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First published online 12 February 2003
doi: 10.1242/jcs.00354

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Microtubule-dependent redistribution of the type-1 inositol 1,4,5-trisphosphate receptor in A7r5 smooth muscle cells

¹ Laboratory of Physiology, CME/VIB04, K.U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium
² Laboratory for Neuronal Membrane Trafficking, CME/VIB04, K.U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium

View larger version (77K): [in a new window]   Fig. 1. Subcellular localization of IP3R1 and IP3R3 in A7r5 cells. Cells were fixed, permeabilized and incubated with (A,B,D,E) Rbt03 polyclonal antibody (1/750) or (C,F) MMAtype 3 monoclonal antibody (1/100), and subsequently treated with anti-rabbit FITC (1/750) or anti-mouse FITC (1/500) secondary antibodies, respectively. IP3R localization was investigated in (A-C) resting cells or. (D-F) after stimulation with AVP (3 µM, 5 hours). A flattened Z-stack image of the confocal images of IP3R1 was generated (A) before and (D) after stimulation with AVP (3 µM). As a control, cells were incubated either (G) with the pre-immune serum of Rbt03 (1/750) or (H) with PBS before treatment with secondary antibodies. Bar, 10 µm.

View larger version (12K): [in a new window]   Fig. 2. Time course of IP3R1 redistribution. The percentage of cells with IP3R1 located in the perinculear region (squares) and in the cytoplasm (circles) after addition of 3 µM AVP is depicted for different time periods. The dashed line represents the removal of AVP (3 µM) after 2 hours, after which the cells where incubated in PBS without Ca2+ for the indicated time periods. Each result is the mean±s.e.m. from three independent experiments. The s.e.m. is not indicated when smaller than the symbol.

View larger version (16K): [in a new window]   Fig. 3. Effect of Ca2+-mobilizing agents on IP3R1 redistribution. The percentage of cells with a perinuclear IP3R1 is depicted before and after incubation with AVP (3 µM), thapsigargin (1 µM) and CPA (50 µM) for 5 hours. Each result is the mean±s.e.m. from three independent experiments. *Significantly different from the control.

View larger version (67K): [in a new window]   Fig. 4. Visualization of the microtubular and actin cytoskeleton. Cells were fixed, permeabilized and incubated (A,C,E) with anti- tubulin (1/2000) or (B,D,F) with rhodamine-phalloidin (1 unit/ml). (A,C,E) Anti-mouse FITC (1/400) was used as secondary antibody. The microtubular and actin cytoskeleton was visualized (A,B) before and after (C,D) AVP (3 µM) or (E,F) OAG (50 µM) treatment for 5 hours. Bar, 10 µm.

View larger version (20K): [in a new window]   Fig. 5. Effect of nocodazole and taxol on IP3R1 redistribution. The percentage of cells with a perinuclear IP3R1 distribution is shown after incubation with AVP (3 µM) for 2 hours in the absence or presence of nocodazole (50 µM) or in the absence or presence of taxol (1 µM). Each result is the mean±s.e.m. from three independent experiments. *Significantly different from the control.

View larger version (144K): [in a new window]   Fig. 6. Endoplasmic reticulum structure of A7r5 cells. ER structure was visualized in cells transfected with the pEYFP-ER vector, which encodes an ER-targeted yellow fluorescent fusion protein. The general ER morphology of (A) unstimulated and (B) AVP (3 µM, 5 hours) stimulated cells did not show significant differences. PDI was visualized (C) before and (D) after stimulation with AVP (3 µM, 2 hours) using the monoclonal anti-PDI antibody (1/100) and anti-mouse FITC (1/750). Bar, 5 µm.

View larger version (44K): [in a new window]   Fig. 7. Visualization of SERCA pumps in A7r5. Cells were fixed, permeabilized and incubated with AS809-27 polyclonal antibody (1/300). Anti-rabbit FITC (1/750) was used as secondary antibody. SERCA was visualized in (A) resting cells and (B) after stimulation with AVP (3 µM, 2 hours). Bar, 10 µm.