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First published online 2 March 2004
doi: 10.1242/jcs.00987

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ß₃ integrin phosphorylation is essential for Arp3 organization into leukocyte _Vß₃-vitronectin adhesion contacts

¹ Department of Cell and Developmental Biology SUNY Upstate Medical University, 750 East Adams St, Syracuse, NY 13210, USA
² Biosource International, Hopkinton, MA 01748, USA

View larger version (21K): [in a new window]   Fig. 1. Inhibition of adhesion by ß3 cytoplasmic tail peptido-mimetics. KVß3 WT or CSß3 (A) and murine BMM or human MDM (B) cells were adhered to vitronectin in the presence (PMA) or absence (control) of PMA (10 ng/ml), and either anti-ß3 antibody (5 µg/mL), TAT-Yp (10 mM) or TAT-Y/F (10 mM) fusion peptides for 1 hour at 37°C as described in Materials and Methods. Data represent the mean±s.d. from triplicate wells from three separate experiments, presented as percent adhesion. (C) K562 cells expressing wild-type, Y747F, Y759F or Y747,759F mutant Vß3 were incubated without (untreated) or with MnCl (2 mM) and sodium pervanadate (100 µM) for 10 minutes at 37°C. Whole cell lysates and ß3 immunoprecipitates were separated by SDS-PAGE and western blotted with anti-ß3 Y747 PSSA and HRP-conjugated secondary antibody or secondary alone (2° alone).

View larger version (84K): [in a new window]   Fig. 2. Diminishing ß3 Y747 PSSA staining during adhesion. KVß3 WT (A-C,E-G,I,J) and KVß3 Y747,759F (D,H) cells were adhered to vitronectin-coated coverslips in the presence of PMA (10 ng/ml) for 10 (A,D,E,H,I) 30 (B,F) or 70 (C,G,J) minutes. Cells were stained with rhodamine phalloidin to detect actin organization (A-D) and also ß3 Y747 PSSA with FITC-conjugated secondary antibody (E-H) as described in Materials and Methods. I(1-5) and J(1-5) show replicate merged images of KVß3 WT cells following adhesion for 10 (I) or 70 (J) minutes followed by costaining with rhodamine phalloidin (red) and ß3 Y747 PSSA (green). Bar, 5 µm.

View larger version (61K): [in a new window]   Fig. 3. Sustained ß3 Y747 phosphorylation during ROCK inhibition. KVß3 WT cells were adhered to vitronectin-coated coverslips in the presence of Rho kinase (ROCK) inhibitor, Y27632 (10 µM) (A-C) or Y27632 and PMA (10 ng/ml) (D-F) for 10 (A,D), 30 (B,E) or 70 (C,F) minutes. Cells were stained for ß3 Y747 PSSA and FITC-conjugated secondary antibody as described in Materials and Methods. Bar, 5 µm.

View larger version (58K): [in a new window]   Fig. 4. ß3 Y747 PSSA localization in murine BMMs. Wild-type murine BMMs were adhered to vitronectin-coated coverslips in the presence of PMA (10 ng/ml) (A,B) or Y27632 (10 µM) (C,D). Cells were stained with ß3 Y747 PSSA antibody and FITC-conjugated secondary antibody as described in Materials and Methods at 10- (A,C) and 30-minute (B,D) adhesion time points. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 5. Arp3 organizes in adherent KVß3 WT cells. KVß3 Y747,759F (A-D) and KVß3 WT (E-L) cells were adhered to vitronectin- (A-C,E-L) or fibronectin-coated (D) coverslips in the presence of PMA alone (10 ng/ml) (A-G) or in combination with Y27632 (10 µM) (H), TAT-Yp (100 µM) (I,K) or TAT-Y/F (100 µM) (J,L) peptides for 10 (A,E), 30 (B,D,F) or 70 (C,G-L) minutes. Cells were stained with Arp3 antibody and FITC-conjugated secondary antibody (A-J) or rhodamine phalloidin (K,L) as described in Materials and Methods. Bar, 5 µm.

View larger version (51K): [in a new window]   Fig. 6. Phosphorylated Pyk2 colocalizes with ß3 in mature adhesion contacts. KVß3 WT (A-I) and KVß3 Y747,759F (J-R) cells were adhered to vitronectin-coated coverslips in the presence of PMA (10 ng/ml) for 10, 30 or 70 minutes. Cells were double-labeled as described in Materials and Methods with anti-ß3 monoclonal antibody (A-C,J-L) and Pyk2 Y402 PSSA (D-F,M-O) with TRITC-conjugated anti-mouse (red, ß3) and FITC-conjugated anti-rabbit (green, Pyk2 Y402 PSSA) secondary antibodies. Merging of images (G-I,P-R) reveals the organization of Pyk2 Y402 PSSA into podosomes (F) and colocalization of total ß3 and Pyk2 Y402 PSSA (I) after 70 minutes of adhesion in KVß3 WT cells, but not in KVß3 Y747,759F cells (O,R). Bar, 5 µm.

View larger version (75K): [in a new window]   Fig. 7. Pyk2, Arp3 and vinculin are present in Vß3 adhesion contacts. KVß3 WT cells were adhered to vitronectin-coated coverslips for 70 minutes in the presence of PMA (10 ng/ml). Cells were double-labeled as described in Materials and Methods for vinculin and Arp3 (A-C), Pyk2 and Arp3 (D-F), or vinculin and Pyk2 Y402 PSSA (G-I) antibodies with TRITC-conjugated anti-mouse and FITC-conjugated anti-rabbit secondary antibodies. Merging of images (C,F,I) reveals colocalization of the respective proteins in adhesion contacts. Bar, 5 µm.

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