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First published online 15 April 2003
doi: 10.1242/jcs.00424

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Mechanical constraint imposed on plasma membrane through transverse phospholipid imbalance induces reversible actin polymerization via phosphoinositide 3-kinase activation

¹ CNRS-UMR 5539, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France
² INSERM U563, CHU Purpan, place du Dr Blayac, 31059 Toulouse Cedex 03, France

View larger version (107K): [in a new window]   Fig. 1. Scanning electron micrographs showing the representative morphology for resting platelets (a); platelets incubated with 1% (0.2)PS of total amount of PL for 2 and 30 minutes, respectively (b,c); platelets incubated with 1% of (0.2)PC for 2 and 30 minutes respectively (d,e); stimulated platelets with the Ca2+-ionophore A23187 (1 µM) pre-incubated with 100 µg/ml of calpeptin (f); platelets pre-treated with cytochalasin D for 15 minutes and then incubated with (0.2)PS for 2 minutes (g); platelets pre-treated with cytochalasin D for 15 minutes and then incubated with (0.2)PC for 2 minutes (h); platelets pre-treated with nocodazole for 15 minutes and then incubated with (0.2)PC for 2 minutes (i). Bars, 1 µm.

View larger version (95K): [in a new window]   Fig. 2. Confocal microscopy micrographs showing the G-actin (green) and the F-actin (red) staining of resting platelets (a-c); platelets incubated with 1% excess of (0.2)PC for 2 minutes (d-f) and 30 minutes (g-i); platelets stimulated with the Ca2+-ionophore A23187 (1 µM) pre-incubated with 100 µg/ml of calpeptin (j-l). Bars, 2 µm.

View larger version (17K): [in a new window]   Fig. 3. Induction of actin polymerization by excess PL in resting platelets. Actin filament content in different platelets extracts was determined by the Dnase 1 inhibition assay as indicated in Materials and Methods. Ctrl, the actin filament content in resting platelets; PC 2min and PC 30min, the actin filament content in platelets incubated with (0.2)PC for 2 and 30 minutes, respectively; PS 2min and PS 30min, the actin filament content in platelets incubated with (0.2)PS for 2 and 30 minutes, respectively; Calp+A23, the actin filament content in platelets pre-treated with 100 µg/ml calpeptin for 30 minutes and then activated with the calcium ionophore A23187 (1 µM). Black and hatched bars represent the actin filament content in the absence or presence of 50 µM cytochalasin D (CD), respectively. The data are expressed as the mean of eight individual experiments ± s.d.

View larger version (53K): [in a new window]   Fig. 4. PI 3-kinase involvement on filopodia extension and actin polymerization induced by excess PL. Scanning electron micrographs showing the representative morphology for resting platelets (a); resting platelets incubated with 1% of DLPC for 2 minutes (b); resting platelets pre-treated with 100 nM wortmannin for 15 minutes and then incubated with 1% of DLPC for 2 minutes (c). Bar, 1 µm. (d) Actin filament content in different cell extracts as measured by the Dnase 1 inhibition assay. Ctrl, the actin filament content in resting platelets; WCtrl, the actin filament content in platelets pre-incubated with 100 nM wortmannin for 15 minutes; DLPC, the actin filament content in resting platelets incubated with 1% of DLPC for 2 minutes; WDLPC, the actin filament content in resting platelets pre-incubated with 100 nM wortmannin for 15 minutes; LYDLPC, the actin filament content in resting platelets pre-incubated with 50 µM LY294002 for 15 minutes before addition of PC; PMA, the actin filament content in platelets stimulated with 100 nM PMA for 20 minutes; WPMA, the actin filament content in resting platelets before addition of PMA. (e) The quantification of PtdIns(3,4)P2 in resting platelets (Ctrl); in platelets incubated with DLPC for 2 minutes (DLPC), in platelets pre-incubated with 100 nM wortmannin (WDLPC) or 50 µM LY294002 (LYDLPC) 15 minutes before addition of DLPC; and in platelets stimulated with 1 Unit/ml of thrombin (Thb). The data are expressed as the mean of four individual experiments ± s.d.

View larger version (40K): [in a new window]   Fig. 5. Reversible translocation of PI 3-kinase to the plasma membrane and phosphorylation of Akt upon addition of excess phospholipids. (A) Superimposed optical sections (focused on the middle of the cell body) of PI 3-kinase (green) and F-actin (red) recorded by confocal microscopy. (B,C) Immunoblotting of phosphorylated Akt of total platelets lysates treated in different conditions. (B) (lane 1) Control; (lanes 2 and 3) in the presence of excess PC at 1 and 5 minutes, respectively; (lanes 4 and 5) the same conditions as lane 2 but in the presence of 100 nM wortmannin and 50 µM LY294002, respectively; (lanes 6 and 7) in the presence of excess PS at 1 and 30 minutes, respectively; (lane 8) the same conditions as lane 6 but in the presence of 100 nM wortmannin; (lane 9) platelets activated by 1 U/ml of thrombin. (C) Platelets in the presence of excess PC after a 1 minute (lane 1) and 30 minute (lane 2) incubation. The data are representative of three individual experiments. Bar, 1.5 µm (A).

View larger version (42K): [in a new window]   Fig. 6. DIC microscopy micrographs of untreated L929 fibroblasts (a), L929 fibroblasts incubated for 3 hours at 37°C in the presence of 2.5 µg/ml nocodazole (b), and L929 fibroblasts incubated for 3 hours at 37°C in the presence of 2.5 µg/ml nocodazole and subsequently treated with 50 µM DLPC (<2% of endogenous phospholipids) for 3 minutes (c). Cells were then fixed for microscopic observations. Bars, 10 µm.

View larger version (49K): [in a new window]   Fig. 7. Fluorescent microscopy micrographs of untreated L929 fibroblasts (a); L929 fibroblasts incubated for 3 hours at 37°C in the presence of 2.5 µg/ml nocodazole (b); L929 fibroblasts incubated for 3 hours at 37°C in the presence of 2.5 µg/ml nocodazole and subsequently treated with 50 µM DLPC (<2% of endogenous phospholipids) for 3 minutes (c); cells incubated for 3 hours at 37°C in the presence of nocodazole 2.5 µg/ml and 100 nM wortmannin for 20 minutes at 37°C, and then treated with DLPC 50 µM for 3 minutes (d). Cells were fixed, permeabilized and reacted with 0.5 µM phalloidine-rhodamine (F-actin labelling). Bars, 10 µm.