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First published online 13 June 2006
doi: 10.1242/jcs.03024

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Rac1 and Rac2 regulate macrophage morphology but are not essential for migration

Ann P. Wheeler^1,2,*, Claire M. Wells¹, Stephen D. Smith^1,3, Francisco M. Vega¹, Robert B. Henderson⁴, Victor L. Tybulewicz⁴ and Anne J. Ridley^1,3,

¹ Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House Street, London, W1W 7BS, UK
² MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, WC1E 6BT, London, UK
³ Department of Biochemistry and Molecular Biology, University College London, Gower Street, WC1E 6BT, London, UK
⁴ Division of Immune Cell Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

View larger version (22K): [in a new window]   Fig. 1. Rho GTPase expression in BMMs derived from Wt, Rac2–/– and Rac1/2–/– mice. (A) To determine the expression profile of Rac isoforms in BMMs, RT-PCR was performed with isoform-specific primers to Rac1, Rac2 and Rac3 on cDNA derived from Wt, Rac2–/– and Rac1/2–/– BMM mRNA. PCR was carried out for 30 cycles. (B) As a positive control for mRNA extraction and cDNA synthesis, RT-PCR was performed with primers to CDK4 on BMM-derived cDNA. PCR was carried out for 30 cycles.

View larger version (50K): [in a new window]   Fig. 2. Rac1 and Rac2 affect cytoskeletal organization in macrophages. (A) Wt, Rac2–/– and Rac1/2–/– BMMs in macrophage growth medium were fixed and stained with TRITC-phalloidin and anti-ß-tubulin antibodies to show F-actin and microtubules. Confocal images are shown of the basal plane of cells. (B) Quantification of F-actin levels in Wt, Rac2–/– and Rac1/2–/– BMMs from TRITC-phalloidin staining of paraformaldehyde-fixed cells (see Materials and Methods). Bars, 10 µm. (C) Quantification of dorsal ruffling. BMMs were stimulated with CSF-1 and stained as in (B). Cells were considered to be ruffling if more than one dorsal ruffle was present. *P<0.05 and **P<0.01 compared with levels in Wt BMMs, n=30 (Student's t-test). (D) Dorsal ruffling in response to CSF-1 stimulation in Wt, Rac2–/– and Rac1/2–/– BMMs. BMMs were starved of CSF-1, then stimulated with CSF-1 for 30 minutes, fixed and stained with TRITC-phalloidin to show F-actin. Confocal images were taken through the medial plane of cells to show membrane ruffles; arrows indicate membrane ruffles.

View larger version (45K): [in a new window]   Fig. 3. Rac2–/– and Rac1/2–/– macrophages have altered morphology. Wt, Rac2–/– and Rac1/2–/– BMMs in growth medium were fixed and stained for F-actin and paxillin or ß-tubulin. (A) The spread area of the basal surface of BMM was measured (see Materials and Methods). (B) The elongation ratio is the ratio of the cell length (longest chord through the cell) to the width (longest axis perpendicular to the longest chord). (C) Confocal images illustrating the stellate, elongated and migratory morphological classification groups. BMMs were stained for F-actin (red) and ß-tubulin (green). (D) Quantification of BMM morphology. BMM shape was defined according to the three classification groups shown in C. Results are shown as mean ± s.e.m. of 150 cells from each population over three separate experiments. *P<0.05 vs Wt BMMs (Student's t-test).

View larger version (88K): [in a new window]   Fig. 4. Rac1 affects podosome assembly. Wt, Rac1–/–, Rac2–/– and Rac1/2–/– BMMs in macrophage growth medium were stained for F-actin (red in merged image) and paxillin (green in merged image). A higher magnification image of podosomes in shown in the top right hand corner of Wt and Rac1–/– BMM images. Bars, 10 µm.

View larger version (59K): [in a new window]   Fig. 5. Rac1/2–/– BMMs have altered migratory properties. (A) Cell migration was followed by time-lapse microscopy for 6.5 hours. Migration tracks of cells that moved more than 30 µm from their origin are shown in the vector plots. The origin of each cell is at the intersection of the x and y axis. (B) Quantification of macrophage displacement (net distance moved from origin). The percentage of Wt, Rac2–/– and Rac1/2–/– BMMs that had moved the indicated distance (displacement) from their origin after 6.5 hours is shown. (C) Graph showing the directional persistence (track length/displacement) of Wt, Rac2–/– and Rac1/2–/– BMMs. *P<0.05 vs Wt BMMs (Student's t-test). Data are pooled from three independent experiments; n=120 cells for each genotype. (D-F) Representative phase-contrast images from time-lapse movies of (D) Wt, (E) Rac2–/–, (F) Rac1/2–/– BMMs; elapsed time is indicated in minutes.

View larger version (29K): [in a new window]   Fig. 6. Rac1 is required for invasion through Matrigel. BMMs were plated onto Transwell filters (A) or Matrigel-coated Transwell filters (B). The number of cells on the lower side of the filters was counted after 24 hours. Results are from three independent experiments, data are means ± s.e.m.