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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02374

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PLC1 is essential for early events in integrin signalling required for cell motility

¹ Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London, SW3 6JB, UK
² Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, SM2 5NG, UK

View larger version (61K): [in a new window]   Fig. 1. (A) Time-lapse video microscopy of human umbilical vein endothelial cells (HUVECs) and cancer cell lines BE and DU145. Cells plated on Matrigel were allowed to attach for 1 hour and then analysed for 16 hours to establish elongation and motility profiles. Images shown were taken from recordings at 1 (a), 2 (b), 4 (c), 8 (d) and 16 (e) hours after plating (left panel). Individual cell elongation was measured from several recorded images using frame-by-frame analysis over 16 hours and a mean average percentage elongation post-plating calculated and reported with standard deviations (n=30; right panel). (B) Serum-starved cells were plated on growth factor-reduced Matrigel with serum as a chemoattractant. Invasion into this matrix was measured over 48 hours. The leading edge of migration of cells was recorded by Z-section confocal microscopy and the invasion distance of individual cells calculated from several reconstructed images using velocity software and reported along with standard deviations (n=10; left panel). Three-dimensional reconstruction of BE invasion profile at 24 and 48 hours using Velocity software (right panel).

View larger version (60K): [in a new window]   Fig. 2. (A) PLC1-specific siRNA depletion of PLC1 protein levels. Cells were treated with siRNA probes for 72 hours and processed for western blotting. Protein levels of PLC1 were compared with control blots against GAPDH and PLC2 or PLC1. (B) PLC1 activity is inhibited by U73122 but not by U73343. Purified PLC1 activity was measured in the presence of the inhibitor (10 µM) using a PtdIns(4,5)P2 hydrolysis assay. Results are expressed as the average PtdIns(4,5)P2 hydrolysis from four experiments with standard deviations shown. (C) Left panel; cells pre-treated with siRNA [BE (), DU145 ()] or U73122 [BE (), DU145 ()] were plated on plastic and the number of viable adherent cells counted at various time points and compared with matched controls (scrambled siRNA or U73343). Right panel; HUVECs cells were pre-treated with either siRNA (solid bar) or U73122 (hatched bar) and viable adherent cell counted after 16 hours and compared with controls (scramble siRNA or U73343). Results shown are an average of four experiments and are shown with standard deviations. (D) Cell elongation is inhibited by U73122 treatment or siRNA, and the inhibition can be rescued by expression of rat PLC1. Pre-treated [siRNA (PLC1 or scrambled), U73122 (2 µM) or U73343 (2 µM)] BE (top panels) or DU145 (bottom panels) cells were plated on Matrigel and the level of elongation analysed by phase contrast microscopy. BE cells were also transfected with GFP in the presence or absence of siRNA or with GFP-PLC1 in the in the presence of siRNA as indicated in the middle panel. Representative images are shown and average percentage cell elongation, which was analysed from 20 independent cell images is reported with standard deviations; BE: U73343 166.2±18.5%, U73122 4.3±14.4%, scrambled siRNA 151.1±25.9%, PLC1 siRNA 11.6±10.4%, scrambled siRNA+GFP 154.9±29.2%, PLC1 siRNA+GFP 13.4±11.8%, PLC1 siRNA+GFP-PLC1 151.8±23.6%; DU145: U73343 46.4±16.8%, U73122 2.9±6.9. (E) HUVEC cell elongation and network formation is inhibited by siRNA or U73122. Pre-treated [siRNA (PLC1 or scrambled), U73122 (1 µM) or U73343 (1 µM)] HUVECs were plated on Matrigel and elongation analysed by fluorescent microscopy. (F) Cell invasion is inhibited by PLC1 siRNA or U73122. Serum-starved pre-treated (siRNA, U73122 or U73343) cells were plated onto Matrigel-coated Transwell chambers and invasion through the filter and into the matrix, towards serum (10%), was measured by fluorescent microscopy after 48 hours with the average percentage (number) of invading cells compared with control (untreated, 100%) sample from five experiments displayed with standard deviations: 1, control; 2, PLC1 siRNA; 3, scrambled siRNA; 4, U73122 (2 µM); 5, U73343 (2 µM).

View larger version (53K): [in a new window]   Fig. 3. U73122 and siRNA inhibit actin protrusions. BE cells treated with siRNA or U73122 were plated on Matrigel for the times shown, fixed and then co-stained with Texas-Red Phalloidin (F-actin: red) and FITC -tubulin antibody (Tubulin: green). Stains were visualized by confocal microscopy. Arrow heads represent F-actin protrusions.

View larger version (48K): [in a new window]   Fig. 4. (A) Inhibition of integrin ß1 or Src-family kinases diminishes Src(Y416)- and PLC1(Y783)-activating phosphorylations. BE cells were plated on Matrigel and either left untreated (control) or treated with either blocking ß1 integrin (4B4) antibody (10 µg/ml), Src inhibitor PP2 (10 µM) or the control compound PP3 (10 µM) for 8 hours. Cells were recovered from the Matrigel and processed for western blotting using antibodies to phosphorylated or unphosphorylated forms of PLC1 and Src as indicated. An inhibitor of EGF receptor AG1478 (10 µM) did not prevent PLC1 phosphorylation in this type of experiment (not shown). (B) Stimulation of integrin ß1 enhances Src (Y416) and PLC1 (Y783) phosphorylation. Serum-starved BE cells were stimulated with either Mn2+ (5 µM) or the activating integrin ß1 antibody (TS2/16 10 µg/ml) for 30 minutes and then lysed and processed for western blotting using antibodies to phosphorylated or unphosphorylated forms of PLC1 and Src as indicated. (C) PLC1, Src and GIT1 co-immunoprecipitation is integrin ß1 dependent. BE cells, either untreated (–) or treated (+) with the integrin ß1 blocking antibody 4B4 (10 µg/ml), were plated on Matrigel for 8 hours, then recovered from Matrigel, lysed and immunoprecipitation (IP) was performed with either PLC1 or Src antibodies as indicated at the top. Subsequent western blotting (Wb) of these samples was performed using different PLC1 or Src antibodies or the GIT1 antibody as indicated on the left. In the same experiment, EGF receptor could not be detected in the immunoprecipitates (not shown). (D). Src-family kinases Src, Fyn and Lck can directly phosphorylate the PLC1(Y783) site. Purified PLC1 was incubated with purified Src-family kinases, indicated at the top, in an in vitro phosphorylation assay for 30 minutes. PLC1(Y783) phosphorylation was analysed using specific antibodies whereas autophosphorylation of Src kinases was verified using general anti-phosphotyrosine antibodies by western blotting (top panels). Time course of Src phosphorylation of PLC1(Y783). Src phosphorylation of PLC1 was analysed using a time-course in vitro phosphorylation assay followed by western blotting. Amount of phosphorylation was quantified by densitometry (bottom panel).

View larger version (29K): [in a new window]   Fig. 5. Blocking integrin ß1 or Src activity inhibits cell elongation and invasion. (A) BE cells were plated on Matrigel and treated with either blocking ß1 integrin antibody (4B4; 10 µg/ml), Src inhibitor (PP2, 10 µM), Src inhibitor control (PP3, 10 µM) or an inhibitor of EGF receptor (AG1487, 10 µM) for 8 hours. Elongation profiles were then analysed by phase contrast microscopy. Representative images are shown and average percentage cell elongation, which was analysed from 20 independent cell images, is reported with standard deviations; Control 161.2±17.8%, anti-ß1 3.6±8.9%, PP3 153.3±24.7%, PP2 11.8±12.3%, AG1827 155.8±21.9%. (B) HUVECs were plated on Matrigel and treated as in part A. Elongation profiles were analysed by fluorescent microscopy. (C) Serum-starved cells treated as for part A were plated onto Matrigel-coated Transwell chambers and invasion through the filter and into the matrix towards serum (10%) was analysed by microscopy after 48 hours. Results are representative of five independent experiments with the average percentage (number) of invading cells compared with control (untreated, 100%) sample displayed with standard deviations: 1, control; 2, anti-ß1; 3, PP2; 4, PP3.

View larger version (28K): [in a new window]   Fig. 6. (A) Calcium release is stimulated by activation of integrin ß1. FLUO3/AM-labelled serum-starved BE cells were placed in suspension and stimulated with either the activating integrin ß1 antibody (TS2/16 10 µg/ml; top panel) or Mn2+ (5 µM; middle panel). Changes in the intracellular calcium concentration were then measured using a fluorimeter (ex. 490 nm, em. 525 nm). Inhibitors U73122 (2 µM) or PP2 (10 µM) were included in these incubations where indicated. Representative traces are shown, and quantified intracellular calcium concentration, which was analysed from three independent experiments is reported with standard deviations: basal/unstimulated calcium 100 nM; TS2/16 stimulation: control 297.6±12.6 nM, +PP2 109.2±16.9 nM, +U73122 99.5±5.4 nM. Mn2+ stimulation: control 326.5±13.5 nM, +PP2 100.3±13.1 nM, +U73122 90.5±17.4 nM. Fura2/AM labelled BE cells were also plated on Matrigel and calcium release anlaysed by dual-colour real-time fluorescent microscopy at excitation wavelengths of 380 nm (green: low/basal intracellular calcium) and 340 nm (red: high/released intracellular calcium) (emission 510 nm). Sample images at various time points were taken from the videos and are displayed (lower panel). (B) PKC phosphorylation status is unaffected by stimulation or inhibition of integrin ß1. Cells plated on Matrigel were either left untreated (control) or treated with the integrin ß1 blocking antibody 4B4 (10 µg/ml; anti-ß1), PP3 or PP2 for 8 hours, recovered from the matrix and processed for western blotting using antibodies to phosphorylated or unphosphorylated forms of PKC as indicated (left panels). Serum-starved BE cells on plastic were left unstimulated (control) or stimulated with either the activating integrin ß1 antibody (TS2/16 10 µg/ml) or Mn2+ (5 µM) for 30 minutes, lysed and processed for western blotting as when analysed on Matrigel (middle panel). As a control for the antibody specificity, A431 cells grown on plastic were either left unstimulated, stimulated with EGF (100 ng/ml) or stimulated with EGF (100 ng/ml) in the presence of AG1478 (10 µM) inhibitor (right panel).

View larger version (31K): [in a new window]   Fig. 7. Inhibition of Ins(1,4,5)P3 and calcium but not PKC inhibits early changes in cell morphology. (A) BE cells plated on Matrigel and treated for 8 hours with either 2-APB (40 µM) an Ins(1,4,5)P3 receptor antagonist, BAPTA-AM (20 µM), an intracellular calcium-chelating agent, thapsigargin (THAPS; 100 nM) an inhibitor of the sarcoplasmic endoplasmic reticulum calcium ATPase or the PKC inhibitor compounds R0-31-8220 (3 µM) or GF109203X (3 µM) and its structurally related control bisindolylmaleimide V (BiV) (3 µM). Cell elongation was analysed by microscopy with representative images shown and average percentage cell elongation, analysed from 20 independent cell images reported with standard deviations; control 161.2±17.8%, APB-2 7.7±6.4%, BAPTA-AM 1.8±6.5%, thapsigargin 3.2±4.3%, RO-31-8220 45.0±17.9%, GFX109203X 41.0±21.3%, BisV 154±16.7%. (B) HUVECs were plated on Matrigel and treated as for part A. Cell elongation and networking was analysed by fluorescent microscopy. (C) Serum-starved cells treated as for part A were plated onto Matrigel-coated Transwell chambers and invasion through the filter and into the matrix, towards serum (10%) was analysed by microscopy after 48 hours with the average percentage (number) of invading cells compared with control (untreated, 100%) sample from five experiments displayed with standard deviations. 1, control; 2, 2-APB; 3, BAPTA-AM; 4, R0-31-8220; 5, GFX109203X; 6, BiV.

View larger version (27K): [in a new window]   Fig. 8. Model of PLC1 involvement in the ß1 integrin-Src signalling cascade.