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Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts.

Thompson O, Kleino I, Crimaldi L, Gimona M, Saksela K, Winder SJ - PLoS ONE (2008)

Bottom Line: Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src.Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, UK.

ABSTRACT

Background: Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal findings: We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with beta-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions: We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

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Related in: MedlinePlus

Myoblasts form podosomes in response to PDBu.To determine if myoblast cells could form podosomes, myoblast cells were allowed to adhere and spread on various substrates, and stimulated with PDBu for 30 min and fixed and stained for the podosome markers cortactin (green) and F-actin (red). Myoblasts formed peripheral actin and cortactin containing puncta on all substrates tested. An unstimulated cell grown on a glass coverslip is also shown for comparison (A, bottom right panel). These structures are morphologically indistinguishable from podosomes (A). The structures appeared columnar within the cell and cortactin was localised around a more central actin core as seen in the Z section of the zoomed region of B. The identity of the DG and actin-rich puncta as podosomes/invadopodia was further substantiated by the degradation of rhodamine-gelatin beneath them, seen as darker areas of reduced rhodamine gelatin (red in merge, arrowed) that are coincident with DG localisation (green in merge, arrowed) and F-actin (blue in merge). Scale bars 20 µm in A and 10 µm in B, C, 2 µm in zoomed region of B.
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pone-0003638-g002: Myoblasts form podosomes in response to PDBu.To determine if myoblast cells could form podosomes, myoblast cells were allowed to adhere and spread on various substrates, and stimulated with PDBu for 30 min and fixed and stained for the podosome markers cortactin (green) and F-actin (red). Myoblasts formed peripheral actin and cortactin containing puncta on all substrates tested. An unstimulated cell grown on a glass coverslip is also shown for comparison (A, bottom right panel). These structures are morphologically indistinguishable from podosomes (A). The structures appeared columnar within the cell and cortactin was localised around a more central actin core as seen in the Z section of the zoomed region of B. The identity of the DG and actin-rich puncta as podosomes/invadopodia was further substantiated by the degradation of rhodamine-gelatin beneath them, seen as darker areas of reduced rhodamine gelatin (red in merge, arrowed) that are coincident with DG localisation (green in merge, arrowed) and F-actin (blue in merge). Scale bars 20 µm in A and 10 µm in B, C, 2 µm in zoomed region of B.

Mentions: Whilst much is known about the role of dystroglycan in mature differentiated skeletal muscle [28], less is known about the functions of DG in cell adhesion in developing muscle [1]. We therefore examined the role of DG in the formation of cell adhesions during spreading and adhesion of myoblasts. During the early phases of cell spreading, and independent of the substrate on which the cells were spreading on, DG could be seen in relatively large and densely staining puncta (Figure 1). These puncta were often located more centrally, and appeared morphologically distinct from peripheral focal adhesion-like structures. Whilst both α- and β-dystroglycan as well as utrophin co-localised in these puncta, other more traditional adhesion proteins including vinculin, talin and FAK as well as the more generic phospho-tyrosine also typically seen in adhesion structures were also localised in the puncta (Figure 1). Interestingly, neither α- and β-dystroglycan nor utrophin appeared to localise in more peripheral focal adhesion type structures, compare vinculin staining in Figure 1. The dystroglycan-staining puncta do not appear morphologically to resemble focal adhesions and the differential staining of dystroglycan also suggests that the puncta are somehow different to focal adhesions stained by vinculin. Whilst it is possible that the puncta are focal contacts [29], [30] their more central position in the cell and their relatively large size would argue against this. One other adhesion structure that has a larger and rounder morphology is the podosome, typically found in cells of the monocyte lineage, osteoclasts and smooth muscle cells [6]. Myoblasts have not previously been demonstrated to form podosomes. Therefore in order to determine if myoblasts could form podosomes, we subjected well-spread myoblasts to stimulation with the phorbol ester PDBu, in order to activate Src downstream of PKC as part of a pathway leading to podosome formation [31]. Myoblast cells were then co-stained for classical markers of podosomes: actin and cortactin (Figure 2A). Podosomes contain a dense actin-rich core surrounded by a loose meshwork of adhesion proteins and actin binding and regulatory proteins [6]. In this regard the structures formed in myoblast cells appear morphologically similar to podosomes seen in other cell types [2] (Figure 2B) and thus henceforth will be considered as podosomes. Podosome formation was also independent of the substrate that the cells were adhered to. Actin- and cortactin-containing structures formed in a band behind the leading edge of the cell equally well on glass, gelatin, fibronectin and whole or E3 fragment of laminin, compared to the unstimulated cell (Figure 2A). A further characteristic of podosomes and structurally related invadopodia, is the ability to degrade the matrix beneath the adhesion site [6]. The dystroglycan-containing structures in myoblasts appear to share this property too, in that there are zones of clearance of rhodamine-gelatin beneath the dystroglycan-containing adhesion structures (Figure 2C). In addition, DG co-localised in podosomes with plectin and integrin, other proteins known to be resident in podosomes [6], [32] (Figure S1A). Other well-characterised myoblast cells such as C2C12 [33] were also able to form podosomes in response to PDBu (Figure S1B). To ascertain whether or not the localisation of DG to podosomes was unique to myoblasts we also examined the localisation of DG in A7r5 smooth muscle cells in response to PDBu stimulation. A7r5 cells are a well-established model for podosomes and as shown in Figure S1C, DG also colocalised in podosomes with actin, cortactin or vinculin. Thus it appears that myoblasts can form podosomes and that dystroglycan is localised to podosomes along with other well recognised podosome proteins. Furthermore as can be seen from movies of myoblast cells expressing GFP-β-actin (Movie S1, S2, S3), podosome structures and podosome dynamics appear qualitatively similar to GFP-β-actin movies from A7r5 cells for example see [34] and have similar dynamics to DsRed-SM22 (used to label F-actin) podosomes in A7r5 cells [35], [36].


Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts.

Thompson O, Kleino I, Crimaldi L, Gimona M, Saksela K, Winder SJ - PLoS ONE (2008)

Myoblasts form podosomes in response to PDBu.To determine if myoblast cells could form podosomes, myoblast cells were allowed to adhere and spread on various substrates, and stimulated with PDBu for 30 min and fixed and stained for the podosome markers cortactin (green) and F-actin (red). Myoblasts formed peripheral actin and cortactin containing puncta on all substrates tested. An unstimulated cell grown on a glass coverslip is also shown for comparison (A, bottom right panel). These structures are morphologically indistinguishable from podosomes (A). The structures appeared columnar within the cell and cortactin was localised around a more central actin core as seen in the Z section of the zoomed region of B. The identity of the DG and actin-rich puncta as podosomes/invadopodia was further substantiated by the degradation of rhodamine-gelatin beneath them, seen as darker areas of reduced rhodamine gelatin (red in merge, arrowed) that are coincident with DG localisation (green in merge, arrowed) and F-actin (blue in merge). Scale bars 20 µm in A and 10 µm in B, C, 2 µm in zoomed region of B.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572840&req=5

pone-0003638-g002: Myoblasts form podosomes in response to PDBu.To determine if myoblast cells could form podosomes, myoblast cells were allowed to adhere and spread on various substrates, and stimulated with PDBu for 30 min and fixed and stained for the podosome markers cortactin (green) and F-actin (red). Myoblasts formed peripheral actin and cortactin containing puncta on all substrates tested. An unstimulated cell grown on a glass coverslip is also shown for comparison (A, bottom right panel). These structures are morphologically indistinguishable from podosomes (A). The structures appeared columnar within the cell and cortactin was localised around a more central actin core as seen in the Z section of the zoomed region of B. The identity of the DG and actin-rich puncta as podosomes/invadopodia was further substantiated by the degradation of rhodamine-gelatin beneath them, seen as darker areas of reduced rhodamine gelatin (red in merge, arrowed) that are coincident with DG localisation (green in merge, arrowed) and F-actin (blue in merge). Scale bars 20 µm in A and 10 µm in B, C, 2 µm in zoomed region of B.
Mentions: Whilst much is known about the role of dystroglycan in mature differentiated skeletal muscle [28], less is known about the functions of DG in cell adhesion in developing muscle [1]. We therefore examined the role of DG in the formation of cell adhesions during spreading and adhesion of myoblasts. During the early phases of cell spreading, and independent of the substrate on which the cells were spreading on, DG could be seen in relatively large and densely staining puncta (Figure 1). These puncta were often located more centrally, and appeared morphologically distinct from peripheral focal adhesion-like structures. Whilst both α- and β-dystroglycan as well as utrophin co-localised in these puncta, other more traditional adhesion proteins including vinculin, talin and FAK as well as the more generic phospho-tyrosine also typically seen in adhesion structures were also localised in the puncta (Figure 1). Interestingly, neither α- and β-dystroglycan nor utrophin appeared to localise in more peripheral focal adhesion type structures, compare vinculin staining in Figure 1. The dystroglycan-staining puncta do not appear morphologically to resemble focal adhesions and the differential staining of dystroglycan also suggests that the puncta are somehow different to focal adhesions stained by vinculin. Whilst it is possible that the puncta are focal contacts [29], [30] their more central position in the cell and their relatively large size would argue against this. One other adhesion structure that has a larger and rounder morphology is the podosome, typically found in cells of the monocyte lineage, osteoclasts and smooth muscle cells [6]. Myoblasts have not previously been demonstrated to form podosomes. Therefore in order to determine if myoblasts could form podosomes, we subjected well-spread myoblasts to stimulation with the phorbol ester PDBu, in order to activate Src downstream of PKC as part of a pathway leading to podosome formation [31]. Myoblast cells were then co-stained for classical markers of podosomes: actin and cortactin (Figure 2A). Podosomes contain a dense actin-rich core surrounded by a loose meshwork of adhesion proteins and actin binding and regulatory proteins [6]. In this regard the structures formed in myoblast cells appear morphologically similar to podosomes seen in other cell types [2] (Figure 2B) and thus henceforth will be considered as podosomes. Podosome formation was also independent of the substrate that the cells were adhered to. Actin- and cortactin-containing structures formed in a band behind the leading edge of the cell equally well on glass, gelatin, fibronectin and whole or E3 fragment of laminin, compared to the unstimulated cell (Figure 2A). A further characteristic of podosomes and structurally related invadopodia, is the ability to degrade the matrix beneath the adhesion site [6]. The dystroglycan-containing structures in myoblasts appear to share this property too, in that there are zones of clearance of rhodamine-gelatin beneath the dystroglycan-containing adhesion structures (Figure 2C). In addition, DG co-localised in podosomes with plectin and integrin, other proteins known to be resident in podosomes [6], [32] (Figure S1A). Other well-characterised myoblast cells such as C2C12 [33] were also able to form podosomes in response to PDBu (Figure S1B). To ascertain whether or not the localisation of DG to podosomes was unique to myoblasts we also examined the localisation of DG in A7r5 smooth muscle cells in response to PDBu stimulation. A7r5 cells are a well-established model for podosomes and as shown in Figure S1C, DG also colocalised in podosomes with actin, cortactin or vinculin. Thus it appears that myoblasts can form podosomes and that dystroglycan is localised to podosomes along with other well recognised podosome proteins. Furthermore as can be seen from movies of myoblast cells expressing GFP-β-actin (Movie S1, S2, S3), podosome structures and podosome dynamics appear qualitatively similar to GFP-β-actin movies from A7r5 cells for example see [34] and have similar dynamics to DsRed-SM22 (used to label F-actin) podosomes in A7r5 cells [35], [36].

Bottom Line: Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src.Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, UK.

ABSTRACT

Background: Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal findings: We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with beta-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions: We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

Show MeSH
Related in: MedlinePlus