Research article

Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy

Minttu Marttila, Elina Lemola, William Wallefeld, Massimiliano Memo, Kati Donner, Nigel G. Laing, Steven Marston, Mikaela Grönholm, Carina Wallgren-Pettersson


NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin–myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.

  • actin binding
  • calcium-sensitivity
  • cap myopathy
  • circular dichroism (CD)
  • nemaline myopathy (NM)
  • β-tropomyosin (β-Tm)


NM (nemaline myopathy) is a genetically heterogeneous congenital myopathy showing wide clinical variability, including different grades of severity and ages of onset [1]. It was described in two separate reports by Shy et al. [2] and Conen et al. [3] in 1963. The diagnostic criteria are muscle weakness and nemaline bodies present in skeletal muscle, with no other simultaneously occurring significant structural abnormalities. NM-causing mutations have been found in seven genes: nebulin (NEB) [4], α-actin (ACTA1) [5], α-Tm (tropomyosin) (TPM3) [6], β-Tm (TPM2) [7], troponin T1 (TNNT1) [8], cofilin-2 (CFL2) [9] and KBTBD13 (Kelch repeat and BTB domain-containing protein 13) [10].

Cap myopathy was first described in 1981 by Fidzianska et al. [11]. It is characterized by the presence of cap-like structures that are located peripherally under the sarcolemma and show abnormal accumulation of sarcomeric proteins [11]. The first genetic causes of cap disease were identified in the TPM2 gene in 2007: a deletion of glutamic acid at position 139 [12] and a change at position 41 of glutamate to lysine [13].

Tms are encoded by four genes, TPM1–4 (α, β, γ and δ), from which at least 20 different isoforms are expressed [14]. In skeletal muscle three Tm isoforms are expressed from the TPM1 (α-Tmfast or α-Tm), TPM2 (β-Tm) and TPM3 (α-Tmslow or γ-Tm) genes [15]. Tms are fibrous molecules composed of two α-helices forming coiled-coil dimers. The dimers polymerize head-to-tail and overlap by eight to nine amino acids. Tm dimers are located in the grooves of F-actin (filamentous actin) filaments providing structural stability and modulating filament function [14]. These interactions are essential for the function of striated muscle. In order to investigate the molecular mechanisms causing NM and cap myopathy, we studied the effects of five patient mutations in β-Tm (the TPM2 gene) on actin binding and thin filament Ca2+ regulation. In addition, protein modification and conformation were studied using MS and CD spectroscopy.

One of the mutations studied (Q147P) causes NM [7], two (K49del and E139del) cause cap myopathy [12,13,16] and one (E41K) is associated with a dominantly inherited myopathy that showed nemaline bodies in one of two biopsies of a mother and caps in her daughter's biopsy [12,13]. The last of the mutations studied (E117K) is associated with an unspecific congenital myopathy, first diagnosed as NM, but, on closer scrutiny, found not to show verifiable nemaline bodies [7].

View this table:
Table 1 Mutations in the TPM2 gene used in the present study and summary of results

~, values equal to WT Tm values; –, values lower than wt; +, values higher than WT; #, not measurable. Calculated WT and mutant Tm α-helical content is in percentage at 20°C and 37°C using Contin-LL software [24,25].



TPM2 cDNA (GenBank® accession number NM_009416) cloned into pGEX4-1 (GE Healthcare) was used as a template for PCR. Forward 5′-GATCCATGGACGCCATCAAGAAGAAG-3′ and reverse 5′-TCGAGTCAGAGGGAAGTGATGTCAT-TG-3′ primers were designed to copy the TPM2 sequence, but to exclude the GST tag. Fusion DNA polymerase (Thermo Fisher Scientific) was used under the following PCR conditions: denaturation at +98°C for 30 s, denaturation at +98°C for 10 s, annealing at +58°C for 30 s, elongation at +72°C for 30 s and final elongation at +72°C for 7 min. The programme was cycled 34 times. Products were cloned into the Zero blunt TOPO vector using a PCR cloning kit (Invitrogen). Plasmids were extracted using QIAprep Spin Miniprep kit (Qiagen). The mutations (Table 1) were introduced into mouse cDNA using the QuikChange® Site-Directed Mutagenesis kit (Stratagene). Plasmids were sequenced using BigDye version 3.1 sequencing chemistry and an ABI 3730 DNA Analyzer (Applied Biosystems). The sequences were analysed using the Sequencher 4.5 software.

Production of WT (wild-type) and aberrant β-Tms

Recombinant β-Tms were produced using a Bac-to-Bac baculovirus expression system (Invitrogen) in Sf9 insect cells. The insect cells were grown in supplemented Grace's Insect Medium (Invitrogen) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) in 500 ml of medium at 27°C.

Extraction of proteins

Proteins were extracted from Sf9 cells using a modification of the method described by Akkari et al. [17]. Extraction was performed in a buffer containing 1 M NaCl, 50 mM imidazole (pH 7), 0.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 5 mM DTT (dithiothreitol) and 1× protease inhibitor (Roche). Proteins were boiled for 10 min in a water bath. The denatured proteins were spun at 9000 rev./min in a Sorval SS-34 rotor for 20 min at +4°C and the supernatant saved. The proteins were precipitated in 70% ammonium sulfate (Fluka) and incubated at room temperature (22°C) in a mixer for 1 h. The samples were spun at 9000 rev./min in a Sorval SS-34 rotor for 20 min at 4°C and the pellets saved. The pellets were dissolved into a buffer containing 400 mM NaCl, 5 mM Pipes and 1 M DTT, pH 7.1. Samples were transferred into Slide-A-Lyzer 10 kDa molecular-mass cut-off dialysis cassettes (Pierce). They were dialysed in 200–500 volumes of the same buffer overnight, with one buffer change. The buffer was changed to 50 mM Na2HPO4/NaH2PO4, 1 M NaCl and 5 mM DTT (pH 4.5), and the proteins were dialysed for 2.5 h in a mixer at 4°C and the buffer changed after 1 h. The precipitated proteins were separated from the buffer by spinning in a Sorval SS-34 rotor at 16000 rev./min for 15 min at 4°C. The pellets were dissolved in a buffer containing 400 mM NaCl, 5 mM Pipes and 1 M DTT (pH 7.1). The samples were dialysed in 200–500 volumes of the same buffer overnight, with one buffer change. The proteins were spun in an Eppendorf centrifuge at 14000 rev./min for 20 min at 4°C. The supernatant contained the purified β-Tms.

Actin binding

Actin-binding assays were performed using Cytoskeleton's Actin Binding Protein Biochem Kit (Cytoskeleton). In a previous actin-binding assay 1.5 μM Tms and 5 μM F-actin were used [18]; however, in the present study we used 10.35 μM Tms and 23 μM F-actin to be able to detect more actin-binding mutants. Tms were allowed to bind to F-actin for 30 min at room temperature. Samples were run in a Beckman Coulter Optima MAX Ultracentrifuge at 60000 rev./min for 1.5 h at 24°C. Pellet and supernatant fractions were separated, analysed by SDS/PAGE (12% gels) and stained with Coomassie Blue. Pellet and supernatant bands were quantified from four experiments carried out in duplicate using ImageJ (NIH).

Mass spectrometry

In-gel digestion and LC-MS/MS (liquid chromatography tandem MS) analysis of the resulting peptides was done at the Protein Chemistry Research Group and Core Facility in the Institute of Biotechnology, University of Helsinki (Helsinki, Finland).

Three-dimensional models

The mutations E41K, K49del and E139del were made to the amino acid sequence of Tm2 (GenBank® accession number P58775.1). The probable secondary structure of the mutants were assigned by homology modelling using the crystal structure corresponding to PDB codes 1C1GA and 2B9CA as templates, following a standard method in Discovery Studio (Accelreys).

Circular dichroism

Tm samples were analysed by CD spectroscopy. Spectral CD measurements were obtained over the far UV wavelength range of 250–190 nm in a buffer containing 0.25 M NaF and 0.02 M Na2HPO4/NaH2PO4 at pH 7.0. CD analysis was done using a J-810 Spectropolarimeter (Jasco) equipped with a Peltier thermostatically controlled cuvette holder, controlled via a PC running Spectra Analysis (Jasco, version 1.53.04) software. One-way ANOVA was conducted for the percentage α-helical content and melting temperature data sets with a Newman–Keuls post-hoc test using GraphPad Prism Version 4. The threshold for significance was set at P<0.05.

IVMA (in vitro motility assay) data acquisition and analysis

Flow cells were constructed from a microscope slide and siliconized coverslip (washed in 0.2% dichloromethylsilane in chloroform). Fluorescently labelled F-actin, Tm and troponin were mixed and incubated at 10× working concentration for 15 min at 4°C. HMM (heavy meromyosin; 100 μl of 0.2 mg/ml) was infused into the flow cell, followed by 100 μl of thin filament mixture (10 nM actin-Φ, troponin and Tm between 10 and 100 nM). The fraction of fluorescently labelled thin filaments moving and their corresponding velocity were analysed in 50 mM KCl, 25 mM imidazole, 4 mM MgCl2, 1 mM EDTA, 5 mM DTT, 0.5 mg/ml BSA, 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 3 mg/ml glucose, 0.5% methylcellulose, 5 mM Ca2+/EGTA buffer, 1 mM MgATP and Tm at appropriate concentration. Filament movement was automatically tracked and recorded as described by Marston et al. [19]. The Ca2+-concentration dependency data were fitted to the 4-parameter Hill equation: y=a+Xmax [Ca2+]n/(EC50+[Ca2+]n).


Production and extraction of recombinant proteins

In order to study the functional differences between WT Tm and Tms containing known human disease-causing mutations, the mutations were introduced into WT β-Tm. Because post-translational modifications, especially N-terminal acetylation, are important for the function of β-Tms [20] we produced Tms in a baculovirus expression system in insect cells (Sf9) [17]. The purified proteins were run in SDS/PAGE, and differences in migration rate were found between the samples (Figure 1). The samples of mutations K49del and E139del run slower in SDS/PAGE.

Figure 1 β-Tms produced in insect cells

SDS/PAGE of recombinant proteins stained with Coomassie Blue. Slower migrating samples are indicated with arrows. M, molecular mass standards (in kDa).

Actin binding of WT and mutant Tms

The WT Tm and Tms with human disease-causing mutations were tested for actin binding (Figures 2A and 2B). Three samples, K49del, E139del and Q147P showed significantly weaker affinity for actin than WT. One sample, E117K, bound actin more strongly, whereas E41K did not show any significant change in actin binding (Figure 2C).

Figure 2 F-actin co-sedimentation assay

(A) Purified actin and Tm, BSA as a negative control or α-actinin as a positive control were incubated together and co-sedimented. The supernatants (S) and pellets (P) were separated, run on SDS/PAGE and Coomassie Blue stained. (B) F-actin co-sedimentation assay with WT Tm and Tm mutants performed as in (A). Molecular masses (in kDa) are given in the centre of each panel. (C) Quantification of actin–Tm co-sedimentation assay. Gels were quantified to determine the mean relative intensities of four independent actin co-sedimentation assays (P/Pwt:S/Swt). Results are means±S.D. of each sample. P values (WT compared with mutant) for the samples are 0.0007, 0.0392, 0.0001, 0.0029, 0.0013 and 0.2417 respectively using one-tailed distribution and the unpaired Student's t test. *Significant difference from the WT.

Functional analysis by IVMA

IVMA analyses the movement of fluorescently labelled thin filaments over a bed of immobilized HMM [21]. Thin filaments were reconstituted using rabbit skeletal actin and troponin, together with the baculovirus-expressed WT or mutant β-Tm homodimers. With WT thin filaments, we observed that both the speed of filament sliding over myosin and the fraction of filaments that are motile are regulated by Ca2+. The troponin–Tm complex inhibits the movement of thin filaments under relaxing conditions (Ca2+ ~ 10−9 M) so that the fraction motile is 10% or less. At activating Ca2+ concentrations (3.5 μM) at least 80% of filaments are motile, like actin alone. The sliding speed also increases 2-fold between relaxing and activating Ca2+ concentrations.

When mutant β-Tm K49del, E139del and Q147P were incorporated into thin filaments, it was found that motility was not switched off in low Ca2+ even with high Tm concentrations. Thin filaments with the WT β-Tm switched off at the standard Tm concentration (Figure 3) indicating that the abnormal function was due to the mutation in Tm. These mutants (K49del, E139del and Q147P) appear to have a lower affinity for actin compared with the WT, as indicated by the direct measurements. We hypothesize that these mutants could be washed away by the last buffers that are infused in the flow cell, leaving motility similar to actin alone. Therefore no curves could be recorded from with these constructs.

Figure 3 Tm Ca2+ sensitivity

The effect of β-Tm mutations E117K (A) and E41K (B) on Ca2+ regulation of thin filament motility. Ca2+-dependence of sliding speed (upper panel) and fraction of filaments motile (lower panel) are plotted at different Ca2+ concentrations. Continuous lines and closed circles represent mutant Tm with skeletal muscle actin and troponin, broken lines and open circles represent WT Tm. Results are means±S.E.M. of five replicate measurements in a single motility cell. The lines are fits to the Hill equation. Ca2+ sensitivity for the fraction motile parameter is EC50=0.060±001 μM for WT and EC50=0.17±0.04 μM for the E117K experiment, EC50=0.36±04 μM for WT and EC50=1.00±0.18 μM for the E41K experiment. EC50 of thin filaments is variable between different troponin preparations.

Thin filaments containing the β-Tm mutations E41K and E117K that bound strongly to actin were able to regulate the filaments, with low Ca2+ concentration causing a complete switch off of their motility.

Four Ca2+ curves were recorded from thin filaments containing E117K Tm (one represented in Figure 3A, lower panel). In comparison with WT thin filaments, motility was generally lower at high Ca2+ concentration (79.5±4.1% compared with 96.2±0.5% for WT, P= 0.006, unpaired t test). The speed of sliding increased slightly with increasing Ca2+ concentration, but it was significantly less than the WT Tm (sliding speed at 3.5 μM Ca2+/sliding speed at 1 nM Ca2+=1.34±0.16 for E117K and 2.04±0.15 for WT Tm, P= 0.02). In addition, the Ca2+ sensitivity of activation was much less (EC50 E117K/EC50 WT Tm=2.44±0.35, P= 0.03) (Figure 3A, upper panel). Thus the E117K mutation is predicted to lead to reduced contractility. Two complete Ca2+ titration curves were recorded with the E41K Tm (Figure 3B). The results were similar to E117K, although maybe more marked in terms of reduced Ca2+ sensitivity (EC50 E41K/EC50 WT Tm=2.77), but the increase in sliding speed was similar in E41K and WT.

MS and three-dimensional models

Purified β-Tm samples were run on SDS/PAGE gels. Two samples, K49del and E139del, showed slower migration than the WT and other mutant proteins. To find out whether the difference in migration could be due to post-transcriptional modifications, we used in-gel digestion of the proteins followed by LC-MS/MS analysis of the resulting peptides. The mass-spectrometric database ( contains eight phosphorylation sites that have been found in mouse and human Tm. In addition to these we found in our analyses four novel phosphorylation sites. These were present in all samples including the WT. No other significant differences in peptide mass were found (results not shown). The protein modifications caused by the mutations and the N-terminal acetylation of the Tms were verified using LC-MS/MS. Post-transcriptional modifications were not found to be the reason for the difference in migration. Thus it may be due to a change in secondary structure conformation of the K49del and E139del mutants [22,23] as found with the D175N mutation in α-Tm [24]. To study this, three-dimensional models of the differentially migrating mutants were created (Figure 4). The α-helical conformation was changed in the K49del and E139del mutants.

Figure 4 Structural alignments of Tms

Structural alignments of WT and mutant Tm using the Discovery Studio 2.1 program. The mutations K49del (A), E139del (B and C) and E41K (D) were made in rat Tm2 (P58775.1) and were superimposed over the WT structure 1C1GA (A, C and D) or 2B9CA (B). 2B9CA was used as it provided a better resolution. Amino acids before deletions are marked in deletion mutants and E41 in E41K. Amino acids used in comparison are shown.

CD spectra of Tm

Since three-dimensional models indicated that the structure may be affected in the mutants with aberrant migration pattern, to further study the structures of our mutant proteins, and the effect of disease causing mutations on overall secondary structure, CD spectra were produced. At 20°C all recombinant Tm proteins showed CD spectra characteristic of α-helical proteins, featuring a minimum at 222 and 208 nm and a maximum at 190 nm (Figure 5). Table 1 shows the calculated α-helical content of each protein as determined by the Contin-LL method [25,26]. At 20°C WT Tm had a calculated α-helical content of 97.3±2.8%. E117K displayed an α-helical content higher than that of the WT (99.8±0.2%). Two of the mutant proteins displayed lower calculated α-helical content: E139del (79.5±0.7%) and K49del (84.6±3.1%).

Figure 5 CD of Tms

CD spectra (250–190 nm) of Tm measured at 20°C and 37°C.

At 37°C WT and mutant Tm proteins also displayed CD spectra characteristic of α-helical proteins (Figure 6). WT Tm was calculated to be 81.9±1.8% α-helical, whereas the Q147P and E117K showed the highest α-helical contents (88.3±0.8% and 87.9±4.3% respectively). The lowest α-helical contents were seen with the K49del and E139del mutants (65.0±1.6% and 68.2±1.2% respectively). At both of the temperatures used, K49del and E139del displayed lower α-helical content than WT Tm. E117K showed higher α-helical content than WT at both temperatures and Q147P at 37°C.

Figure 6 Thermodynamic analysis of Tms

For the Tm thermodynamic analysis, CD absorption was observed at 222 nm as the temperature was increased from 20°C to 70°C.

Thermodynamic analysis of β-Tm

To assess the thermodynamic properties of WT and mutant β-Tm the CD absorption was observed at 222 nm as the temperature was increased from 20°C to 70°C. This allowed observation of the protein from a completely folded state to a completely unfolded state and allowed the construction of a melt profile. WT and mutant proteins were modelled as a two-state transition from a fully folded (0% unfolded) state to a fully unfolded state (100% unfolded), and this was then plotted against temperature (Figure 6). A best-fit equation was generated from the data and the mid-point of the denaturation, taken as the Tmelt (melting temperature) shown in Table 1. WT Tm was calculated to have a Tm of 40.2±0.0°C. The majority of mutant Tm proteins were calculated to have Tmelt similar to or slightly higher than that of the WT. The K49del mutant was found to have the lowest Tmelt of all samples studied (39.3±0.2°C). The denaturation profile of the E117K mutant appears to be quite different from both the WT and the other mutants, indicating that this may have a different unfolding profile, involving different sequential steps.


Altogether 11 mutations causing NM, cap myopathy or distal arthrogryposis have been described in the TPM2 gene that codes for β-Tm [7,12,13,16,2729]. Included in the present study were five of these, found to cause NM or cap myopathy. In striated muscle, β-Tm is present mainly as a heterodimer with α-Tm in fast muscles and γ-Tm in slow muscles [30]. Tm is almost completely α-helical and forms a parallel coiled-coil dimer. The Tm dimers polymerize with overlapping ends, forming a continuous strand that binds to actin in a slightly curved structure [31]. Tm, troponin and actin interactions regulate the contraction of muscle fibres in response to Ca2+ [14]. Therefore changes in the association with actin could be the cause of muscle weakness in muscle disorders, such as NM and cap myopathy, caused by Tm mutations. In the current models [32], it is proposed that Tm has a 7-fold repeated amino acid sequence motif, the heptad repeat that corresponds to the seven actin monomers covered by one Tm. Each motif is subdivided into α- and β-zones. In the relaxed state, Tm forms contacts with actin through positively charged residues in the N-terminal part of an α-zone and acidic residues on the C-terminal side of an α-zone (Figure 7). Ca2+ regulation is imposed on actin–Tm by the troponin complex that switches actin–Tm between the on and off conformational states. Thus mutations in Tm potentially can cause muscle dysfunction leading to myopathy through a variety of mechanisms, including effects on formation of dimers, end–end interactions, actin binding and the regulatory interaction with troponin.

Figure 7 Human β-Tm sequence

(A) Human β-Tm sequence with mutations (E41K, K49del, E117K, E139del and Q147P) used in the present study and α-zones. The corresponding heptad repeat positions are shown above the sequence. (B) Schematic presentation of the fifth α-zone. Sequence of rat striated muscle α-Tm with the positively charged residue followed by apolar residues marked that are found on the N-terminal side of each α-zone. On the C-terminal side, acidic residues found in each of the seven α-zones are proposed to form close contacts with subdomains 1 and 3 of actin. Corresponding heptad repeat positions are shown above the sequence. Based on data taken from [32].

The a and d residues of the Tm heptad repeat, as in all α-helix heptad repeats, are usually hydrophobic and packed at the interface of the α-helix. The e and g residues stabilize the coiled coil through interchain electrostatic interactions and are often oppositely charged. Residues in the b, c and f positions bind other proteins [33]. The K49del, E117K and Q147P mutations are in e and g positions, probably affecting dimerization. The E139del and E41K mutations are in position f, and may affect the binding of other proteins: E139del the binding of actin and E41K a presumed other thin filament protein.

Defective formation of physiological dimers may affect the relative expression levels of the isoforms. Ochala et al. [34] have shown that the β/α ratio changes expressing the E41K β-Tm mutation and Corbett et al. [15] have shown altered Tm isoform ratios in muscle from M9R γ-Tm mutation patients and in the transgenic mouse model of this mutation.

It is well established that strong end–end interactions, integral αβ repeats and a continuous α-helix are essential for Tm binding to actin [15]. Three of the five β-Tm mutations investigated in the present study did not bind to actin in our assays: K49del, E139del and Q147P. The deletion mutants K49del and E139del shorten the α-zone of the second and fourth repeats respectively. Moreover, the deletions are close to proposed major actin contacts: Lys49 is located after a positively charged residue present in the N-terminal side of the α-zone proposed to form a direct contact with actin, whereas Glu39 is one of the acidic residues of the C-terminal half of the fourth α-zone proposed to form a contact with actin (Figure 7). All the deletion mutations change the heptad repeat sequence subsequent to the deleted amino acid that seems to have a profound effect upon the helical structure of the Tm dimer (Figure 4). In addition, the site-specific disruption of Tm–Tm and Tm–actin contacts and the long-range effect of shortening the α-zone would be expected to reduce the affinity of Tm for actin [35,36] as observed in the present study (Figure 2C). Interestingly, both the mutations that appeared to have caused the histological features of cap disease only (K49del and E139del), led to relatively large drops in α-helical content at both 20°C and 37°C, whereas those causing NM (or NM and cap disease) showed either a slight decrease or an increase in α-helical content (Table 1).

The mutant Q147P has been found to cause NM [7]. The Q147 residue is in position g of the heptad repeat and in the fourth β-zone just on the C-terminal side of the fourth α-zone. The non-polar proline introduced into the heptad repeat position g, normally occupied by a polar residue, should interfere with the local coiled-coil structure. Proline is not present in any WT Tm or in coiled-coil proteins in general, as it produces a kink in the coiled-coil helix [37]. Actin binding of the Q147P mutant was found to be significantly weaker than the binding of actin to WT Tm as might be expected with the disruption of the continuous α-helix [12,38,39].

Although actin–Tm affinity is much reduced in assays at sub-micromolar concentrations, it remains likely that mutant Tm can nevertheless bind to myofibrils in muscles where Tm concentration is approximately 100 μM. Moreover, it is well known that troponin greatly enhances the affinity of Tm for actin [18]. In fact, we were able to obtain some regulation of thin filaments in the motility assay with the weak-binding Tm mutants (K49del, E139del and Q147P) by raising the Tm and troponin concentrations. It remains to be seen whether the stoichiometry of Tm is deficient in the muscles of patients with these mutations.

E41K and E117K mutants bind to actin normally and both appear to directly affect the thin filament regulatory system. These mutations had profound effects upon Ca2+ regulation in thin filaments with a large reduction in Ca2+-sensitivity and for E117K a much lower increase in sliding speed (i.e. cross-bridge turnover rate) due to troponin-inactivating conditions, which we have ascribed to the activity of troponin [39,40]. It is likely that the mutation can disrupt the delicate co-operative and allosteric interactions of the thin filament regulatory system. Although Glu17 is not in a region of Tm known to interact with troponin T, there is ample evidence for long-range propagation of structural and functional effects of mutations in Tm [41,42]. The mutation E117K is the one mutation to have stronger affinity for actin than WT Tm. We speculate that the higher affinity for actin might explain the absence of nemaline bodies in this patient. Tighter binding could result in the sarcomeric proteins remaining in the thin filaments and the Z disc, and not forming nemaline bodies. The contractile deficiency at the level of the troponin switch parallels that previously reported for CFTD (congenital fibre type disproportion) mutations, noted for muscle weakness without nemaline bodies [43,44].

Previous studies have addressed the pathogenic background of TPM2 mutants that cause pathologies other than cap and nemaline body formation. Analysis of alterations in contractile properties due to a TPM2 mutation, R133W, causing distal arthrogryposis in an affected mother and daughter [27], indicated slowing of cross-bridge attachment and acceleration of detachment. Thus the authors speculated that the alteration in myosin–actin kinetics induced by the mutation would cause fewer myosin molecules to be in the strong actin-binding state. This was suggested to be the cause of muscle weakness in the patients [45]. In another study using troponin-exchanged skinned fibres, the R91G mutant in TPM2 caused a significant increase in Ca2+ sensitivity. R91G β-Tm was found to have a lower actin affinity than WT and form a less stable coiled coil [45]. In the related TPM3 gene, the M9R mutation causing NM was found to have a similar effect on actin binding [46]. In the present study, one of the mutants (E117K) showed stronger actin binding and higher α-helical content compared with the WT. This mutation caused neither cap formation nor any verifiable nemaline body formation and may be classified as CFTD. Three of the mutants causing nemaline body or cap formation showed weak actin binding, and two of these also had low α-helical content. Thus we speculate that a defective α-helical content and less effective actin binding might contribute to the formation of nemaline bodies and caps, possibly through the disorganization of interrelated sarcomeric proteins. In other cases, abnormal actin–Tm or troponin–Tm interactions may be the underlying cause of the patients' muscle weakness.

In summary, actin binding was weak in three of five mutants suggesting that abnormal binding between actin and aberrant Tm is the pathogenetic mechanism causing muscle weakness in these patients. Ca2+ activation of contractility was defective in the two other mutations, suggesting an alternative cause of contractile dysfunction. However, in view of the histological and clinical overlap described in previously published cases [13,27,47,48,50], these hypotheses need to be further explored.


Carina Wallgren-Pettersson was responsible for planning the project and for clinical correlations in the paper. Minttu Marttila performed actin-binding experiments, three-dimensional models and mass spectrometry and wrote the paper. Elina Lemola was responsible for protein production and actin-binding experiments. Kati Donner produced three tropomyosin constructs. Mikaela Grönholm, a senior protein expert, planned parts of the experimental work and wrote the paper. Nigel Laing and Steven Marston wrote the paper. William Wallefeld performed circular dichroism experiments and wrote the paper. Massimiliano Memo performed in vitro motility experiments and wrote the paper.


This work was supported by the Association Française contre les Myopathies, the Sigrid Jusélius Foundation, the Academy of Finland, the Finska Läkaresällskapet and the Medicinska understödsföreningen Liv och Hälsa r.f. (to M.M. and C.W.P.); an Australian National Health and Medical Research Council Fellowship [grant number 403904 (to N.G.L.)]; and the British Heart Foundation (to S.M. and M.M.). The University of Helsinki is a partner of TREAT-NMD (European Commission, 6th Framework Programme, proposal no. 036825;


We thank Dr Nigel Clarke and Nancy Mokbel for helpful comments on the paper, Katarina Pelin for help on the project, Saara Ollila and Mari Korhonen for helping with the insect cell cultures, Jaakko Sarparanta for advice on the protein work, Pertteli Salmenperä for discussing the migration differences, Per Harald Jonson for help with the three-dimensional models and Tuula Nyman and Gunilla Rönnholm for MS data.

Abbreviations: CFTD, congenital fibre type disproportion; DTT, dithiothreitol; F-actin, filamentous actin; HMM, heavy meromyosin; IVMA, in vitro motility assay; LC-MS/MS, liquid chromatography tandem MS; NM, nemaline myopathy; Tmelt, melting temperature; Tm, tropomyosin; WT, wild-type


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