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Desmin mutations in the terminal consensus motif prevent synemin-desmin heteropolymer filament assembly
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Research Article
Desmin mutations in the terminal consensus motif prevent synemin-desmin heteropolymer filament assembly Oussama Chourbagi a , Francine Bruston a , Marianna Carinci a , Zhigang Xue b , Patrick Vicart a , Denise Paulin b , Onnik Agbulut a,⁎ a
University Paris Diderot-Paris 7/CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, F-75013, Paris, France b UPMC-Paris 6, UR-4, Laboratoire de Génétique et Physiopathologie des Tissus Musculaires, F-75005, Paris, France
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Disorganization of the desmin network is associated with cardiac and skeletal myopathies characterized
Received 6 August 2010
by accumulation of desmin-containing aggregates in the cells. Multiple associations of intermediate
Revised version received
filament proteins form a network to increase mechanical and functional stability. Synemin is a desmin-
7 January 2011
associated type VI intermediate filament protein. Neither its impact on desmin network nor how it
Accepted 10 January 2011
integrates into desmin filament is yet elucidated. To gain more insight into the molecular basis of these
Available online 22 January 2011
processes, we coexpressed synemin with different desmin mutants in ex vivo models. The screening of fourteen desmin mutants showed that synemin with desmin mutants revealed two behaviors. Firstly,
Keywords:
synemin was co-localized in desmin aggregates and its coexpression decreased the number of cells
Intermediate filaments
containing aggregates. Secondly, synemin was excluded from the aggregates, then synemin had no
Desmuslin
effect on desmin network organization. Among fourteen desmin mutants, there were only three
Salt-bridges
mutants, p.E401K, p.R406W and p.E413K, in which synemin was not found in aggregates. This behavior
Desminopathy
was correlated to the abnormal salt-bridges of desmin-dimer as seen in silico constructs. Moreover, desmin constructs in silico and published results in literature have predicted that the salt-bridges absence in the desmin filament building prevent longitudinal annealing and/or radial compaction. These results suggest that the state of desmin-filament assembly is crucial for synemin anchorage and consequently might involve mechanical and functional stability of the cytoskeletal network. © 2011 Elsevier Inc. All rights reserved.
Introduction Intermediate filaments (IFs) are primary determinants of the cell's architecture and plasticity [1]. They are defined as part of a large gene family that encodes polymerizing proteins into individual 10 nm filaments [2]. The 52 kDa protein desmin is the constitutive
subunit of the type III intermediate filaments in all muscle tissues. Desmin, which is essential for tensile strength and muscle integrity, forms a three-dimensional scaffold around the myofibril Z-disc and connects the entire contractile apparatus to the subsarcolemmal cytoskeleton, the nuclei and other cytoplasmic organelles. There is also a significant amount of desmin in
⁎ Corresponding author at: University Paris Diderot-Paris 7, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, 4, rue Marie Andrée Lagroua Weill-Hallé (case 7006), 75 205 Paris cedex 13 France. Fax:+33 1 5727 7966. E-mail addresses: [email protected] (O. Chourbagi), [email protected] (F. Bruston), [email protected] (M. Carinci), [email protected] (Z. Xue), [email protected] (P. Vicart), [email protected] (D. Paulin), [email protected] (O. Agbulut). Abbreviations: IFs, intermediate filaments; ULFs, unit-length filaments; DMEM, Dulbecco's modified Eagle medium; CMV promoter, cytomegalovirus promoter; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.01.013
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neuromuscular [3,4] and myotendinous junctions [5,6] which allows the anchoring of myofibrils to the junctional sarcolemma. Disorganization of the desmin network is associated with cardiac and skeletal myopathies and a loss of force generation in smooth muscles [7]. In addition, mutations in the desmin gene can lead to desminopathy, a subgroup of the genetically and clinically heterogeneous group of myofibrillar myopathies. Desmin deposits, which are observed in patients suffering from desminopathy, were sometimes associated with abnormal aggregates of other cytoskeletal proteins such as synemin, nestin, plectin and αB-crystallin localized between myofibrils and beneath the sarcolemma [8,9]. The molecular architecture of desmin filaments is not entirely known [10,11]. It seems acquired that the unit-length filaments (ULFs) represent the precursors during filament formation. Each ULF of 16 nm, is comprised of the association of four octamers [12], that simultaneously undergo longitudinal annealing and radial compaction, finally result in a 10 nm diameter filament. Some desmin mutants belonging to the rod domain assemble into seemingly normal IFs as p.E245D [13], while other mutants interfere with the assembly process at distinct stages such as tetramer formation [10,14], ULFs formation [15], filament elongation [16] or IF maturation. As generally known, mutations located in the rod domain were disturbed coiled-coil formation via proline substitution or highly conserved heptad repeat pattern modification or critical intra-helical and inter-helical salt-bridge modifications [10,15]. As generally undersood, the degree of desmin filament assembly perturbation of variant desmin mutants cannot directly be correlated with the severity of the disease [17]. Synemin is a large type VI IF protein (150–230 kDa) found in all four classes of muscle cells [18–22] presenting a central rod domain flanked by a very short N-terminal head and a long C-terminal tail (Fig. 1). Synemin has been shown to be unable to self-assemble and is necessarily assembled in heteropolymers [23,24]. In muscles,
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synemin is associated with desmin [18]. As generally assumed, multiple associations of IF proteins to form a network increase its mechanical stability and give structural and functional versatility to the IF cytoskeleton [19]. Synemin interacts with type III IFs through the head and rod domains [22], and through its long C-terminal interacts with membrane proteins [25]. Via its interaction with cytoskeletal proteins dystrophin, utrophin and vinculin, synemin is able to link these heteropolymers to adherens-type junctions within striated muscle cells suggesting that it plays an important role in component assembly of the cytoskeleton [5,26,27] and in morphogenesis [28]. Synemin could participate in focal adhesion dynamics and be essential for cell adhesion and migration [29]. The present study was designed first to evaluate the impact of synemin on pathological desmin network organization and second to gain further insights upon molecular mechanisms involved in the process of synemin integration into desmin filaments. Thus, we performed cotransfections of synemin with wild-type desmin or with different desmin mutants in ex vivo models (C2C12, a mouse derived-myoblast; SW13, human adrenocortical carcinoma cells) and examined various cellular and molecular parameters. In conclusion, we suggest that the state of desmin-filament assembly appears crucial for synemin anchorage for the building of the cytoskeletal network.
Material and methods Cloning and site-directed mutagenesis Eukaryotic expression vector pcDNA3 (Invitrogen, Germany) under control of CMV promoter containing the cDNA of the human synemin H isoform [23] and the full-length human desmin cDNA with the Myc tag integrated in 5' were used. Mutations were
Fig. 1 – Desmin (A) and synemin (B) tripartite structure and location of fourteen desmin mutants examined in this study. Tripartite structure comprising a central α-helical rod domain flanked by non-helical head and tail domains. The central rod domain, formed by four α-helical segments (1A, 1B, 2A, 2B) separated by three short polypeptide linkers (L1, L12, L2). Desmin central α-helical rod domain, maintains a seven residue repeat pattern (heptad) that guides two polypeptides to form a homopolymeric coiled-coil dimer, the elementary unit of the desmin network. The heptad repeats were marked as abcdefg. The most conserved motif in the C-terminal part of the 2B segment was shown in insert.
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introduced into plasmid vectors by site-directed mutagenesis using the Stratagene QuikChange kit (Germany). Sequencing was done by Genoscreen (France). The positive clones obtained were grown overnight in a large volume of sterile LB medium with ampicillin, under agitation at 37 °C. Plasmids were extracted using the Qiagen Maxiprep kit (Qiagen, Germany).
Cell culture Mouse myoblasts, C2C12 (desmin+, vimentin+) and human adrenocortical carcinoma cells, SW13 (desmin-, vimentin-) were grown at 37 °C and 5% CO2 in DMEM (Dulbecco's modified Eagle medium, Invitrogen) supplemented with 20% (for C2C12) or 5% (for SW13) of fetal calf serum (Invitrogen), and penicillin G (50 U/ ml final) and streptomycin (50 ng/g final).
Transient transfection and immunostaining C2C12 and SW13 cells grown on Petri dish plated on coverslips at densities of 103-104/cm2 were transiently transfected using jetPEI (polyplus-transfection kit) according to the manufacturer's protocol (Ozyme). Two days after transfection, cells were processed for immunocytochemistry. Briefly, cells were fixed and permeabilized in methanol-acetone (70:30) for 15 min at –20 °C. After rehydration, specimens were blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS) for 20 min. Cells plated on coverslips were incubated with monoclonal anti-desmin antibody (clone D33, 1:100, Dako) or monoclonal anti-Myc tag antibody (clone 9E10, diluted 1:200, Santa Cruz Biotechnology) with or without the polyclonal rabbit anti-synemin antibody (dilution 1:300, [20]) for 90 min at room temperature. The binding of primary antibodies was detected by incubation for one hour with Alexa Fluor conjugated anti-mouse IgG or anti-rabbit IgG (1:1000). Finally, cells were washed in PBS and mounted with VECTASHIELD® HardFSet™ Mounting Medium with DAPI (4,6-diamidino- 2-phenylindole; Vector Laboratory, USA) for nuclear staining. Images were taken with a confocal microscope (Leica DM2500 TCS SPE).
Immunodetection quantification, cell counts Desmin network was visualized with immunostaining using antidesmin and anti-Myc antibodies and than cotransfected cells with synemin were counted and categorized into two categories; cells containing filamentous network (category 1), cell containing aggregates (category 2). The counting was performed in a blinded manner. All experiments were carried out in three, or more, independent transfections and for each situation at least two hundred transfected cells were counted in blinded manner. It should be noted that in this case SW13 cells are the best model to examine this process since the absence of endogenous IFs network in the cells prevents the adverse effects of this endogenous proteins on synemin-desmin interaction.
GROMOS96 43B1 parameters set, without reaction field, from Swiss-PdbViewer 4.0.1 (http://spdbv.vital-it.ch/download.html).
Results Fourteen desmin mutants that are responsible for desminopathy were used in this study (Fig. 1A). It should be noted that to distinguish endogenous desmin expressed in C2C12 and transfected constructs, a myc-tag was introduced in all desmin constructs. Using immunostaining, desmin mutants were sub-grouped according to their capacity to form a filamentous network and/or aggregates (Table 1, Fig. 2). Among the fourteen desmin mutants, four are able to form filamentous structures (Table 1, Fig. 2B). Three mutants, p.Δ114E, p.A357P and p.L370P, located in the rod domain presented a dominant effect in the endogenous network and only formed big aggregates throughout the cytoplasm (Fig. 2C). The other desmin mutants, p.Q389P, p.D399Y, p.E401K, p.R406W, p.E413K, p.T453I and p.S460I were able to integrate preexisting desmin network and also form desmin containing aggregates concentrated around the nuclei and throughout the cytoplasm (Fig. 2D).
Desmin mutations in the terminal consensus motif prevent synemin localization in the desmin aggregates In order to define the localization and distribution of the synemin with different desmin mutants, we cotransfected synemin with wild-type desmin or foorteen desmin mutants in C2C12 cells and immunostaining was performed. Our results demonstrated that synemin is colocalized with endogenous and Myc tagged wildtype desmin network (Figs. 3A-C). The screening of fourteen desmin mutants shows that synemin is colocalized with all of the desmin mutants which are able to form filamentous structures (Table 1). However, with the mutants that form aggregates two situations were observed (Fig. 3). Firstly, synemin is able to integrate into a preexisting desmin network and is also localized in desmin containing aggregates caused by p.D399Y (Figs. 3D-F), p. Δ114E, p.A357P, p.L370P, p.Q389P, p.T453I and p.S460I (Figs. 3G-I) desmin mutants (Table 1). Secondly, synemin is able to integrate
Table 1 – Mutated desmin organization in C2C12 and correlation with synemin localization. Immunodetection of synemin and Myc-desmin of fourteen desmin mutants highlights the absence of synemin in aggregates from p. E401K, p.R406W and p.E413K mutants. Desmin IF state in C2C12
The crystal structure of human vimentin coil-coiled 2B fragment (1gk6) was extracted from Protein Data Bank (http://www.rcsb. org/pdb/) and used to construct chimeric desmin models (WT and mutants) with PyMOL software (DeLano Scientific LLC). For energy minimization, computations were done in vacuo with the
Synemin-desmin colocalization* in network in aggregates
Filament Aggregate
Molecular model and secondary structure predictions
Mutant
Filament and aggregate
p.T442I, p.K449T, p.I451M, p.V469M p. E114del, p.A357P, p.L370P p.Q389P, p.D399Y, p.T453I, p.S460I p.E401K, p.R406W, p.E413K
+
b
a
+
+
+
+
−
* Presence (+) or absence (−) of synemin in network or aggregates; a, absence of network; b, no aggregates were observed.
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Fig. 2 – Functional analysis of different desmin mutants in C2C12 cells. Immunostaining of Myc-tagged desmin (red fluorescence) 48 hours after transfection of wild-type desmin (A); p.I451M (B); p.A357P (C) and p.R406W (D) desmin mutants. Note filamentous network organization in C2C12 cells when transfected with p.I451M desmin mutant. In contrast, p.R406W and p.A357P desmin mutants forms desmin containing aggregates which are distributed throughout the cytoplasm. Moreover, p.A357P presented a dominant effect in the endogenous network and only formed big aggregates throughout the cytoplasm. Nuclei were stained in blue by DAPI. Bar: 5 μm.
into a preexisting network but is totally excluded from desmin containing aggregates caused by p.E401K (Figs. 3J-L), p.R406W (Figs. 3M-O), p.E413K (Figs. 3P-S) desmin mutants (Table 1). Taken together, the screening of fourteen desmin mutants showed that in three mutants, p.E401K, p.R406W and p.E413K, synemin is totally excluded from the desmin containing aggregates. It should be noted that vimentin which is a one of the abundant IF in C2C12 [30,31] is located with all desmin aggregates provoked by different desmin mutants (data not shown). The difference of synemin's behavior with desmin mutants raises the question of the function of desmin residue or its position in interaction with synemin. The replacement of the arginine (R) positive residue by a tryptophan (W) neutral apolar residue at the position 406 or the replacement of the glutamic acid (E) negative residue by a lysine (K) positive residue at the position 401 abolished E401-R406 inter-helical salt-bridge which is important for the network via monomer/dimer formation or stabilization [10]. In addition, we emphasize that the salt-bridge E401-R406 within the desmin dimer is crucial for synemin-desmin heteropolymer filament assembly. In order to confirm this hypothesis we examined the inter-helical salt-bridge between E401 and R406 residues using in silico modeling and transfection studies.
The salt-bridge E401-R406 within the desmin dimer is crucial for synemin-desmin heteropolymer filament assembly In silico chimeric constructs of wild-type desmin, replacing residue E401 or R406 by other residues that have the same charge or not, have been established by using the energy minimization method.
The atomic distances were calculated between different desmin residues. Fig. 4 shows the distances calculated for different residues located at the position 406 of monomer 2 and 401 of monomer 1 (R406K, W, F; E401K, N, D). The modeling predicts that the inter-helical salt-bridge between E401 residue of monomer 1 and R406 residue of monomer 2 is present in the wild-type desmin dimer with an inter-side chain distance of 2.8 angstrom (Å) (Fig. 4A). The modeling of mutated desmin for R406 residue, shows that ionic bond between 401-406 residues is suppressed when the arginine (R) positive residue is replaced by a tryptophan (W) or a phenylalanine (F), neutral residues (Figs. 4B-C). Interestingly, the replacement of the arginine by a similar charge residue lysine (K), also suppressed ionic bond between the 401406 residues. In spite of a similar charge, the lysine side chain is shorter than the arginine's, which leads to a higher inter-side chain distance (4.8 Å) (Fig. 4D). The modeling of mutated desmin for E401 residue (monomer 1), shows that the replacement of a glutamic acid (E) negative residue by a lysine positive residue or an asparagin (N) neutral residue suppressed the ionic bond between 401-406 (Fig. 4E). In contrast, replacement of the glutamic acid (E) negative residue by a similar charge residue, aspartic acid (D), has a predicted distance of 3.7 Å (Fig. 4F). In order to examine the role of the salt-bridge E401-R406 in the integration process of synemin in desmin network, we carried out site-directed mutagenesis on the amino acids of the highly conserved region 401-EIATYRKLLEGEE-413 replacing the residue E401 by K, N, D or the residue R406 by W, F, K residues. Each of six desmin mutants and synemin were cotransfected in the C2C12 cells
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Fig. 3 – Immunofluorescence detection of synemin and mutated desmin in C2C12 cells. Immunostaining of Myc-tagged desmin (red fluorescence, A, D, G, J, M, P) and synemin (green fluorescence, B, E, H, K, N, R) 48 hours after cotransfection of synemin with wild-type desmin (A-C); synemin with p.D399Y (D-F); p.S460I (G-I); p.E401K (J-L); p.R406W (M-O) and p.E413K (P-S) desmin mutant. Combined red and green fluorescence in images were made. Nuclei were stained in blue by DAPI. Note an extensive filamentous network with perfect desmin-synemin colocalization between wild-type desmin and synemin (C). Ruptures in the filamentous network and aggregates appeared with all desmin mutants (arrows). Note the absence of synemin in aggregates caused by p.E410K, p.R406W and p.E413K desmin mutants (arrow head, K-L, N-O, R-S). Bar: 5 μm.
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Fig. 4 – In silico modeling of inter-helical salt-bridge between the 401 and 406 residues. The crystal structure of coiled coil 2B fragment of human vimentin (1gk6) was downloading from the protein data bank (PDB). Different chimeric desmin dimer mutants were established with PyMOL software using computations with GROMOS96 implementation of Swiss-PDB viewer 4.0.1 for energy minimization. Blue and red residues represent positive and negative net charges, respectively. The residues located at the positions 401 (monomer 1) and 406 (monomer 2) are drawn in a stick representation. Dot line represents the salt-bridges in wild type (A) and p.E401D desmin dimer (F). Distances were expressed as Angstroms.
and the results were summarized in Table 2 and Fig. 5. At the position 406 in desmin, when the arginine (R) positive residue is replaced by a tryptophan (W) or a phenylalanine (F), neutral residues, or a lysine (K) positive residue, it prevents the synemindesmin co-localization in the aggregates (Figs. 5A-C). It should be noted that the modeling in silico predicted that the ionic interaction between 401-406 residues was abolished with these mutations.
The replacement of the E401 negative residue by a positive lysine (K) residue or an asparagin (N) neutral residue also prevents the synemin-desmin colocalization in the aggregates probably due to the loss of charges. By contrast, the replacement of the glutamic acid (E401) negative residue by a similar charge residue, aspartic acid (E401D), shows the presence of synemin in the aggregates formed by E401D (Figs. 5D-F).
Table 2 – Salt-bridge state 401-406 of desmin dimer after various mutations obtained by site-directed mutagenesis. Immunostaining of Myc-desmin and synemin confirm the strong correlation between the presence of salt-bridge and the synemin-desmin colocalization in the aggregates. Atomic distances were done by PyMOL software. Mutation
Monomer 1 E401
Monomer 2 R406
none D N K none K F W
Residue charge and polarity Negative polar Negative polar Neutral polar Positive polar Positive polar Positive polar Neutral apolar Neutral apolar
* Presence (+) or absence (−) of synemin in the aggregates.
Salt-bridge Predicted interaction E401-R406 D401-R406 No No E401-R406 E401-K406 No No
Atomic distance (Å) 2.8 3.7 No No 2.8 4.8 No No
State Existing Preserved No No Existing Corrupted No No
*Synemin localization + + − − + − − −
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Fig. 5 – Immunostaining of synemin and mutated desmin in C2C12 cells. Immunostaining of Myc-tagged desmin (red fluorescence, A, D) or synemin (green fluorescence B, E) in C2C12 cells after cotransfection of synemin with different desmin mutants; p.R406K (A-C) and p. E401D (D-F). Combined red and green fluorescence in images were made. Nuclei were stained in blue by DAPI. Ruptures in the filamentous network and aggregates appeared with all desmin mutants (arrows). Note the absence of synemin in aggregates caused by p. R406K desmin mutant (arrow head, B-C). Note the synemin's presence in the aggregates caused by p.E401D desmin mutant (E). Bar: 5 μm.
Strong correlation between experimental data concerning the synemin-desmin colocalization in the aggregates and in silico predictions on the breaking of the salt-bridge between 401-406 residues, leads to conclude that the ionic bond interaction E401R406 within the desmin dimer is crucial for the synemin-desmin heteropolymer filament assembly.
Concerning E413K, using in silico modeling, we show that intrastrand salt-bridge between K407 and E413 is also broken and the distance between K407-E413 increases from 2.8 to 5.5 Å (Fig. 6). Our results show that the tail is probably maintained by this saltbridge in the wild-type desmin, whereas in the mutant p.E413K, tail dimer is free and consequently takes more space which may
Fig. 6 – In silico modeling of salt-bridges for E413K mutation. The crystal structure of coiled coil 2B fragment of human vimentin (1gk6) was downloading from the protein data bank (PDB). p.E413K mutated desmin dimer was established with PyMOL software using computations with GROMOS96 implementation of Swiss-PDB viewer 4.0.1 for energy minimization. Blue and red residues represent positive and negative net charges, respectively and drawn in a stick representation. Dot line represents the intra-strand salt-bridges in normal (A) and pathologic dimer (B). Distances were expressed as Angstroms.
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accumulate in the next step of filament assembly and subsequently disrupt the radial compaction of ULFs.
Synemin decreases desmin-aggregate formation To investigate the effect of synemin on desmin mutants and particularly to examine if the expression of synemin can improve the desmin network and consequently decrease desmin-aggregate formation in desminopathy, we cotransfected synemin with wild-type desmin or desmin mutants in SW13 cells. The desmin network was visualized with immunostaining and then cotransfected cells were counted and distributed into two categories; 1cells containing filamentous network, 2- cells containing aggregates (Fig. 7). Immunofluorescence microscopy and quantification of transfected cells revealed that when wild-type desmin is expressed into SW13 cells, 75% of transfected cells present a filamentous network and 25% of transfected cells contain irregular bundles of filaments (Fig. 7A). When SW13 cells were cotransfected with desmin and synemin, we found more homogenous labeling of desmin network and less than 10% of transfected cells presented irregularly bundles of filaments suggesting synemin might improve desmin network (Figs. 7DF). Transfection of p.D399Y desmin mutant into SW13 cells revealed that this mutant causes a formation of cytoplasmic aggregates in 85% of transfected cells (category 2) and/or a formation of very short filamentous structures (category 1) (Fig. 7B). Cotransfection of p.D399Y desmin mutant with synemin shows that synemin is co-localized with desmin both in aggregates and in short filamentous structures (Figs. 7G-I). In this situation, desmin aggregates formed by this mutant decrease from 85% to 45% (Fig. 7N). The decrease of number of cell containing desmin aggregates also confirmed with p.Q389P and p. T453I desmin mutants (Fig. 7N). Transfection of p.E401K, p. R406W and p.E413K desmin mutants into SW13 cells shows that these mutants are unable to form a filamentous network, they forms numerous and very small, dot-like aggregates, which are distributed throughout the cytoplasm of 100% transfected cells (category 2, Figs. 7J-L). Interestingly, cotransfection of p.E401K, p. R406W and p.E413K desmin mutants with synemin shows no modifications on the aggregates formed by these mutants (Fig. 7N). Concerning localization of synemin with these mutants, we demonstrated that neither synemin nor desmin mutants is able to form a filamentous network, they form numerous small, dot-like aggregates, which are distributed independently throughout the cytoplasm (Fig. 7M).
Discussion The major finding of this study is to demonstrate that i) synemin integration into desmin network is dependent of desmin filament assembly state, ii) coassembly of synemin with desmin improves desmin network organization and consequently decrease desmin containing aggregates. Concerning the synemin-desmin association, the screening of fourteen desmin mutants shows that desmin-associated protein synemin is perfectly colocalized with all of the desmin mutants which are able to form filamentous structures de novo, but with the mutants that have no capacity to form filamentous structures on their own two behaviors were observed. First, synemin is able
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to integrate into a preexisting network and is also localized in desmin containing aggregates (p.Δ114E, p.A357P, p.L370P, p. Q389P, p.D399Y, p.T453I and p.S460I). Second, synemin is able to integrate into a preexisting network but is totally excluded from desmin containing aggregates (p.E401K, p.R406W, p. E413K). Functional analysis of these three desmin mutants shows that they are unable to form filamentous structures on their own. They form only small aggregates throughout the cytoplasm in SW13 cells. Interestingly, they retain the ability to integrate into preexisting intermediate filament networks in C2C12 cells when the ratio of wild-type desmin is superior to the mutant one. When the amount of desmin mutant becomes superior to wild-type one, desmin filament assembly is perturbed and desmin containing aggregates become to form. In this case, synemin is totally excluded from the desmin containing aggregates. As the varied behavior of synemin in desmin network integration process is dependent on desmin mutants and endogenous versus mutant desmin ratio, we could suggest that synemin fixation site was generated during filament assembly and consequently desmin filament assembly state appears crucial for synemin anchorage. Regarding the molecular structure of these desmin mutants in which synemin is excluded from the aggregates, two mutants were identified in the terminal consensus motif 405-YRKLLEGEE413. This motif, which highly conserved in all types of intermediate filaments in a wide range of species, is crucial for the formation of dimeric complexes and also for the control of filament width during assembly process via intra- and interhelical salt-bridges [32]. Interestingly, this highly conserved terminal consensus motif is not only involved in the formation of the coiled-coil structure but also in its loosening. From the leucine at the position 409, the coiled-coil structure loosens, so that the helices gradually separate and eventually bend away from each other and eventually becoming disordered [33–35]. For these reasons, the mutations located in this region have a drastic effect on network structure organization. Bar et al. studies show that p.R406W and p.E413K desmin mutants exhibit disturbed longitudinal annealing and radial compaction during desmin filament assembly process [36]. Moreover, p.E401K and p.R406W desmin mutants alter the entire dimer interaction via interhelical salt-bridge corruption which causes formation of unstable ULF-like particles, whose longitudinal annealing is slowed drastically, so that eventually small, granular dead-end structures ensue [17]. Dimerization between two α-helices to form coiledcoil is well documented notably for coiled-coil structures and their ionic interactions. All IF proteins exhibit a similar interhelical salt-bridge between glutamic acid (E) and arginine (R) residues, located into the highly conserved amino acid motif of helix 2B (YRKLLEGEE) and this, since in the nuclear lamins [37] via keratins and vimentin [32]. Furthermore, there are disease mutations in all cytoplasmic and nuclear IF proteins that involve this inter-helical salt-bridge [1]. Statistical analysis of the occurrence of this ionic interaction in coiled-coils is characterized for parallel dimeric coiled-coils and the atomic structure is of type i to i + 5 ionic interaction [38]. Similar analysis of intrahelical ionic interactions in α-helices and coiled-coils has been performed confirming that the configurations, which have simultaneously a large probability to form the ionic interaction and a frequent occurrence, are those, which have the most stabilizing effect as 4RE, 3ER and 4ER interactions [39].
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p.E401K and p.R406W desmin mutants also disturb heptads repeat pattern which is important for the formation of coiled-coil structure [40]. It should be noted that the highly conserved inter-helical salt-bridge E401-R406 is formed between a glutamic acid in a g position on one chain and an arginine in an e position on the second chain. Electrostatic interactions arising primarily from residues in positions e and g are known to play a crucial role in specifying the relative alignment and orientation of the two chains within the coiled coil [41]. Pruszczyk et al. suggested that p.E413K mutation also alters electrostatic interactions which are important for the proper dimer–dimer interactions during the assembly process [33]. The authors indicate that p.E413K
mutations generate a new abnormally intra-helical salt-bridge between residue E410 and the mutant K413 which probably increase stiffness and decrease the flexibility of the coiled-coil structure. Moreover, using in silico modeling, we show that intrastrand salt-bridge between K407 and E413 is also broken and the distance between K407-E413 increases from 2.8 to 5.5 Å due to charge repulsion. Our results show that the tail is probably maintained by this salt-bridge in the wild-type desmin, whereas in the mutant p.E413K, tail dimer is free and consequently takes more space which may accumulate in the next step of filament assembly and subsequently disrupt the radial compaction of ULFs.
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Apparently, any change in this highly conserved region can influence filament assembly by disrupting interactions within the dimer and/or above dimeric level. The salt-bridge between 401 and 406 residues is known for its importance in desmin homodimer filament assembly [10]. In wild type desmin state, the negative charged glutamic acid residue at the 401 position of one monomer forms an inter-helical salt-bridge with positive charged arginine at the 406 position in the other monomer. Our results obtained in silico modeling predict that the inter-helical salt-bridge between E401-R406 residues is present in the wildtype desmin dimer with an inter-side chain distance of 2.8 Å. In addition, salt-bridge is also present when glutamic acid (E) at the 401 position is replaced by similarly charged residue aspartic acid (D). By contrast, when we replaced arginine (R) at the 406 position by similarly charged residue lysine (K) is suppressed ionic bond between 401-406 residues. In spite of a similar charge, lysine side chain is shorter than arginine which leads to a higher an inter-side chain distance (4.8 Å). Replacement of glutamic acid (E) at the 401 position or arginine (R) at the 406 position by neutral or opposite charge residue inter-helical salt-bridge is suppressed. Strong correlation between in silico predicts on the breaking of saltbridge between 401-406 residues and experimental data concerning the absence of synemin-desmin association in the aggregates, lead to formulate that the salt-bridge interaction E401R406 within the desmin dimer is crucial for synemin-desmin heteropolymer filament assembly. These findings highlight the crucial role of the inter-helical salt-bridge since it seems to be enough that the salt-bridge rupture prevents the radial compaction and longitudinal annealing and consequently synemindesmin heteropolymer ULF assembly. Moreover, our transfection studies demonstrated that when synemin is localized in the desmin containing aggregates (p.Q389P, p.D399Y, p.T453I) synemin expression drastically decreases cell number containing aggregates, but when synemin is excluded from the aggregates (p.E401K, p.R406W, p.E413K) in this case coexpression of synemin has no effect on desmin network organization. Interestingly, synemin has been shown to be incapable of self-assembly and is necessarily assembled in heteropolymers. For this reason, in muscle, synemin should be associated with desmin to form a filamentous network. But our results demonstrate that coexpression of synemin with wild-type desmin
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into SW13 cells reveals more homogenous labeling of desmin network. It should be noted that synemin and desmin are normally coexpressed in muscle, it is likely that this has physiological significance. Synemin probably provides some required interactions with other structures. Synemin is a desmin-associated type VI IF protein, it interacts with type III IFs with the head and rod domains [21], and through its long C-terminal, interacts with membrane proteins [25] suggesting that synemin plays an important role in component assembly of the cytoskeleton [5,26,27] and in morphogenesis [28]. Coassembly either with vimentin and paranemin also allowed homogenous and extended desmin network [42]. Note that vimentin is able to form an extended network on its own so this was an expected result. However, as paranemin is unable to form a network on its own, these results support the hypothesis that the large intermediate filament proteins provide some required interactions with other structures, strengthening the IF network. Moreover, the presence of a K8/K18 keratin network also appeared to allow extended desmin IFs despite that keratins and desmin do not form copolymers but assemble into distinct networks. Taken together, our results highlight that the state of desmin filament assembly appears crucial for synemin anchorage and consequently might involve mechanical and functional stability of cytoskeletal network. Moreover, in this study we underline the crucial role of the inter-helical and intra-strand salt-bridges located at the terminal consensus motif of desmin since it seems to be enough that the salt-bridge rupture prevents the radial compaction and longitudinal annealing and consequently synemin-desmin heteropolymer ULF assembly.
Acknowledgments This work was supported by the “Association Française contre les Myopathies” (AFM), contract n° 14040, the University Paris Diderot-Paris7, the “Agence Nationale de la Recherche” (ANR), contract n° 06-MRAR-039-01. O.C. was supported by fellowships from the Ministère de la Recherche et Technologie” (MRT) and the AFM. We would like to thank Dr. Bertrand Goudeau for gift of vector constructs of human desmin, Dr. Zhenlin Li, Dr. Pierre Joanne and Valentin Wucher for his helpful advices.
Fig. 7 – Immunofluorescence detection of desmin (A-C) and desmin-synemin (D-M) in SW13 cells and quantification of desmin containing aggregates in various condition (N). Immunostaining of Myc-tagged desmin (red fluorescence) 48 hours after transfection of wild-type desmin (A); p.D399Y (B) and p.R406W (C) desmin mutants in SW13 cells. Immunostaining of desmin (red fluorescence, D, G, J) and synemin (green fluorescence E, H, K) after cotransfection of synemin with wild-type desmin (D-F); p.D399Y (G-I) and p.R406W (J-M) desmin mutants. Combined red and green fluorescence in images were made. Nuclei were stained in blue by DAPI. Note more homogenous labeling of desmin network when desmin is coassembled with synemin (arrow, A, D-F). Note synemin colocalization with D399Y aggregates, while with p.R406W mutant, neither synemin nor desmin mutants is able to form a filamentous network, they form numerous small, dot-like aggregates, which are distributed independently throughout the cytoplasm (J-M). Quantification of desmin containing aggregates in SW13 cells 48 hours after transfection of different desmin mutants with or without synemin (N). Myc-tagged desmin was visualized with immunostaining and then transfected cells were counted and distributed into two categories; 1- cells containing filamentous network, 2- cells containing aggregates. Immunofluorescence microscopy and quantification of transfected cells revealed that synemin decreases desmin aggregates formed by p.Q389P, p.D399Y and p.T453I mutants, but synemin has no effect on desmin aggregates formed by p.E401K, p.R406W and p.E413K. Over 200 cells in random fields that expressed either desmin and/or synemin were examined in blinded manner. Results were expressed as percentage of transfected cell number containing desmin aggregates. Data were expressed as mean ± SD, n > 3. Bar: 5 μm (A-L); 0.8 μm (M).
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Research Article
Desmin mutations in the terminal consensus motif prevent synemin-desmin heteropolymer filament assembly Oussama Chourbagi a , Francine Bruston a , Marianna Carinci a , Zhigang Xue b , Patrick Vicart a , Denise Paulin b , Onnik Agbulut a,⁎ a
University Paris Diderot-Paris 7/CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, F-75013, Paris, France b UPMC-Paris 6, UR-4, Laboratoire de Génétique et Physiopathologie des Tissus Musculaires, F-75005, Paris, France
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Disorganization of the desmin network is associated with cardiac and skeletal myopathies characterized
Received 6 August 2010
by accumulation of desmin-containing aggregates in the cells. Multiple associations of intermediate
Revised version received
filament proteins form a network to increase mechanical and functional stability. Synemin is a desmin-
7 January 2011
associated type VI intermediate filament protein. Neither its impact on desmin network nor how it
Accepted 10 January 2011
integrates into desmin filament is yet elucidated. To gain more insight into the molecular basis of these
Available online 22 January 2011
processes, we coexpressed synemin with different desmin mutants in ex vivo models. The screening of fourteen desmin mutants showed that synemin with desmin mutants revealed two behaviors. Firstly,
Keywords:
synemin was co-localized in desmin aggregates and its coexpression decreased the number of cells
Intermediate filaments
containing aggregates. Secondly, synemin was excluded from the aggregates, then synemin had no
Desmuslin
effect on desmin network organization. Among fourteen desmin mutants, there were only three
Salt-bridges
mutants, p.E401K, p.R406W and p.E413K, in which synemin was not found in aggregates. This behavior
Desminopathy
was correlated to the abnormal salt-bridges of desmin-dimer as seen in silico constructs. Moreover, desmin constructs in silico and published results in literature have predicted that the salt-bridges absence in the desmin filament building prevent longitudinal annealing and/or radial compaction. These results suggest that the state of desmin-filament assembly is crucial for synemin anchorage and consequently might involve mechanical and functional stability of the cytoskeletal network. © 2011 Elsevier Inc. All rights reserved.
Introduction Intermediate filaments (IFs) are primary determinants of the cell's architecture and plasticity [1]. They are defined as part of a large gene family that encodes polymerizing proteins into individual 10 nm filaments [2]. The 52 kDa protein desmin is the constitutive
subunit of the type III intermediate filaments in all muscle tissues. Desmin, which is essential for tensile strength and muscle integrity, forms a three-dimensional scaffold around the myofibril Z-disc and connects the entire contractile apparatus to the subsarcolemmal cytoskeleton, the nuclei and other cytoplasmic organelles. There is also a significant amount of desmin in
⁎ Corresponding author at: University Paris Diderot-Paris 7, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, 4, rue Marie Andrée Lagroua Weill-Hallé (case 7006), 75 205 Paris cedex 13 France. Fax:+33 1 5727 7966. E-mail addresses: [email protected] (O. Chourbagi), [email protected] (F. Bruston), [email protected] (M. Carinci), [email protected] (Z. Xue), [email protected] (P. Vicart), [email protected] (D. Paulin), [email protected] (O. Agbulut). Abbreviations: IFs, intermediate filaments; ULFs, unit-length filaments; DMEM, Dulbecco's modified Eagle medium; CMV promoter, cytomegalovirus promoter; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.01.013
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neuromuscular [3,4] and myotendinous junctions [5,6] which allows the anchoring of myofibrils to the junctional sarcolemma. Disorganization of the desmin network is associated with cardiac and skeletal myopathies and a loss of force generation in smooth muscles [7]. In addition, mutations in the desmin gene can lead to desminopathy, a subgroup of the genetically and clinically heterogeneous group of myofibrillar myopathies. Desmin deposits, which are observed in patients suffering from desminopathy, were sometimes associated with abnormal aggregates of other cytoskeletal proteins such as synemin, nestin, plectin and αB-crystallin localized between myofibrils and beneath the sarcolemma [8,9]. The molecular architecture of desmin filaments is not entirely known [10,11]. It seems acquired that the unit-length filaments (ULFs) represent the precursors during filament formation. Each ULF of 16 nm, is comprised of the association of four octamers [12], that simultaneously undergo longitudinal annealing and radial compaction, finally result in a 10 nm diameter filament. Some desmin mutants belonging to the rod domain assemble into seemingly normal IFs as p.E245D [13], while other mutants interfere with the assembly process at distinct stages such as tetramer formation [10,14], ULFs formation [15], filament elongation [16] or IF maturation. As generally known, mutations located in the rod domain were disturbed coiled-coil formation via proline substitution or highly conserved heptad repeat pattern modification or critical intra-helical and inter-helical salt-bridge modifications [10,15]. As generally undersood, the degree of desmin filament assembly perturbation of variant desmin mutants cannot directly be correlated with the severity of the disease [17]. Synemin is a large type VI IF protein (150–230 kDa) found in all four classes of muscle cells [18–22] presenting a central rod domain flanked by a very short N-terminal head and a long C-terminal tail (Fig. 1). Synemin has been shown to be unable to self-assemble and is necessarily assembled in heteropolymers [23,24]. In muscles,
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synemin is associated with desmin [18]. As generally assumed, multiple associations of IF proteins to form a network increase its mechanical stability and give structural and functional versatility to the IF cytoskeleton [19]. Synemin interacts with type III IFs through the head and rod domains [22], and through its long C-terminal interacts with membrane proteins [25]. Via its interaction with cytoskeletal proteins dystrophin, utrophin and vinculin, synemin is able to link these heteropolymers to adherens-type junctions within striated muscle cells suggesting that it plays an important role in component assembly of the cytoskeleton [5,26,27] and in morphogenesis [28]. Synemin could participate in focal adhesion dynamics and be essential for cell adhesion and migration [29]. The present study was designed first to evaluate the impact of synemin on pathological desmin network organization and second to gain further insights upon molecular mechanisms involved in the process of synemin integration into desmin filaments. Thus, we performed cotransfections of synemin with wild-type desmin or with different desmin mutants in ex vivo models (C2C12, a mouse derived-myoblast; SW13, human adrenocortical carcinoma cells) and examined various cellular and molecular parameters. In conclusion, we suggest that the state of desmin-filament assembly appears crucial for synemin anchorage for the building of the cytoskeletal network.
Material and methods Cloning and site-directed mutagenesis Eukaryotic expression vector pcDNA3 (Invitrogen, Germany) under control of CMV promoter containing the cDNA of the human synemin H isoform [23] and the full-length human desmin cDNA with the Myc tag integrated in 5' were used. Mutations were
Fig. 1 – Desmin (A) and synemin (B) tripartite structure and location of fourteen desmin mutants examined in this study. Tripartite structure comprising a central α-helical rod domain flanked by non-helical head and tail domains. The central rod domain, formed by four α-helical segments (1A, 1B, 2A, 2B) separated by three short polypeptide linkers (L1, L12, L2). Desmin central α-helical rod domain, maintains a seven residue repeat pattern (heptad) that guides two polypeptides to form a homopolymeric coiled-coil dimer, the elementary unit of the desmin network. The heptad repeats were marked as abcdefg. The most conserved motif in the C-terminal part of the 2B segment was shown in insert.
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introduced into plasmid vectors by site-directed mutagenesis using the Stratagene QuikChange kit (Germany). Sequencing was done by Genoscreen (France). The positive clones obtained were grown overnight in a large volume of sterile LB medium with ampicillin, under agitation at 37 °C. Plasmids were extracted using the Qiagen Maxiprep kit (Qiagen, Germany).
Cell culture Mouse myoblasts, C2C12 (desmin+, vimentin+) and human adrenocortical carcinoma cells, SW13 (desmin-, vimentin-) were grown at 37 °C and 5% CO2 in DMEM (Dulbecco's modified Eagle medium, Invitrogen) supplemented with 20% (for C2C12) or 5% (for SW13) of fetal calf serum (Invitrogen), and penicillin G (50 U/ ml final) and streptomycin (50 ng/g final).
Transient transfection and immunostaining C2C12 and SW13 cells grown on Petri dish plated on coverslips at densities of 103-104/cm2 were transiently transfected using jetPEI (polyplus-transfection kit) according to the manufacturer's protocol (Ozyme). Two days after transfection, cells were processed for immunocytochemistry. Briefly, cells were fixed and permeabilized in methanol-acetone (70:30) for 15 min at –20 °C. After rehydration, specimens were blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS) for 20 min. Cells plated on coverslips were incubated with monoclonal anti-desmin antibody (clone D33, 1:100, Dako) or monoclonal anti-Myc tag antibody (clone 9E10, diluted 1:200, Santa Cruz Biotechnology) with or without the polyclonal rabbit anti-synemin antibody (dilution 1:300, [20]) for 90 min at room temperature. The binding of primary antibodies was detected by incubation for one hour with Alexa Fluor conjugated anti-mouse IgG or anti-rabbit IgG (1:1000). Finally, cells were washed in PBS and mounted with VECTASHIELD® HardFSet™ Mounting Medium with DAPI (4,6-diamidino- 2-phenylindole; Vector Laboratory, USA) for nuclear staining. Images were taken with a confocal microscope (Leica DM2500 TCS SPE).
Immunodetection quantification, cell counts Desmin network was visualized with immunostaining using antidesmin and anti-Myc antibodies and than cotransfected cells with synemin were counted and categorized into two categories; cells containing filamentous network (category 1), cell containing aggregates (category 2). The counting was performed in a blinded manner. All experiments were carried out in three, or more, independent transfections and for each situation at least two hundred transfected cells were counted in blinded manner. It should be noted that in this case SW13 cells are the best model to examine this process since the absence of endogenous IFs network in the cells prevents the adverse effects of this endogenous proteins on synemin-desmin interaction.
GROMOS96 43B1 parameters set, without reaction field, from Swiss-PdbViewer 4.0.1 (http://spdbv.vital-it.ch/download.html).
Results Fourteen desmin mutants that are responsible for desminopathy were used in this study (Fig. 1A). It should be noted that to distinguish endogenous desmin expressed in C2C12 and transfected constructs, a myc-tag was introduced in all desmin constructs. Using immunostaining, desmin mutants were sub-grouped according to their capacity to form a filamentous network and/or aggregates (Table 1, Fig. 2). Among the fourteen desmin mutants, four are able to form filamentous structures (Table 1, Fig. 2B). Three mutants, p.Δ114E, p.A357P and p.L370P, located in the rod domain presented a dominant effect in the endogenous network and only formed big aggregates throughout the cytoplasm (Fig. 2C). The other desmin mutants, p.Q389P, p.D399Y, p.E401K, p.R406W, p.E413K, p.T453I and p.S460I were able to integrate preexisting desmin network and also form desmin containing aggregates concentrated around the nuclei and throughout the cytoplasm (Fig. 2D).
Desmin mutations in the terminal consensus motif prevent synemin localization in the desmin aggregates In order to define the localization and distribution of the synemin with different desmin mutants, we cotransfected synemin with wild-type desmin or foorteen desmin mutants in C2C12 cells and immunostaining was performed. Our results demonstrated that synemin is colocalized with endogenous and Myc tagged wildtype desmin network (Figs. 3A-C). The screening of fourteen desmin mutants shows that synemin is colocalized with all of the desmin mutants which are able to form filamentous structures (Table 1). However, with the mutants that form aggregates two situations were observed (Fig. 3). Firstly, synemin is able to integrate into a preexisting desmin network and is also localized in desmin containing aggregates caused by p.D399Y (Figs. 3D-F), p. Δ114E, p.A357P, p.L370P, p.Q389P, p.T453I and p.S460I (Figs. 3G-I) desmin mutants (Table 1). Secondly, synemin is able to integrate
Table 1 – Mutated desmin organization in C2C12 and correlation with synemin localization. Immunodetection of synemin and Myc-desmin of fourteen desmin mutants highlights the absence of synemin in aggregates from p. E401K, p.R406W and p.E413K mutants. Desmin IF state in C2C12
The crystal structure of human vimentin coil-coiled 2B fragment (1gk6) was extracted from Protein Data Bank (http://www.rcsb. org/pdb/) and used to construct chimeric desmin models (WT and mutants) with PyMOL software (DeLano Scientific LLC). For energy minimization, computations were done in vacuo with the
Synemin-desmin colocalization* in network in aggregates
Filament Aggregate
Molecular model and secondary structure predictions
Mutant
Filament and aggregate
p.T442I, p.K449T, p.I451M, p.V469M p. E114del, p.A357P, p.L370P p.Q389P, p.D399Y, p.T453I, p.S460I p.E401K, p.R406W, p.E413K
+
b
a
+
+
+
+
−
* Presence (+) or absence (−) of synemin in network or aggregates; a, absence of network; b, no aggregates were observed.
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Fig. 2 – Functional analysis of different desmin mutants in C2C12 cells. Immunostaining of Myc-tagged desmin (red fluorescence) 48 hours after transfection of wild-type desmin (A); p.I451M (B); p.A357P (C) and p.R406W (D) desmin mutants. Note filamentous network organization in C2C12 cells when transfected with p.I451M desmin mutant. In contrast, p.R406W and p.A357P desmin mutants forms desmin containing aggregates which are distributed throughout the cytoplasm. Moreover, p.A357P presented a dominant effect in the endogenous network and only formed big aggregates throughout the cytoplasm. Nuclei were stained in blue by DAPI. Bar: 5 μm.
into a preexisting network but is totally excluded from desmin containing aggregates caused by p.E401K (Figs. 3J-L), p.R406W (Figs. 3M-O), p.E413K (Figs. 3P-S) desmin mutants (Table 1). Taken together, the screening of fourteen desmin mutants showed that in three mutants, p.E401K, p.R406W and p.E413K, synemin is totally excluded from the desmin containing aggregates. It should be noted that vimentin which is a one of the abundant IF in C2C12 [30,31] is located with all desmin aggregates provoked by different desmin mutants (data not shown). The difference of synemin's behavior with desmin mutants raises the question of the function of desmin residue or its position in interaction with synemin. The replacement of the arginine (R) positive residue by a tryptophan (W) neutral apolar residue at the position 406 or the replacement of the glutamic acid (E) negative residue by a lysine (K) positive residue at the position 401 abolished E401-R406 inter-helical salt-bridge which is important for the network via monomer/dimer formation or stabilization [10]. In addition, we emphasize that the salt-bridge E401-R406 within the desmin dimer is crucial for synemin-desmin heteropolymer filament assembly. In order to confirm this hypothesis we examined the inter-helical salt-bridge between E401 and R406 residues using in silico modeling and transfection studies.
The salt-bridge E401-R406 within the desmin dimer is crucial for synemin-desmin heteropolymer filament assembly In silico chimeric constructs of wild-type desmin, replacing residue E401 or R406 by other residues that have the same charge or not, have been established by using the energy minimization method.
The atomic distances were calculated between different desmin residues. Fig. 4 shows the distances calculated for different residues located at the position 406 of monomer 2 and 401 of monomer 1 (R406K, W, F; E401K, N, D). The modeling predicts that the inter-helical salt-bridge between E401 residue of monomer 1 and R406 residue of monomer 2 is present in the wild-type desmin dimer with an inter-side chain distance of 2.8 angstrom (Å) (Fig. 4A). The modeling of mutated desmin for R406 residue, shows that ionic bond between 401-406 residues is suppressed when the arginine (R) positive residue is replaced by a tryptophan (W) or a phenylalanine (F), neutral residues (Figs. 4B-C). Interestingly, the replacement of the arginine by a similar charge residue lysine (K), also suppressed ionic bond between the 401406 residues. In spite of a similar charge, the lysine side chain is shorter than the arginine's, which leads to a higher inter-side chain distance (4.8 Å) (Fig. 4D). The modeling of mutated desmin for E401 residue (monomer 1), shows that the replacement of a glutamic acid (E) negative residue by a lysine positive residue or an asparagin (N) neutral residue suppressed the ionic bond between 401-406 (Fig. 4E). In contrast, replacement of the glutamic acid (E) negative residue by a similar charge residue, aspartic acid (D), has a predicted distance of 3.7 Å (Fig. 4F). In order to examine the role of the salt-bridge E401-R406 in the integration process of synemin in desmin network, we carried out site-directed mutagenesis on the amino acids of the highly conserved region 401-EIATYRKLLEGEE-413 replacing the residue E401 by K, N, D or the residue R406 by W, F, K residues. Each of six desmin mutants and synemin were cotransfected in the C2C12 cells
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Fig. 3 – Immunofluorescence detection of synemin and mutated desmin in C2C12 cells. Immunostaining of Myc-tagged desmin (red fluorescence, A, D, G, J, M, P) and synemin (green fluorescence, B, E, H, K, N, R) 48 hours after cotransfection of synemin with wild-type desmin (A-C); synemin with p.D399Y (D-F); p.S460I (G-I); p.E401K (J-L); p.R406W (M-O) and p.E413K (P-S) desmin mutant. Combined red and green fluorescence in images were made. Nuclei were stained in blue by DAPI. Note an extensive filamentous network with perfect desmin-synemin colocalization between wild-type desmin and synemin (C). Ruptures in the filamentous network and aggregates appeared with all desmin mutants (arrows). Note the absence of synemin in aggregates caused by p.E410K, p.R406W and p.E413K desmin mutants (arrow head, K-L, N-O, R-S). Bar: 5 μm.
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Fig. 4 – In silico modeling of inter-helical salt-bridge between the 401 and 406 residues. The crystal structure of coiled coil 2B fragment of human vimentin (1gk6) was downloading from the protein data bank (PDB). Different chimeric desmin dimer mutants were established with PyMOL software using computations with GROMOS96 implementation of Swiss-PDB viewer 4.0.1 for energy minimization. Blue and red residues represent positive and negative net charges, respectively. The residues located at the positions 401 (monomer 1) and 406 (monomer 2) are drawn in a stick representation. Dot line represents the salt-bridges in wild type (A) and p.E401D desmin dimer (F). Distances were expressed as Angstroms.
and the results were summarized in Table 2 and Fig. 5. At the position 406 in desmin, when the arginine (R) positive residue is replaced by a tryptophan (W) or a phenylalanine (F), neutral residues, or a lysine (K) positive residue, it prevents the synemindesmin co-localization in the aggregates (Figs. 5A-C). It should be noted that the modeling in silico predicted that the ionic interaction between 401-406 residues was abolished with these mutations.
The replacement of the E401 negative residue by a positive lysine (K) residue or an asparagin (N) neutral residue also prevents the synemin-desmin colocalization in the aggregates probably due to the loss of charges. By contrast, the replacement of the glutamic acid (E401) negative residue by a similar charge residue, aspartic acid (E401D), shows the presence of synemin in the aggregates formed by E401D (Figs. 5D-F).
Table 2 – Salt-bridge state 401-406 of desmin dimer after various mutations obtained by site-directed mutagenesis. Immunostaining of Myc-desmin and synemin confirm the strong correlation between the presence of salt-bridge and the synemin-desmin colocalization in the aggregates. Atomic distances were done by PyMOL software. Mutation
Monomer 1 E401
Monomer 2 R406
none D N K none K F W
Residue charge and polarity Negative polar Negative polar Neutral polar Positive polar Positive polar Positive polar Neutral apolar Neutral apolar
* Presence (+) or absence (−) of synemin in the aggregates.
Salt-bridge Predicted interaction E401-R406 D401-R406 No No E401-R406 E401-K406 No No
Atomic distance (Å) 2.8 3.7 No No 2.8 4.8 No No
State Existing Preserved No No Existing Corrupted No No
*Synemin localization + + − − + − − −
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Fig. 5 – Immunostaining of synemin and mutated desmin in C2C12 cells. Immunostaining of Myc-tagged desmin (red fluorescence, A, D) or synemin (green fluorescence B, E) in C2C12 cells after cotransfection of synemin with different desmin mutants; p.R406K (A-C) and p. E401D (D-F). Combined red and green fluorescence in images were made. Nuclei were stained in blue by DAPI. Ruptures in the filamentous network and aggregates appeared with all desmin mutants (arrows). Note the absence of synemin in aggregates caused by p. R406K desmin mutant (arrow head, B-C). Note the synemin's presence in the aggregates caused by p.E401D desmin mutant (E). Bar: 5 μm.
Strong correlation between experimental data concerning the synemin-desmin colocalization in the aggregates and in silico predictions on the breaking of the salt-bridge between 401-406 residues, leads to conclude that the ionic bond interaction E401R406 within the desmin dimer is crucial for the synemin-desmin heteropolymer filament assembly.
Concerning E413K, using in silico modeling, we show that intrastrand salt-bridge between K407 and E413 is also broken and the distance between K407-E413 increases from 2.8 to 5.5 Å (Fig. 6). Our results show that the tail is probably maintained by this saltbridge in the wild-type desmin, whereas in the mutant p.E413K, tail dimer is free and consequently takes more space which may
Fig. 6 – In silico modeling of salt-bridges for E413K mutation. The crystal structure of coiled coil 2B fragment of human vimentin (1gk6) was downloading from the protein data bank (PDB). p.E413K mutated desmin dimer was established with PyMOL software using computations with GROMOS96 implementation of Swiss-PDB viewer 4.0.1 for energy minimization. Blue and red residues represent positive and negative net charges, respectively and drawn in a stick representation. Dot line represents the intra-strand salt-bridges in normal (A) and pathologic dimer (B). Distances were expressed as Angstroms.
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accumulate in the next step of filament assembly and subsequently disrupt the radial compaction of ULFs.
Synemin decreases desmin-aggregate formation To investigate the effect of synemin on desmin mutants and particularly to examine if the expression of synemin can improve the desmin network and consequently decrease desmin-aggregate formation in desminopathy, we cotransfected synemin with wild-type desmin or desmin mutants in SW13 cells. The desmin network was visualized with immunostaining and then cotransfected cells were counted and distributed into two categories; 1cells containing filamentous network, 2- cells containing aggregates (Fig. 7). Immunofluorescence microscopy and quantification of transfected cells revealed that when wild-type desmin is expressed into SW13 cells, 75% of transfected cells present a filamentous network and 25% of transfected cells contain irregular bundles of filaments (Fig. 7A). When SW13 cells were cotransfected with desmin and synemin, we found more homogenous labeling of desmin network and less than 10% of transfected cells presented irregularly bundles of filaments suggesting synemin might improve desmin network (Figs. 7DF). Transfection of p.D399Y desmin mutant into SW13 cells revealed that this mutant causes a formation of cytoplasmic aggregates in 85% of transfected cells (category 2) and/or a formation of very short filamentous structures (category 1) (Fig. 7B). Cotransfection of p.D399Y desmin mutant with synemin shows that synemin is co-localized with desmin both in aggregates and in short filamentous structures (Figs. 7G-I). In this situation, desmin aggregates formed by this mutant decrease from 85% to 45% (Fig. 7N). The decrease of number of cell containing desmin aggregates also confirmed with p.Q389P and p. T453I desmin mutants (Fig. 7N). Transfection of p.E401K, p. R406W and p.E413K desmin mutants into SW13 cells shows that these mutants are unable to form a filamentous network, they forms numerous and very small, dot-like aggregates, which are distributed throughout the cytoplasm of 100% transfected cells (category 2, Figs. 7J-L). Interestingly, cotransfection of p.E401K, p. R406W and p.E413K desmin mutants with synemin shows no modifications on the aggregates formed by these mutants (Fig. 7N). Concerning localization of synemin with these mutants, we demonstrated that neither synemin nor desmin mutants is able to form a filamentous network, they form numerous small, dot-like aggregates, which are distributed independently throughout the cytoplasm (Fig. 7M).
Discussion The major finding of this study is to demonstrate that i) synemin integration into desmin network is dependent of desmin filament assembly state, ii) coassembly of synemin with desmin improves desmin network organization and consequently decrease desmin containing aggregates. Concerning the synemin-desmin association, the screening of fourteen desmin mutants shows that desmin-associated protein synemin is perfectly colocalized with all of the desmin mutants which are able to form filamentous structures de novo, but with the mutants that have no capacity to form filamentous structures on their own two behaviors were observed. First, synemin is able
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to integrate into a preexisting network and is also localized in desmin containing aggregates (p.Δ114E, p.A357P, p.L370P, p. Q389P, p.D399Y, p.T453I and p.S460I). Second, synemin is able to integrate into a preexisting network but is totally excluded from desmin containing aggregates (p.E401K, p.R406W, p. E413K). Functional analysis of these three desmin mutants shows that they are unable to form filamentous structures on their own. They form only small aggregates throughout the cytoplasm in SW13 cells. Interestingly, they retain the ability to integrate into preexisting intermediate filament networks in C2C12 cells when the ratio of wild-type desmin is superior to the mutant one. When the amount of desmin mutant becomes superior to wild-type one, desmin filament assembly is perturbed and desmin containing aggregates become to form. In this case, synemin is totally excluded from the desmin containing aggregates. As the varied behavior of synemin in desmin network integration process is dependent on desmin mutants and endogenous versus mutant desmin ratio, we could suggest that synemin fixation site was generated during filament assembly and consequently desmin filament assembly state appears crucial for synemin anchorage. Regarding the molecular structure of these desmin mutants in which synemin is excluded from the aggregates, two mutants were identified in the terminal consensus motif 405-YRKLLEGEE413. This motif, which highly conserved in all types of intermediate filaments in a wide range of species, is crucial for the formation of dimeric complexes and also for the control of filament width during assembly process via intra- and interhelical salt-bridges [32]. Interestingly, this highly conserved terminal consensus motif is not only involved in the formation of the coiled-coil structure but also in its loosening. From the leucine at the position 409, the coiled-coil structure loosens, so that the helices gradually separate and eventually bend away from each other and eventually becoming disordered [33–35]. For these reasons, the mutations located in this region have a drastic effect on network structure organization. Bar et al. studies show that p.R406W and p.E413K desmin mutants exhibit disturbed longitudinal annealing and radial compaction during desmin filament assembly process [36]. Moreover, p.E401K and p.R406W desmin mutants alter the entire dimer interaction via interhelical salt-bridge corruption which causes formation of unstable ULF-like particles, whose longitudinal annealing is slowed drastically, so that eventually small, granular dead-end structures ensue [17]. Dimerization between two α-helices to form coiledcoil is well documented notably for coiled-coil structures and their ionic interactions. All IF proteins exhibit a similar interhelical salt-bridge between glutamic acid (E) and arginine (R) residues, located into the highly conserved amino acid motif of helix 2B (YRKLLEGEE) and this, since in the nuclear lamins [37] via keratins and vimentin [32]. Furthermore, there are disease mutations in all cytoplasmic and nuclear IF proteins that involve this inter-helical salt-bridge [1]. Statistical analysis of the occurrence of this ionic interaction in coiled-coils is characterized for parallel dimeric coiled-coils and the atomic structure is of type i to i + 5 ionic interaction [38]. Similar analysis of intrahelical ionic interactions in α-helices and coiled-coils has been performed confirming that the configurations, which have simultaneously a large probability to form the ionic interaction and a frequent occurrence, are those, which have the most stabilizing effect as 4RE, 3ER and 4ER interactions [39].
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p.E401K and p.R406W desmin mutants also disturb heptads repeat pattern which is important for the formation of coiled-coil structure [40]. It should be noted that the highly conserved inter-helical salt-bridge E401-R406 is formed between a glutamic acid in a g position on one chain and an arginine in an e position on the second chain. Electrostatic interactions arising primarily from residues in positions e and g are known to play a crucial role in specifying the relative alignment and orientation of the two chains within the coiled coil [41]. Pruszczyk et al. suggested that p.E413K mutation also alters electrostatic interactions which are important for the proper dimer–dimer interactions during the assembly process [33]. The authors indicate that p.E413K
mutations generate a new abnormally intra-helical salt-bridge between residue E410 and the mutant K413 which probably increase stiffness and decrease the flexibility of the coiled-coil structure. Moreover, using in silico modeling, we show that intrastrand salt-bridge between K407 and E413 is also broken and the distance between K407-E413 increases from 2.8 to 5.5 Å due to charge repulsion. Our results show that the tail is probably maintained by this salt-bridge in the wild-type desmin, whereas in the mutant p.E413K, tail dimer is free and consequently takes more space which may accumulate in the next step of filament assembly and subsequently disrupt the radial compaction of ULFs.
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Apparently, any change in this highly conserved region can influence filament assembly by disrupting interactions within the dimer and/or above dimeric level. The salt-bridge between 401 and 406 residues is known for its importance in desmin homodimer filament assembly [10]. In wild type desmin state, the negative charged glutamic acid residue at the 401 position of one monomer forms an inter-helical salt-bridge with positive charged arginine at the 406 position in the other monomer. Our results obtained in silico modeling predict that the inter-helical salt-bridge between E401-R406 residues is present in the wildtype desmin dimer with an inter-side chain distance of 2.8 Å. In addition, salt-bridge is also present when glutamic acid (E) at the 401 position is replaced by similarly charged residue aspartic acid (D). By contrast, when we replaced arginine (R) at the 406 position by similarly charged residue lysine (K) is suppressed ionic bond between 401-406 residues. In spite of a similar charge, lysine side chain is shorter than arginine which leads to a higher an inter-side chain distance (4.8 Å). Replacement of glutamic acid (E) at the 401 position or arginine (R) at the 406 position by neutral or opposite charge residue inter-helical salt-bridge is suppressed. Strong correlation between in silico predicts on the breaking of saltbridge between 401-406 residues and experimental data concerning the absence of synemin-desmin association in the aggregates, lead to formulate that the salt-bridge interaction E401R406 within the desmin dimer is crucial for synemin-desmin heteropolymer filament assembly. These findings highlight the crucial role of the inter-helical salt-bridge since it seems to be enough that the salt-bridge rupture prevents the radial compaction and longitudinal annealing and consequently synemindesmin heteropolymer ULF assembly. Moreover, our transfection studies demonstrated that when synemin is localized in the desmin containing aggregates (p.Q389P, p.D399Y, p.T453I) synemin expression drastically decreases cell number containing aggregates, but when synemin is excluded from the aggregates (p.E401K, p.R406W, p.E413K) in this case coexpression of synemin has no effect on desmin network organization. Interestingly, synemin has been shown to be incapable of self-assembly and is necessarily assembled in heteropolymers. For this reason, in muscle, synemin should be associated with desmin to form a filamentous network. But our results demonstrate that coexpression of synemin with wild-type desmin
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into SW13 cells reveals more homogenous labeling of desmin network. It should be noted that synemin and desmin are normally coexpressed in muscle, it is likely that this has physiological significance. Synemin probably provides some required interactions with other structures. Synemin is a desmin-associated type VI IF protein, it interacts with type III IFs with the head and rod domains [21], and through its long C-terminal, interacts with membrane proteins [25] suggesting that synemin plays an important role in component assembly of the cytoskeleton [5,26,27] and in morphogenesis [28]. Coassembly either with vimentin and paranemin also allowed homogenous and extended desmin network [42]. Note that vimentin is able to form an extended network on its own so this was an expected result. However, as paranemin is unable to form a network on its own, these results support the hypothesis that the large intermediate filament proteins provide some required interactions with other structures, strengthening the IF network. Moreover, the presence of a K8/K18 keratin network also appeared to allow extended desmin IFs despite that keratins and desmin do not form copolymers but assemble into distinct networks. Taken together, our results highlight that the state of desmin filament assembly appears crucial for synemin anchorage and consequently might involve mechanical and functional stability of cytoskeletal network. Moreover, in this study we underline the crucial role of the inter-helical and intra-strand salt-bridges located at the terminal consensus motif of desmin since it seems to be enough that the salt-bridge rupture prevents the radial compaction and longitudinal annealing and consequently synemin-desmin heteropolymer ULF assembly.
Acknowledgments This work was supported by the “Association Française contre les Myopathies” (AFM), contract n° 14040, the University Paris Diderot-Paris7, the “Agence Nationale de la Recherche” (ANR), contract n° 06-MRAR-039-01. O.C. was supported by fellowships from the Ministère de la Recherche et Technologie” (MRT) and the AFM. We would like to thank Dr. Bertrand Goudeau for gift of vector constructs of human desmin, Dr. Zhenlin Li, Dr. Pierre Joanne and Valentin Wucher for his helpful advices.
Fig. 7 – Immunofluorescence detection of desmin (A-C) and desmin-synemin (D-M) in SW13 cells and quantification of desmin containing aggregates in various condition (N). Immunostaining of Myc-tagged desmin (red fluorescence) 48 hours after transfection of wild-type desmin (A); p.D399Y (B) and p.R406W (C) desmin mutants in SW13 cells. Immunostaining of desmin (red fluorescence, D, G, J) and synemin (green fluorescence E, H, K) after cotransfection of synemin with wild-type desmin (D-F); p.D399Y (G-I) and p.R406W (J-M) desmin mutants. Combined red and green fluorescence in images were made. Nuclei were stained in blue by DAPI. Note more homogenous labeling of desmin network when desmin is coassembled with synemin (arrow, A, D-F). Note synemin colocalization with D399Y aggregates, while with p.R406W mutant, neither synemin nor desmin mutants is able to form a filamentous network, they form numerous small, dot-like aggregates, which are distributed independently throughout the cytoplasm (J-M). Quantification of desmin containing aggregates in SW13 cells 48 hours after transfection of different desmin mutants with or without synemin (N). Myc-tagged desmin was visualized with immunostaining and then transfected cells were counted and distributed into two categories; 1- cells containing filamentous network, 2- cells containing aggregates. Immunofluorescence microscopy and quantification of transfected cells revealed that synemin decreases desmin aggregates formed by p.Q389P, p.D399Y and p.T453I mutants, but synemin has no effect on desmin aggregates formed by p.E401K, p.R406W and p.E413K. Over 200 cells in random fields that expressed either desmin and/or synemin were examined in blinded manner. Results were expressed as percentage of transfected cell number containing desmin aggregates. Data were expressed as mean ± SD, n > 3. Bar: 5 μm (A-L); 0.8 μm (M).
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