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First published online 10 June 2003
doi: 10.1242/jcs.00606

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LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment

Andrew Smith^?,*, Madelon Bracke^?,*,, Birgit Leitinger^?,, Joanna C. Porter^?,¶ and Nancy Hogg^?,**

^? Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, Lincoln's Inn Fields, London WC2A 3PX, UK
^* These authors contributed equally to this work

View larger version (13K): [in a new window]   Fig. 1. Inhibition of myosin activation and its effects on LFA-1-mediated T cell attachment to ICAM-1. (A) Attachment of T cells to ICAM-1 with and without Mg2+/EGTA stimulation compared with attachment in the presence of Mg2+/EGTA plus BDM (myosin ATPase inhibitor, 20 mM, n=6, P=0.02). (B) The effect on T cell attachment to ICAM-1 of MLCK inhibitor ML-7 and ROCK inhibitor Y-27632 (0-50 µM). (C) The effect of various peptides on T cell attachment. Antennapedia linked to a 13-mer peptide containing the auto-inhibitory site of MLCK (ant-MLCK-pep), and, as controls, a scrambled version of the same peptide (ant-scram-pep) and antennapedia only (ant-pep) (representative of 5 experiments). Values are expressed in A and B as the mean±s.e.m. and in C as the mean±s.d.

View larger version (33K): [in a new window]   Fig. 2. Identification of MLCK and ROCK in human T cells. Western blot analysis of T cells probed with mAbs specific for MLCK (lane 2) and ROCK I (lane 3). Mouse lung lysate was used as a positive control for the smooth muscle and non-smooth muscle forms of MLCK (lane 1). Molecular weight markers of 220 kDa (myosin) and 97kDa (phosphorylase A) are indicated.

View larger version (82K): [in a new window]   Fig. 3. LFA-1-mediated migration of T cells on ICAM-1. Time lapse video microscopy of human T cells treated with Mg2+/EGTA showing a sequence of initial contact with and migration on ICAM-1. Cells and medium were preheated to 37°C prior to recording. See Movie 1 for further examples of attachment, polarization and migration. The white arrows at time 0 and 45 seconds highlight the cells that have made initial contact with ICAM-1. Bar, 20 µm.

View larger version (59K): [in a new window]   Fig. 4. Effect of inhibiting MLCK activity on T cell attachment and migration on ICAM-1. Phase images of T cells on (A) ICAM-1, (B) ICAM-1 in the presence of Mg2+/EGTA, (C) preincubated with 20 µM ML-7 and plated onto ICAM-1 with Mg2+/EGTA. Bar, 10 µm. Motility and migration as determined by time lapse microscopy (scale in µm). (D) T cells on ICAM-1 in the presence of Mg2+/EGTA (average speed=10.3 µm/minute; scale in µm). (E) T cells plated onto ICAM-1 in Mg2+/EGTA and allowed to migrate for 20 minutes followed by the addition of 20 µM ML-7 for 70 minutes (average speed=0.71 µm/minute). Green and red dots represent the starting and stopping positions of each individual T cell.

View larger version (28K): [in a new window]   Fig. 5. Time lapse analysis of the effect of MLCK inhibition on morphology of the migrating T cell. ML-7 at a final concentration of 20 µM was added to T cells polarized on ICAM-1 in the presence of Mg2+/EGTA, and the morphological response recorded by time lapse microscopy over 30 minutes. Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 6. Effect of inhibiting CaM activity on T cell attachment and migration on ICAM-1. (A) Proportion of T cells in presence of Mg2+/EGTA bound to ICAM-1 over an increasing dose range of CaM inhibitor, W-7 (n=4). (B) Motility and migration as determined by time lapse microscopy (scale in µm). As in Fig. 4, Mg2+/EGTA-stimulated T cells were plated onto ICAM-1 minus or plus pre-incubation with 50 µM W-7 and allowed to migrate for 30 minutes (average speed of 7.85±0.15 versus 1.53±0.05 µm/minute). Green and red dots represent the starting and stopping positions of each individual T cell. (C) Phase images of Mg2+/EGTA-stimulated T cells plated onto ICAM-1 and allowed to migrate for 30 minutes followed by the addition of 50 µM W-7 for 10 minutes. Bar, 20 µm.

View larger version (68K): [in a new window]   Fig. 7. Effect of inhibiting ROCK activity on T cell attachment and migration on ICAM-1. Phase images of Mg2+/EGTA-stimulated T cells after 20 minutes of migration. (A) On ICAM-1 or pre-incubated with 10 µM Y-27632. (B) T cells, pre-incubated with C3 exoenzyme overnight at 37°C prior to plating on ICAM-1. (C) T cells were allowed to adhere to ICAM-1 in presence of Mg2+/EGTA for 20 minutes followed by the addition of 10 µM Y-27632. The morphological effects were recorded by time lapse microscopy over 15 minutes (bar, 20 µm). (D) T cells on ICAM-1 in the presence of Mg2+/EGTA (average speed=12.6±0.22 µm/minute). T cells were allowed to adhere for 20 minutes followed by the addition of 10 µM Y-27632 for 70 minutes (average speed=4.81±0.13 µm/minute; scale in µm). Green and red dots represent the starting and stopping positions of each individual T cell.

View larger version (15K): [in a new window]   Fig. 8. Effect of MLCK, CaM and ROCK inhibitors on cell spreading and morphology. (A) Mg2+/EGTA-stimulated T cells were allowed to migrate on ICAM-1 or BSA for 20 minutes in the presence of either ML-7 (25 µM), W-7 (50 µM) or Y-27632 (20 µM) prior to fixation, permeabilization and staining for F-actin (n=6). Cell area was recorded by a LSC (6000 cells per cover slip). (B) T cell length was measured after incubation with either ML-7 (25 µM), W-7 (50 µM), Y-27632 (20 µM) or C3 exoenzyme (20 µg/ml) (n=18-34). All measurements were taken after the T cells were allowed to migrate for 20 minutes on ICAM-1 in the presence of Mg2+/EGTA. The dotted line represents the average diameter of T cells in suspension (included as a reference point). All results are expressed as an mean±s.d. P<0.01(***).

View larger version (82K): [in a new window]   Fig. 9. Subcellular localization of MLCK, ROCK and myosin compared to actin in T cells migrating on ICAM-1. MLCK (A) and ROCK (B) were detected with mAbs specific for MLCK and ROCK I, followed by Alexa 488-goat anti-mouse IgG and counter-stained with Alexa 546-phalloidin to visualize F-actin. Images are displayed as gradient maps where red represents high and blue low fluorescence intensity. (C) Myosin (green) and F-actin (red) were merged to determine colocalization (yellow) within an Mg2+/EGTA-stimulated T cell on ICAM-1. (A,B) Images represent projections onto the x-y plane of all individual optical sections taken along the z-axis using maximal fluorescence values. Image C represents a single section in the x-y plane.

View larger version (14K): [in a new window]   Fig. 10. Effect of MLCK and ROCK inhibitors on migration of the T cell population. (A) Average speed of Mg2+/EGTA-stimulated T cells on ICAM-1 in the presence of inhibitors 25 µM ML-7, 50 µM W-7, 20 µM Y-27632 (all n=15) or no inhibitor (n=55). (B) The T cells were plated on ICAM-1 in the presence of Mg2+/EGTA and allowed to migrate for 20 minutes. Inhibitor treatment was initiated and the T cells were scored after a further 10 minutes. `Lamellar protrusion' describes membrane ruffling at either the leading edge or, in the case of stationary cells, around the cell circumference. `Tail anchored' describes polarized cells that failed to translocate a full cell length. n=50-70 per treatment group.