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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

DG over-expression abolishes podosome formation.Quantification of dystroglycan and Tks5 protein levels in control myoblast cells (wild-type), cells stably expressing GFP-tagged full-length DG (DG-GFP), shRNA against DG (knockdown) or a control shRNA (sense). Dystroglycan levels for the four cell lines are shown in A, with corresponding Tks5 levels in B. Whilst dystroglycan levels were increased by 2.3-flod and decreased by half in overexpressing and knockdown cells respectively, Tks5 levels were reduced by approximately 33% in both cases. Wild-type, sense, knockdown and myoblasts transiently expressing a myc-tagged DG construct were induced to form podosomes with PDBu and the number of cells exhibiting podosomes counted following fixation and staining. DG depletion caused a modest reduction in cells with podosomes compared to control, whereas DG over-expression completely abolished podosome formation. (C,D; mean±SEM).
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pone-0003638-g005: DG over-expression abolishes podosome formation.Quantification of dystroglycan and Tks5 protein levels in control myoblast cells (wild-type), cells stably expressing GFP-tagged full-length DG (DG-GFP), shRNA against DG (knockdown) or a control shRNA (sense). Dystroglycan levels for the four cell lines are shown in A, with corresponding Tks5 levels in B. Whilst dystroglycan levels were increased by 2.3-flod and decreased by half in overexpressing and knockdown cells respectively, Tks5 levels were reduced by approximately 33% in both cases. Wild-type, sense, knockdown and myoblasts transiently expressing a myc-tagged DG construct were induced to form podosomes with PDBu and the number of cells exhibiting podosomes counted following fixation and staining. DG depletion caused a modest reduction in cells with podosomes compared to control, whereas DG over-expression completely abolished podosome formation. (C,D; mean±SEM).

Mentions: If as proposed Tks5 is a ‘master regulator’ of podosome assembly [21], and DG also associates with Tks5, DG may also have a regulatory role in the podosome assembly process. In order to address this we examined myoblast cells either overexpressing or depleted for DG (Figure 5A–C). As determined by assessing the characteristic actin morphology of podosomes and compared to control cells, siRNA-mediated depletion of DG to 50% of endogenous levels in myoblast cells had a modest but statistically significant effect on the number of cells producing podosomes in response to PDBu. By contrast, over-expression of DG (tagged with either Myc or GFP) dramatically and completely abolished the ability of myoblasts to form podosomes following PDBu stimulation (Figure 5D). In both dystroglycan knockdown and dystroglycan overexpressing cells there was a reduction in Tks5 to an approximately equal level. But despite this reduction in Tks5, the levels of reduction did not correlate with the change in podosome levels in these cells. Thus the effect on podosome assembly of altering DG expression levels either up or down was not a simple consequence of affecting Tks5 levels in the cell (Figure 5B,C) as Tks5 levels were equivalent in both knockdown and overexpressing cells. We hypothesised therefore, that in over-expressing DG we are potentially titrating out factors such as Tks5 that are crucial to podosome formation. The relative levels of Tks5 and dystroglycan would therefore appear to be as important in the regulation of podosome assembly as the actual levels of each protein. From this one could propose that podosome formation should be restored in cells overexpressing DG by the concomitant overexpression of Tks5 in the same cell, restoring the ratio of dystroglycan to Tks5. By co-transfecting myoblasts with Tks5-GFP and DG-Myc we could observe a restoration of podosome formation in response to PDBu, but only once Tks5 overexpression reached some threshold level of expression that balanced the level of DG-Myc overexpression and permitted podosome formation (Figure 6A). However, if DG-myc levels predominated over Tks5-GFP, podosome assembly is inhibited (Figure 6B). Quantification of the relative fluorescence intensity for the two fluors (GFP for Tks5 and rhodamine for DG-Myc) per µm2 of cell area in the cells in Figure 6A,B revealed that a fluorescence ratio of DG∶Tks5 of 1∶0.6 was permissive for podosome formation, whereas a DG∶Tks5 fluorescence ratios of 1∶0.47 or 1∶0.2 was inhibitory to podosome formation. These ratios represent ratios of fluorescence and not ratios of the actual proteins, but nonetheless changing the amount of dystroglycan relative to Tks5 appears to alter the ability of cells to form podosomes in a ‘dose-dependent’ manner (Fig. 6C). These data suggest the existence of a finely tuned regulatory balance between dystroglycan and Tks5 in podosome assembly.


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)

DG over-expression abolishes podosome formation.Quantification of dystroglycan and Tks5 protein levels in control myoblast cells (wild-type), cells stably expressing GFP-tagged full-length DG (DG-GFP), shRNA against DG (knockdown) or a control shRNA (sense). Dystroglycan levels for the four cell lines are shown in A, with corresponding Tks5 levels in B. Whilst dystroglycan levels were increased by 2.3-flod and decreased by half in overexpressing and knockdown cells respectively, Tks5 levels were reduced by approximately 33% in both cases. Wild-type, sense, knockdown and myoblasts transiently expressing a myc-tagged DG construct were induced to form podosomes with PDBu and the number of cells exhibiting podosomes counted following fixation and staining. DG depletion caused a modest reduction in cells with podosomes compared to control, whereas DG over-expression completely abolished podosome formation. (C,D; mean±SEM).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572840&req=5

pone-0003638-g005: DG over-expression abolishes podosome formation.Quantification of dystroglycan and Tks5 protein levels in control myoblast cells (wild-type), cells stably expressing GFP-tagged full-length DG (DG-GFP), shRNA against DG (knockdown) or a control shRNA (sense). Dystroglycan levels for the four cell lines are shown in A, with corresponding Tks5 levels in B. Whilst dystroglycan levels were increased by 2.3-flod and decreased by half in overexpressing and knockdown cells respectively, Tks5 levels were reduced by approximately 33% in both cases. Wild-type, sense, knockdown and myoblasts transiently expressing a myc-tagged DG construct were induced to form podosomes with PDBu and the number of cells exhibiting podosomes counted following fixation and staining. DG depletion caused a modest reduction in cells with podosomes compared to control, whereas DG over-expression completely abolished podosome formation. (C,D; mean±SEM).
Mentions: If as proposed Tks5 is a ‘master regulator’ of podosome assembly [21], and DG also associates with Tks5, DG may also have a regulatory role in the podosome assembly process. In order to address this we examined myoblast cells either overexpressing or depleted for DG (Figure 5A–C). As determined by assessing the characteristic actin morphology of podosomes and compared to control cells, siRNA-mediated depletion of DG to 50% of endogenous levels in myoblast cells had a modest but statistically significant effect on the number of cells producing podosomes in response to PDBu. By contrast, over-expression of DG (tagged with either Myc or GFP) dramatically and completely abolished the ability of myoblasts to form podosomes following PDBu stimulation (Figure 5D). In both dystroglycan knockdown and dystroglycan overexpressing cells there was a reduction in Tks5 to an approximately equal level. But despite this reduction in Tks5, the levels of reduction did not correlate with the change in podosome levels in these cells. Thus the effect on podosome assembly of altering DG expression levels either up or down was not a simple consequence of affecting Tks5 levels in the cell (Figure 5B,C) as Tks5 levels were equivalent in both knockdown and overexpressing cells. We hypothesised therefore, that in over-expressing DG we are potentially titrating out factors such as Tks5 that are crucial to podosome formation. The relative levels of Tks5 and dystroglycan would therefore appear to be as important in the regulation of podosome assembly as the actual levels of each protein. From this one could propose that podosome formation should be restored in cells overexpressing DG by the concomitant overexpression of Tks5 in the same cell, restoring the ratio of dystroglycan to Tks5. By co-transfecting myoblasts with Tks5-GFP and DG-Myc we could observe a restoration of podosome formation in response to PDBu, but only once Tks5 overexpression reached some threshold level of expression that balanced the level of DG-Myc overexpression and permitted podosome formation (Figure 6A). However, if DG-myc levels predominated over Tks5-GFP, podosome assembly is inhibited (Figure 6B). Quantification of the relative fluorescence intensity for the two fluors (GFP for Tks5 and rhodamine for DG-Myc) per µm2 of cell area in the cells in Figure 6A,B revealed that a fluorescence ratio of DG∶Tks5 of 1∶0.6 was permissive for podosome formation, whereas a DG∶Tks5 fluorescence ratios of 1∶0.47 or 1∶0.2 was inhibitory to podosome formation. These ratios represent ratios of fluorescence and not ratios of the actual proteins, but nonetheless changing the amount of dystroglycan relative to Tks5 appears to alter the ability of cells to form podosomes in a ‘dose-dependent’ manner (Fig. 6C). These data suggest the existence of a finely tuned regulatory balance between dystroglycan and Tks5 in podosome assembly.

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