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First published online 21 October 2008
doi: 10.1242/jcs.027680

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PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics

¹ Ludwig Institute for Cancer Research, University College London, 91 Riding House Street, London W1W 7BS, UK
² Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57, Huddinge, Sweden
³ Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
⁴ King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London SE1 1UL, UK

View larger version (36K): [in this window] [in a new window]   Fig. 1. PAK1 regulates CSF1-induced MAPK activation but not macrophage differentiation or chemotaxis. (A) Lysates from WT and PAK1–/– BMMs were immunoblotted for PAK1 and PAK2 using a group-1-specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was detected as a loading control. Gr, cells in growth medium. (B) Flow cytometry analysis of WT and PAK1–/– BMM surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. (C) WT BMMs were stimulated with 33 ng/ml CSF1, and lysates were immunoblotted for Thr423-P-PAK1 (P-PAK1) and β-actin as a loading control. (D) WT and PAK1–/– BMMs were stimulated with 33 ng/ml CSF1 and lysates were immunoblotted for Ser473-P-Akt, Thr202/Tyr204-P-ERK1/2, Thr180/Tyr182-P-p38 and Ser298-P-MEK1/2 levels. β-actin was detected as a loading control. Western blots are representative of three separate experiments. (E) To investigate chemotaxis, 1x105 WT or PAK1–/– BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean ± s.e.m. of three experiments performed in triplicate.

View larger version (53K): [in this window] [in a new window]   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.

View larger version (86K): [in this window] [in a new window]   Fig. 3. PAK1 is required for lamellipodial stability during spreading. (A) WT and PAK1–/– BMMs were plated onto glass-bottomed culture dishes in growth medium and cell spreading was visualised by time-lapse microscopy. Images at the indicated time points of the movies are shown. Scale bars: 1 µm. (B) The length and the perimeter of WT and PAK1–/– lamellipodia was determined using movies as in A. Data shown are the mean ± s.e.m; n=145 and n=225 lamellipodia from WT and PAK1–/– BMMs, respectively, from six separate experiments. **P<0.01 and ***P<0.001, Student's t-test. (C) Kymographs of extending lamellipodia. The final frame from the movie used (left panels) indicates the region of kymograph production. Right panels show kymographs; the inset shows the kymograph with the membrane edge highlighted (white line). (D) Quantification of the number of lamellipodia per cell observed at the specified times in movies. Mean ± s.e.m.; n=9 cells from six separate experiments for both WT and PAK1–/– BMMs.

View larger version (32K): [in this window] [in a new window]   Fig. 4. PAK1 promotes ERK1/2 activation at the cell periphery. (A) WT and PAK1–/– BMMs were adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for Thr202/Tyr204-P-ERK1/2, Thr180/Tyr182-P-p38, Ser298-P-MEK1/2 and total ERK1/2. Densitometry quantification of phosphorylated ERK1/2 and p38 levels equalised to total ERK1/2 protein levels are shown (a.u., arbitrary units). Data are mean ± s.d. of two separate experiments. (B) WT and PAK1–/– BMMs were plated onto glass coverslips for 10 minutes in growth medium and were stained using an ERK1/2 antibody and TRITC-phalloidin to visualise F-actin. Cells were imaged by confocal microscopy. ERK1/2 localisation was quantified by determining the number of cells with ERK1/2 staining at the periphery. The mean ± s.d. is shown for two separate experiments; n=60 (WT) and n=45 (PAK1–/–). (C) BMMs were stained with a Thr202/Tyr204-P-ERK1/2 antibody (P-ERK1/2) and TRITC-phalloidin (F-actin). Localisation of ERK1/2-P was quantified by determining the number of cells with staining at the cell periphery. The mean ± s.d. is shown for two separate experiments; n=39 (WT) and n=62 (PAK1–/–). (D) WT and PAK1–/– BMMs were kept in suspension or adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for Ser217/221-P-MEK1/2, and total ERK1/2 as a loading control. Densitometry quantification of phosphorylated MEK1/2 equalised to total ERK1/2 protein levels is shown (a.u., arbitrary units). Data are mean ± s.d. of two separate experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Inhibition of ERK1/2 activation promotes macrophage spreading. WT BMMs were plated on glass coverslips in the presence or absence of 1 µg/ml U0126. (A) Representative images of cells stained for F-actin. (B) Cell spread area was quantified at the indicated time points. The mean spread area ± s.e.m. from three separate experiments is shown. ***P<0.001 comparing WT+U0126 to untreated WT; n=50 cells per time point per experiment. Scale bars: 5 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Inhibition of ERK1/2 activation reduces lamellipodial stability. (A) WT BMMs in growth medium were pretreated with 1 µg/ml U0126 for 1 hour and plated on to glass-bottomed tissue culture dishes. Time-lapse microscopy was used to monitor cell spreading. Frames from the indicated time points are shown. Arrows indicate lamellipodia. Scale bar: 20 µm. (B) Quantification of the number of lamellipodia per cell observed in the time-lapse movie frames specified. Data are the mean ± s.e.m. of three separate experiments. (C) Representative kymograph of a lamellipodium in a U0126-treated cell. The membrane edge is highlighted (white line).

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