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First published online April 28, 2005
doi: 10.1242/10.1242/jcs.02314

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BCL6 suppresses RhoA activity to alter macrophage morphology and motility

¹ Department of Developmental and Molecular Biology,
² Department of Cell Biology
³ Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (105K): [in a new window]   Fig. 1. Bcl6–/– macrophages have a morphology distinct from that of Bcl6+/+ BMM. BMM were plated on tissue culture plastic (A,B) and fibronectin-coated coverslips (C,D) and grown to subconfluence in the presence of CSF1 before fixation. Phase-contrast (A,B) and scanning EM (C,D) images of Bcl6+/+ (A,C) and Bcl6–/– BMM (B,D). (E) Polarization indices of Bcl6+/+ (+/+) and Bcl6–/– (–/–) BMM (Polarization index=ratio of maximum cell length to maximum cell width, n>30; error bars represent s.e.m.; *P<0.001 significantly different from the index in Bcl6+/+ BMM cells). (F) Numbers of pseudopodia per cell in Bcl6+/+ and Bcl6–/– BMM (for maximum cell length/maximum cell width ratio 1.5, pseudopodia count=1; n=100). Bar, 20 µm (A,B); 10 µm (C,D).

View larger version (116K): [in a new window]   Fig. 4. Spreading is delayed in Bcl6+/+ macrophages and is associated with altered focal complex remodeling. Bcl6+/+ and Bcl6–/– BMM were plated on fibronectin-coated coverslips for the indicated times, fixed and examined by confocal microscopy. (A) BMM fixed 1, 2, 4, 6, 8 and 24 hours after plating and examined by interference reflection microscopy. (B) BMM fixed 3 hours after plating and examined by IRM and immunofluorescence staining for phosphoY118 paxillin. (C) Analysis of mean cell footprint areas at 15 and 30 minutes, 1, 2, 4, 6, 8 and 24 hours (Bcl6+/+, black, Bcl6–/–, gray; mean±s.e.m. of cells in six fields). Bar, 10 µm.

View larger version (86K): [in a new window]   Fig. 2. Bcl6–/– macrophages display altered focal complex and F-actin structures. Bcl6+/+ (A,C,E,G,I,K) and Bcl6–/– BMM (B,D,F,H,J,L) were plated on fibronectin-coated coverslips, grown to subconfluence in the presence of CSF1, then starved of CSF1 for 24 hours prior to re-stimulation for 20 minutes. Immunofluorescence samples (A-H) were stained for Y118 paxillin (C,D; green in merged fields G,H) and F-actin (E,F; red in merged fields). Arrows indicate dense collections of phospho-paxillin-rich point contacts, closed arrowheads indicate focal complexes that subtend F-actin bundles, and open arrowheads indicate circumferential focal complexes with overlying F-actin belts. Inserts in H highlight the focal complexes and F-actin structures. (I,J) Representative cytoskeletal replicas. (K,L) Enlarged regions framed in I and J. Bar, 10 µm (A-H); 500 nm (I,J).

View larger version (16K): [in a new window]   Fig. 3. Bcl6–/– macrophages have reduced motility. Approximately 1x105 BMM were added to 8 µm pore-size inserts in either the presence (chemokinesis, black bars) or absence (chemotaxis, gray bars) of CSF1 and then placed in CSF1-containing wells for 3 hours. Migrated cells were scored and normalized to the total cell number in an insert-free well. The means of four fields per insert were averaged and the results shown are representative of three independent experiments (mean±s.e.m.). Differences in proliferation rates (unpublished data) would not have significantly contributed to the results of this 3-hour experiment.

View larger version (22K): [in a new window]   Fig. 5. RhoA is hyper-activated in Bcl6–/– macrophages. (A) A GST-Rhotekin-RBD pull-down assay was performed on WT and Bcl6–/– BMM stimulated with CSF1 for 0 and 20 minutes. Eluted proteins were examined by western blotting for associated active RhoA. Bottom panel demonstrates equivalent presentation of input protein as determined by RhoA western blotting of 30 µg RIPA lysates from the above samples. (B) Bar graph summarizes the results from three independent GST-Rhotekin-RBD pull-down assays (mean±s.d.; *P<0.05 significantly different from intensity in Bcl6+/+ BMM at 0 or 20 minutes; **P<0.05, significantly different from intensity in Bcl6+/+ BMM at 0 minute).

View larger version (66K): [in a new window]   Fig. 6. Reconstitution of BCL6 expression and RhoA inhibition each cause phenotypic reversion of ventral surface adhesion structures and repolarization. (A-D) A BCL6 expression construct was retrovirally transduced into Bcl6–/– BMM and the cells sorted for GFP expression 36 hours post-infection. GFP-positive cells were plated on fibronectin-coated coverslips for 24 hours before fixation and staining for Y118 paxillin (C) and visualization of GFP expression (B). (D) Quantification of polarization indices for Bcl6+/+, Bcl6–/– BMM and BCL6-reconstituted Bcl6–/– BMM (error bars, s.e.m.; *P<0.02 significantly different from index in Bcl6+/+ BMM cells). (E-J) Bcl6+/+ and Bcl6–/– BMM were treated with either TBS buffer (control, E,F), Tat-tagged C3 transferase (G,H) or the ROCK1 inhibitor, Y27632 (I,J) before fixation and staining for Y118 paxillin. Treatment with another ROCK1 inhibitor, HA 1077, produced similar results to Y27632 treatment (not shown). Bar, 10 µm.

View larger version (102K): [in a new window]   Fig. 7. RhoGAP and RasGAP levels are reduced at the plasma membrane in Bcl6–/– macrophages. RhoGAP (C and D) and RasGAP (E and F) colocalize at the plasma membrane and membrane localization of each was reduced in Bcl6–/– BMM. Arrowheads highlight plasma membrane ruffles, visualized by phase contrast (A and B) and F-actin staining (G and H). Bar, 10 µm.

View larger version (88K): [in a new window]   Fig. 8. Surface CSF1R expression and colocalization with RhoGAP is reduced in Bcl6–/– macrophages. RhoGAP (C and D) colocalizes with the CSF1R (E and F) at the plasma membrane. Localization of RhoGAP and the CSF1R to the plasma membrane both appear reduced in Bcl6–/– BMM. Up to 20-25% of BMM may be binucleate. Arrowheads highlight plasma membrane ruffles. (G) CSF1R western blot analysis showed reduced expression of the mature 165 kDa isoform in Bcl6–/– BMM and the presence of a potential proteolytic cleavage product with molecular mass of 100 kDa. Lower panel, GAPDH loading control. (H) FACS analysis of surface CSF1R expression in vector-transduced Bcl6+/+ and Bcl6–/– BMM, and BCL6-reconstituted Bcl6–/– BMM. Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 9. Overexpression of the CSF1R corrects the phenotype of Bcl6–/– BMM cultured in CSF1. (A) FACS analysis of surface CSF1R expression in vector-transduced Bcl6+/+ and Bcl6–/– BMM, and CSF1R-transduced Bcl6–/– BMM. (B) Polarization indices of vector-transduced Bcl6+/+ and Bcl6–/– BMM, and GFP-expressing CSF1R-transduced Bcl6–/– BMM (error bars, s.e.m.; *P<0.02 significantly different from index in Bcl6+/+ BMM). (C-I) Bcl6–/– BMM overexpressing the CSF1R, as judged by high GFP levels (C, cell in the lower left corner), showed reversion of adhesion structures from peripheral focal complexes typical of Bcl6–/– BMM (E) to widespread ventral surface point contacts (F, cell in the lower left corner), similar to those seen in Bcl6+/+ BMM (D). Corresponding phase-contrast images are also shown (G-I). Bar, 10 µm.

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