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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02939

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Inhibition of calpain stabilises podosomes and impairs dendritic cell motility

¹ Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London, SE1 1UL, UK
² Advanced Science and Technology Laboratory, AztraZeneca Charnwood, Loughborough, LE11 5RH, UK
³ Molecular Immunology Unit, Institute of Child Health, University College London, WC1N 1EH, UK

View larger version (31K): [in a new window]   Fig. 1. Calpain 1 and 2 are expressed in DCs. DCs plated overnight on poly-L-lysine-coated glass coverslips were fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and double stained to detect calpain 1 (green) and actin (red) (A) or calpain 2 (green) and actin (red) (B). Arrowheads in A indicate the colocalisation of calpain 1 and actin at the edges of the podosome core at the rear of the podosome cluster. Magnifications of the boxed areas in A and B with calpain staining and double staining are shown on the right. Bars, 10 µm. (C) Western blots of calpain 1 and calpain 2 in total lysates of DCs. The positions of molecular size markers (in kDa) are indicated.

View larger version (15K): [in a new window]   Fig. 2. Calpain regulates podosome disassembly in DCs. (A) Calpain activity in the presence of inhibitors was detected. As expected, inhibition of calpain activity was found using the calpain inhibitors ALLM (50 µM), ALLN (50 µM) or the calpastatin peptide (Calp Pept, 50 µM) but not using cathepsin inhibitor 1 (Cathep. Inh,10 µM), proteosome inhibitor 1 (Proteo. Inh, 10 µM) or the scrambled version of the calpastatin peptide (Calp Pept NC, 50 µM). (B) The percentage of DCs with podosomes was not affected by treatment with calpain inhibitors. Inhibition of cathepsin B and proteosome resulted in DC podosome loss. Results are mean values ± s.e.m. from 50 cells with three coverslips examined per experiment. *P<0.05 and ***P<0.005 compared with values in untreated cells (Student's t-test).

View larger version (66K): [in a new window]   Fig. 3. Treatment of DCs with calpain inhibitors results in accumulation of ß2 integrin subunits, actin, vinculin and talin in podosomes. DCs plated overnight on poly-L-lysine-coated glass coverslips were left untreated or were treated with ALLM, ALLN, calpastatin peptide or the scrambled version of the calpastatin peptide (Calp Pept NC) and then fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and triple stained to detect ß2 integrin subunit (green), actin (red) and vinculin (blue) (A-E). Inhibition of calpain with ALLM (B), ALLN (C) or the calpastatin peptide (E) resulted in an exaggerated accumulation of ß2 integrin subunits, vinculin and actin in DC podosomes, compared with untreated cells (A) or cells treated with the scrambled version of the calpastatin peptide (D). ß2 integrin subunit, actin and vinculin single staining from the boxed areas in main panels is shown magnified below. Quantification of the fluorescent intensity of the staining of ß2 integrin (F), vinculin (G) and actin (H). Sizes of the actin core of podosomes of untreated and treated DC (I). Alternatively, DCs were stained to detect talin (green) and actin (red) (J-N). Inhibition of calpain with ALLM (K), ALLN (L) or the calpastatin peptide (N) induced an exaggerated accumulation of talin in DC podosomes, compared with untreated cells (J) or cells treated with the scrambled version of the calpastatin peptide (M). Single staining is shown to the right of panels J-N. (O) Quantification of the fluorescent intensity of the staining of talin. The micrographs are representative of the cytoskeletal organisation of DCs detected in three independent experiments. Bars, 10 µm. The graphs illustrate the mean percentage of cells ± s.e.m. from three experiments with 20 cells per treatment, per experiment. **P<0.01, ***P<0.005 compared with values in the untreated cells (Student's t-test).

View larger version (11K): [in a new window]   Fig. 4. Adhesion turnover and motility of DCs are regulated by calpain. (A) DCs were plated on poly-L-lysine-coated coverslips overnight and then mounted in viewing chambers to measure the adhesion turnover index using IRM time-lapse video. Values are mean ± s.e.m. of ten cells per treatment group from three experiments. (B) Phase-contrast time-lapse videos were recorded to measure the velocity of DCs in the viewing chambers. Values are mean ± s.e.m. percentage of 20 cells per treatment group from three experiments. *P<0.05, **P<0.01, ***P<0.005 compared with values in untreated cells (Student's t-test).

View larger version (14K): [in a new window]   Fig. 5. Calpain regulates spreading and adhesion of DCs. Using phase-contrast video time-lapse microscopy, untreated and ALLM or ALLN treated cells were filmed for 240 minutes while spreading on poly-L-lysine coated dishes. (A) Area of cell spread on the substratum measured using motion analysis software. The graphs illustrate the mean percentage of cells ± s.e.m. from three experiments. The area of treated cells was significantly different to that in untreated cells (P<0.005) by ANOVA analysis. (B) Percentage of adhesion of DCs on poly-L-lysine-coated wells 30 minutes after plating. The graphs illustrate the mean percentage of cells ± s.e.m. from three experiments *P<0.05 compared with untreated cells (Student's t-test). ALLM and ALLN treatments resulted in decreased area of spread and adhesion efficiency.

View larger version (95K): [in a new window]   Fig. 6. Inhibition of calpain results in accumulation of podosome components in spreading DCs. Untreated and ALLM- or ALLN-treated DCs were allowed to spread on poly-L-lysine-coated coverslips for 10 minutes (A,B,C), 15 minutes (D,E,F) or 2 hours (panels G,H,I). At these time points cells were fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and stained to detect ß2 integrin subunits, vinculin and actin. The main images show the distribution of actin and inserts the localisation of ß2 integrin subunits (green), vinculin (red) and the colocalisation with actin (blue). Five minutes after plating, untreated cells developed peripheral lamellae and a central cluster of podosomes (A). At 15 minutes, the central cluster of podosomes displayed some asymmetry of distribution towards the extending region of lamellae (D, arrowhead). By 2 hours, the majority of untreated DCs were polarised with a cluster of podosomes behind the leading edge (G). Inhibition of calpain resulted in increased accumulation of ß2 integrin subunits, vinculin and actin in podosomes. At 15 minutes, cells failed to develop major lamellar extensions and only peripheral spikes were seen (E,F). By 2 hours, ALLM- and ALLN-treated cells displayed podosomes richer in ß2 integrin subunits, vinculin and actin (panels H,I). The micrographs are representative of the cytoskeletal organisation of DC detected in three independent experiments. Bars, 10 µm.

View larger version (32K): [in a new window]   Fig. 7. Calpain activity is required for transendothelial migration of DC. After incubation of CFSE-labelled DCs on a monolayer of SVEC 4-10 endothelial cells for 1 hour, co-cultures were washed once with PBS and fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and stained to detect actin filaments. (A) Percentage of DC transmigrated, transmigrating or retained on the apical surface of the monolayer. (B) Spread area of DCs under the monolayer in untreated (black), ALLM-treated (grey) and ALLN-treated (white) cells. Values are mean ± s.e.m. of 50 measurements from three coverslips. (C) Skewed confocal micrographs of optical sections at the apical and basal poles of endothelial cell monolayers. CFSE-labelled DCs are green and the distribution of actin filaments is shown in red. Treatment of DCs with calpain inhibitors reduced the transmigration of DCs across the endothelial cells and reduced the spread area under the endothelial cell monolayer. The graphs illustrate the mean percentage of cells ± s.e.m. from three experiments. *P<0.05, **P<0.01 compared with levels in untreated cells (Student's t-test). The micrographs are representative of the cytoskeletal organisation of DCs detected in three independent experiments.

View larger version (77K): [in a new window]   Fig. 8. Calpain cleaves the podosome proteins WASP, Pyk2 and talin. Total lysates of untreated and ALLM- or ALLN-treated plated DCs were separated by SDS-PAGE and western blotted. Immunoblotting with the corresponding antibodies showed that WASP, Pyk2 and talin but not vinculin or Src were cleaved by calpain. Two calpain cleavage products of WASP of 40 kDa and a 45 kDa (asterisks) were detected. One cleavage product of Pyk2 (50 kDa, asterisk) and three of talin (70 kDa, 90 kDa and 190 kDa, asterisks) were detected.

View larger version (61K): [in a new window]   Fig. 9. WASP is required for formation of dynamic DC podosomes and location of WASP in DC is regulated by calpain activity. WASP-null DCs expressing eGFP-WASP were plated on poly-L-lysine-coated coverslips overnight and then left untreated (A,B) or treated with the calpain inhibitor ALLM (C,D) and mounted in viewing chambers to measure adhesion turnover index using confocal microscopy. Micrographs of the interference reflection signal (A,C) and eGFP-WASP location (B,D) were taken 20 seconds apart. Bars, 10 µm. (E) Quantification of adhesion turnover of WASP-null DCs expressing eGFP constructs in the presence or absence of calpain inhibitors. The graphs illustrate the mean percentage of cells ± s.e.m. of 20 cells per treatment group from three experiments. Significant differences were observed at *P<0.05 and **P<0.01 as indicated.

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