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First published online 18 January 2005
doi: 10.1242/jcs.01661

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Expression of junctional adhesion molecule-A prevents spontaneous and random motility

¹ Laboratory of Systems Biology, Department of Immunology and Cell Biology, Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milano, Italy
² Department of Vascular Biology, FIRC Institute of Molecular Oncology, 20139 Milano, Italy
³ Laboratory of Vascular Biology, Department of Immunology and Cell Biology, Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milano, Italy
⁴ Department of Biomolecular and Biotechnological Sciences, School of Sciences, University of Milan, 20133 Milano, Italy

View larger version (85K): [in a new window]   Fig. 1. JAM-A expression. Expression of JAM-A was evaluated by FACS analysis using anti-JAM-A mAb BV12 (shaded area). Other anti-JAM-A antibodies (mAbs BV19 and BV11) gave similar results. The white area indicates negative control antibodies. Results are expressed as arbitrary units of fluorescence (on the x-axis) and as cell count (on the y-axis).

View larger version (47K): [in a new window]   Fig. 2. JAM-A expression reduces the number of membrane protrusions. (A) Cells were plated onto fibronectin-coated coverslips (for 2 hours), stained with FITC-phalloidin and examined by IF microscopy. (B) Individual cells were analyzed for measuring the distribution of the number of long protrusions (i.e. length >2 µm) per cell. Results are derived from three experiments.

View larger version (51K): [in a new window]   Fig. 3. JAM-A expression prevents cell motility. (A) Cells were plated onto gold particles, incubated for 24 hours and fixed. Individual cells were analyzed for measuring the distribution of (B) particle-free area and (C) number of tracks per cell. Results are derived from three experiments.

View larger version (60K): [in a new window]   Fig. 4. JAM-A expression enhances microtubule polymerization. Cells were incubated for 16 hours in either the absence (Control/C) or the presence (Nocodazole/N) of 10 µM nocodazole. In some samples, nocodazole was removed after the 16 hours incubation, and cells incubated for an additional 2 hours (Nocodazole withdrawal/NW). Using the anti--tubulin mAb B-5-1-2, microtubules were analyzed by IF microscopy (A), and NP40-insoluble tubulin was analyzed by western blotting (B). In (B), ZO-1 is shown as a loading control for NP40-insoluble material upon nocodazole withdrawal.

View larger version (85K): [in a new window]   Fig. 5. JAM-A expression stabilizes microtubules. Cells were plated onto fibronectin-coated coverslips and incubated for 90 minutes at 37°C. Then, after the addition of 10 nM taxol (where indicated), cells were further incubated for 30 minutes at 37°C. Finally, cells were incubated at either 37°C or 4°C for an additional 30 minutes, stained with either anti--tubulin mAb B-5-1-2 (A) or anti-acetylated--tubulin mAb 6-11B-1 (B).

View larger version (59K): [in a new window]   Fig. 6. JAM-A expression increases the number of focal adhesions. Cells were plated onto fibronectin, incubated for 2 hours, and then stained with either anti-vinculin mAb hVIN-1 (A) or anti-paxillin mAb 349 (B). (C) Individual cells were analyzed for measuring the distribution of the number of vinculin-based focal adhesions per cell; results are derived from three experiments. In (D), cells were plated onto fibronectin for 30 minutes, incubated for an additional 90 minutes in either the absence (Control) or presence of 10 nM taxol, 40 mM lithium and 20 µM PKC-PS, and finally stained with anti-vinculin mAb hVIN-1.

View larger version (111K): [in a new window]   Fig. 7. JAM-A expression causes morphological changes in directionally migrating cells and in cell groups. In (A), confluent cell monolayers were scratch-wounded and incubated for an additional 24 hours. In (B), cells were seeded at high density and incubated for 2 hours to allow the formation of small groups of contacting cells. Finally, cells were stained with crystal violet (Phase contrast), FITC-phalloidin (F-actin), and anti-vinculin mAb hVIN-1 (Vinculin).

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