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S100A13 and S100A6 exhibit distinct translocation pathways in endothelial cells

¹ Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstr. 75, CH-8032 Zurich, Switzerland
² Department of Biochemistry, 30, quai Ernest Ansermet, CH-1211 Genève 4, Switzerland

View larger version (36K): [in a new window]   Fig. 1. Purification of recombinant S100A13. (A) Recombinant S100A13 was purified using the pPROEX HTb expression system and Ni-NTA affinity chromatography. 20 µl of protein extract from transformed DH5 cells was loaded before (lane 1) and 3 hours after (lane 2) IPTG induction. Lane 3 shows the purified fusion protein S100A13 from the Ni-NTA column. Protein extract was loaded on a 14% SDS Tricine-PAGE under reducing condition, followed by Coomassie G-250 staining. (B) Purified S100A13 from the Ni-NTA column is shown in lane 1. The fusion protein was then digested by TEV protease to remove the histidine tag as shown in lane 2, and S100A13 purified by gel filtration is shown in lane 3.

View larger version (11K): [in a new window]   Fig. 2. Calcium binding to S100A13 and the effect of zinc. Flow dialysis at 25°C in 50 mM Tris HCl, pH 7.5, 150 mM KCl, with (circles; two different experiments) or without 100 µM zinc (triangles). Protein concentration is 25 µM. The lines connecting the symbols were generated using the Adair equation for four sites with the constants (translated to stoichiometric constants as described in the Materials and Methods) indicated in Table 1.

View larger version (112K): [in a new window]   Fig. 3. Subcellular localization of S100 proteins in endothelial cells (ECV). Several S100 proteins were found in ECV, and different subcellular distributions of the proteins was observed by staining with the appropriate antibodies. Rabbit anti-S100 antibodies were used at a 1:1000 dilution, and a goat Cy3-conjugated anti-rabbit antibody was used for visualization under the 40x oil objective lens in the Zeiss light microscope with a filter of 450 to 490 nm. Nuclear localization was observed in cells stained with S100A13 (A), S100A1 (C), S100A4 (E) and S100A5 (F) antibodies; perinuclear stainings were observed with S100A6 (B) and S100B (G) antibodies. A very faint staining was seen with the S100A3 antibody (D). H shows one example of background staining with pre-absorbed S100A6 antibody, and no obvious staining was observed. The same observation was made with S100A13 and the other pre-absorbed S100 antibodies, indicating the specificity of the antibodies used. Bar, 15 µm.

View larger version (17K): [in a new window]   Fig. 4. Western blot analysis of S100 proteins expressed in endothelial cells (ECV). 35 µg of total protein extract was loaded in each lane of a pre-cast 4-20% Tris-glycine gradient SDS PAGE gel and blotted onto the nitrocellulose membrane. Various rabbit anti-S100 antibodies were used as primary antibodies, and signals were detected by horseradish-peroxidase-conjugated goat anti-rabbit antibody and enhanced chemiluminescence. The following S100 proteins were identified in ECV — S100A13 (lane 1), S100A6 (lane 2), S100A1 (lane 3), S100A4 (lane 5), S100A5 (lane 6) and S100B (lane 7). S100A3 was not detected in this cell line as shown in lane 4. # indicates monomeric protein, and the dimeric and polymeric forms are seen above these.

View larger version (75K): [in a new window]   Fig. 5. Localization and translocation of endogenous S100A13 in endothelial cells (ECV). (A) S100A13 protein was mainly localized in the nucleus of the untreated cells. Similar nuclear localization of S100A13 was also observed in cells treated with brefeldin A (C) and bafilomycin A (E) before angiotensin II stimulation. By contrast, small vesicles at the cellular periphery (indicated by arrows) were evident after angiotensin II stimulation (B); and treatment with amlexanox (D) or demecolcine (F) before addition of angiotensin II had no effect on the translocation of the protein. Areas with a series of vesicles were enlarged by 20% and presented in the small squares at the right hand corners (B,D,F). The samples were analyzed by confocal microscopy. Bar, 15 µm (F).

View larger version (22K): [in a new window]   Fig. 8. Quantification of S100 protein translocation. Cells producing vesicles containing either S100A13 or S100A6 were counted and divided by the total cell counts (approximately 200 cells were counted for each experiment), and the columns represent the mean of three experiments. (A) Percentage of cells with S100A13 translocating in vesicles in response to angiotensin II alone or after treatment with the drugs indicated. * indicates a statistically significant difference between cells stimulated with angiotensin II alone and treated with brefeldin A or bafilomycin A (P<0.05). (B) Percentage of cells with S100A6 translocating in vesicles in response to angiotensin II and the drugs indicated. * indicates a statistically significant difference between cells treated with angiotensin II alone and with amlexanox (P<0.1). The P value was calculated by using Student-t-variant R. A. Fisher and F. Yates.

View larger version (102K): [in a new window]   Fig. 6. Localization and translocation of endogenous S100A6. (A) S100A6 was mainly distributed around the cell nuclei and in the cytoplasm of ECV cells; S100A6 translocated in the form of vesicles (indicated by arrows) in cells stimulated by angiotensin II (C); (B) Fewer cells with vesicle formation were observed after amlexanox treatment. However, S100A6 translocation in angiotensin-II-stimulated cells was not affected by brefeldin A (D) nor by demecolcine (E) or bafilomycin A (F). All the samples were analyzed by confocal microscopy. Bar, 15 µm.

View larger version (77K): [in a new window]   Fig. 7. Effects of the inhibitory drugs used to study the various translocation pathways. Cells were left untreated (A,C,E), incubated with brefeldin A (B), amlexanox (D) or demecolcin (F) and stained with mouse anti-ß-COP (A,B) and mouse anti-tubulin (E,F) antibodies. Phalloidin-TRITC was used to localize actin stress fibers (C,D). The scale bar in B applies for both A and B; the scale bar in D applies for C and D; and the scale bar in F applies for E and F. All the scale bars are 15 µm.

View larger version (37K): [in a new window]   Fig. 9. S100A13 and S100A6 are contained in distinct vesicles. (A) S100A13 distribution in cells stimulated by angiotensin II and visualized by a Cy2-conjugated anti-goat antibody. (B) S100A6 distribution in cells stimulated by angiotensin II and visualized by Cy3-conjugated anti-rabbit antibody. (C) A superimposed image of A and B. The sample was analyzed by confocal microscopy. Bar, 15 µm. Arrows indicate vesicles containing S100A13 and arrowheads indicate vesicles with S100A6.

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