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Fig. 2. PAK1 regulates macrophage spreading but not migration speed. (A) WT and PAK1^–/– BMMs were seeded onto tissue culture plastic in growth medium. Upper panels are representative micrographs of migrating BMMs 8 hours after seeding. Scale bars: 100 µm; arrows indicate round, flat cells; arrowheads indicate lamellipodia on migrating cells. Cell migration was observed by time-lapse microscopy for 8 hours. Mean migration speed at each time point was determined from cell tracks, n=70 and n=60 for WT and PAK1^–/– BMMs, respectively. Results are representative of three separate experiments (lower panels). (B) WT and PAK1^–/– BMMs were allowed to adhere to uncoated glass coverslips in macrophage starvation medium supplemented with 33 ng/ml CSF1 for the indicated times. Cells were stained for F-actin. Scale bars: 10 µm. (C) The images in B were analysed to quantify the spread area of WT and PAK1^–/– BMMs. Data are the mean ± s.e.m. of three separate experiments; n=50 cells per time point per experiment. (D) Quantification of WT and PAK1^–/– BMM spread area as determined in C, using images of cells 24 hours after adhesion. Data are the mean ± s.e.m. of three separate experiments; n=50 cells per experiment. (E) PAK1^–/– BMMs nucleofected with GFP or GFP-PAK1 were allowed to adhere to uncoated glass coverslips in growth medium for the indicated times. Cells were stained for F-actin; Scale bar: 10 µm. The spread area of GFP- and GFP-PAK1-expressing cells was determined. Data are the mean ± s.d. of two separate experiments; n=20 cells per experiment.

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