Figure 3: PI3K activities required for persistent migration but not directional sensing.
(a) Time-lapse images showing directed migration and accumulation of T cells, untreated or treated with 100 nM wortmannin, in hCCL5**-containing media towards the uncaging light path patterned as a line. Open arrowheads: polarized cells with a shape index >1.5. Scale bar, 50 μm. (b) Schematic diagram of single T-cell chemotaxis assay by two-photon uncaging of hCCL5**. The focal point (∼0.078 μm2, crossed circles) always placed on one side of the cell. Angle θ is between the current instantaneous velocity vector and the velocity vector at the first time point after uncaging. The centroid positions of the cell are denoted with the red and green dots. Time-lapse images of T cells that, in response to active CCL5, generate new pseudopods (c, ‘N-turn’) or extend existing pseudopods towards the uncaging light focus (d, ‘U-turn’) or remain uncommitted for a prolonged period (e, ‘C-turn’); cell shapes outlined in colours of different warmth to indicate time and pseudopod dynamics. See corresponding Supplementary Movie 7–9. Scale bar, 10 μm. (f,g) Kinetic decay curves of cosine (θ). For each cell, values of cosine (θ) before it reaches zero were used in calculation. Cells whose cosine (θ) remained positive throughout the intial 200 s on uncaging were excluded. T cells untreated or treated with wortmannin (100 nM) at 10% (f) or 80% laser power output for uncaging (g); left and middle: curves for individual cells (coloured) and mean±s.e.m. (black); right: comparison of untreated and treated; *P<0.05, **P<0.01, by two-tailed t-tests.
