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Human Molecular Genetics Advance Access originally published online on April 5, 2007
Human Molecular Genetics 2007 16(11):1351-1358; doi:10.1093/hmg/ddm085
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The pathomechanism of filaminopathy: altered biochemical properties explain the cellular phenotype of a protein aggregation myopathy

Thomas Löwe1,{dagger}, Rudolf A. Kley2, Peter F.M. van der Ven1,3, Mirko Himmel1,{ddagger}, Angela Huebner4, Matthias Vorgerd2 and Dieter O. Fürst1,3,*

1 Department of Cell Biology, University of Potsdam, 14476 Potsdam-Golm, Germany, 2 Department of Neurology, Neuromuscular Center Ruhrgebiet, Ruhr-University Bochum, 44789 Bochum, Germany, 3 Department of Molecular Cell Biology, University of Bonn, 53121 Bonn, Germany and 4 Children's Hospital, Technical, University Dresden, 01307 Dresden, Germany

* To whom correspondence should be addressed at: Department of Molecular Cell Biology, University of Bonn, Ulrich-Haberland-Str. 61a, 53121 Bonn, Germany. Tel: +49 228735301; Fax: +49 228735302; Email: dfuerst{at}uni-bonn.de

Received December 19, 2006; Revised March 19, 2007; Accepted March 28, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myofibrillar myopathy (MFM) is a pathologically defined group of hereditary human muscle diseases, characterized by focal myofibrillar destruction and cytoplasmic aggregates that contain several Z-disc-related proteins. The previously reported MFM-associated mutation (8130G -> A; W2710X) in the filamin C gene (FLNC) leads to a partial disturbance of the secondary structure of the dimerization domain of filamin C, resulting in massive protein aggregation in skeletal muscle fibers of the patients. Here, we provide a thorough characterization of the biochemical, biophysical and cellular properties of the mutated filamin C polypeptide. Our experiments revealed that the mutant dimerization domain is less stable and more susceptible to proteolysis. As a consequence, it does not dimerize properly and forms aggregates in vitro. Furthermore, the expression of mutant filamin in cultured cells results in the formation of protein aggregates. The mutant filamin does not associate with wild type filamin. These findings are of great importance to explain the pathomechanism of this disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myofibrillar myopathies (MFM) are a group of rare neuromuscular disorders morphologically characterized by myofibrillar disintegration and cytoplasmic accumulation of several Z-disk proteins in the muscle fibers of the patients (1,2). Various genes have been associated with this disease: the genes encoding desmin (3,4) (DES, OMIM 125660 [OMIM] ), myotilin (5) (MYOT, OMIM 604103 [OMIM] ), ZASP (6) (LDB3, OMIM 605906 [OMIM] ), {alpha}B-crystallin (7,8) (CRYAB, OMIM 123590 [OMIM] ) and filamin C (9) (FLNC, OMIM 102565 [OMIM] ). Although all these proteins have in common that they are associated with the sarcomeric Z-disc, the mechanisms that lead to the similar morphological features, including the aggregation of these proteins, have remained poorly understood. In the case of desmin, it seems that the mutated protein variants exert a dominant negative effect and induce a collapse of the desmin cytoskeleton (10–13). In contrast, the myotilin mutations are point mutations clustered in proximity to the {alpha}-actinin binding region (5,14,15), but apparently binding is not affected (16). Even though the ZASP mutations locate within a motif that is important for the association with {alpha}-actinin, neither the binding between both proteins seems affected, nor is ZASP destabilized (17). Known {alpha}B-crystallin mutations result in the expression of a mutant form with a single amino acid exchange (R120G) or C-terminally truncated protein variants (7,8). The R120G missense mutation forms aggregates together with desmin upon expression in BHK21 cells and cardiomyocytes, but again, the underlying mechanism has remained unclear (7,18,19).

We have recently described a mutation in FLNC that is causative for a novel form of MFM (9). This gene encodes the striated muscle specific-isoform of the filamin family. The filamins have an actin-binding domain (ABD) at their amino-terminus that is followed by 24 immunoglobulin (Ig-)-like domains. The C-terminal Ig-like domain 24, is responsible for the ability of the protein to dimerize (20). This explains its actin filament bundling and cross-linking activities. Apart from their role in organizing the actin filament system, filamins interact with a plethora of cellular proteins of great functional diversity, indicating that they are multifunctional signaling adapter proteins (21,22). Diverse nonsense, missense and splice variant mutations in the non-muscle-specific genes encoding filamin A and filamin B are causative for a wide range of human diseases, which especially affect the brain and the skeleton (23–28).

Mice lacking filamin A due to the spontaneous occurrence of a stop codon in domain 22 (Y2388X) show both, midline skeletal defects, and cardiac malformations (29). Similarly, the ablation of exons 41–48 of the FLNC gene, i.e. the region encoding the C-terminal 5 domains of filamin C, results in neonatal lethality due to severe defects in myogenesis and myotube structure (30). Thus far, none of the described phenotypes could be linked to a specific molecular mechanism.

In this report, we have analyzed the functional consequences of a mutation recently described by us resulting in a deletion of the C-terminal 16 amino acids, which are part of the dimerization region of filamin C (9). This mutation is not only unique because it was the first one to be described in the FLNC gene, but it is also the first mutation in a dimerization domain of all the filamins. We show that the mutation destabilizes the structure of the domain, prevents proper dimerization and results in aggregation of the mutant protein.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The W2710X mutation renders filamin C less stable and enhances aggregate formation
Our previous work using circular dichroism (CD) spectroscopy indicated a disturbance of the secondary structure of the typical ß-sheet conformation of the mutated Ig-like domain 24 of filamin C (9). Interestingly, all mutant constructs could only be purified in significantly lower amounts, and at the same time more of the purified protein precipitated upon dialysis. This was further analyzed by measuring the thermodynamic stability using far UV CD spectroscopy. The optimal wavelength for performing this experiment, i.e. the wavelength that gives the biggest difference in absorption between the native and the unfolded protein, was determined to be 220 nm (not shown). For the subsequent thermal denaturation experiments, the sample was heated in the cuvette and data were collected as described in Materials and Methods. The observed reduction in signal amplitude correlated to the increase in temperature and the concomitant loss of secondary structure and the transition to random-coil. This heat-dependent denaturation was irreversible, since aggregated protein was evident after cooling. As a measure for thermal stability, therefore, only melting points could be used. The results of these measurements unequivocally demonstrated a strongly reduced stability of the mutated dimerization domain in comparison to the wild type protein (Fig. 1). Thus, the melting point of the mutant single Ig-like domain 24 was ~20°C lower than the one of the wild type counterpart, implying that the loss of secondary structure causes the reduced stability of the mutated domain. Using a construct comprising domains 23 and 24, this difference was somewhat less pronounced, supporting our previous observation that domain 23 and/or the hinge region between both domains stabilize the formed filamin dimers (20).


Figure 1
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  		Figure 1. CD-spectroscopy of wild type and mutant filamin constructs. Bacterially expressed wild type and mutant filamin (FLN) C domains 24 and 23–24 were thermally denatured and analyzed by CD-spectroscopy at a wavelength of 220 nm. Signals were recorded over a temperature range from 10 to 90°C (abscissa). The ordinates give the ellipticity ({Theta}) and the dotted lines indicate melting points (Tm). Note that the mutant constructs (indicated by the frayed symbols) denature at significantly lower temperatures, indicating a decreased stability caused by the W2710X mutation.

 
Improperly folded and instable proteins are often more prone to degradation by proteolytic enzymes. The fact that it was more difficult to obtain sufficient amounts of purified mutant protein may already be taken as an indication for reduced stability. In order to prove this assumption, corresponding wild type and mutant protein fragments comprising domains 23–24 were treated with the protease thermolysin, an enzyme that preferentially cleaves proteins prior to leucine and phenylalanine residues. Gel electrophoretic analysis of the resulting digests revealed that the mutant protein was completely digested already after an incubation time of 30 min (Fig. 2). Thus, both the altered spectroscopic properties and the proteolytic digest indicate that the described mutation renders filamin C less stable in solution.


Figure 2
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  		Figure 2. Protein stability analyzed by thermolysin digestion. Wild type (FLNC d23–24) and mutant (FLNC d23–24mut) filamin C fragments were treated with the protease thermolysin for 1 to 60 min, as indicated above each lane. Samples were analyzed by polyacrylamide gel electrophoresis. Note that the mutant protein was already completely digested after an incubation time of 30 min while a significant portion of the wild type variant was still intact after 60 min of incubation, indicating less stable folding of the mutant protein.

 
The formation of filamin dimers is disturbed by the mutation
Since we previously mapped the dimer formation of filamins to their C-terminal Ig-like domains (20) and the W2710X mutation truncates precisely this region, we compared the dimer formation capability of wild type and mutant dimerization domain 24, and found that the mutant dimerization domain forms dimers very inefficiently when compared with the wild type domain. Instead, the major part of the polypeptides was detected in large aggregates (9). We have now extended this work and investigated whether wild type filamin was able to form hetero-oligomers with mutant filamin and whether the observed filamin aggregates contained both wild type and mutant filamins. For these assays, the two filamin C domains 23–24 (FLNC d23–24) variants were expressed as fusion proteins with two distinct immunotags. The purified proteins were mixed, allowed to dimerize and cross-linked. Interestingly, the mutant filamin C was almost exclusively detected in high molecular mass aggregates, whereas the wild type variant was mainly found in dimers (Fig. 3), indicating that the aggregation of mutant filamin does not disturb the dimerization of wild type filamin.


Figure 3
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  		Figure 3. Chemical cross-linking experiments using wild type (d23–24) and mutant (d23–24mut) filamin constructs. Assays were performed in the absence (–) or presence (+) of EGS in PBS or cross-linking (CL) buffer and reaction mixtures were subsequently analyzed by SDS–PAGE (A) and Western blotting (B). Without the cross-linker EGS, only monomers (m) of the constructs were detected. In the presence of EGS, the wild type variant of the construct was mainly found in the dimer form (d), while the mutant construct was detected in complexes with higher molecular mass representing trimers (t) and aggregated (a) oligomers (A). An identical gel was transferred to nitrocellulose, and the formation of dimers, trimers and bigger aggregates was confirmed by using specific antibodies against the EEF-immunotag of the constructs (B). When differentially tagged wild type and mutant constructs were mixed in the assays and detected individually (det.) after cross-linking, we found that the wild type construct was not included in the aggregates of mutant filamin (C). (D) and (E) show analytical gel chromatography that was performed to analyze the molecular mass of proteins in the native state. Samples were applied to a Superdex 75 HR 10/30 column, and the retention times were related to standard proteins to estimate the molecular mass of proteins and protein complexes. Dotted lines give the chromatograms of the mutant proteins, and the full lines indicate the migration of wild type constructs. Note, the greatly decreased retention times for the mutant proteins.

 
Since chemical cross-linking is a more ‘static’ method that can hardly detect dynamic changes, we also assessed dimer formation under more dynamic conditions by performing analytical gel chromatography. Retention times for the wild type constructs d24 and d23–24 fitted well to the expected values for dimers of these polypeptides. In contrast, the mutant constructs were exclusively recovered in a single, broad peak with a maximum corresponding to a molecular mass indicative of oligomer (at least octamers up to 13mers) formation (Fig. 3D and E). These results clearly show a distinct molecular behavior of wild type and mutant filamins.

Protein binding properties of the filamin C carboxy-terminus are not altered by the mutation
The function of the filamins relies to a large degree on their ability to dimerize and on the property to bind and bundle F-actin. The effect of the mutation on these properties of filamin C was investigated by cosedimentation assays using wild type or mutant filamin C, respectively, together with filamentous actin. Since it is impossible to express molecules with molecular masses as high as that of filamin in bacteria, constructs were designed that would allow the expression of smaller protein variants (mini-filamins), in which the ABD of filamin C (amino acids 1–268) was fused N-terminally to the wild type (amino acids 2497–2725) or mutant (amino acids 2497–2709) domains 23–24. Both constructs were expressed as GST fusion proteins to enable purification. The GST part of the fusion protein was subsequently removed by proteolysis, and the purified mini-filamins were used in high speed (100 000 g, Fig. 4A) and low speed (10 000 g, Fig. 4B) cosedimentation assays. Under both conditions, both the mutant and the wild type constructs were found to cosediment with actin filaments. Binding curves could not significantly discriminate between both protein variants, indicating, as expected, that actin binding is not grossly altered by this filamin mutation.


Figure 4
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  		Figure 4. Functional analysis of actin and sarcoglycan binding. Actin co-sedimentation assays were performed with mini-filamins (‘F’ in (A) and (B)) in which the ABD of filamin C was linked to wild type or mutant domains 23–24. High-speed sedimentation assays (A) showed that both variants co-sedimented identically and thus were able to bind F-actin (‘A’ in (A) and (B)). Note increased proteolysis in the supernatant of the reaction with the mutant variant. In low speed co-sedimentation assays (B) both variants of the mini-filamin were found in the pellet, indicating that the mutant mini-filamin has not lost its ability to cross-link actin filaments. Note increased proteolysis in the supernatant of the reactions with the mutant variant. Control lanes (ctr) assured that F-actin was only found in the supernatant and did not sediment under these conditions. To analyze the ability of the mutant and wt filamin C variants to bind sarcoglycans ({gamma}-SG and {delta}-SG, respectively) dot-blot overlays were performed (C). T7-tagged wild type (d23–24) or mutant (d23–24mut) filamin C was spotted onto a nitrocellulose filter together with BSA as a control (immob), and the filter was overlaid with bacterially expressed and purified EEF-tagged cytoplasmic tails of both sarcoglycans. Binding of the protein fragments was detected using an antibody to the EEF-tag. Note that the sarcoglycans bind both filamin variants but not BSA.

 
Representative for many protein interactions in the C-terminal portion of filamin, we chose to investigate whether the mutation had any effect on the capacity of filamin C to interact with the transmembrane proteins {gamma}- and {delta}-sarcoglycan. This was investigated using dot-blot overlay experiments. Equal quantities of native wild type and mutant filamin C d23–24 were spotted onto nitrocellulose membranes together with BSA as a control, and the membranes were overlaid with the recombinantly expressed cytoplasmic tails of both sarcoglycans that carried an EEF-immunotag. These experiments indicated that the two sarcoglycans bound to both the wild type and the mutant filamin C variants, but not to BSA (Fig. 4C). These results demonstrate that mutant filamin C is still able to bind the sarcoglycans in a comparable way, indicating that the last 16 amino acids of filamin C are not involved in the binding to these transmembrane proteins.

The formation of filamin aggregates observed in diseased muscle fibers is reflected in transient transfections
To analyze whether the mutation would directly result in protein aggregation in living cells, wild type and mutant filamin C constructs were transiently expressed in PtK2 cells. Although these cells are of non-muscle origin, they are more readily transfectable than muscle cells and exhibit an elaborate actin cytoskeleton. This makes them an excellent tool to study properties of transiently expressed actin-binding proteins, with the disadvantage of the lack of muscle-specific proteins that might influence particular properties of filamin C. The full-length filamin C cDNA we cloned did not lead to significant expression levels. In order to increase the transfection efficiency, we designed smaller constructs which would contain all known filamin C binding sites known to date. Thus, the ABD of filamin C was fused to domains 15–24. For the specific detection of the transfected filamin constructs and to discriminate them from the intrinsic protein, they were provided with a myc-tag. Subsequent to the transfection, the cells were fixed and co-stained with a myc-tag antibody to detect the mini-filamins and an antibody specific for {alpha}-actinin to visualize the actin cytoskeleton. The wild type filamin construct was always found to associate with the stress fibers of the cells in a punctate fashion, where it colocalized with {alpha}-actinin (Fig. 5B). Transfected mutant filamin constructs were largely revealed in a similar pattern, but in addition most of the transfected cells displayed brightly fluorescent aggregates of the transiently expressed protein (Fig. 5A). Interestingly, and in agreement with the aggregates in the muscles of the patients suffering from filaminopathy, {alpha}-actinin was excluded from these aggregates (Fig. 5A).


Figure 5
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  		Figure 5. Transient transfections of PtK2 cells with myc-tagged mutant (A) and wild type (B) variants of mini-filamins comprising the ABD of filamin C fused to the immunoglobulin domains 15–24. Transfected cells were double-stained with an antibody specific for the myc-tag (red) and an antibody recognizing {alpha}-actinin (green). The right panel shows the merged pictures. Note that both constructs are efficiently targeted to the dense bodies of the stress fibers where they colocalize with {alpha}-actinin (arrows). Only the mutant construct is also found in cytoplasmic aggregates that are devoid of {alpha}-actinin (arrowheads).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
One of the hallmarks of the pathology of MFM is the finding that the myofibrillar Z-disc is the earliest site of pathological changes. This is followed by gross disorganization of fiber architecture and the formation of large protein aggregates (1,2). Genetic analyses have revealed mutations in several Z-disc-associated protein-encoding genes as the cause of this group of neuromuscular diseases: desmin (3), {alpha}B-crystallin (7,8), myotilin (5), ZASP (6) and filamin C (9), all leading to similar structural features. The pathomechanisms that induce the aggregation of Z-disc and sarcolemma-related proteins is largely unknown. Whereas it was shown that mutant desmin variants disturb intermediate filament assembly in vitro as well as in vivo (10–13), resulting in aggregation of the mutant protein together with wild type desmin and other proteins, our understanding of the alterations of the distinct biochemical and cellular properties of the other mutant proteins has remained unclear.

We have recently reported an MFM-associated mutation in the FLNC gene that causes a truncation of the C-terminal 16 amino acids of the filamin C protein (9). Since the atomic structure of this domain was unraveled (31), we can predict that the mutation removes the last ß-strand from the terminal Ig-like domain (Fig. 6). In the wild type protein, this truncated ß-strand directly interacts with ß-strand A, which is important for the coherence of the two ß-sheets that comprise an Ig-like domain. Consequently, this would render the mutant protein less stable. Our initial CD-spectroscopy had indeed indicated that the mutant domain 24 exhibits a less stable secondary structure, but obviously some tertiary structure is retained (9). Thermal denaturation experiments have now clarified how this loss of secondary structure translates into distinct molecular properties of the mutant domain 24. While the wild type domain was found to be denatured at a temperature of 77.2°C, the melting point of the mutant domain was ~58.2°C. This highly reduced stability against thermal denaturation highlights the reduced structural stability of the mutant protein and will result in increased exposure of hydrophobic residues. One consequence may be that such regions are more prone to proteolytic cleavage, and in line with this, the mutant domain is clearly more susceptible to proteolysis by the protease thermolysin. Therefore, it is an interesting possibility that in the muscles of filaminopathy patients, the incorrect folding of the dimerization domain not only leads to aggregation, but might also render the protein more prone to proteolysis. In this context, it is important to note that filamin C is a substrate of the proteases calpain 1 and 3, which cleave filamin C in the hinge region between domains 23 and 24 (32,33). However, due to the lack of an antibody that specifically recognizes Ig-domain 24 of filamin C, it is presently not clear whether increased proteolysis also occurs in vivo.


Figure 6
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  		Figure 6. Sketch illustrating the effect of the mutation. Topology of filamin C domain 24 as derived from the crystal structure of the domain (31) aligned to the amino acid sequence (A) and in a two-dimensional view (B). ß-strands C and D form the interface between dimerized domains. Strand G directly interacts with strand A, which is important for the coherence of the two ß-sheets that comprise an Ig-like domain. The position of the mutation is marked with an asterisk.

 
Altered molecular characteristics of the mutant filamin C are very likely to have a strong impact on the organization of its many ligands. Indeed, within the muscles of the filaminopathy patients, we find large aggregates that apart from desmin (a hallmark for the protein aggregates found in MFM) also contain the filamin ligands myotilin, {gamma}- and {delta}-sarcoglycans (9) and Xin (Kley et al., manuscript in preparation). This finding is remarkable, because in normal adult skeletal muscle, the localizations of Xin and the sarcoglycans are highly restricted to the myotendinous junctions and the sarcolemma, respectively (34–36).

The described altered biochemical properties of the mutant filamin C protein would, in theory, allow for the following scenario: the less stable secondary structure of domain 24 might result in weaker dimer formation and thus destabilize actin-based structures. Alternatively, the mutant protein could directly form aggregates, leading to almost unaltered actin meshwork geometries based on the intact protein. Our cross-linking experiments clearly support the second possibility by demonstrating a surprising molecular self-sorting that separates a wild type from a mutant protein pool (Fig. 3C). We assume that the same situation occurs in vivo, which would mean that the aggregates that are found in the patient muscle cells at least initially contain solely the mutated protein. The presently available antibodies do, unfortunately, not allow addressing this question directly. Filamin- and Z-disc-associated proteins are subsequently recruited into these aggregates, thereby gradually destabilizing tissue homeostasis. This may at least in part explain why the onset of this disease is relatively late. In line with this, transfected mutant filamin constructs were revealed both along stress fibers (like the wild type protein) and in aggregates, without causing obvious adverse effects in these cells (Fig. 5).

The aggregates of the transfected filamin constructs share another important property with those of the patients, inasmuch as they contain only a very specific, limited number of proteins and not ‘any’ Z-disc-associated protein. For instance, the filamin ligands xin (35), myotilin (37) as well as {gamma}- and {delta}-sarcoglycan (38) are recruited into aggregates, whereas the two major Z-disc proteins, {alpha}-actinin and titin, are not [(9) and Kley et al., manuscript in preparation]. The latter may even shed some doubt on the physiological relevance of the interaction of filamin with titin that was reported recently (39). These findings also underline the fact that filamin is a dual compartment protein that is found both in myofibrils and in the sarcolemmal dystrophin-glycoprotein complex (37,38,40). As a result, the connection of the myofibrillar cytoskeleton to the extracellular matrix may be weakened and dystrophin-associated signaling might be affected.

In summary, our biochemical and cellular data provide a first mechanistic explanation for filaminopathy and evidence to suggest a novel pathogenic process in muscle disorders with abnormal protein aggregation. Introduction of this filamin C mutation into mice may therefore provide an interesting animal model for MFM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cloning and mutagenesis
For expression studies in eukaryotic cells, a partial filamin C cDNA was cloned into the pEGFP C2 vector (Clontech): the portion encoding the ABD (amino acids 1-268) was combined with the fragment encoding wild type or mutant Ig-like domains 15 to 24 (amino acids 1631–2725 and 1631–2709, respectively). Both sequences, and sequences mentioned below are according to Entrez Acc. No Q14315 [GenBank] (NCBI). To avoid tag-induced alterations, this ‘mini-filamin’ was expressed with the short myc-tag at its amino terminus. The integrity of the constructs was verified by DNA sequencing (Agowa, Berlin, Germany).

Protein expression and purification
Protein expression was performed essentially as described previously (20). Truncated cDNA fragments were cloned either into the prokaryotic expression vector pET23-EEF (resulting in fusion proteins carrying a C-terminal His6-tag and an EEF-immunotag) (37), or into pET23-T7 (resulting in fusion proteins carrying an N-terminal T7-tag and a C-terminal His6-tag) (41), or into pGEX-6P1 (enabling expression of GST-fusion proteins; Amersham).

His-tagged proteins were expressed in E. coli BL21-CodonPlus(DE3)-RIL (Novagen) and purified using the QiaExpress kit as described by the manufacturer (Qiagen). GST-tagged proteins were expressed in E. coli as described by the manufacturer (Amersham). For purification, the recombinant protein was bound to Glutathione-Uniflow Resin columns (Clontech). After washing, GST was cleaved from the proteins using PreScission Protease (Amersham), and purified according to the recommendations of the manufacturer.

{gamma}- and {delta}-sarcoglycan fragments encoding the N-terminal 39 and 38 amino acids, respectively, were cloned in pET23-EEF and expressed as described earlier.

Protein concentrations were determined by measuring the specific absorbances at 280 nm.

Protein chemical methods
Skeletal muscle actin was purified from chicken breast muscle acetone powder and co-sedimentation assays were performed under the conditions described previously (42).

Secondary structure of expressed proteins was analyzed by CD-spectroscopy essentially under the conditions described (20). Ten scans were averaged and the recorded ellipticity was plotted against the wavelength.

Chemical cross-linking of recombinant mutant and wild type filamin fragments was performed as described (20). For heterodimerization experiments, both fragments were incubated separately for 30 min at 0°C in a buffer containing 600 mM NaCl to assure that filamins are in monomer state, and equal quantities of mutant and wild type filamin d23–24 carrying a T7- or an EEF-tag, respectively, were mixed and incubated for 5 min at 37°C. Subsequently, 1.3 mM ethylene glycol bis(succinimidylsuccinate) (EGS) was added and the mixture was incubated for a further 20 min at 37°C. Analysis was carried out by SDS–PAGE and Western blotting.

To investigate the susceptibility of mutant and wild type filamin constructs to proteolytic digestion, selected fragments were incubated with the endopeptidase thermolysin. Ten microns purified protein solubilized in 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0, were incubated with 10 µg/ml thermolysin (Sigma) at 37°C. After different incubation times, the reaction was stopped by addition of 0.2 volumes of 5 x SDS sample buffer, and the samples were analyzed by SDS–PAGE.

Analytical gel filtration was used to analyze the molecular mass of recombinant proteins in the native state. A Superdex 75 HR 10/30 column (Pharmacia) was equilibrated with running buffer (50 mM Na-phosphate, 300 mM NaCl, pH 7.4), and 100 µl of the protein samples (concentration 100 µM), dialyzed overnight against running buffer, were applied to the column. Proteins were separated at a flow rate of 0.8 ml/min (ÄKTA FPLC, Amersham Biosciences). The molecular mass of recombinant filamin fragments was determined using a calibration curve established with standard proteins (BSA, chymotrypsin, ovalbumin, RNase A).

Dot blot protein binding assays
Purified recombinant proteins (and BSA as a control) were spotted on nitrocellulose membranes (BA-85, Schleicher and Schüll). After air drying, the strips were blocked with 4% (w/v) low fat milk powder in TBST. Individual strips were overlaid with the respective protein in blocking solution for 90 min at room temperature, or overnight at 4°C. After three washes with TBST, bound protein was immunodetected with antibodies specific for the respective immunotag (see below).

Antibodies
Since recombinant proteins were either tagged N-terminally with a peptide of the bacteriophage T7 major capsid protein (T7-tag) or C-terminally with the amino acids EEF, fusion proteins could be detected either by a mAb specific for the T7 tag (Novagen) or by the rat mAb YL1/2 (43). The latter antibody was a kind gift of Dr. J. Wehland, Braunschweig, Germany, respectively. A mAb specific for the myc-tag (Clone 9E10, a kind gift of Dr Paula Salmikangas, Helsinki, Finland) was used to detect mini-filamins in transfected cells. The mAb BM75.2 (Sigma) was used to stain {alpha}-actinin.

Immunodetection
Nitrocellulose membranes were blocked and incubated using standard procedures. Peroxidase-conjugated secondary antibodies were purchased from Jackson Immuno Research Laboratories. Conjugated enzymes were detected by enhanced chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and Kodak XAR-351 film.

Transient transfection of PtK2 cells
PtK2 cells (kidney cells from Potorous tridactylis; ATCC Number CCL-56) were cultured and transfected as described before (42). Forty-eight to seventy-two hours post transfection, cells were fixed and double-stained with an antibody specific for the myc-tag of the recombinant proteins, and an antibody specific for {alpha}-actinin to identify stress fibers using standard procedures (40).


   ACKNOWLEDGEMENTS
 
We thank A. Freiberg and Dr R. Seckler (University of Potsdam) for help with CD spectra and helpful discussions. D.O.F., PvdV, M.V. and A.H. are members of the German Network on Muscular Dystrophies (MD-NET), supported by the Bundesministerium für Forschung und Bildung (BMBF), (No. 01GM0601). M.V. and R.K. are supported by WiMed, Kliniken Bergmannsheil Bochum and FoRUM, Ruhr-University Bochum. A.H. was supported by the MeDDrive program of the Medical Faculty, Technical University Dresden, Germany.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Centre for Molecular Neurobiology (ZMNH), University of Hamburg, 20251 Hamburg, Germany. Back

{ddagger} Present address: Interdisciplinary Centre for Clinical Research (IZKF), Cell Migration Group, 04103 Leipzig, Germany. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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R. A. Kley, Y. Hellenbroich, P. F. M. van der Ven, D. O. Furst, A. Huebner, V. Bruchertseifer, S. A. Peters, C. M. Heyer, J. Kirschner, R. Schroder, et al.
Clinical and morphological phenotype of the filamin myopathy: a study of 31 German patients
Brain, December 1, 2007; 130(12): 3250 - 3264.
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