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First published online 19 February 2008
doi: 10.1242/jcs.021006

Journal of Cell Science 121, 784-795 (2008)
Published by The Company of Biologists 2008
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Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region

Josephine C. Adams1,2,*, Amber A. Bentley1, Marc Kvansakul3,{ddagger}, Deborah Hatherley4 and Erhard Hohenester3

1 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
2 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
3 Division of Cell and Molecular Biology, Imperial College London, Biophysics Section, Blackett Laboratory, London, SW7 2AZ, UK
4 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK


Figure 1
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  		Fig. 1. Retention of endogenous and heterologously expressed TSP1 in the ECM of cultured cells. (A) Analysis of ECM isolation conditions. Confluent 4-day cultures of COS-7 cells were fixed in 2% paraformaldehyde and permeabilised in 0.5% Triton X-100 (PFA), or extracted as indicated, then stained with FITC-phalloidin to visualise F-actin or DAPI to visualise DNA. (B) Production of endogenous ECM by C2C12 and COS-7 cells. Confluent 4-day cultures were extracted with 20 mM NH4OH and stained as indicated for components of the insoluble ECM. (C) Biochemical analysis. NH4OH-insoluble ECM from 4-day cultures of the indicated cells was solubilised in hot SDS-PAGE sample buffer containing 100 mM DTT, resolved on 10% polyacrylamide gels and analysed by immunoblotting for fibronectin (FN) (COS-7 and C2C12 cells) or TSP1 (control COS-7 cells and COS-7 transfected to express human TSP1). (D) COS-7 cells were transfected with human TSP1, cultured for 3 days and then fixed in 2% paraformaldehyde, permeabilised and stained for human TSP1 to visualise the intracellular pool, or extracted with 20 mM NH4OH and the insoluble ECM stained for human TSP1. Right panel shows a single array of puncta; arrows indicate examples of individual puncta. Scale bars: 25 µm (A); 10 µm (B,D).

 

Figure 2
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  		Fig. 2. Evolutionary conservation of ECM retention activity in trimeric and pentameric TSPs. (A) Schematic diagrams of Ciona TSPA, Drosophila TSP and COMP/TSP5. (B) Retention of the indicated TSPs in the ECM of COS-7 cells. Experimental conditions were as for Fig. 1B; TSPs were expressed with V5-epitope tags and detected with FITC-tagged V5 antibody. Scale bars: 20 µm. (C,D) Quantification of the relative expression (C) and ECM retention (D) of TSPs. Each column represents the mean of three to five experiments; bars represent s.e.m.

 

Figure 3
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  		Fig. 3. TSP1 domain constructs. (A) Schematic diagrams of the TSP1 domain proteins. (B,C) Proteins were collected from conditioned medium of COS-7 cells on heparin-Sepharose (B) or Talon metal affinity resin (C) and analysed by SDS-PAGE and immunoblotting under reducing (R) or non-reducing (NR) conditions. Molecular mass markers are in kDa.

 

Figure 4
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  		Fig. 4. Retention of TSP1 in ECM depends on the C-terminal region in trimeric form. (A) Retention of the indicated TSP1 domain deletion proteins in NH4OH-insoluble ECM of COS-7 cells detected by indirect immunofluorescence with antibodies to human TSP1 (upper panels) or direct fluorescence of mRFP (lower panels). (B-E) Quantification of the relative expression (B,C) or ECM retention (D,E) of TSP1 domains, as detected by immunofluorescence (B,D) or mRFP (C,E). Each column represents the mean from 3-16 independent experiments, bars represent s.e.m. (F) Biochemical analysis of mRFPovTSP1C from COS-7 ECM. (G) Time course of ECM incorporation of mRFPovTSP1C. mRFPovTSP1C was expressed in COS-7 cells for 18 hours, then cells replated and extracted with 20 mM NH4OH from 1 hour to 3 days later. Arrows indicate initial deposits of mRFPovTSP1C. The mean cell area at each time point, as measured from ten phalloidin-stained cells in each sample, is given below each panel. Colour panels show X-Y confocal projections of ECM puncta (upper panel; arrow indicates area of merged puncta) or intracellular distribution of mRFPovTSP1C after 3 days (lower panel). (H) Confocal X-Z sections show distribution of mRFPovTSP1C in ECM (upper panel) or paraformaldehyde-fixed cells (lower panel) at 3 days. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Contributions of the RGD motif and L-lectin domain to ECM deposition. (A) Structural similarity of TSP1 C-terminal globular domain to L-type lectins. Left, TSP1 type35-7GCT crystal structure (PDB1ux6) (Kvansakul et al., 2004Go) with the type 3 repeats in yellow, the L-lectin domain in orange and calcium ions as blue spheres. The remaining type 3 repeats and two EGF domains in light grey were modelled from the homologous TSP2 structure (PDB1yo8) (Carlson et al., 2005Go). The RGD and DDD motifs in TSP1 (see text) are shown in atomic detail. The putative CD47-binding motifs (Gao et al., 1996Go) are in green. Right, crystal structure of VIP36 carbohydrate-recognition domain in complex with calcium (blue sphere) and a mannose trisaccharide (carbon and oxygen atoms shown in green and red, respectively) (PDB2e6v) (Satoh et al., 2007Go). The side chain of Asp131 (see text) is shown in atomic detail. (B,C) Cell attachment to wild-type or mutant TSP1 C-terminal monomer fragments. C2C12 and HASMC (B), or strain-matched wild-type or CD47-null mouse fibroblasts (C) were adhered to surfaces coated with 1 µM of each protein for 1 hour under serum-free conditions. Each column represents the mean from three experiments, bars=s.e.m. (D,E) Quantification of the relative expression (D) or ECM retention (E) of wild-type and mutant mRFPovTSP1C proteins. Each column represents the mean of three independent experiments; bars represent s.e.m. *P=0.02; **P=0.009.

 

Figure 6
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  		Fig. 6. Analysis of the role of CD47 in TSP1 C-terminal region interactions. (A) CD47 is not essential for ECM retention of mRFPovTSP1C. NH4OH-insoluble ECMs were analysed after 4 days. Scale bar: 10 µm. (B-D) Surface plasmon resonance analysis of CD47 interaction with SIRP{alpha} (B,C) or type35-7GCT (D).

 

Figure 7
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  		Fig. 7. Role of β1 integrins in ECM retention of mRFPoTSP1C. (A,B) Quantification of relative expression (A) or ECM retention (B) of mRFPo or mRFPovTSP1C by GD25 and GD25β1A cells. Each column represents the mean of three experiments; bars represent s.e.m. Insets in B show representative confocal images of mRFPoTSP1C puncta in the ECMs of GD25 and GD25β1A cells. (C) Partial codistribution of fibronectin and mRFPovTSP1C in the ECM of GD25β1A cells. Top row, arrows indicate absence of colocalised fibronectin. Middle and bottom rows, arrows indicate areas of colocalisation. Scale bars: 10 µm.

 

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© The Company of Biologists Ltd 2008