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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01545

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PTEN regulates motility but not directionality during leukocyte chemotaxis

Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, UAM Campus de Cantoblanco, 28049 Madrid, Spain

View larger version (34K): [in a new window]   Fig. 1. Biochemical characterization of PTEN chimeras. (A) Extracts from Jurkat cells transfected with mock, N- or C-terminus PTEN chimeras or a bicistronic plasmid encoding PTEN or PTEN-C/S and GFP, were assayed for in vitro lipid phosphatase activity using PtdIns(3,4,5)P3 as a substrate in a Malachite Green-based assay. The phosphate released (measured in pmoles) by the mock cell lysate was considered the reference unit. A western blot showing the corresponding cell extract PTEN levels is shown at the right. Data are representative of three separate experiments. (B) Extracts from unstimulated and CXCL12 (100 nM)-stimulated mock, PTEN-GFP- or PTEN-C/S-GFP-Jurkat cells were analyzed sequentially with anti-phosphoAKT and AKT Ab, and with anti-PTEN as a protein expression control. After densitometry, AKT phosphorylation was determined as the phospho-AKT:AKT ratio (x10) (n=3). (C) The AKT phosphorylation index was also determined using extracts from unstimulated and CXCL12-stimulated mock, GFP-PTEN or PTEN-GFP-Jurkat cells as above (n=2).

View larger version (62K): [in a new window]   Fig. 2. The PTEN-GFP chimera does not polarize during chemotaxis. (A) In chemotaxis assays, a passive fMLP diffusion gradient was established near HL60 cells co-expressing PTEN-GFP and RFP. Dark-phase and red and green fluorescence were recorded in a confocal microscope; the first 20 frames were recorded at 5-second intervals and remaining frames every 20 seconds. Dark-phase, green fluorescence, and red and green merge are shown; a video with single-color recording is provided (see supplementary material, Movie 1). Asterisk indicates pipette tip. The cell is representative of 28 out of 30 cells analysed. Bar, 10 µm. (B) Red and green fluorescence scanning using line `a' of cells in A. (C) Red and green fluorescence scanning using line `b' of cells in A. (D) HL60 cells co-expressing PTEN-GFP and RFP-GPI were analyzed in chemotaxis assays towards fMLP as in A. Dark-phase, green fluorescence, and red and green channel merge are shown; see supplementary material Movie 2. Dark-phase images are shown at a lower magnification to indicate the position of the chemoattractant source. The cell is representative of 25 out of 25 cells analysed. Bar, 10 µm. (E) Red and green fluorescence scanning of cells in panel D.

View larger version (96K): [in a new window]   Fig. 3. GFP-PTEN remains evenly distributed during chemotaxis. (A) HL60 cells co-expressing GFP-PTEN and RFP were analyzed in chemotaxis assays towards fMLP. Dark-phase, green fluorescence, and red and green channel merge are shown; see supplementary material, Movie 3. The asterisk indicates the pipette tip. The cell is representative of 14 out of 15 cells analysed. Bar, 10 µm. (B) Red and green fluorescence scanning of cells in panel A.

View larger version (64K): [in a new window]   Fig. 4. PTEN-C/S-GFP does not polarize during chemotaxis. (A) HL60 cells co-expressing PTEN-C/S-GFP and RFP-GPI were analyzed in chemotaxis assays towards fMLP as in Fig. 2A. Dark-phase, green fluorescence, and red and green channel merge are shown; see supplementary material, Movie 4. The asterisk indicates the pipette tip. The cell is representative of 20 out of 22 cells analysed. Bar, 10 µm. (B) Red and green fluorescence scanning of cells in panel A.

View larger version (39K): [in a new window]   Fig. 5. PTEN chimeras are found mostly in the cytosol of Jurkat cells. Unstimulated or CXCL12 (100 nM)-stimulated mock-, PTEN-GFP- and PTEN-C/S-GFP-expressing Jurkat cells were fractionated in density gradients to isolate membrane (M) and cytosolic (C) fractions, and western blots were performed with anti-PTEN, -p85 PI3K, -transferrin receptor (TfR) and -tubulin antibodies (n=3). Relative accumulation of p85 and PTEN-GFP in the membrane and cytosolic fractions during a CXCL12 stimulation time course was calculated by densitometry.

View larger version (41K): [in a new window]   Fig. 6. Endogenous PTEN is distributed homogeneously in the cytosol of HL60 cells. (A) Immunofluorescence analysis with anti-PTEN antibody (red) to show the endogenous PTEN location in unstimulated or fMLP-stimulated HL60 cells. The cells are representative of 25 out of 25 cells analysed. Bar, 10 µm. (B) Immunofluorescence of mock- and PTEN-GFP-transfected CEM cells was performed to demonstrate anti-PTEN antibody (red staining) specificity. Bar, 10 µm. (C) Analysis of PTEN and p85 in the membrane and cytosolic fractions of fMLP-stimulated HL60 cells was performed as described in Fig. 5.

View larger version (38K): [in a new window]   Fig. 7. PTEN regulates actin polymerization but not cell orientation. (A) HL60 cells were untransfected (N.T.) or transfected with control or PTEN-specific siRNA. PTEN and tubulin (loading control) were analyzed by western blot. The graph at the right shows the quantification of PTEN estimated by densitometry using tubulin values for normalization. (B) The cells in panel A were analyzed for chemotaxis toward fMLP in transwells. Cells (2x105) were added to the upper chamber; the number of migrated cells in the lower well was determined by FACS and depicted as a percentage of migrating cells. (C) Jurkat cells were transfected with a bicistronic plasmid encoding PTEN or PTEN-C/S and GFP. PTEN and GFP expression were analyzed by western blot. Equal protein amounts of a GFP-transfected HL60 cell lysate were included in the western blot to compare relative amounts of endogenous (HL60) and ectopically expressed PTEN (Jurkat). (D) The cells (2x105) in panel C were analyzed for chemotaxis toward CXCL12 in transwell assays; GFP-positive cells in the lower well were determined by FACS; results are expressed as the percentage of migrating cells. (E) The cells in panel C were analyzed for chemotaxis toward CXCL12 as in Fig. 2A; dark-phase and green fluorescence were recorded at 23-second intervals. Migration parameters for 20 cells/condition were determined with specific software. Representative paths of three cells are shown; average speed is shown of all cells analyzed. *, start point; , pipette tip. (F) CXCL12-induced actin polymerization was measured by FACS in mock, PTEN-, and PTEN-C/S-expressing cells described in panel C, using Alexa-fluor 633-phalloidin.

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