Department of Developmental Anatomy, Osaka Prefecture College of Health Sciences, 3-7-30 Habikino, Habikino, Osaka 583-8555, Japan

View larger version (111K): [in a new window]   Fig. 1. S1 fiber formation in ARL cells at various states of confluency. ARL cells were cultured at a low cell density for 3 days (A) or in a confluent state for 3 weeks (B), and stained with anti-S1 protein antibody McAb 351 (A1, B1), monoclonal anti-vimentin antibody V9 (B2), or double-stained with McAb 351 (A2) and a polyclonal anti-vimentin antibody (A3). At low cell densities, S1 proteins are present not only in the nucleus, but also in the cytoplasm in association with VFs. (C) ARL cells were cultured for 3 days to 40% confluency (lane 1) and for 2 weeks at a full confluency (lane 2). Total proteins equivalent to 0.2 µg cellular DNA were analyzed by immunoblotting using McAb 351. Lane 3, reference S1 proteins isolated from rat liver nuclei. Bar, 25 µm.

View larger version (55K): [in a new window]   Fig. 2. Absence of S1 fibers in vimentin-deficient cells. Vimentin-deficient tTA-1 mouse fibroblasts (–/–), and control vimentin (+/+) MFT-6 fibroblasts were double-stained with anti-S1 protein antibody McAb 351 (left) and an anti-vimentin antibody (middle). Images were obtained by confocal microscopy. Merged images are shown on the right.

View larger version (39K): [in a new window]   Fig. 3. Protein crosslinking experiment. Proteins in ARL cells were crosslinked with DTBP and resolved, without prior reduction of disulfide bonds by SDS-PAGE (1D). The sample-lane was excised, incubated with DTT to cleave DTBP and subjected to the second dimension SDS-PAGE (2D). Blotted proteins were stained with Coomassie Brilliant Blue (A), destained and probed with McAb 351 (B) and finally reprobed with an anti-vimentin antibody (C). Non-crosslinked proteins form a curved diagonal in 2D (A); S1 proteins C2 and D2 (B) and vimentin (C) are seen on horizontal lines at the monomer sizes: they were crosslinked to the products of various sizes. Their non-crosslinked forms are seen on the left as strongly stained spots.

View larger version (32K): [in a new window]   Fig. 4. Immunoprecipitation of vimentin with McAb 351. ARL cell extract was immunoprecipitated with control Tris-buffered saline (lane 1), control antibody I-65 against toxoplasma membrane (lane 2), control 3F7 against an RNA-binding protein RBP-MS (lane 3) or McAb 351 against S1 proteins C2 and D2 (lane 4) and the precipitated proteins were immunoblotted for vimentin. Lane 5, reference total proteins from ARL cells. Positions of molecular weight protein markers (M) are indicated on the right-hand side of the blot.

View larger version (53K): [in a new window]   Fig. 5. S1 fibers are unrelated to cell proliferation and cellular metabolic activity. (A) RNA and DNA synthesis activities. ARL cells cultured to a 50-60% confluency (low density cells) or for 1 and 3 weeks at full confluency were incubated for 45 minutes in a fresh medium containing [3H]uridine or [3H]thymidine. Incorporated radioactivities were determined with respect to DNA, and compared with the low density cell values (100%). (B) ARL cells at about 20% confluency were further incubated for 3 days in the presence or absence of 20 µg/ml aphidicolin (aphid) or 6 mM hydroxyurea (HU) and stained with McAb 351. (C) ARL cells grown in complete medium were rinsed three times with serum-free medium, and further incubated for 75 hours in the presence (+) or absence (–) of 10% fetal calf serum. Cells were stained with McAb 351. Bar, 50 µm.

View larger version (91K): [in a new window]   Fig. 6. Scratch-wound experiments. The center areas of 3-week confluent monolayers of ARL cells grown on coverslips were removed by scratching with a pipette tip, and the incubation was continued in fresh medium for 30 hours. Cells were stained with McAb 351 (A), or double-stained with McAb 351 (B1) and an anti-vimentin antibody (B2). (C) After scratching, ARL cells were further incubated in the presence or absence of aphidicolin (20 µg/ml) for 30 hours. Cells were stained with McAb 351. In all panels, right-hand areas correspond to the marginal regions of cleared areas made by scratching. Bar, 50 µm.

View larger version (65K): [in a new window]   Fig. 7. S1 fibers in animal tissues. (A) A section of the cerebellum from an adult rat was double-stained with an anti-vimentin antibody (a, red) and McAb 351 (b, green). Purkinje cells are seen on the diagonal, and the granule cells on the right-hand side. Bar, 50 µm. (B) White blood cells from an adult rat were incubated at 37°C for 20 minutes between a slide glass and a sheet of Saran wrap in the presence (2-4) or absence (1, 5) of fMLP. The cells were stained with McAb 351 (a), DAPI (1b-3b), and an anti-vimentin antibody (4b, 5b). Panels 1, monocytes; panels 2-5, neutrophils. Bar, 20 µm.

View larger version (108K): [in a new window]   Fig. 8. Appearance of S1 proteins in the cytoplasm of migratory fibroblasts. (A-H) Fibroblasts in the healing tissue of gastric ulcer (left panels) and those in the sham-operated intact tissue of the stomach (right panels) were stained with McAb 351 (A-D) and with an anti-vimentin antibody (E-H). Nuclei (arrows) were counterstained with Methyl Green. Spindle-shaped fibroblasts were cut along the short (A,B,E,F) and long axes (C,D,G,H). In addition to the nucleus, the cytoplasm was stained with McAb 351 in ulcerated healing tissue (A,C compare with B,D). Vimentin was present in the cytoplasm of both ulcerated (E,G) and intact (F,H) tissues, with more vimentin in the former. (I) Vimentin mRNA levels during gastric ulcer healing. Total RNA from gastric tissues on day 5 after ulcer formation was analyzed by Northern blotting for vimentin and GAPDH. One representative autoradiogram of six determinations is shown. Bar, 12.5 µm.

View larger version (108K): [in a new window]   Fig. 9. Effects of actinomycin D and okadaic acid on S1 fiber formation. (A) ARL cells grown to 65% confluency were incubated in the presence of actinomycin D (5 µg/ml) for up to 9 hours and immunostained with McAb 351. (B) ARL cells cultured for 2 weeks at full confluency were treated with 50 nM okadaic acid (+OA) for 3 hours, and immunostained with McAb 351. Arrowheads indicate S1 fibers. Bar, 25 µm.

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