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Blood, Vol. 107, Issue 4, 1627-1635, February 15, 2006
Rho GEF Lsc is required for normal polarization, migration, and adhesion of formyl-peptide–stimulated neutrophils
Blood Francis et al.
107: 1627
Supplemental materials for: Francis et al
Methods and Materials Southern blot of genomic tail DNA
Tail genomic DNA was extracted, digested with ScaI or BgllI, and subjected to Southern blotting with a 3’- or 5’-probe, respectively, using standard methods1. Isolation of mouse splenocytes
The spleen was removed, dissociated in RPMI media supplemented with 25 mM HEPES, 10% FBS, 1% L-Glutamine, 100 IU/ml penicillin, and 100 µm/ml streptomycin, the suspension was passed through a 70 µm strainer (Becton Dickinson), cells were collected by centrifugation at 400 g for 10 min at 4°C, and then re-suspended in the same media. RT-PCR amplification of Lsc partial cDNAs from splenocyte RNA
Primers corresponding to exonic sequence separated by at least one intron for Lsc (f-gatctgatctctgaggatgtccag, r-gaagttccttcccgacttcttgtc) and primers corresponding to exonic sequence for mouse GAPDH (f-tgaaggtcggtgtgaacggatttggc, r-catgtgggccatgaggtccaccac) were used to amplify partial cDNAs from Lsc KO and WT mouse splenocyte first-strand cDNA by PCR (25 cycles) on a RoboCycler Gradient 96 Temperature Cycler (Stratagene). The reactions were subjected to electrophoresis on a 2% agarose gel. Western blotting
A total of 106 neutrophils or splenocytes were suspended in 1 mL of cold lysis buffer2, and subjected to 20 strokes in a Dounce homogenizer (Wheaton, Millville, NJ). A 25 µL aliquot of the lysate was subjected to Western blotting with the M-19 anti-Lsc antibody and HRP-conjugated anti-goat IgG antibody using previously described methods2. Activated Rho GTPase pull-down assays
Activated RhoA, Cdc42, Rac1, and Rac2 were measured in ∼ 2 - 4 × 106 suspended bone marrow neutrophils using the corresponding EZ-Detect Activation Kit (Pierce). These kits are modified forms of standard pull-down assays3. The pull-down sample and a standardized volume of the corresponding crude lysate were subjected to Western blotting with a monoclonal mouse IgG anti-RhoA,B,C antibody (Pierce), anti-Cdc42 antibody (Pierce), anti-Rac1 antibody (Pierce), or anti-Rac2 antibody (Santa Cruz Biotechnology) and with an HRP-conjugated goat anti-mouse IgG antibody (Bio-rad) according to the manufacturer’s instructions. The immunoblots were developed with the ECL Plus detection system (Amersham Biosciences). Images were collected and the band intensities quantitated with a Storm 860 Imaging System (Amersham Biosciences). For each Rho GTPase (RhoA, Cdc42, Rac1, or Rac2), the Rho GTPase activity at each time point, in each experiment, was calculated as the band intensity corresponding to the activated Rho GTPase/ band intensity corresponding to the total Rho GTPase in a fixed volume of lysate. This Rho GTPase activity was then normalized to the mean Rho GTPase activity in WT neutrophils at time = 0 s for presentation and statistical comparison. Assays for RhoA, Rac1, and Rac2 activity were performed in triplicate, and assays for Cdc42 activity were performed in duplicate. Integrin expression
Bone marrow neutrophils in adhesion buffer were incubated with 10 µM fMLP or vehicle (DMSO) alone for 30 min at 37°C, washed once in FACS buffer (PBS without Ca2+ or Mg2+ supplemented with 2% heat-inactivated FCS and 0.09% sodium azide), incubated with anti-CD12/32 antibody for 20 min at 4°C, labeled with the indicated fluorophore-conjugated antibodies for 30-45 min at 4°C, washed once in FACS buffer, and immediately subjected to analysis on a FACScan analyzer (Becton Dickinson). Forward- and side-scatter gates were established with anti-Gr1-labeled neutrophils and were identical for WT and Lsc KO neutrophils. At least 104 gated events were recorded in each experiment. Complete peripheral blood counts
Approximately 750 µL of venous blood was obtained from 11 gender-matched mice (18-20 weeks old) of each genotype by retro-orbital phlebotomy and cell counts were obtained using a Cell-Dyn 3700 hematologic analyzer (Abbott Diagnostics, Abbott Park, IL). E. coli Peritonitis
E. coli were obtained from ATCC (#25922), propagated in Luria-Bertani broth, and maintained as a glycerol stock. E. coli were grown in Luria-Bertani broth overnight, washed once in PBS, and 1 × 106 cfu in 200 µl were injected into the peritoneal cavity of WT or Lsc KO mice with a 25 gauge needle. The mice were given food and water ad libitum, and at 12 h after the injection, the animals were sacrificed by cervical dislocation, and the peritoneal cavity was lavaged with 5 ml of PBS. The leukocyte count and differential leukocyte fractions were obtained from a mixed sample of the lavage fluid using a Cell-Dyn 3700 hematologic analyzer (Abbott Diagnostics), and used to calculate the total neutrophil count in the 5 ml peritoneal lavage. Chemical Dermatitis
For each mouse, 10 µL of 4% croton oil in acetone and 10 µL of acetone alone were applied to both the inner and outer surface of the right and left ear pinnae, respectively. After 6 h, the mice were sacrificed, a 6-mm diameter punch biopsy of each ear was obtained, weighed, and assayed immediately for myeloperoxidase activity by placing the tissue disc in 400 µL of PBS and subjecting it to 60 strokes in a 1 mL Dounce homogenizer (Wheaton), sonication for 20 s, 3 freeze-thaw cycles in liquid nitrogen, and then sonication for 20 s. The cell homogenate was then subjected to centrifugation at 10,000 g for 20 min at 4°C, 40 µL of the supernatant was incubated with 360 µL of myeloperoxidase assay buffer (50 mM sodium phosphate buffer pH 6.0, 0.4 mg/mL O-phenylenediamine, 0.05% hydrogen peroxide) at room temperature for 20 min, 100 µL of stop buffer (0.4 M H2SO4) was added, and the OD490 of the mixture was measured. Data were summarized as the mean +/- SEM ratio of the OD490 reading for the ear exposed to croton oil normalized to the reading for the ear exposed to acetone. To measure the cell count, only the inner surface of the pinnae were painted as above, after 6 h the ears were harvested and fixed in 4% PFA overnight, transferred to 70% ethanol overnight, a 6-mm punch biopsy from each ear was embedded in paraffin, and 10 µm sections were obtained and stained with hematoxylin and eosin. Cells in nine cross-sectional fields for each ear were counted manually by an observer blinded to the genotype of the mice. Data were summarized as the mean ratio +/- SEM of the number of cells counted per field in the ear exposed to croton oil normalized to the number of cells per field in the ear exposed to acetone. Cell culture, differentiation, and transfection
HL-60 cells (ATCC #CCL-240, Manassas, VA) were maintained, differentiated, and transfected using previously published methods4. Immunostaining HL-60 cells and neutrophils
HL-60 cells: DMSO-differentiated (6 days) HL-60 cells transfected with the FLAG.Lsc-pBJ1 plasmid2 were allowed to recover for 24 h, adhere to fibronectin-coated cover slips for 2 h, incubated with vehicle (DMSO) or 10 µM fMLP for 5 min, fixed in 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton, labeled with Alexa 594-conjugated phalloidin (Molecular Probes), M2 anti-FLAG antibody, and Alexa 488-conjugated goat anti-mouse IgG antibody. Bone marrow neutrophils: Bone marrow neutrophils were isolated, allowed to adhere to a glass cover slip for 30 min, fixed and permeabilized as described for HL-60 cells, and then labeled with Alexa 594-conjugated phalloidin, rabbit IgG C-23 anti-Sos1 antibody (Santa Cruz Biotechnology) and Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes). Confocal microscopy of HL-60 cells and neutrophils
Confocal images were obtained with an Axiovert 100M inverted microscope (Zeiss, Thornwood, NY) equipped with an LSM 510 laser scanning system (Zeiss) using an oil-immersion 63 × 1.4 numerical aperture plan Apochromat objective (Zeiss) at room temperature. Excitation was achieved with a 25 mW argon laser emitting at 488 nm, and a 1.0 mW helium/neon laser emitting at 543 nm. 505-530 nm and 560-615 nm band pass filters were used to collect Alexa 488 and Alexa 594 emissions, respectively. The optical section thickness was 0.5 µm. References 1. Nagy A, Gertsenstein, M., Vintersten, K., and Behringer, R. Manipulating the Mouse Embryo (ed 3rd.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2003
2. Eisenhaure TM, Francis SA, Willison LD, Coughlin SR, Lerner DJ. The Rho Guanine Nucleotide Exchange Factor Lsc Homo-oligomerizes and Is Negatively Regulated through Domains in Its Carboxyl Terminus That Are Absent in Novel Splenic Isoforms. J Biol Chem. 2003;278:30975-30984
3. Benard V, Bokoch GM. Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 2002;345:349-359
4. Srinivasan S, Wang F, Glavas S, Ott A, Hofmann F, Aktories K, Kalman D, Bourne HR. Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J Cell Biol. 2003;160:375-385
Files in this Data Supplement:
- Figure S1. Lsc is specifically enriched at the leading and trailing edge of fMLP-stimulated myeloid HL-60 cells (JPG, 12.6 KB)
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Confocal images of HL-60 cells expressing FLAG epitope-tagged Lsc that were adhered to fibronectin-coated glass cover slips, incubated with vehicle alone (-fMLP) or 10 µM fMLP (+fMLP) for 5 min, fixed, permeabilized, and then labeled with mouse anti-FLAG antibody (green) and phalloidin (red). Lsc is enriched in the leading edge (solid arrowhead) and, to a lesser extent, in the trailing edge of formyl-peptide-stimulated HL-60 cells. The distribution of Lsc in unstimulated and stimulated HL-60 cells is nearly identical to that in neutrophils (Figure 1). Bars represent 10 µm.
- Figure S2. Sos1 is a Rho GEF that is not enriched at the leading or trailing edges of fMLP-stimulated neutrophils (JPG, 23.7 KB)
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(A) Confocal images of mouse neutrophils prepared as described in Figure 1 and lebeled with anti-Sos1 antibody (green) and phalloidin (red). Sos1 is a Dbl-Rho GEF with a PH domain that, unlike Lsc, is distributed diffusely in the cytoplasm of unstimulated neutrophils, and is not enriched in the leading edge (solid arrowhead) or trailing edge of fMLP-stimulated neutrophils. Photomicrographs representative of n = 2 independent experiments. Bars represent 10 µm. (B) Quantitative comparison of Lsc and Sos1 enrichment in the leading edge of fMLP-stimulated mouse neutrophils. Neutrophils were prepared, stimulated with fMLP, labeled with phalloidin and either anti-Lsc or anti-Sos1 antibody, and imaged as described in Figure 1 and Figure S2A. Rho GEF distribution was scored as enriched at the leading edge if the majority of the Rho GEF signal overlapped with the organized F-actin network at the leading edge. Twenty cells labeled with each antibody were scored for leading edge enrichment, and the number of enriched cells in each group was compared using a Chi-square test. Lsc is enriched at the leading edge of most neutrophils, while Sos1 is not.
- Video 1. Wild-type bone marrow neutrophils stimulated with fMLP (MOV, 2.07 MB)
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DIC images (25× objective) of neutrophils adhered to a glass cover slip and exposed to 10 µM fMLP in a Zigmond chamber were acquired at 15 s intervals for 15 min. The set of 61 images acquired during the 15 min experiment were assembled into a Quicktime video formatted at 650 × 515 pixels playing 7.5 frames per second. This video is representative of 6 similar videos acquired in 3 independent experiments. (See Materials and Methods for details)
- Video 2 (MOV, 1.84 MB)
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Lsc KO bone marrow neutrophils stimulated with fMLP. Images were acquired and assembled into a video as described in the legend for Video 1. This video is representative of 6 similar videos acquired in 3 independent experiments. (See Materials and Methods for details).
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