QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00997

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wells, C. M. Articles by Ridley, A. J. Search for Related Content PubMed PubMed Citation Articles by Wells, C. M. Articles by Ridley, A. J. Social Bookmarking                         What's this?

Rac1-deficient macrophages exhibit defects in cell spreading and membrane ruffling but not migration

Claire M. Wells^1,*,, Marita Walmsley^2,, Steen Ooi², Victor Tybulewicz² and Anne J. Ridley^1,3,

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

View larger version (31K): [in a new window]   Fig. 1. Rac isoform expression in bone marrow-derived macrophages (BMMs). (A) RT-PCR to establish primer specificity. RT-PCR was performed on plasmid DNA containing Rac1, Rac2 or Rac3 cDNA using the Rac1/2/3 isoform-specific primers as described in Materials and Methods. (B) RT-PCR to establish Rac isoform expression in BMMs. RT-PCR was performed on cDNA derived from Wt BMM RNA using the same primer sets as in A). As a control for the presence of genomic DNA in the cDNA preparation RT-PCR was also carried out on cDNA prepared in the absence of reverse transcriptase. (C) Analysis of Rac1 and Rac2 protein expression levels in BMMs. Lysate from Wt BMMs and recombinant Rac1 (top) and Rac2 (bottom) protein (lanes 1 and 2; 1.5 and 2.5 ng, respectively) were resolved by SDS-PAGE, western blotted and probed with anti-Rac1 (top) or anti-Rac2 (bottom) antibody. Blots were re-probed with anti-ß-tubulin antibody. (D) Bands on autoradiographs of three western blots similar to those in C were quantified using Kinetic Imaging software. The level of Rac1 or Rac2 protein in BMMs was compared with the level of recombinant Rac1 or Rac2 (lane 1, 1.5 ng).

View larger version (25K): [in a new window]   Fig. 2. Characterisation of Rac1 deletion. (A) Southern blot of DNA cut with BamHI, derived from the bone marrow of a Rac1flox/flox (1) and a Rac1flox/floxMx1-Cre (2) mouse that had been injected with polyIC as described in the Materials and Methods. Bone marrow was harvested 2 days after the last polyIC injection. The Southern blot was probed with a DNA fragment from intron 3 of Rac1 which hybridises to a 2.9 kb and a 1.5 kb fragment in the Rac1flox and Rac1 alleles respectively, as shown (Walmsley et al., 2003). The number below lane 2 shows the percentage deletion of the Rac1flox allele. Typical deletion of the Rac1flox allele in bone marrow was 90% or higher, and persisted for at least 3 months following polyIC injection (not shown). (B) Analysis of Rac1 and Rac2 protein expression in Wt and Rac1–/– BMMs. Recombinant Rac1 (Rac1 Con.) and Rac2 (Rac2 Con.) proteins (1.5 ng) purified from E. coli were resolved by SDS-PAGE and immunoblotted with anti-Rac2 or anti-Rac1 antibodies (left blot) to demonstrate the specificity of the antibodies. A western blot of lysates from growing Wt, WtCre and Rac1–/– BMMs was probed sequentially with anti-Rac2, anti-Rac1 and anti-ß-tubulin antibodies (right blot). (C) Analysis of Cdc42 activity in growing cells. WtCre and Rac1–/– BMMs were maintained in growth medium. Cells were lysed and GST-WASP-CRIB was used to precipitate endogenous GTP-bound Cdc42. Immunoblots were performed using an anti-Cdc42-specific antibody. Immunoblots of whole cell lysates (WCL) retained from the GST-WASP-CRIB pull-down lysates were performed using the same antibody. Autoradiographs of the GST-WASP-CRIB pull-down and the WCL blots were quantified using kinetic imaging software, and the level of GTP-bound Cdc42 normalised to protein expression levels in the WCL. The results shown are the mean±s.e.m. of three independent experiments. (D) Analysis of Cdc42 activity during CSF1 stimulation. WtCre and Rac1–/– BMMs were maintained in growth medium (G), starved of CSF-1 overnight (S) or starved of CSF-1 overnight and re-stimulated with recombinant mCSF-1 (30 ng/ml) for 2 minutes (2'). Cells were lysed and GST-WASP-CRIB was used to precipitate endogenous GTP-bound Cdc42. Immunoblots were performed using an anti-Cdc42-specific antibody. Immunoblots of whole cell lysates (WCL) retained from the GST-WASP-CRIB pull-down lysates were performed using the same antibody. Autoradiographs of the GST-WASP-CRIB pull-down and the WCL blots were quantified using kinetic imaging software, and the level of GTP-bound Cdc42 normalised to Cdc42 expression levels in the WCL. The results shown are the mean±s.e.m. of three independent experiments.

View larger version (47K): [in a new window]   Fig. 3. Rac1–/– BMMs have altered morphology. (A) Growing BMMs derived from Wt and Rac1–/– mice were stained for F-actin with TRITC-phalloidin. Scale bar: 10 µm. (B) Quantification of adhesive area. Growing Wt, WtCre and Rac1–/– cells were stained for F-actin, and total area of adhesion was measured (see Materials and Methods). The results shown are mean ± s.e.m. of 100 cells from each population over three separate experiments. (C) Quantification of cell shape. Growing Wt, WtCre and Rac1–/– cells were stained for F-actin, and elongation ratio (ratio of the longest to the shortest cell axes) was measured (see Materials and Methods). The results shown are mean ± s.e.m. of 100 cells from each population over three separate experiments. Statistical significance compared to Wt values was calculated using Student's t-test, ***P<0.001.

View larger version (40K): [in a new window]   Fig. 4. Rac1–/– BMMs have a reduced ruffling response to CSF-1. CSF-1-starved (A,B) and CSF-1-stimulated BMMs (C,D) were fixed and stained for F-actin with TRITC-phalloidin. Arrows indicate membrane ruffles. (Inset) An example of a Rac1–/– BMM that has a full ruffling response to CSF-1. Scale bar: 10 µm. (E,F) Quantification of changes in adhesive area (E) and cell elongation ratio (ratio of the longest to the shortest cell axes; F) in growing, starved and CSF-1 stimulated WtCre and Rac1–/– BMMs. The results shown are mean±s.e.m. of 100 cells from each population over two separate experiments. Statistical significance compared to WtCre growing values was calculated using Student's t-test, *P<0.05; ***P<0.001.

View larger version (24K): [in a new window]   Fig. 5. Effects of N17Cdc42, N17Rac1 and WtRac1 on BMMs. (A) Quantification of the ruffling response to CSF-1. CSF-1-starved control (Wt or WtCre) or Rac1–/– cells were microinjected with 100 ng/µl Flag-tagged N17Cdc42, N17Rac1 or WtRac1 cDNA, as indicated below each bar. The cells were incubated for 3 hours prior to re-stimulation with 30 ng/ml CSF-1 for 5 minutes. Following stimulation, the cells were fixed and stained for F-actin. The ruffling response to CSF-1 was scored as described in Materials and Methods. Black bars, uninjected control and Rac1–/– cells. (B-E) Ruffling response in microinjected Rac1–/– cells. Cells were microinjected with 100 ng/µl Flag-tagged N17Cdc42 or WtRac1 cDNA. The cells were incubated for 3 hours prior to re-stimulation with 30 ng/ml CSF-1 for 5 minutes. Following stimulation, the cells were fixed and stained for F-actin. Arrows indicate membrane ruffling. Scale bar: 10 µm. (F) Quantification of cell elongation ratio following exogenous expression of N17Rac1 and WtRac1 as described above. The elongation ratio (ratio of the longest to the shortest cell axes) was then measured (see Materials and Methods). The results shown are mean±s.e.m. of 60 cells from each population over at least three separate experiments. Statistical significance compared to WtCre values was calculated using Student's t-test, ***P<0.001.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter