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Human Molecular Genetics, 2004, Vol. 13, No. 15 1657-1668
DOI: 10.1093/hmg/ddh170
Human Molecular Genetics, Vol. 13, No. 15 © Oxford University Press 2004; all rights reserved

Ezrin-dependent regulation of the actin cytoskeleton by ß-dystroglycan

H.J. Spence1, Y.-J. Chen2, C.L. Batchelor3, J.R. Higginson2,3, H. Suila4, O. Carpen4 and S.J. Winder3,*

1CRUK Beatson Laboratories, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK, 2Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK, 3Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK and 4Department of Pathology and Neuroscience Program, Biomedicum, 5th floor and Helsinki University Central Hospital, Haartmaninkatu, PO Box 63 (Haartmaninkatu 8), University of Helsinki, 00014 Helsinki, Finland

Received April 6, 2004; Accepted May 21, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dystroglycan is part of an adhesion receptor complex linking the extracellular matrix to the actin cytoskeleton. Previous studies have implicated dystroglycan in basement membrane formation and as a crucial link between dystrophin and laminin in muscle. We report here a further novel function for dystroglycan which appears to be in addition to its role as an adhesion molecule. ß-dystroglycan has been localized to microvilli structures in a number of cell types where it associates with the cytoskeletal adaptor ezrin, through which it is able to modulate the actin cytoskeleton and induce peripheral filopodia and microvilli. Ezrin is able to interact with dystroglycan through a cluster of basic residues in the juxtamembrane region of dystroglycan, and mutation of these residues both prevents ezrin binding and the induction of actin-rich surface protrusions. These studies reveal novel functions and additional signalling roles for dystroglycan, raising the possibility of new avenues for therapeutic intervention in diseases such as Duchenne muscular dystrophy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dystroglycan is a heterodimeric cell adhesion molecule mediating a link between the extracellular matrix and actin cytoskeleton (reviewed in 1). Previous studies have suggested that dystroglycan is necessary for basement membrane assembly (2,3) and crucial to muscle integrity (4). In all cases, {alpha}-dystroglycan is presumed to bind laminin in the extracellular matrix and to the extracellular face of ß-dystroglycan (5). On the intracellular face, ß-dystroglycan interacts with utrophin or dystrophin (6,7) and via them F-actin (8,9). The role for dystroglycan as part of the dystrophin–glycoprotein complex of muscle is well established (10). In muscle, where dystroglycan associates predominantly with dystrophin and associated proteins (5), loss of dystroglycan concomitant with mutations in dystrophin is also associated with the loss of the entire subsarcolemmal actin network (11) with consequent muscle fragility and necrosis as typified in Duchenne muscular dystrophy. The role for dystroglycan in basement membrane assembly, however, has recently been called into question (4,12,13). Rather than an essential factor for basement membrane assembly, dystroglycan is proposed to act in a more generic adhesion role in concert with integrins in maintaining cell survival (12). Chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy, but the muscle basement membrane is still formed (4). In zebrafish embryos lacking dystroglycan, basement membrane formation is not compromised, but morphant embryos do exhibit a severe muscular dystrophy phenotype (13). More recently, it has been suggested that dystroglycan may perform additional functions and have alternate intracellular binding partners. Cell adhesion to laminin substrates can induce the tyrosine phosphorylation of ß-dystroglycan (6,14) resulting in a loss of association with utrophin or dystrophin (15,16). Furthermore, whilst phosphorylation of ß-dystroglycan on tyrosine reduces its ability to interact with utrophin or dystrophin, the ability to interact with caveolin is not altered (14,17) suggesting a potential mechanism whereby dystroglycan may associate with alternate protein complexes at the cell membrane such as the recently described interaction with components of the ERK MAP kinase cascade (18).

We report here the association of dystroglycan in a different protein complex at the cell membrane and a novel function for dystroglycan which appears to be in addition to its role as an adhesion molecule. ß-dystroglycan has been localized to microvilli structures in a number of cell types where it associates with the cytoskeletal adaptor ezrin, through which it is able to modulate the actin cytoskeleton.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-dystroglycan-ezrin binding
Dystroglycan is synthesized as a single precursor peptide that is post-translationally cleaved to yield the mature extracellular {alpha}- and transmembrane ß-dystroglycan proteins (Fig. 1A) (19,20). Sequence analysis of dystroglycan shows no significant overall homology with any other proteins in the databases, with the exception of homologs from other species and regions predicted to form the signal sequence and the transmembrane region. A recent report has suggested that {alpha}-dystroglycan contains two cadherin-like domains (21) and short regions of the ß-dystroglycan cytoplasmic domain have homology to a number of actin-binding proteins (22), beyond this, however, no other extensive homologies exist. Examination of the sequence of the cytoplasmic region of ß-dystroglycan by eye, revealed a small region of similar character to the juxtamembrane region of a number of other cell adhesion receptors (Fig. 1B). Furthermore, it has been demonstrated in other cell adhesion receptors that these basic juxtamembrane regions are binding sites for ezrin/radixin/moesin (ERM) family proteins (23–26). We therefore carried out GST-pulldown and immunoprecipitation experiments to determine if ezrin was able to interact with ß-dystroglycan. Addition of a GST-fusion with the cytoplasmic domain of ß-dystroglycan to extracts from Cos-7 cells followed by western blotting for ezrin revealed a clear band of ~80 kDa in the GST-dystroglycan pulldown (Fig. 1C, lane 2), whereas GST alone did not pulldown ezrin (Fig. 1C, lane 1), similar data were also obtained with Hela cell extracts (data not shown). This result is consistent with the hypothesis that dystroglycan is able to interact with ezrin. In order to determine if the ezrin–dystroglycan interaction was able to occur in a cellular context, immunoprecipitates from C2/C4 cell lysates using ß-dystroglycan antisera were probed for ezrin, again a specific band at ~80 kDa was observed (Fig. 1C, lane 4), consistent with the ability of dystroglycan to associate with ezrin in the cell. Immunoprecipitation with an irrelevant antiserum, pan-ERK, did not immunoprecipitate ezrin (Fig. 1C, lane 5) under these conditions. Thus it would appear that ezrin is able to associate with ß-dystroglycan, possibly via a cluster of basic residues in the juxtamembrane region of the cytoplasmic tail of ß-dystroglycan.



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  		Figure 1. Dystroglycan domain organization and ezrin association. (A) A schematic showing the domain organization of the dystroglycan precursor polypeptide (upper) and the proteolytically cleaved mature {alpha}-dystroglycan and ß-dystroglycan proteins (lower). SS, signal sequence; {alpha}-DG, {alpha}-dystroglycan; e, extracellular; tm, transmembrane; c, cytoplasmic domains of ß-dystroglycan. (B) The amino acid sequences of the cytoplasmic juxtamembrane regions of a number of cell adhesion receptors including; ß-dystroglycan, CD44, CD43, ICAM-1 and ICAM-2 with basic regions suggested to be involved in binding to ERM family members underlined (23–26). The positions of conserved residues shown to be involved in a structure of the radixin FERM domain complexed with an ICAM-2 peptide are in grey boxes. (C) Western blots for ezrin show that ezrin interacts with dystroglycan by both GST-pulldown and immunoprecipitation. Lane 1, GST alone and lane 2, GST-ß-dystroglycan GST-ß-DG specifically pulls down an ezrin immunoreactive band of ~80 kDa (lane 2). Lane 3 shows the ezrin band in total cell extract used in the immunoprecipitation as a control, and lane 4 shows the ezrin immunoprecipitated from a C2/C4 cell extract with a ß-dystroglycan antiserum. Immunoprecipitation with panERK serum (lane 5) under these conditions did not result in ezrin immunoprecipitation.

 
Ezrin colocalizes with ß-dystroglycan in membrane extensions
The ability of ß-dystroglycan to associate biochemically with ezrin suggested that the two proteins may colocalize in a distinct cellular compartment. ERM proteins, and ezrin in particular, are known to be involved in the formation of microvilli structures in a wide number of cell types (27–30). We therefore examined the localization of ezrin and ß-dystroglycan in epithelial cells, including Hela, A431 (data not shown) and JEG3 choriocarcinoma (Fig. 2). As expected, ezrin was found localized to microvilli structures in these cells (Fig. 2B), furthermore ß-dystroglycan also localized to microvilli and colocalized extensively with ezrin (Fig. 2A, D and inset). Confocal sections of JEG3 cells further demonstrated the apical localization of both ezrin and ß-dystroglycan to microvilli-like structures (Fig. 2E–H). As determined by fine confocal sectioning the colocalization of ezrin and ß-dystroglycan was most evident at the base of the microvilli. Some colocalization was also seen in cell contact regions visualized by submembranous F-actin accumulation (Fig. 2E–H). Interestingly, the basal surface of JEG-3 cells stained positively for ß-dystroglycan but not ezrin. Furthermore, as previously demonstrated in REF52 fibroblast cells (22), DG-GFP was both localized to and induced actin-rich surface protrusions, including filopodia and microvilli-like structures, in a number of other cell types of both mesenchymal and epithelial origin including Cos-7, Hela and A431 cells (Fig. 3).



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  		Figure 2. Colocalization of dystroglycan and ezrin. JEG3 cells grown on glass coverslips were stained for ezrin and ß-dystroglycan and analysed by immunofluorescence (A, B, D), DIC (C) or confocal (E–H) microscopy. (A–D) Cells double stained for ezrin (A) and ß-dystroglycan (B) show extensive colocalization of the proteins at the apical surface, especially in microvilli as seen in the merged image and in insets [(D) ezrin: green; ß-dystroglycan: red; areas of colocalization: yellow]. Some of the microvilli are shown by arrows. (C) DIC image. Bar=10 µm. (E–H) Series of confocal images from basal to the apical surface. The cells were triple-stained for ß-dystroglycan (E) red, ezrin (F) green and F-actin (G) blue and merged image (H). ß-dystroglycan and ezrin colocalize especially at the base of microvilli. On the other hand, ezrin is virtually absent from the basal surface of the cells, which are positive for ß-dystroglycan (red). Sections are from basal; left, to apical; right. Bar=50 µm.

 


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  		Figure 3. Dystroglycan-GFP induces and is localized to filopodia and microvilli-like structures. Epifluorescent images of REF52, Cos-7, Hela and A431 cells (A–D, respectively) transfected with dystroglycan-GFP. Dystroglycan-GFP induced the formation, and was localized to peripheral filopodia structures (arrowheads in A, B, D), dorsal microvilli-like structures (arrows in B–D) or both (B, D). The images of Hela (C) and A431 cells (D) were taken in a focal plane near to the dorsal surface of the cells with consequent blurring of the cell edges. Cells transfected with GFP alone did not exhibit any alteration in morphology or staining of filopodia or microvilli (22) and data not shown.

 
Mutation of the putative binding site for ezrin on dystroglycan prevents surface protrusions
The binding of ezrin by ß-dystroglycan and their presence in microvilli-like structures raised the possibility that ß-dystroglycan might participate in the formation of cortical actin-rich structures. We have previously shown that dystroglycan expression in REF52 cells led to a marked alteration of cell morphology, with the induction of numerous actin-rich surface protrusions around the cell periphery and on the dorsal surface, and a dramatic reorganization of the actin cytoskeleton (Fig. 3) (22). With increasing levels of ß-dystroglycan-GFP expression, there was an increase in both the number of actin-rich surface protrusions and in the extent of the remodelling of the actin cytoskeleton including the appearance of microvilli-like structures. These changes were in part attributed to a direct interaction between ß-dystroglycan and actin (22), though it is equally possible in light of the data presented above that dystroglycan could recruit ezrin to the membrane resulting in a similar phenotype. In order to further substantiate the link between dystroglycan, ezrin and actin-rich surface protrusions, we generated dystroglycan constructs containing an altered putative ezrin binding site (Fig. 4A). As expected, this construct was unable to interact with ezrin (Fig. 4B). GST-pulldown experiments with a GST-dystroglycan fusion protein lacking the putative ezrin binding site were unable to pulldown ezrin from Cos-7 cell extracts, whereas a GST-fusion to the complete dystroglycan cytoplasmic domain did pulldown ezrin (Fig. 4B; see also Fig. 1D, lane 2). GST alone was not able to pulldown ezrin (Fig. 4B; see also Fig. 1D, lane 1). Deletion of the ezrin binding site in the context of a dystroglycan GST-fusion protein clearly prevented the ability of ezrin to interact with the cytoplasmic domain of dystroglycan. However, in order to address a role for ezrin binding to this region and the ability of dystroglycan to induce actin-rich surface protrusions, we generated a full-length GFP-tagged dystroglycan construct with a similar mutation in the ezrin binding site (DG{Delta}E-GFP; Fig. 4A). When compared to the surface protrusion phenotype of wild-type dystroglycan-GFP expression (Fig. 4C) with the presence of numerous protrusions at the cell periphery and on the apical surface of the cell; REF52 cells transiently transfected with the ezrin binding site-deleted dystroglycan-GFP construct had a completely smooth morphology, with no peripheral or apical protrusions apparent (Fig. 4D). Taken together these data substantiate the importance of the link between ß-dystroglycan and ezrin in actin-rich surface protrusion formation.



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  		Figure 4. Substitution of the putative ezrin binding sites prevents dystroglycan-induced filopodia. (A). Wild-type dystroglycan cytoplasmic domain juxtamembrane sequence (DG) and the sequence mutated in the putative ezrin binding motif either as a GST fusion protein (GST-DG{Delta}E) or as a full-length GFP construct (DG{Delta}E-GFP). (B) GST-pulldown with GST alone, a construct with an altered potential ezrin binding site (GST-DG{Delta}E) and wild-type dystroglycan (GST-DG) western blotted for ezrin. Only GST-DG is able to interact with ezrin. (C) Projection of stacked confocal images of REF52 cells expressing either DG-GFP (C) or DG{Delta}E-GFP (D) visualized by GFP fluorescence. Numerous protrusions are apparent around the cell periphery (arrows in C) and on the surface of the cell (arrowheads). When the putative ezrin binding site is mutated, surface protrusions are absent (D). (E) Quantification of phenotypes observed in (C) and (D). Data are mean ± standard deviation.

 
Compared to REF52 cells transfected with DG-GFP which exhibit numerous actin-rich surface protrusions (Figs 3A and 5A–C and insets), REF52 cells transiently transfected with DG{Delta}E-GFP have an actin cytoskeleton more typical of untransfected cells, with no actin-rich filopodia and a normal complement of actin stress fibres (Fig. 5D–F). Furthermore, actin rich surface protrusions appear to be specifically associated with an increase in dystroglycan expression. Expression of ezrin alone (Fig. 5G–I), whilst localized to some actin containing surface protrusions, does not induce a large number of microvilli-like structures as seen with DG-GFP expression (Fig. 5A–C).



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  		Figure 5. Induction of actin-rich surface protrusions by dystroglycan, but not ezrin or a dystroglycan mutant. REF52 cells expressing DG-GFP (A–C), DG{Delta}E-GFP (D–F) or over-expressing wild-type ezrin (G–I) stained for actin with rhodamine phalloidin (A, D, G), GFP signal for dystroglycan constructs (B, E), ezrin with monoclonal 3C12 and FITC conjugated secondary antibody (H) and respective merged images (C, F, I). Compared to cells expressing DG-GFP with numerous actin-rich protrusions around the cell periphery (arrowed in insets in A, B) and dystroglycan-containing surface projections (B, C), cells expressing either DG{Delta}E-GFP (D–F) or ezrin (G–I) do not exhibit the numerous actin protrusions and there are relatively few surface projections.

 
We next investigated whether the apparent lack of actin-rich surface protrusion formation by the DG{Delta}E-GFP construct was actually a consequence of this construct failing to target ezrin to the cytoskeletal fraction. We transiently expressed DG-GFP or DG{Delta}E-GFP in Cos-7 cells, which also exhibit DG-GFP-induced microvilli formation (Fig. 3B), and determined the proportion of total cellular ezrin associated with membrane fraction, cytosolic fraction and triton-insoluble cytoskeletal fraction (31). In all assays shown here, no ezrin was recovered in the membrane fraction which is therefore not represented in the figures. Despite a transfection efficiency of less than 50%, it is clear that the amount of ezrin associated with the triton-insoluble cytoskeletal fraction, as determined by ratio from the sum of all fractions, when either DG-GFP or DG{Delta}E-GFP are expressed, is significantly different (Fig. 6A and B). By comparison with cells transfected with DG-GFP, DG{Delta}E-GFP caused a reduction in the amount of ezrin associated with the actin cytoskeleton from 62 to 40% (P=0.03) which was matched by a marked reduction in actin-rich surface protrusions in REF52 cells (Figs 4D and 5D–F), with wild-type levels (16±0% of transfected cells), compared to 85.5±3.5% of cells transfected with the DG-GFP construct (Fig. 4E). These data confirm a role for ezrin binding to dystroglycan in the induction of actin-rich surface protrusions in these cells.



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  		Figure 6. Deletion of the putative ezrin binding sites prevents ezrin association with the actin cytoskeleton. Cos-7 cells transiently expressing dystroglycan-GFP (~50% efficiency as judged by GFP signal) show an increased level of ezrin recruitment to the triton-insoluble cytoskeletal fraction compared to cells transiently expressing DG{Delta}E-GFP. (A) A representative western blot of ezrin in cytoplasmic (C) and triton insoluble–cytoskeletal (T) fractions. (B) Quantification of this and similar data by densitometric scanning. Dystroglycan-GFP expressing cells show a significantly increased association of ezrin with the cytoskeletal fraction as compared to cells expressing DG{Delta}E-GFP. Data are mean±standard deviation, * denotes statistical significance at P=0.03 Student's t-test.

 
Mutations in dystroglycan or ezrin prevent actin rich protrusions
As indicated above, ezrin expression in itself is insufficient to induce actin-rich surface protrusions (Fig. 5G–I); however, when ezrin is expressed in the presence of DG-GFP, cells typically exhibit a large number of actin-rich surface protrusions (Figs 7B and 8A; and unpublished data). This provided a rationale to examine the role of ezrin phosphorylation in the induction of actin-rich surface protrusions by dystroglycan. ERM proteins are known to be phosphorylated and activated at a conserved threonine residue (T567), mutation of T567 to alanine (T567A) prevents ezrin phosphorylation and renders the protein inactive and unable to bind F-actin. Coexpression of VSVG-tagged ezrin constructs (32), either wild-type or containing a T567A substitution with DG-GFP or DG{Delta}E-GFP, could therefore be used to elucidate the role of ezrin phosphorylation in dystroglycan-induced actin-rich surface protrusions. Western blotting for VSVG-tagged ezrin revealed that, in the presence of dystroglycan, a significant proportion of the VSVG-ezrin was present in the triton-insoluble cytoskeletal fraction of cell extracts (Fig. 7A). When either dystroglycan ezrin binding site mutant was coexpressed with wild-type VSVG-ezrin, or when wild-type dystroglycan was expressed with VSVG-ezrin T567A, nearly all of the VSVG-ezrin was recovered in the cytoplasmic fraction (Fig. 7A). As seen in previous experiments, the presence of ezrin in the cytoskeleton fraction was correlated with the presence of actin-rich surface protrusions, which was confirmed when doubly-transfected cells staining for both VSVG and GFP were scored for the presence of surface protrusions (Fig. 7B). Expression of either the mutated T567A phosphorylation site in ezrin, the ezrin binding site in dystroglycan, or both, caused a sequential reduction in the number of cells with surface protrusions implicating both dystroglycan and ezrin phosphorylation in their formation. DG-GFP and DG{Delta}E-GFP were both targeted to the cell membrane as expected, whereas only wild-type but not T567A ezrin was targeted to the membrane (Fig. 8A–D). Furthermore, there was only a significant colocalization of the two proteins and the formation of microvilli-like structures when DG-GFP and wild-type ezrin were expressed together in the same cell (Fig. 8A and E–G) and not when either DG{Delta}E-GFP, ezrin T567A or both were present (Fig. 8B–D).



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  		Figure 7. Both a functional ezrin binding site in dystroglycan and phosphorylation site in ezrin are required for ezrin translocation and actin protrusion formation. Cos-7 cells coexpressing either DG-GFP (DG) or DG{Delta}E-GFP (DG{Delta}) and VSVG-ezrin (E) or VSVG-ezrin T567A (E T-A) were subjected to ezrin fractionation assay (A) and assessed for the number of surface protrusions by fluorescence microscopy as described in Materials and Methods (B). The amounts of VSVG-tagged ezrin construct present in the cytoplasmic (C) and triton insoluble–cytoskeletal (T) fractions was determined by western blotting for the VSVG tag (A). Considerably more VSVG-ezrin was recovered in the triton insoluble cytoskeletal fraction in the presence of DG-GFP as compared to DG{Delta}E-GFP, and the VSVG-ezrin T-A mutant was recovered mainly in the cytoplasmic fraction in the presence of DG-GFP. Quantification of these and other data indicate that the introduction of an altered ezrin binding site into dystroglycan, a mutated phosphorylation site into ezrin, or both sequentially reduce the number of surface protrusions present on cells (B). Data are mean±standard deviation; all data are significantly different from each other at P=0.048 or better by Student's t-test.

 


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  		Figure 8. Morphology of cells coexpressing dystroglycan and ezrin constructs. Cos-7 cells co-expressing the indicated combinations of DG-GFP (DG), DG{Delta}E-GFP (DG{Delta}), VSVG-ezrin (E) and VSVG-ezrin T567A (E T-A) stained for ezrin using the VSVG tag (red) or dystroglycan using the intrinsic GFP signal (green). Only in the presence of the wild-type constructs (A) is there an extensive colocalisation (yellow) and a significant number of surface protrusions apparent (arrows). When either mutant (B, C) or both mutants (D) were present, the cells show very few surface protrusions when compared with DG-GFP (A). A confocal section through the dorsal surface of the cells in A is shown as the separated and merged images to highlight the high degree of overlap (G) between the expressed DG-GFP (F) and VSVG-ezrin (E) in surface protrusions.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
These studies indicate a novel function for dystroglycan in both the regulation of the actin cytoskeleton, transduction of signals from the extracellular matrix to the cytoskeleton and regulation of cell morphology. As we have shown, not only is dystroglycan able to directly interact with the actin cytoskeleton independently of utrophin (22), but also via ezrin, a member of the ERM family of proteins. Furthermore, the dystroglycan- and ezrin-dependent interaction with the cytoskeleton appears to occur specifically at the surface of cells where the two proteins are found colocalized in actin-rich filopodia- and microvilli-like structures. These findings have wide implications for our understanding of dystroglycan function in a variety of muscular dystrophies, during Mycobacterium leprae infection where dystroglycan/laminin complex acts as a receptor for the leprosy bacterium, as well as in a more fundamental role in the regulation and organization of the actin cytoskeleton and cellular morphology.

Dystroglycan localizes with ezrin in actin-rich surface protrusions
Previous studies in a number of cell and tissue types have shown dystroglycan to be associated with basement membrane structures. Deletion of dystroglycan in mice leads to peri-implantation defects associated with the lack of Reichert's membrane formation (3), and dystroglycan has been suggested to be required for basement membrane assembly in embryoid bodies (2), though more recently this finding has been called into question (12). Nevertheless, it is clear that dystroglycan is a vital part of a protein complex involved in linking the actin cytoskeleton to laminin in the extracellular matrix found at the basal aspect of many cells (reviewed in 1). In muscle tissues, in particular, the role of dystroglycan and the dystrophin glycoprotein complex is crucial to the maintenance of sarcolemmal integrity (reviewed in 33,34), but dystroglycan is also important in non-muscle tissues. Dystroglycan forms a distinct complex with utrophin in epithelial cells (35) where it has been shown to be involved in epithelial development (36). Even within the same tissue there are distinct glycoforms of dystroglycan indicating that there may be more than one dystroglycan complex capable of performing distinct functions within that tissue or cell (37). For the first time we demonstrate the presence of a distinct dystroglycan complex involved in an extra-basal function and presumably not involving the previously characterized cytoskeletal anchors for dystroglycan [i.e. dystrophin or utrophin (22)].

The association of dystroglycan with ezrin in the microvilli of epithelial cells and the presence of dystroglycan and ezrin in cortical actin-rich protrusions in other cells types point to a completely novel function for dystroglycan, both in the formation of cellular structures and regulation of the actin cytoskeleton. Furthermore, these functions clearly do not depend on the presence of previously identified cytoskeletal anchors for dystroglycan such as utrophin (6) as protrusions were evident in cells derived from a utrophin knockout mouse that completely lack utrophin (22,38). The colocalization of endogenous dystroglycan and ezrin in microvilli-like structures and coimmunoprecipitation and GST-pulldown of dystroglycan and ezrin from cell extracts suggest a direct role for dystroglycan in the formation of microvilli-like structures. Dystroglycan is therefore one of a small but growing number of transmembrane proteins that associate with ezrin in surface structures including CD44, ICAM-1, ICAM-2 and L-selectin (23–26). In most cases, the recruitment of activated ezrin to these cell adhesion receptors has been reported to induce the formation of microvilli structures (23,25,26), dystroglycan would also appear to be no exception in this regard. The association of ezrin with cell adhesion receptors is thought to occur through clusters of basic residues in the juxtamembrane region of the cytoplasmic tail of the receptor which are also found in dystroglycan. Deletion or alteration of the character of these sequences in dystroglycan was sufficient to prevent the association of ezrin with dystroglycan and prevented dystroglycan-induced actin-rich protrusions. A recent structure of the radixin FERM domain complexed with an ICAM-2 peptide has shed further light on this type of interaction (39). Whilst the basic residues apparently contribute to the overall affinity of the ICAM-2 peptide for the FERM domain, other conserved non-polar and hydrophobic residues c-terminal of the basic residues also contributed to binding (39). Three of the four most critical residues for FERM binding are also conserved in dystroglycan (Fig. 1B), mutation of some of these residues, as we have done here, would be expected to cause a dramatic reduction in affinity for the ezrin FERM domain as our functional data indicate.

It may be possible for binding of ezrin to the cluster of positively charged residues in the juxtamembrane region of dystroglycan to occur simultaneously with utrophin or dystrophin binding to the carboxy-terminal 20 residues of dystroglycan. Previous studies have shown the carboxy-terminal 20 residues of dystroglycan, containing a WW domain binding motif, to be involved in the interaction with dystrophin and utrophin (6,7,40,41), though it has been suggested that the positively charged juxtamembrane sequence might facilitate the interaction of the dystrophin EF-hand region with dystroglycan (40). The binding sites for ezrin and dystrophin/utrophin are therefore well separated, ~100 residues apart, and given the lack of secondary structure in the cytoplasmic tail of dystroglycan (6), the tail may act as a flexible scaffold for binding partners such as ezrin and dystrophin/utrophin to interact with the actin cytoskeleton. If dystrophin/utrophin and ezrin can interact with dystroglycan simultaneously however, they clearly do not require the presence of one another as dystroglycan was still able to induce a protrusion phenotype in the absence of utrophin and dystrophin (22).

Regulation of dystroglycan-induced actin-rich structures
Deletion mutagenesis of dystroglycan clearly demonstrated a requirement for both the cytoplasmic domain of ß-dystroglycan and its correct localization to the plasma membrane in the induction of surface protrusions (22), and substitution of a stretch of basic residues in the juxtamembrane region of dystroglycan prevented ezrin binding and surface protrusion formation. To date, the majority of data support the conformational activation of ezrin, requiring the release of the interaction between the amino and carboxy-terminal ERM association domains, the N-ERMAD and C-ERMAD, respectively, in order for either domain to bind to another protein. Phosphorylation of a conserved threonine in the C-ERMAD of ERM proteins by PKC-{theta} or Rho kinase results in the relief of the autoinhibitory interaction between the N-ERMAD and C-ERMAD (42–44). In ezrin, radixin and moesin, the site phosphorylated is a conserved threonine, T567, T564 or T558, respectively. Ezrin phosphorylation was clearly required for the induction of filopodia and microvilli-like structures by dystroglycan, as coexpression of an ezrin T567A construct that could not be phosphorylated, prevented the dystroglycan-induced structures. Dystroglycan-induced surface protrusions were also shown to be inhibited by dominant-negative Cdc42 (22), suggesting a role for Cdc42 mediated signalling in the formation of dystroglycan-induced surface structures. The Cdc42 and Rac (p21) activated kinase PAK, has been demonstrated to be involved in the phosphorylation of merlin at S518 in its C-terminal domain (45), a site known to be involved in activating merlin (46), but this site is not conserved in other ERM proteins. One Cdc42 effector that has been demonstrated to phosphorylate ERM proteins is the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) (47,48). Cdc42 activation of MRCK led to the production of prominent filopodial structures in Hela cells with concomitant reorganization of the actin cytoskeleton (47), and in NIH3T3 cells phosphorylated ERM proteins accumulated in filopodia in a Cdc42 and MRCK-dependent manner. A similar mechanism involving Cdc42 and MRCK could be involved in the dystroglycan- and ezrin-dependent formation of microvilli and filopodia. Further work will be required to elucidate the precise signalling mechanisms involved in the dystroglycan-mediated induction of filopodia and microvilli.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Expression constructs
A full-length mouse dystroglycan cDNA encoding {alpha}- and ß-dystroglycan (generously provided by Derek Blake, Oxford) was cloned into the pEGFP vector (Clontech) using Sma1 and Sal1 restriction sites engineered by PCR as described previously (22). Mutation of the ezrin binding site (RKKRK to RENGK) in full-length dystroglycan-GFP was carried out using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturers instructions. GST-dystroglycan (residues 775–897) was produced by PCR and subcloned through pTopo into pGEX-4T1 using EcoR1 restriction sites. A GST-dystroglycan construct (residues 775–897) containing mutations in the ezrin binding site (RKKRK to AAAAA) was produced by PCR, including EcoR1 and Xho1 restriction sites and cloning into pGEX-4T1. GST and GST-dystroglycan proteins were produced by expression in Escherichia coli BL21(DE3) and purified on glutathione-Sepharose (Amersham Biosciences) according to the manufacturers instructions. A vesicular stomatitis virus G protein (VSVG) tagged ezrin construct was generously provided by Monique Arpin (Paris) (32), the T567A mutation was introduced into this construct using site-directed mutagenesis as above.

Cell culture
Cos-7, JEG3, C2/C4 and REF52 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum or 20% for C2/C4 cells, and maintained in a 5% CO2 atmosphere. Hela and A431 cells were maintained as above but using RPMI1640 medium in place of DMEM and 5% serum. Transient transfection of various expression constructs was achieved by introduction into sub-confluent cell cultures using lipofectamine (Invitrogen; 6 µl/ml) and a final DNA concentration of 2 µg/ml in Optimem (Invitrogen) over 5 h. Cells were transferred to media with serum and cultured for a further 15 h prior to assay.

Immunofluorescence microscopy
Cells were grown on glass coverslips and fixed for 10 min in 3.7% paraformaldehyde and permeabilized for 1 min in 0.1% Triton X-100 in phosphate buffered saline (PBS), alternatively cells were fixed and permeabilized at –20°C in methanol for 10 min. Fixed cells were blocked in 5% FCS, 1% BSA in PBS for 1 h at room temperature and then incubated with primary antibody alone or in combination. Rhodamine or fluorescein-conjugated (Vector Laboratories) or Alexa 488 and Alexa 594-conjugated (Molecular Probes) species-specific secondary antisera were used at 1 : 100 for 1 h. Actin filaments were visualized with TRITC-phalloidin or Alexa 660-conjugated phalloidin (Molecular Probes; 1 µg/ml for 60 min). Coverslips were mounted in Vectashield (Vector Laboratories) and images captured digitally by cooled CCD camera on an Olympus Provis epifluorescence microscope or Leica DMIRE2. Confocal analysis was performed with Leica SP2 confocal microscope equipped with Ar, Kr and HeNe-lasers. Cells were determined to have an actin-rich surface protrusion phenotype if the cells had more than 30 surface protrusions over the whole surface (22).

Protein immunoblotting
Sub-confluent dishes of cells were washed twice with ice cold PBS and lysed with modified RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EGTA, 0.1% SDS, 1% sodium deoxycholate, 1% NP40, 10 mM sodium pyrophosphate, 0.5 mM sodium fluoride, 1 mM PMSF, 10 mg/ml each of aprotinin, leupeptin and benzamidine and 100 mM sodium vanadate), for 15 min on ice. The insoluble material was pelleted at 14 000g for 30 min at 4°C. Protein concentrations were measured by the Micro BCA method (Pierce). For direct detection of proteins, 5 µg of total cell lysate were separated by SDS–PAGE, transferred to PVDF and probed with an anti-ezrin antiserum (1 : 8000) (25). Detection was by reaction with horseradish peroxidase-conjugated secondary antibody and visualisation was by ECL as per manufacturer's instructions (Amersham Biosciences).

Immunoprecipitation, pulldown and cytoskeleton assays
Immunoprecipitation of ezrin with ß-dystroglycan antiserum was performed essentially as described previously (6,15,49). Briefly, clarified cell extract in RIPA buffer (200 µg) was incubated for 2 h at 4°C with ß-dystroglycan monoclonal serum 43DAG/8D5 (1 : 10, a generous gift from Louise Anderson, Newcastle), or for control experiments a pan-ERK serum (1 : 100, Transduction Laboratories). Immunoprecipitates were collected by a further 1 h incubation with protein A-Sepharose (Amersham Biosciences) followed by extensive washing with RIPA buffer. Immune complexes were solubilized in SDS sample buffer prior to electrophoresis, transfer and western blotting.

GST pulldown assays were performed using 500 µg of cell extract and 10 µg of GST, GST-ß-dystroglycan or GST-ß-dystroglycan mutant. Samples were incubated for 1 h at 4°C followed by a further incubation of 1 h with glutathione-Sepharose. The glutathione-Sepharose beads were washed extensively and bound proteins eluted in SDS sample buffer prior to electrophoresis, transfer and western blotting.

The translocation of ezrin, from cytoplasm to cytoskeleton, was assessed by the fractionation of Cos-7 cells transiently expressing GFP DG{Delta}E-GFP or DG-GFP, with or without VSVG-ezrin, as described previously (31). Briefly, transiently transfected Cos 7 cells were homogenized in 10 mM Hepes pH 7.4, 1 mM Na3VO4, 2.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 100 µg/ml CHAPS and 1 mM PMSF, and centrifuged for 20 min at 18 000 r.p.m. Soluble material was removed (‘membrane’ fraction) and the pellet homogenized in Triton X-100 lysis buffer (25 mM MES pH 6.4, 1 mM Na3VO4, 2.5 mM MgCl2, 3 mM EGTA, 0.5% Triton X-100, 10% glycerol, 1 mM PMSF) and centrifuged at 4°C for 20 min at 14 000 r.p.m. The soluble (cytoplasmic) fraction and the insoluble (cytoskeletal) fraction and earlier membrane fractions were solubilized in SDS sample buffer. In each experiment, equivalent volumes of fractions were run on SDS–PAGE, ezrin was never found to localize to the membrane fraction in any of the experiments presented and is therefore not represented. As all of the fractions from the assay are run on the gel, the total amount of ezrin is proportional to the sum of the individual bands, furthermore and as a consequence, protein loading does not need to be standardized as it is the ratio between the individual fractions in any one sample that is important. Quantification of ezrin in the various fractions are by densitometric scanning of westerns blots for ezrin or in some experiments the VSVG tag and presented as % ezrin in cytoskeleton derived by ratio from the sum of the densities of the bands.


   ACKNOWLEDGEMENTS
 
We are grateful to Louise Anderson and Clare Isacke for their generous gifts of antisera, Derek Blake for mouse dystroglycan cDNA and Monique Arpin for the VSVG-ezrin construct. We are also grateful to Margaret Frame, Kathryn Ayscough and members of the Winder laboratory for helpful discussions and critical reading of the manuscript. This work was supported by grants from the Royal Society, Tenovus Scotland, MRC and Wellcome Trust to S.J.W. and from the Academy of Finland, Sigrid Juselius Foundation, US Army Neurofibromatosis Research grant DAMD17-00-0550 and Helsinki University Central Hospital Funds to OC.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 1142222332; Fax: +44 1142765413; Email: s.winder{at}sheffield.ac.uk


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 DISCUSSION
 MATERIALS AND METHODS
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