Supporting Online Material for Sequential Regulation of DOCK2 Dynamics by Two Phospholipids during Neutrophil Chemotaxis Akihiko Nishikimi, Hideo Fukuhara, Wenjuan Su, Tsunaki Hongu, Shunsuke Takasuga, Hisashi Mihara, Qinhong Cao, Fumiyuki Sanematsu, Motomu Kanai, Hiroshi Hasegawa, Yoshihiko Tanaka, Masakatsu Shibasaki, Yasunori Kanaho, Takehiko Sasaki, Michael A. Frohman, Yoshinori Fukui*
*To whom correspondence should be addressed. E-mail:[email protected] Published 26 March 2009 on Science Express DOI: 10.1126/science.1170179
This PDF file includes Materials and Methods Figs. S1 to S17 References
Supporting Online Material (SOM) Materials and Methods Mice DOCK2–/–, DOCK2-GFP, and PI3K–/– mice have been described elsewhere (S1–S3). The animals were maintained in specific pathogen-free conditions in the animal facility of Kyushu University. All experiments were done in accordance with the guidelines of the Committee of Ethics of Animal Experiments, Kyushu University. Reagents L--phosphatidic acid (PA) and phosphatidylinositol 4, 5-bisphospahte [PI(4,5)P2] was obtained from Avanti Polar Lipid (Alabaster, AL). L--phosphatidylcholine (PC), L--phosphatidylethanolamine (PE), L--phosphatidyl-L-serine (PS), oleoylL--lysophosphatidic acid (LPA), and 1-oleoyl-2-acetyl-sn-glycerol (DAG) and pertussis toxin were obtained from Sigma-Aldrich (St. Louis, MO). R59 022 was obtained from Tocris Bioscience (Ellisville, MO). Alexa Fluor 546-conjugated phalloidin, BODIPY FL dye–labeled PA, CellMask™ Orange plasma membrane stain, and anti-GFP antibody were obtained from Invitrogen (Carlsbad, CA). Anti-GST antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Akt and anti-phospho-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA). N-formyl-Met-Leu-Phe (fMLP) was obtained from Nacalai Tesque (Kyoto, Japan). Recombinant mouse C5a and CXCL2 were obtained from R&D systems (Minneapolis, MN). Plasmid construction For expression of N-terminally-tagged GFP- or DsRed monomer-fusion proteins in mammalian cells, pCI-GFP and pCI-DsRed vectors were created by subcloning a cDNA encoding EGFP or DsRed monomer (Clontech, Palo Alto, CA) into pCI (Promega, Madison, WI), respectively. The cDNAs encoding mouse DOCK2 (S1), PLD2 (S4), PLD2 K758R (S4), and the PH domain of mouse SOS (residues 423-551) (S5) were subcloned
into pCI-GFP or pCI-DsRed. To express DOCK2-PH in which the C-terminal region of DOCK2 is replaced by the PH domain of SOS, cDNAs encoding the amino acid residues 1-1626 of DOCK2-9A and the SOS-PH were subcloned into pCI-GFP. The construct to express PAK-RBD-GFP (S6) was created by inserting the cDNAs encoding PAK-RBD (residues 65-150 of mouse PAK1) and EGFP into pCI vector. To bacterially express recombinant proteins encoding GST at the N-terminus and His tag at the C-terminus, pET-GST-His vector was created by subcloning the genes encoding GST and 6x His into pET22b (Novagen, Madison, WI). The cDNAs encoding the C-terminal region of DOCK2 (residues 1615-1828) (S1), DOCK180 (residues 1611-1865) (S7) and DOCK5 (residues 1616-1848) (S7), or PLC-PH (residues 1-137) (S8) and lactadherin-C2 (residues 308-463) (S9) were amplified with PCR and subcloned into pET-GST-His. GST-Akt-PH in pGEX-6T vector has been previously described (S2). Site-directed or deletion mutagenesis was performed using the method of inverse PCR. Neutrophil isolation Bone marrow (BM) neutrophils were used in this study. To prepare BM neutrophils, BM cells were isolated from femurs and tibias of mice, and layered onto the discontinuous Percoll (GE Healthcare, Chalfont St Giles, UK) gradient. After centrifugation, cells at the 62/81% interface were recovered and washed twice with Hank’s balanced salt solution (HBSS; Invitrogen). More than 90% of the recovered cells were Gr-1+Mac-1+ mature neutrophils. Cell culture HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% FCS and maintained in 5% CO2 at 37°C. Transfection BM neutrophils were electroporated with the specified plasmid DNA (5 μg) using the Human Monocyte Nucleofector kit (Amaxa Biosystems, Cologne, Germany) as previously
described (S2). Cells were then suspended in human monocyte medium (Amaxa Biosystems) supplemented with 10% FCS and maintained in 5% CO2 at 37°C for 2-3 h before assay. Transient transfection into HEK293T cells was performed with polyethylenimine as described elsewhere (S10). Cell labeling and fluorescence microscopy BM neutrophils (1 x 106 / ml) suspended in HBSS were stimulated with 10 μ fMLP, 25 nM C5a, 5 nM CXCL2 or 10 μg/ml phospholipids at 37°C for the specified times. Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then stained with Alexa Fluor 546-conjugated phalloidin. In some experiments, cells were treated with 0.2% 1-butanol, 0.2% 2-butanol, 750 nM FIPI, 10 μ R59 022 or 25–100 nM wortmannin before stimulation. The plasma membrane staining was performed by incubating cells in a solution of CellMask™ orange plasma membrane stain (0.1 μg/ml) diluted in Ca2+- and Mg2+-free HBSS supplemented with 0.5% BSA for 60 s at room temperature. Cells were then washed with the same buffer twice before stimulation. For pertussis toxin treatment, cells were suspended in human monocyte medium supplemented with 10 % FCS, and cultured for 3 h in 5% CO2 at 37°C in the presence of the toxin (500 ng/ml) before stimulation. All fluorescent images were taken using a laser scanning confocal microscope (LCM510 meta; Carl Zeiss, Oberkochen, Germany). To analyze the effect of PLD2 on DOCK2 localization, HEK293T cells expressing GFP-tagged DOCK2 and DsRed-tagged PLD2 were fixed with 4% paraformaldehyde for 10 min, washed with PBS, and stained with DAPI (1 μg/ml) for 30 min at room temperature. Microscopic analysis was performed with a laser scanning confocal microscope (LCM510 meta). Cell fractionation Cell fractionation was performed as described previously (S11). Briefly, transfected cells were suspended in Buffer A (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM NaF, 1 mM Na3VO4, complete protease inhibitors), and subjected to a single freeze/thaw cycle followed
by centrifugation at 16,000 x g for 10 min. The supernatant was collected and used as the ‘cytosolic’ fraction. The pellet was washed with Buffer A before extraction with Buffer A containing 1% Triton X-100, and the clarified supernatant was used as the ‘membrane’ fraction. Chemotaxis assay To stimulate neutrophils by a point source of fMLP, a custom-made micropipette (Femtotips; Eppendorf, Hamburg, Germany) containing 10 μM fMLP in HBSS was placed in proximity of the cells with micromanipulator, and a chemotactic gradient was generated with passive diffusion from the tip. Images were taken at 5-s intervals for 60 s. In some experiments, cells were treated with 0.4% 1-butanol, 0.4% 2-butanol or 750 nM FIPI before fMLP stimulation. All fluorescent images were taken using a laser scanning confocal microscope (LCM510 meta). To evaluate motility during chemotaxis, neutrophils were allowed to migrate in an EZ-Taxiscan chamber (Effector Cell Institute, Tokyo, Japan) along an fMLP gradient (0–50 μM). Phase contrast images of chemotaxing cells were acquired at 60-s intervals over 15 min. In some experiments, transfected neutrophils were placed in an EZ-Taxiscan chamber on a heated stage (37°C), and GFP-positive cells were analyzed with a fluorescence microscope (model IX70, Olympus, Tokyo, Japan) equipped with 20X objective and CoolSNAP HQ/OL camera (Photometrics, Waterloo, Canada). Images were imported as stacks to ImageJ (NIH), and analyzed with the manual tracking and the chemotaxis and migration tools. Lipid micelle preparation Preparation of lipid micelles was performed as described (S5) with a slight modification. Briefly, PA, PC, PS and PE were dissolved in chloroform (0.5 mg) and dried in Speed Vac concentrator (Savant Instruments, Farmingdale, NY). The lipid powder was resuspended in HBSS (1 ml), and the solution was sonicated for 1 min, snap-frozen in liquid nitrogen, and thawed in a 37 °C incubator. The freeze and thaw cycle was repeated at least eight times until the lipid mixture became semi-transparent.
Synthesis of PIP3-coupled beads A solution of phosphoramide (2) (S12) (322.5 mg, 0.359 mmol) in dichloromethane (9.0 ml) and 1 H-tetrazole (37.6 mg, 0.537 mmol) was added to alcohol (1) (S13) at 0°C. After the reaction mixture was stirred for 2 h at room temperature, MCPBA (142.0 mg, 0.823 mmol) was added in one portion at -78°C, and the suspension was allowed to warm to room temperature over 4 h. The reaction was quenched with 10% aqueous sodium sulfite (5 ml). Usual workup and purification yielded 3 (257.8 mg, 73%) as colorless oil. 10% Palladium on carbon (61.1 mg, 0.0572 mmol) was added to a solution of 3 (56.0 mg, 0.0286 mmol) in tert-butanol (9.6 ml), water (1.6 ml), and NaHCO3 (24.1 mg, 0.286 mmol). The suspension was hydrogenated at 2 Mpa and 30°C for 48 h. The mixture was filtrated by CeliteTM, rinsed with 50% tert-butanol in water (5 ml x 3), and concentrated (without purification). Affi-Gel 10 (12 ml slurry, 0.172 mmol) was filtered and washed with ice-water (20 ml). The wet gel slurry was transferred to a solution of 4 (0.0286 mmol) and NaHCO3 (24.1 mg, 0.286 mmol) in water (25 ml). The reaction mixture was stirred for 18 h at 0°C. 1 M ethanolamine in H2O (2.5 ml) was added, and stirred for 1 h at 0°C. The reaction mixture was filtered and washed with ice-water (25 ml). OC OC 15 H 31 Bn O
OC 11 H22 N H 2
N (i -P r)2
1 H- tetra zo l e
CH 2 C l2 , rt, th en mC P BA - 78 °C to rt 73 %
Bn O OH (Bn O)2 (O )PO 1
OB n O P(O) (OB n) 2 OP (O)( OBn )2
NaO O H OO P O (N aO) 2 (O)P O 4
OC OC 15 H 31
B nO O P
OH OP (O) (ON a) 2 OP( O)(O Na )2
OC 11 H22 N H 2
H 2 (2 Mp a ) Pd /C, N a HC O 3
O t-B uOH / H 2 O, 48 h 97%
OH OP (O) (OBn )2 OP( O)(O Bn )2
(B nO )2 (O)P O
OC OC 15 H 31 OC 11 H22 N H 2
Affi-G el 10 N a H CO 3 H 2O 4 oC , 4 8 h
OC OC 15H 31
Na O H OO (N a O)2 (O) PO 5
OC 11 H 22 N H
OH OP(O )(ON a )2 OP (O) (ON a) 2
Lipid binding assay Lipids (a mixture of PC, PE and indicated phospholipids) were incubated in TBS (20 mM Tris-HCl, 0.15 M NaCl, pH 7.5) at 37°C for 1 h followed by vigorous vortexing for 10 min. The liposomes were precipitated at 18,000 x g for 10 min and washed twice with ice-cold TBS. Liposomes (200 μg) were mixed with purified protein (5 μg) in TBS to make 1 ml of
solution. The mixture was incubated for 2 h at room temperature and washed twice with ice-cold TBS buffer followed by centrifugation at 18,000 x g at 4°C. The binding proteins were immunoblotted using anti-GST antibody. To assay proteins expressed in HEK293T cells for lipid binding, cells transfected with the specified plasmid DNAs were suspended in 3 ml of TBS supplemented with 1 mM EDTA and 1 mM PMSF, and sonicated on ice. Insoluble debris was removed by centrifugation at 18,000 x g for 10 min at 4°C followed by ultracentrifugation at 100,000 x g for 30 min at 4°C. The liposome binding assay was performed at 4°C as described above, except that 1 mM EDTA, 1 mM PMSF and 0.005% NP-40 were added to the reaction and the washing solutions. In some experiments, lipid binding assay was performed using PIP3 -coupled affinity beads or PIP strips (Echelon Biosciences, Salt Lake City, UT) as described (S2, S14). Measurements for PA and PIP3 BM neutrophils were labeled with 32Pi and stimulated with 25 nM C5a. Cellular PA and PIP3 levels were measured by thin layer chromatography as previously described (S15). Statistical analysis Statistical analysis was performed using analysis of variance (ANOVA) followed by the two-tailed multiple t-test with Bonferroni correction (Fig. 1C and Fig. 4B), two-tailed Student’s t-test (Fig. 1, D and F, fig. S2, fig. S4 and fig. S9), or Kruskal Wallis H-test followed by Mann-Whitney U-test with Bonferroni correction (Fig. 4, C and D).
Fig. S1. DOCK2 accumulates preferentially at the leading edge membrane in response to a chemoattractant. After labeling of the plasma membrane (PM) with CellMaskTM Orange, neutrophils from DOCK2-GFP mice were stimulated with C5a (25 nM) for the indicated times. Scale bar, 5 μm. Each profile indicates the intensity of DOCK2 (green) and CellMaskTM Orange (magenta), which was generated along the line from the origin (left) to the arrowhead (right).
Fig. S2. Chemoattractant-induced membrane translocation, but not polarized localization, of DOCK2 depends on PI3K. Neutrophils from PI3K+/+ DOCK2-GFP and PI3K–/– DOCK2-GFP mice were exposed to a uniform concentration of (A) C5a (25 nM) or (B) fMLP (10 μM), and analyzed for DOCK2 and F-actin localization at the indicated times. Scale bar, 5 μm. The graphs indicate the percentage of neutrophils exhibiting membrane translocation of DOCK2 at 15 s or polarized accumulation of DOCK2 at 30 s and 120 s. Data are the mean ± SD of triplicate experiments, in each of which at least 100 cells were analyzed. **P < 0.01.
Fig. S3. Treatment with 1-butanol selectively inhibits accumulation of DOCK2 at the pseudopods. Neutrophils from DOCK2-GFP mice were stimulated with (A) fMLP (10 μM), (B) C5a (25 nM) or (C) CXCL2 (5 nM) in the presence of 0.2% 1-butanol or 2-butanol. Cells were then fixed at the indicated times and stained with phalloidin. Scale bar, 5 μm.
Fig. S4. Treatment with a DGK inhibitor does not affect DOCK2 localization. Neutrophils from DOCK2-GFP mice were exposed to a uniform concentration of C5a (25 nM) in the presence of R59 022 (10 μM). DMSO (vehicle) was used as a control. Cells were fixed at the indicated times and stained with phalloidin. Scale bar, 5 μm. The graph indicates the percentage of neutrophils exhibiting polarized accumulation of DOCK2 at 30 s. Data are the mean ± SD of triplicate experiments, in each of which at least 100 cells were analyzed.
Fig. S5. Treatment with 1-butanol causes extremely thin lamellae. Neutrophils from DOCK2-GFP mice were stimulated with a micropipette containing 10 μM fMLP in the presence of 0.4% 1-butanol or 2-butanol. Representative images are shown of at least 16 cells analyzed per group. Scale bar, 5 μm.
Fig. S6. PLD-generated PA controls localization of activated Rac in neutrophils. After transfection of PAK-RBD-GFP, WT neutrophils were stimulated with C5a (25 nM) in the presence of FIPI (750 nM), vehicle (DMSO), 1-butanol (0.2%), or 2-butanol (0.2%). Cells were then fixed at the indicated times and analyzed for localization of PAK-RBD and F-actin. Scale bar, 5 μm.
Fig. S7. Exogenously added PA is incorporated into the plasma membrane. Neutrophils were treated with micelles containing 0.2% BODIPY-FL-labeled PA for the indicated times and analyzed for localization of exogenously added PA. Scale bar, 5 μm.
Fig. S8. Exogenously added PA induces actin polymerization in neutrophils. WT neutrophils were stimulated with varied phospholipids (10 μg/ml). Cells were then fixed at the indicated times and stained with phalloidin. Scale bar, 5 μm.
Fig. S9. PA-induced actin polymerization is insensitive to pertussis toxin (PTx). WT neutrophils were incubated in the presence of PTx (500 ng/ml) or equivalent volume of distilled water (DW) for 3 h at 37 °C. Cells were then stimulated with (A) PA (10 μg/ml) or (B) fMLP (10 μM) for 120 s and stained with phalloidin. Scale bar, 5 μm. The graphs indicate the percentage of neutrophils exhibiting polarized F-actin accumulation. Data are the mean ± SD of triplicate experiments, in each of which at least 100 cells were analyzed. **P < 0.01.
Fig. S10. Exogenously added PA does not induce Akt phosphorylation. WT Neutrophils were stimulated with C5a (25 nM) or PA (10 μg/ml) for the indicated times and analyzed for Akt phosphorylation at Ser473 and Thr308.
Fig. S11. Treatment with wortmannin suppresses PA-induced membrane accumulation of DOCK2. Neutrophils from DOCK2-GFP mice were stimulated with PA (10 μg/ml) in the presence of wortmannin or vehicle (DMSO), and fixed at the indicated times. Scale bar, 5 μm.
Fig. S12. The C-terminal region of DOCK2 selectively interacts with PA. (A) Bacterially-expressed GST-fusion C-terminal fragment of DOCK2 (DOCK2-C) was applied to PIP strips containing various phospholipids as indicated for lipid overlay assay. Abbreviations: S1P,
GST-DOCK2-C and GST-tagged PH domain of Akt (Akt-PH) were incubated with PIP3-coupled or control beads, and the bound protein was detected with anti-GST antibody. (C) GST-DOCK2-C was pulled down with PC- and PE-based vesicles containing varying amounts of PI(4,5)P2 or PS, and the bound protein was detected with anti-GST antibody. PLC-PH or lactadherin-C2 was used as a positive control for PI(4,5)P2 or PS binding, respectively (S8, S9).
Fig. S13. The 9A mutations do not affect GEF activity of DOCK2. HEK293T cells were transfected with GFP-DOCK2 (WT and 9A) constructs, and GTP-bound form of Rac (GTP-Rac) was pulled down from the cell lysates using GST-fusion PAK-RBD. Precipitated Rac was detected by immunoblotting using anti-Rac antibody. Total Rac or transfected proteins were detected with anti-Rac or anti-GFP antibody, respectively.
Fig. S14. DOCK2-PH chimeric protein binds effectively to PA. GFP-tagged DOCK2-WT or DOCK2-PH was transiently expressed in HEK293T cells, and the cell extracts were pulled down with PC- and PE-based vesicles containing varying amounts of PA. Detection was performed with anti-GFP antibody.
Fig. S15. SOS-PH accumulates preferentially at the leading edge in response to a chemoattractant. After expression of GFP-SOS-PH as a fluorescent probe for PA, neutrophils from WT and DOCK2–/– mice were stimulated with a micropipette containing 10 μM fMLP. Intracellular localization of SOS-PH was analyzed with time-lapse video microscopy. Scale bar, 5 μm.
Fig. S16. Kinetics of PIP3 and PA production in neutrophils stimulated with C5a. Neutrophils from WT and DOCK2–/– mice were labeled with 32Pi and stimulated with 25 nM C5a. Phospholipids were extracted at the indicated times and separated on thin layer chromatography (TLC) plate to be determined for the radioactivities in PA and PIP3 fractions.
A !! ! ! !
Fig. S17. Comparison of the C-terminal region of the CDM family proteins and their capacity for PA binding. (A) Multiple sequence analysis of the C-terminal regions. The C-terminal regions of DOCK2, DOCK180 and DOCK5, all of which are known to function as Rac GEFs (S7, S16), were aligned with ClustalW. Black shadings indicate residues identical to DOCK2. Asterisks indicate residues involved in PA binding for DOCK2. (B) GST-fusion C-terminal fragment of DOCK2, DOCK180 and DOCK5 were bacterially produced and pulled down with PC- and PE-based vesicles containing varying amounts of PA. Detection was performed with anti-GST antibody.
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