S1P induces pressurized, bleb-based migration. (a and b) Representative migratory responses for S1P versus chemokine. WT naive CD4⁺ T cells (previously cultured overnight in media with 10% CS-FBS) were imaged by DIC microscopy on VCAM-coated chambers before and after stimulation (Stim) with S1P (100 nM; a), CCL19 (100 ng/ml, not shown), or CXCL12 (100 ng/ml; b). (c) Representative cell showing the rapid formation and transient nature of blebs in migratory cells following S1P treatment. (a–c) Time is in min:s and relative to point of stimulation; scale bars, 5 μm. Full arrows indicate direction of migration, arrowheads indicate lamellipodia, and asterisks indicate blebs. (d) Bleb quantitation following S1P or chemokine stimulation as in a. Blebs were manually counted from DIC movies of control, S1P-, or chemokine-stimulated cells on VCAM. Each dot corresponds to a cell pooled from three independent experiments. Means (horizontal bars) of S1P, CCL19, and CXCL12 groups were compared with control using a Kruskal-Wallis (nonparametric) one-way ANOVA. (e) Intracellular pressure following S1P or chemokine stimulation. WT naive CD4⁺ T cells (previously cultured overnight in media with 10% CS-FBS) were added to poly-L-lysine–coated plates and left untreated or stimulated with S1P (100 nM) or CCL19 (100 ng/ml). Pressure was measured by the servo null method, collecting readings from as many cells as possible within a 12-min window after stimulation. Where indicated, cells were pretreated with BBS (50 µM) for at least 30 min before stimulation. Each dot corresponds to a cell, pooled from four independent experiments. Means (horizontal bars) of all groups compared with each other using a one-way ANOVA with a Tukey correction for multiple comparisons. IC, intracellular; n.s., P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.