Limits...
Role of WASP in cell polarity and podosome dynamics of myeloid cells.

Monypenny J, Chou HC, Bañón-Rodríguez I, Thrasher AJ, Antón IM, Jones GE, Calle Y - Eur. J. Cell Biol. (2010)

Bottom Line: Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment.In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process.We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell & Molecular Biophysics, King's College London, London SE1 1UL, UK.

Show MeSH

Related in: MedlinePlus

Soluble factors induce DC polarisation and podosome formation. Spleen derived mouse DCs were collected from cultures in RPMI supplemented with 10% FCS, GM-CSF and TGFβ1 and washed 3 times with RPMI to discard supplemented soluble factors. DCs were then resuspended in RPMI (A) or RPMI supplemented with 100 ng/ml SDF1α (B), 100 ng/ml osteopontin (C) or 10% Foetal Calf Serum (FCS) (D) and allowed to spread on poly-l-lysine coated coverslips for 4 h. DCs were fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and double-stained to detect the distribution of β2 integrin (green) and actin (red). Images show the merged confocal images of the distribution of both β2 integrin and actin. Magnifications of the boxed areas on the left or at the bottom of the images show the distribution of β2 integrins and actin separately. The majority of DCs plated with RPMI only developed β2 integrin containing focal contacts or rosettes of podosomes (A). SDF1α, osteopontin and FCS promoted cell polarisation (elongated morphology with a distinct leading edge sustained by a cluster of podosomes) and formation of regular clusters of podosomes with the characteristic honeycomb distribution of β2 integrins surrounding actin puncta (B–D). Bar 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3037472&req=5

fig0010: Soluble factors induce DC polarisation and podosome formation. Spleen derived mouse DCs were collected from cultures in RPMI supplemented with 10% FCS, GM-CSF and TGFβ1 and washed 3 times with RPMI to discard supplemented soluble factors. DCs were then resuspended in RPMI (A) or RPMI supplemented with 100 ng/ml SDF1α (B), 100 ng/ml osteopontin (C) or 10% Foetal Calf Serum (FCS) (D) and allowed to spread on poly-l-lysine coated coverslips for 4 h. DCs were fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and double-stained to detect the distribution of β2 integrin (green) and actin (red). Images show the merged confocal images of the distribution of both β2 integrin and actin. Magnifications of the boxed areas on the left or at the bottom of the images show the distribution of β2 integrins and actin separately. The majority of DCs plated with RPMI only developed β2 integrin containing focal contacts or rosettes of podosomes (A). SDF1α, osteopontin and FCS promoted cell polarisation (elongated morphology with a distinct leading edge sustained by a cluster of podosomes) and formation of regular clusters of podosomes with the characteristic honeycomb distribution of β2 integrins surrounding actin puncta (B–D). Bar 10 μm.

Mentions: During their life span myeloid cells traffic through tissues with different physical and biochemical compositions likely to modulate podosome organisation and dynamics (Cavanagh and von Andrian, 2002). It has recently been shown that the rigidity of the substratum regulates podosome organisation in non-myeloid cells (Collin et al., 2008) as well as myeloid cells (Van et al., 2010) indicating that podosomes can work as mechanosensors. In vitro, both soluble factors present in serum and integrin ligands participate in the regulation of podosome formation and their structural integrity (Calle et al., 2006a; Linder, 2009). DCs plated on poly-l-lysine (a substratum lacking integrin binding sites) or the integrin ligand fibronectin (a ligand for β1 and β2 integrins (Anceriz et al., 2007; van den Berg et al., 2001; Humphries et al., 2006)) in the absence of serum will attach but subsequently fail to form a distinct leading edge or fully assembled podosomes. Instead they mainly assemble β2 integrin containing focal contacts (Figs. 2A and 3A–C). Additionally, between 20% and 30% of the cells develop rings of fused podosome-like structures (Fig. 2A) instead of regularly assembled clusters of discrete podosomes. However, when the serum-free culture medium is supplemented with chemotactic factors such as SDF1α (Vecchi et al., 1999) or osteopontin (Weiss et al., 2001), between 75 and 85% of DCs polarise; forming a leading edge sustained by podosomes similarly to DCs cultured with 10% serum (Fig. 2B–D). These observations indicate that DC chemotactic factors such as SDF1α and osteopontin trigger polarisation and podosome assembly in coordination with the extending leading edge whereas the presence of integrin ligands alone is unable to initiate this process (Fig. 3). We have previously reported that for DCs cultured in the presence of serum, coating the substratum with integrin ligands such as fibronectin or ICAM-1 (the chief ligand for β2 integrins (Humphries et al., 2006)) promotes accumulation of F-actin into podosome cores and recruitment of β2 integrin subunits and vinculin to the podosome ring. Additionally, fibronectin or ICAM-1 promote the stabilisation of podosomes as determined by live interference reflection microscopy (Chou et al., 2006), which correlates with the observed accumulation of podosomal components (Calle et al., 2006a). Taken together, our data support a model of DC migration where chemotactic factors trigger cell polarisation and initiation of podosomes to sustain the leading edge for migration. The presence of integrin ligands from the extracellular matrix or the surface of neighbouring cells promotes maturation and stabilisation of podosomes by inducing further recruitment of structural and regulatory podosomal components. As a result, the leading edge becomes stabilised in the direction of chemoattractant source.


Role of WASP in cell polarity and podosome dynamics of myeloid cells.

Monypenny J, Chou HC, Bañón-Rodríguez I, Thrasher AJ, Antón IM, Jones GE, Calle Y - Eur. J. Cell Biol. (2010)

Soluble factors induce DC polarisation and podosome formation. Spleen derived mouse DCs were collected from cultures in RPMI supplemented with 10% FCS, GM-CSF and TGFβ1 and washed 3 times with RPMI to discard supplemented soluble factors. DCs were then resuspended in RPMI (A) or RPMI supplemented with 100 ng/ml SDF1α (B), 100 ng/ml osteopontin (C) or 10% Foetal Calf Serum (FCS) (D) and allowed to spread on poly-l-lysine coated coverslips for 4 h. DCs were fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and double-stained to detect the distribution of β2 integrin (green) and actin (red). Images show the merged confocal images of the distribution of both β2 integrin and actin. Magnifications of the boxed areas on the left or at the bottom of the images show the distribution of β2 integrins and actin separately. The majority of DCs plated with RPMI only developed β2 integrin containing focal contacts or rosettes of podosomes (A). SDF1α, osteopontin and FCS promoted cell polarisation (elongated morphology with a distinct leading edge sustained by a cluster of podosomes) and formation of regular clusters of podosomes with the characteristic honeycomb distribution of β2 integrins surrounding actin puncta (B–D). Bar 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037472&req=5

fig0010: Soluble factors induce DC polarisation and podosome formation. Spleen derived mouse DCs were collected from cultures in RPMI supplemented with 10% FCS, GM-CSF and TGFβ1 and washed 3 times with RPMI to discard supplemented soluble factors. DCs were then resuspended in RPMI (A) or RPMI supplemented with 100 ng/ml SDF1α (B), 100 ng/ml osteopontin (C) or 10% Foetal Calf Serum (FCS) (D) and allowed to spread on poly-l-lysine coated coverslips for 4 h. DCs were fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and double-stained to detect the distribution of β2 integrin (green) and actin (red). Images show the merged confocal images of the distribution of both β2 integrin and actin. Magnifications of the boxed areas on the left or at the bottom of the images show the distribution of β2 integrins and actin separately. The majority of DCs plated with RPMI only developed β2 integrin containing focal contacts or rosettes of podosomes (A). SDF1α, osteopontin and FCS promoted cell polarisation (elongated morphology with a distinct leading edge sustained by a cluster of podosomes) and formation of regular clusters of podosomes with the characteristic honeycomb distribution of β2 integrins surrounding actin puncta (B–D). Bar 10 μm.
Mentions: During their life span myeloid cells traffic through tissues with different physical and biochemical compositions likely to modulate podosome organisation and dynamics (Cavanagh and von Andrian, 2002). It has recently been shown that the rigidity of the substratum regulates podosome organisation in non-myeloid cells (Collin et al., 2008) as well as myeloid cells (Van et al., 2010) indicating that podosomes can work as mechanosensors. In vitro, both soluble factors present in serum and integrin ligands participate in the regulation of podosome formation and their structural integrity (Calle et al., 2006a; Linder, 2009). DCs plated on poly-l-lysine (a substratum lacking integrin binding sites) or the integrin ligand fibronectin (a ligand for β1 and β2 integrins (Anceriz et al., 2007; van den Berg et al., 2001; Humphries et al., 2006)) in the absence of serum will attach but subsequently fail to form a distinct leading edge or fully assembled podosomes. Instead they mainly assemble β2 integrin containing focal contacts (Figs. 2A and 3A–C). Additionally, between 20% and 30% of the cells develop rings of fused podosome-like structures (Fig. 2A) instead of regularly assembled clusters of discrete podosomes. However, when the serum-free culture medium is supplemented with chemotactic factors such as SDF1α (Vecchi et al., 1999) or osteopontin (Weiss et al., 2001), between 75 and 85% of DCs polarise; forming a leading edge sustained by podosomes similarly to DCs cultured with 10% serum (Fig. 2B–D). These observations indicate that DC chemotactic factors such as SDF1α and osteopontin trigger polarisation and podosome assembly in coordination with the extending leading edge whereas the presence of integrin ligands alone is unable to initiate this process (Fig. 3). We have previously reported that for DCs cultured in the presence of serum, coating the substratum with integrin ligands such as fibronectin or ICAM-1 (the chief ligand for β2 integrins (Humphries et al., 2006)) promotes accumulation of F-actin into podosome cores and recruitment of β2 integrin subunits and vinculin to the podosome ring. Additionally, fibronectin or ICAM-1 promote the stabilisation of podosomes as determined by live interference reflection microscopy (Chou et al., 2006), which correlates with the observed accumulation of podosomal components (Calle et al., 2006a). Taken together, our data support a model of DC migration where chemotactic factors trigger cell polarisation and initiation of podosomes to sustain the leading edge for migration. The presence of integrin ligands from the extracellular matrix or the surface of neighbouring cells promotes maturation and stabilisation of podosomes by inducing further recruitment of structural and regulatory podosomal components. As a result, the leading edge becomes stabilised in the direction of chemoattractant source.

Bottom Line: Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment.In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process.We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell & Molecular Biophysics, King's College London, London SE1 1UL, UK.

Show MeSH
Related in: MedlinePlus