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First published online 17 January 2006
doi: 10.1242/jcs.02755

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ARAP3 is essential for formation of lamellipodia after growth factor stimulation

The Inositide Laboratory, The Babraham Institute, Cambridge, CB2 4AT, UK

View larger version (55K): [in a new window]   Fig. 1. ARAP3-deficient cells do not form protrusive lamellipodia. (A) Western blots of lysates of stable PAE cell lines expressing a scrambled control RNAi oligonuleotide (clone NA1) or olignucleotides directed against different parts of porcine ARAP3 (clones 52B3, 32D6) were probed for ARAP3 (top panel) and subsequently reprobed for ß-COP as a loading control (bottom panel). Careful analysis of blots from several similar experiments leads us to estimate that the reduction in ARAP3 expression in the 52B3 and 32D6 lines is approximately 85%. The original blot had 25 lanes, out of which the shown ones were not immediately adjacent. They were cut and pasted for ease of viewing. (B) Cells of the stable RNAi PAE cell lines were seeded onto coverslips, starved for 16 hours, stimulated for 5 minutes either with PDGF or with its vehicle (as indicated) and fixed. Cells were stained for F-actin. An arrow points to a 52B3 cell carrying a typical small actin-rich protrusion. The photographs shown are representative examples from four independent experiments. (C) NA1, 52B3 and 32D6 cells were treated as above and 120-150 cells per coverslip were counted and scored according to their phenotypes: black bars, no ruffles or lamellipodia; striped bars, protrusive lamellipodia; grey bars, uniform ruffles along entire cell periphery; unfilled bars, dorsal ring ruffles. (D) NA1 and 52B3 cells were treated as in (B) and stained for paxillin. Arrows point to typical clustered focal adhesions in 52B3 cells. Bars in (B) and (D), 20 µm.

View larger version (54K): [in a new window]   Fig. 2. ARAP3-deficient cells do not polarise well. NA1 or 52B3 cells were seeded onto coverslips and left to grow until confluent. Confluent monolayers were scratched with a sterile pipette tip and left to migrate to close the `wound'. (A) Cells were fixed 4.5 hours after scratching, and stained to visualise F-actin. (B) Cells were fixed 3 hours after scratching and stained to visualise -tubulin. Data similar to the photographs shown (A,B) were obtained in two further experiments. A thin broken white line indicates the orientation of the `wound' in each photograph. Bar, 20 µm. (C) Cells were fixed 0.5, 3 and 9 hours after scratching and stained for giantin with a nuclear counterstain. Cells in which the Golgi apparatus lay within the third of the cell facing the `wound' were counted as polarised. Black bars, NA1 control cells; unfilled bars, 52B3; grey bars, 32D6 ARAP3 RNAi cells. The graph integrates data from three independent experiments.

View larger version (81K): [in a new window]   Fig. 3. The ARAP3-knockdown phenotype can be rescued and mimicked. (A) 52B3 cells were seeded onto glass coverslips, starved for 16 hours and then microinjected with the injection marker biotin-dextran and pCMV3(EE) (dextran) or with biotin-dextran and pCMV3(EE)hARAP3 (hARAP3). Where indicated, cells were stimulated with PDGF for 5 minutes prior to fixation. Cells were stained for the presence of the injection marker (not shown), for F-actin (left) or for paxillin (right). The rescue result shown was observed in three independent experiments in 35% of ARAP3-injected cells. 45% of hARAP3-injected cells displayed the ARAP3-overexpression phenotype with retractions and loss of adhesion that has been previously described (Krugmann et al., 2004). (B) PAE cells were transiently transfected with pcDNA3-HA-L67Arf6 and/or pRK5-Myc-L63RhoA and the empty vector where applicable, and seeded onto glass coverslips. Cells were serum-starved for 16 hours prior to stimulation with PDGF for 5 minutes or its vehicle (indicated PDGF and starved). Cells were fixed and stained for the HA and Myc epitope tags (not shown; in cells co-transfected with Arf6 and RhoA constructs, in excess of 90% of cells expressed both plasmids if they expressed L67Arf6) or for the HA or Myc epitope tag together with filamentous actin. Arrows indicate Arf6-typical, actin-rich protrusions. The shown cells are representative examples; experiments were repeated three times. Bars, 20 µm.

View larger version (20K): [in a new window]   Fig. 4. ARAP3-deficient cells have increased activities of RhoA. (A) NA1 and 52B3 cells were transiently transfected with a Myc-tagged ROCK construct. After 16 hours of serum starvation, cells were or were not stimulated with PDGF or its vehicle for 1-15 minutes and lysed. ROCK protein was immunoprecipitated using an anti-Myc antibody and subsequently used for an in vitro kinase assay. The graph represents data pooled (means ± s.d.) from three independent experiments, which were all done in duplicate. The PDGF-stimulated increase in ROCK activity in the 52B3 cells is statistically significant (*P=0.05 in a paired Student's t-test). (B) NA1 and 52B3 cells were seeded into tissue culture dishes, serum starved for 16 hours and lysed straight away or after stimulation with PDGF or its vehicle for 1-10 minutes. Lysates were subjected to western blotting and probed with an anti phospho-MYPT antibody and subsequently with a control antibody (ß-COP) as a loading control. The shown blot is a representative example from three independent experiments.

View larger version (36K): [in a new window]   Fig. 5. ARAP3-deficient cells display increased Arf6 and altered Rac activities. (A) NA1 and 52B3 cells were seeded into tissue culture dishes, and either left to grow or subjected to serum starvation for 16 hours as indicated. Cells were lysed and lysates used for Arf `pull-down' assays using GST-GGA beads as bait. (B) Cells were seeded and starved as in (A) and subsequently stimulated with PDGF or its vehicle for 1, 5 and 10 minutes prior to cell lysis. For ease of comparison, basal levels were adjusted to 1. The graphs shown in (A) and (B) represent pooled data (means ± s.d.) from four independent experiments all of which were carried out in duplicate. (C) NA1 and 52B3 cells were seeded in tissue culture dishes, treated as in (A), and used for Rac `pull-down' assays using GST PAK-CRIB immobilised on Sepharose beads as bait. The shown graph represents pooled data (means ± s.d.) from four independent experiments. (D) Cells were treated as in (B) and used for Rac `pull-down' assays; basal levels were again adjusted to 1. The shown graph represents (E) HA-tagged, N27Arf6 or L67Arf6 transfected PAE, NA1 or 52B3 cells (as indicated) were seeded onto pooled data (means ± s.d.) from five independent experiments. (E) HA-tagged, N27Arf6 or L67 Arf6 transfected PAE, NA1 or 52B3 cells (as indicated) onto glass coverslips and serum starved for 16 hours. Cells were fixed, stained using an antibody for Rac1 and visualised by confocal microscopy. Photographed slices were 0.8 µm thick. Bar, 20 µm.

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