Brain Advance Access published online on October 10, 2007
Brain, doi:10.1093/brain/awm247
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Mechanisms underlying intranuclear rod formation
1Institute for Neuromuscular Research, 2Oncology Research Unit, Children's Hospital at Westmead, 3Discipline of Paediatrics and Child Health, University of Sydney, 4Muscle Development Unit, Children's Medical Research Institute, Sydney, NSW 2145, Australia and 5Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA
Correspondence to:
Prof. Kathryn N. North, Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia E-mail: kathryn{at}chw.edu.au
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Summary |
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Specific mutations within the
-skeletal actin gene (ACTA1) result in intranuclear rod myopathy (IRM), characterized by rod-like aggregates containing actin and -actinin-2 inside the nucleus of muscle cells. The mechanism leading to formation of intranuclear aggregates containing sarcomeric proteins and their impact on cell function and contribution to disease pathogenesis is unknown. In this study, we transfected muscle and non-muscle cells with mutants of -skeletal actin (Val163Leu, Val163Met) associated with intranuclear rod myopathy. By live-cell imaging we demonstrate that nuclear aggregates of actin form within the nuclear compartment, rather than entering the nucleus after formation in the cytoplasm, and are highly motile and dynamic structures. Thus, the nuclear environment supports the polymerization of actin and the movement and coalescence of the polymerized actin into larger structures. We show that the organization of actin within these aggregates is influenced by the binding of -actinin, and that -actinin is normally present in the nucleus of muscle and non-muscle cells. Furthermore, we demonstrate that, under conditions of cell stress (cytoskeletal disruption and ATP depletion), WT skeletal actin forms aggregates within the nucleus that are similar in morphology to those formed by the mutant actin, suggesting a common pathogenic mechanism for aggregate formation. Finally, we show that the presence of intranuclear actin aggregates significantly decreases the mitotic index and hence impacts on the function of the cell. Intranuclear aggregates thus likely contribute to the pathogenesis of muscle weakness in intranuclear rod myopathy.
Key Words:
intranuclear rod myopathy; nuclear aggregates; -skeletal actin; -actinin
Abbreviations:
ACTA1, (-skeletal actin gene; CFTD, congenital fibre-type disproportion; IRM, intranuclear rod myopathy; LMB, leptomycin B; NES, nuclear export sequences; NLS, nuclear localization sequence.
Received May 24, 2007. Revised August 14, 2007. Accepted September 17, 2007.
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Introduction |
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Mutations in the gene encoding
-skeletal actin (ACTA1) are responsible for a number of congenital myopathy subtypes including nemaline myopathy (Nowak et al., 1999
; Ilkovski et al., 2001), intranuclear rod myopathy (IRM) (Sparrow et al., 2003; Hutchinson et al., 2006), myopathy associated with accumulation of actin (actin myopathy) (Nowak et al., 1999; Sparrow et al., 2003), core myopathy (Kaindl et al., 2004) and congenital fibre-type disproportion (CFTD) (Laing et al., 2004). The variety of changes in muscle histology likely result from fundamental differences in the way that ACTA1 mutations disrupt muscle function (Sparrow et al., 2003; Ilkovski et al., 2004; Clarke et al., 2007). IRM associated with ACTA1 mutations is characterized by congenital onset muscle weakness and pathologically by the presence of nuclear aggregates in skeletal muscle, that are highly enriched for both filamentous actin and the actin-binding protein at the Z-line, -actinin-2 (Goebel and Warlo, 1997; Hutchinson et al., 2006).
Mutations of amino acid Val163 in ACTA1 have been identified in a three generation family (Val163Met) (Hutchinson et al., 2006) and in two unrelated patients (Val163Leu) (Goebel et al., 1997a; Nowak et al., 1999) with IRM. In each of the affected individuals, rods occurred almost exclusively within the nuclei of their muscle cells, with a lack of other myopathic features at the light microscopy level, suggesting that this particular mutation predisposes to intranuclear rod formation.
Many questions remain to be answered concerning the mechanisms underlying the formation of nuclear aggregates containing actin and -actinin, and their contribution to muscle weakness in IRM. Finding accumulations of sarcomeric proteins within the nucleus is an unusual observation, as both, -skeletal actin and actinin-2 have not been shown to localize to the nucleus. It is thus not known whether the nuclear aggregates form within the nucleus or whether they enter the nucleus after forming within the cytoplasm. We have utilized IRM mutants of -skeletal actin (Val163Leu, Val163Met) in order to provide further insight into the mechanisms of intranuclear rod formation and their effect on cell function.
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Material and Methods |
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Unless otherwise specified, all cell culture reagents were purchased from Invitrogen and all molecular biology reagents were obtained from Roche.
Cell culture
Mouse C2C12 myoblasts were cultured in 40% high glucose Dulbecco's Modified Eagle Medium (DMEM) and 40% F-12 Nutrient Mixture (HAM) with L-glutamine, supplemented with foetal bovine serum (FBS) and horse serum (HS), each to 10%. Mouse NIH3T3 fibroblasts were cultured in high glucose DMEM, supplemented with FBS to 10%. For transfections and drug treatments, cells were cultured on collagen/matrigel coated thermanox coverslips (Nunc) in 24-well plates.
Constructs
Generation of the WT-actinEGFP and V163L/M-actinEGFP constructs has been previously described (Ilkovski et al., 2004). All actin constructs were generated in the pEGFP-N1 backbone (Clontech). To generate the V163L/M-actinuntagged constructs, a segment of the actin cDNA containing the V163M/L mutations was excised from V163L/M-actinEGFP with PpuMI and XhoI and subcloned into WT-actinuntagged that had also been digested with the same enzymes.
Transfections
C2C12 and NIH3T3 cells were trypsinized and plated on thermanox coverslips (Nunc) 1 day prior to transfection in growth medium. The cells were washed twice with phosphate-buffered saline (PBS) and medium without antibiotics was added 30 min prior to transfection. The cells were transfected at 70–90% confluence using Lipofectamine2000TM, according to the manufacturer's instructions. Briefly, per 2 cm2 culture area, 1.2 µg of DNA and 3.6 µl lipid were prepared in 0.1 ml of OptimemTM and incubated in 0.6 ml of growth medium without antibiotics. Cells were transfected for 6 h, washed twice in PBS and replenished with growth medium.
Cell treatments
LMB (Sigma) was diluted at 2.5, 5 or 10 ng/ml of culture medium and added to the culture wells overnight. LMB activity was tested using the Rev-NES-GFP expression system. The pRev(NES)-GFP plasmid has been described previously (Henderson and Eleftheriou, 2000). In untreated cells expressing the construct, Rev-NES-GFP localized to the cytoplasm, whereas in cells treated with LMB for 3 h or overnight, it localized exclusively to the nucleus.
Cytochalasin D (final concentration 1 µM; Sigma) and latrunculin A (final concentration 5 µM; Sigma) were diluted in culture medium and added to the cells for 45 min–1 h.
For ATP depletion, cells were washed with PBS and incubated in ATP-depletion medium (10 mM sodium azide, 6 mM 2-deoxyglucose in PBS) for 30 min at 37°C without CO2 supply. Following cell treatments, the coverslips were briefly washed in PBS and fixed.
Fixation and immunostaining of cultured cells
C2C12 and NIH3T3 cells grown on thermanox coverslips (Nunc) were fixed in PBS containing 3% paraformaldehyde and permeabilized in 0.1% triton-X 100 for 20 min at room temperature (RT). Samples were washed three times in PBS, then incubated in blocking buffer (PBS plus 2% bovine serum albumin) for 10 min at RT before immunostaining as described previously (Ilkovski et al., 2004). After immunostaining, samples were washed three times in PBS and mounted on 22 x 50 mm2 glass coverslips using FluorsaveTM mounting reagent (Calbiochem).
Antibodies and fluorophores
Primary antibodies: (mAb) -actinin (1:300) (Sigma), (mAb) emerin (1:100) (Novacastra Laboratories Ltd), (pAb) -actinin-2 (4B2, 1:4000) and -actinin-4 (6A2, 1:600) (Dr Alan Beggs, Harvard Medical School), (mAb) actin (C4, 1:200) (BD Biosciences Pharmigen). Secondary antibodies: Cy3-conjugated goat anti-mouse or anti-rabbit IgG (1:250), Cy5-conjugated donkey anti-mouse IgG (1:200) (Jackson ImmunoResearch Laboratories. Inc.), Alexa Fluor 488 goat anti-mouse or anti-rabbit IgG (1:200) (Molecular Probes). For some experiments, TRITC-phalloidin (1:500) (Sigma) and/or ToPro3 iodide (1:200) (Molecular Probes) were added with the secondary antibody.
Extraction of pelleted and soluble protein pools
Transfected cells were rinsed twice in PBS and then scraped in extraction buffer [50 mM MES pH 6.8, 1 mM EGTA pH 8.0, 50 mM KCl, 1 mM MgCl2, 0.5% Triton X-100, protease inhibitor (PI) cocktail from Sigma was added immediately prior to use (1:500)], followed by 1 h ultracentrifugation at 100 000g at 4°C to get pools enriched for filamentous actin, which likely contains the intranuclear aggregates (the pellet, P), and globular actin (the soluble fraction, S). The pelleted and soluble fractions were then separated and 200 µl of the soluble fraction was transferred to a microfuge tube containing 50 µl of 5x SDS sample buffer [312.5 mM Tris pH 6.8, 10% SDS, 50% glycerol, 250 mM DTT, PI cocktail (1:500) and bromophenol blue (BPB)]. The pellet was re-suspended in 1x SDS sample buffer [62.5 mM Tris pH 6.8, 6% SDS, 10% glycerol, 50 mM DTT, PI cocktail (1:500), BPB] and briefly sonicated. All samples were heat-inactivated for 4 min at 94°C and stored at –20°C.
Western blot
Samples were thawed and heated to 94°C for 1 min immediately prior to loading on 5% stacking, 9% resolving SDS–PAGE gels. Western blot was performed as described previously (Cooper et al., 2003).
Imaging
Confocal microscopy was performed using a Leica TCS SP2 Scanning Confocal Microscope equipped with HCX Plan Apo (PH3) 40x/1.25 and 63x/1.32 oil immersion objective lenses. EGFP or alexa, Cy3 or TRITC and Cy5 or ToPro3 were excited at 488, 543 and 633 nm, respectively. For live-cell imaging experiments, cells transfected on 42 mm collagen/matrigel-coated circular glass coverslips were transferred at 6 h after transfection to microscope incubator and scanning stage equipped with temperature regulator 37-2 digital and CTI controller 3700 CO2 IR sensor. The temperature was maintained at 37°C and CO2 at 5%. Images were merged using Leica LCS software and figures were assembled using Adobe Photoshop.
The mitotic index was determined by counting the number of transfected cells in mitosis as a proportion of the total number of transfected cells in randomly chosen fields at 40x magnification. A total of 6919 C2C12 cells were counted from 20 coverslips and 2809 NIH3T3 cells from 12 coverslips. Results are expressed as mean ± SEM; statistical significance was determined by non-parametric 2-tailed Mann–Whitney U-test.
Electron microscopy
Cultured cells were fixed in situ with modified Karnovski's fixative (2.5% glutaraldehyde, 4% paraformaldehyde solution in 0.1 M cacodylate buffer, pH 7.4) for 1 h. Cells were post-fixed with 2% osmium tetroxide, dehydrated through an ascending series of ethanol and embedded in Spurr's epoxy resin. Sections were cut with DIATOME diamond knife on Leica ULTRACUT S ultramicrotome at 70 nm thickness, double contrasted with uranyl acetate and lead citrate. The sections were viewed and photographed with Philips CM120 BioTwin transmission electron microscope.
Statistical analysis
The statistical significance was assessed by non-parametric two-tailed Mann–Whitney U-test.
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Results |
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Mutant
-skeletal actin forms filamentous aggregates with -actinin inside the nucleus of different cell typesPatients bearing mutations within ACTA1 at position Val163 exhibit an IRM, with electron dense aggregates positive for filamentous actin (phalloidin) and
-actinin-2 within the nuclei of their skeletal muscle fibres (Goebel and Warlo, 1997; Hutchinson et al., 2006). Transfection studies using -skeletal actin constructs bearing these mutations (V163L, V163M) and tagged with a green fluorescent protein (V163L/M-actinEGFP) or untagged (V163L/M-actinuntagged), revealed striking intranuclear accumulations of actin, both needle-like and star-like in C2C12 myoblasts (Ilkovski et al., 2004; Fig. 1) and in NIH3T3 fibroblasts (Fig. 1), that closely resembled the intranuclear rod phenotype observed in the patient muscle. WT-actinEGFP, however, localizes to the cytoplasmic microfilaments and does not form intranuclear aggregates (See Supplementary materiall). We performed electron microscopy on C2C12 and NIH3T3 cells transiently expressing V163L-actinEGFP or V163L-actinuntagged and demonstrated that the mutant -skeletal actin aggregates are surrounded by a double nuclear membrane (Fig. 1A) and are thus located inside, rather than above or below the plane of the nucleus. Higher magnification showed that the aggregates have a filamentous appearance (Fig. 1A), which in combination with the phalloidin labelling demonstrates that -skeletal actin can exist in a filamentous form inside the nucleus.
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Muscle and non muscle-specific isoforms of
-actinin labelled a subset of intranuclear aggregates of actin formed when V163L/M-actinuntagged was transfected in muscle (C2C12 myoblasts) and non-muscle (NIH3T3 fibroblasts) cells (Fig. 1B–D). Staining of C2C12 cells expressing V163L/M-actinuntagged with phalloidin and an antibody against -actinin-2 and -actinin-4 showed that both -actinin isoforms accumulate with filamentous actin inside the nucleus (Fig. 1B and data not shown). -Actinin-4 also accumulated with filamentous actin in the nuclei of NIH3T3 cells expressing V163L/M-actinuntagged (Fig. 1C and D). The formation of intranuclear aggregates of mutant (V163L/M) -skeletal actin and -actinin inside the nucleus of a variety of cell types, suggests that the intracellular environment necessary for intranuclear aggregate formation is not cell-type specific.
The aggregates co-labelled by -actinin have a distinct morphology to those not labelled by -actinin. The -actinins were only present within aggregates with a ‘star-like’ shape, localizing to an intensely fluorescent focus from which the phalloidin-labelled actin filaments radiate (Fig. 1B–D, insets). These star-like aggregates were often accompanied by numerous smaller focal aggregates containing both -actinin and filamentous actin (Fig. 1D, inset 1). The needle-like aggregates did not label with antibodies against the -actinins (Fig. 1D, inset 2). Thus, the -actinins likely cross-link the intranuclear aggregates and may influence their morphology.
Intranuclear mutant actin aggregates form inside the nucleus
To address the question whether rods are produced within the nuclei or enter the nuclei after cytoplasmic formation, we performed live-cell imaging of NIH3T3 fibroblasts expressing V163L-actinEGFP and demonstrate that the intranuclear aggregates form inside the nucleus. Diffuse V163L-actinEGFP fluorescence first appeared in the cytoplasm at 
6 h after transfection with the first detectable nuclear fluorescence appearing as bright puncta at 15–16 h after transfection, concurrent with a rapid increase in V163L-actinEGFP expression in the cytoplasm (Fig. 2A; see Video 1 Supplementary materiall). With time the bright puncta increased in number and size inside the nucleus, they joined upon contact and their morphology increasingly resembled needle-shaped aggregates (Fig. 2A). The live-cell imaging results were replicated in C2C12 myoblasts (data not shown). Analysis of pelletable (P) and soluble (S) actin pools in C2C12 myoblasts transfected with WT-actinEGFP or V163L-actinEGFP revealed significantly higher levels of pelletable V163L-actinEGFP (49% total actin) compared to WT-actinEGFP (26% total actin) at 15 h after transfection (Fig. 2B), consistent with the timing of appearance of first fluorescent puncta inside the nucleus (Fig. 2A).
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Intranuclear mutant actin aggregates are dynamic structures
Live-cell imaging also revealed that the intranuclear aggregates containing V163L-actinEGFP are highly dynamic structures. Besides being able to fuse, the needle-shaped aggregates can move rapidly and bend inside the nucleus (Fig. 2C; see Video 2 Supplementary material). Interestingly, the bending of aggregates on opposite sides of the nucleus was synchronous and their movement was restricted to one area of the nucleus and appeared to occur around the point of bending. This suggests that the aggregates may be tethered to a nucleoskeleton.
-Actinin-2 normally resides within the nucleus
-Actinin has not previously been shown to reside within the nucleus—yet it is a major constituent of intranuclear rods in patient muscle and in our cell culture model of intranuclear rod formation. To determine whether -actinin normally resides within the nucleus, we examined the effect of blocking nuclear export on nuclear aggregate formation. We demonstrate that endogenous -actinin-2 can accumulate within the nucleus and its export is sensitive to leptomycin B (LMB), which blocks the CRM1/exportin-mediated pathway. In response to LMB treatment of C2C12 cells, endogenous -actinin-2 formed predominantly focal aggregates that did not label with phalloidin in the area of the nucleus of 20% of cells (Fig. 3A). In a subset of these cells, -actinin-2 formed needle-like aggregates, reminiscent of rods seen in association with the ACTA1 mutants (Fig. 3B).
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The actin proteins contain two nuclear export sequences (NES) and ß-actin has been shown to accumulate within the nucleus and form paracrystalline structures in response to LMB (Wada et al., 1998
) or depletion of the nuclear exporter, exportin 6 (Exp6) (Stuven et al., 2003; Bohnsack et al., 2006). We hypothesized that if -skeletal actin also resides within the nucleus and its export is mediated through CRM1, then LMB treatment would lead to its accumulation within the nucleus. However, immunostaining of LMB-treated C2C12 cells with phalloidin and the C4 antibody, which recognizes all isoforms of actin, failed to show accumulation of actin within the nucleus (data not shown). In addition, LMB treatment of C2C12 and NIH3T3 cells expressing WT-actinEGFP did not result in observable intranuclear aggregates (Fig. 3C). Therefore, if intranuclear aggregates of -skeletal actin result from blocked nuclear export, then this is not mediated through CRM1 in the cell lines used in this study.
Wild-type -skeletal actin forms intranuclear aggregates in response to actin microfilament disruption and ATP depletion
Various types of cell stress lead to the formation of actin-containing aggregates in the cytoplasm or in the nucleus (Nishida et al., 1987; Minamide et al., 2000). Since previous studies have focused on non-muscle actins (Nishida et al., 1987; Minamide et al., 2000), we aimed to determine whether WT -skeletal actin can form intranuclear aggregates in response to cytoskeletal disruption and ischaemia. C2C12 myoblasts were transfected with WT-actinEGFP and treated with 1 µM cytochalasin D (CD) or 5 µM Latrunculin A (LatA) (model of cytoskeletal disruption) or incubated in ATP-depletion medium for 30 min (ischemia). Needle-like aggregates of WT -skeletal actin formed inside the nucleus in response to all treatments, resembling those formed by expression of mutant (V163L/M) actin (Fig. 4). The experiment was also performed in NIH3T3 fibroblasts and similar results were obtained (data not shown). Thus, actin microfilament disruption, ischaemia and the presence of mutations at V163 lead to a common effect of -skeletal actin accumulation inside the nucleus.
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Intranuclear aggregates affect mitotic index
Accumulation of actin in the nucleus leads to decreased cell proliferation and may be toxic at the cellular level (Perrimon et al., 1989
; Wada et al., 1998; Stuven et al., 2003). Here we demonstrate that the intranuclear aggregates due to mutations at position V163 in -skeletal actin lead to reduced mitotic index. By counting the number of cells in mitosis for cells transfected with WT-actinEGFP or V163L-actinEGFP at different time-points after transfection, we found that an increase in the number of transfected cells with intranuclear rods is associated with reduced proportion of cells in mitosis (the mitotic index). At 24 h and 48 h after V163L-actinEGFP transfection, 37% and 81% of the transfected C2C12 myoblasts respectively (31% and 74% in NIH3T3 fibroblasts) contained intranuclear rods and the mitotic index was reduced by 50% and 97%, respectively (55% and 85% in NIH3T3 fibroblasts) (Fig. 5 and data not shown). Replication of the results in NIH3T3 fibroblasts demonstrates that the reduced mitotic index is not a result of myoblast differentiation. In addition, the aggregate-containing cells were not apoptotic, as judged by their chromatin arrangement on electron microscopy (Fig. 1A). Furthermore, there was no change in the number of nuclei per cell in the aggregate-containing cells. A small proportion of mitotic cells expressing V163L-actinEGFP contained aggregates. These aggregates were small in size and were pushed to the side of the condensed DNA during mitosis (Fig. 5C), suggesting that the size of the aggregates could be a factor in determining the extent to which normal cellular processes are affected. Figure 5D shows that the large and ‘star-shaped’ intranuclear aggregates displace the DNA.
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Discussion |
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In one of the first descriptions of IRM, Goebel et al. (1997b
) commented that ‘finding rods within the muscle fibre nuclei is a surprising and spectacular observation. Normal muscle fibre contents of a defined structure are not an intranuclear feature. Thus, encountering rods within nuclei arouses considerable speculation ... Were the rods produced within the nuclei or did they enter the nuclei from the sarcoplasm after cytoplasmic formation?’
In this study, we demonstrate that intranuclear aggregates of mutant -skeletal actin form within the nuclear compartment. On live-cell imaging, the mutant actin first accumulated into small aggregates inside the nucleus that gradually increased in number and size and coalesced into larger aggregates that appear filamentous at the EM level and label with phalloidin (Figs 1 and 2). Thus, the nuclear environment supports the polymerization of actin and the movement and coalescence of the polymerized actin into larger structures. Interestingly, the intranuclear aggregates of mutant actin can form in both muscle and non-muscle cells (Fig. 1). This suggests that the intranuclear aggregates form through a process that does not require muscle lineage- specific proteins and that the ability to form intranuclear aggregates is intrinsic to the mutant protein. In addition, our data demonstrate that intranuclear aggregates may form independently from cytoplasmic aggregates, as no such aggregates formed within the cytoplasm of myoblasts.
The -actinins are a family of actin-binding proteins involved in establishing and maintaining cell structure (Lazarides, 1976; Rajfur et al., 2002; Nakagawa et al., 2004). The actins and -actinins form a variety of dynamic and highly ordered networks in the cytoplasm involved in processes such as cell motility, cell adhesion and cell division. Our results suggest that in the presence of mutations at Val163 in ACTA1, -skeletal actin is able to enter the nucleus and accumulate into aggregates with -actinin (Fig. 1). Unexpectedly, we also demonstrate that -actinin-2 can normally reside within the nucleus (Fig. 3). The -actinins belong to the spectrin protein superfamily; in recent years, a number of other members of this protein superfamily have been shown to localize within the nucleus and are suggested to play a structural role in anchoring other proteins to the nuclear membrane, or perform functions in nuclear processes such as DNA repair (Young and Kothary, 2005). Our finding suggests that, similar to the formation of cytoplasmic rods in nemaline myopathy, -actinin-2 cross-links bundles of mutant actin filaments inside the nucleus into larger aggregates and is thus likely involved in the organization of mutant actin aggregates into a more complex three-dimensional structure, although additional factors may be required in vivo for the formation of nemaline bodies in mature skeletal muscle.
The process that drives the accumulation of mutant actin inside the nucleus remains to be determined. In contrast to -actinin-2, -skeletal actin did not accumulate inside the nucleus in response to inhibition of CRM1-dependent nuclear export. It is still possible, however, that intranuclear aggregates of mutant actin result from blocked export through another exporter, such as exportin-6, which is known to export ß-actin from the nucleus (Stuven et al., 2003; Bohnsack et al., 2006).
Alternately the Val163 mutation may result in increased trafficking of actin into the nucleus. Amino acid 163 lies in the hinge region of the actin monomer and, based on molecular modelling, is likely to affect nucleotide exchange and the folding of the actin monomer (Sparrow et al., 2003; Costa et al., 2004). The mutant actins preferentially localize to the nucleus and their incorporation into cytoplasmic microfilaments is reduced (Fig. 1). Thus the predisposition toward intranuclear rod formation secondary to this mutation may be due to the increased availability of actin monomers in the cytoplasm that cannot properly incorporate into thin filaments, triggering increased trafficking of actin into the nucleus. This possibility is supported by the observation on live cell imaging that the appearance of the first intranuclear aggregates is concurrent with a rapid increase in V163L-actinEGFP expression in the cytoplasm (Fig. 2A; Video 1 Supplementary materiall).
Actin is not known to contain a nuclear localization sequence (NLS) and the mechanism leading to its entry into the nucleus is controversial. Whereas monomeric actin is small enough to enter the nucleus by diffusion (Wada et al., 1998; Bohnsack et al., 2006), cells have very little free actin monomer that is not associated with monomer sequestering proteins; thus, others propose that actin is translocated into the nucleus by a transporter molecule such as cofilin (Nishida et al., 1987; Matsuzaki et al., 1988; Gonsior et al., 1999; Pendleton et al., 2003; Chhabra and dos Remedios, 2005). Previous studies in non-muscle cells suggest that treatment of cells with latrunculin or ATP depletion, leads to cofilin-mediated translocation of ß-actin into the nucleus (Pendleton et al., 2003). In the current study, treatment of both myoblast and fibroblast lines with cytochalasin D, latrunculin A and ATP depletion induced the formation of aggregates inside the nucleus composed of WT -skeletal actin that closely resembled the aggregates formed by the V163L/M actin. This raises the possibility that both the expression of mutant actin in IRM and cell stress (such as disruption of the actin microfilament system and ATP depletion), result in intranuclear rod formation due to a common pathogenic mechanism, such as an increase in ADP-actin monomers in the cytoplasm. Since cofilin has increased binding affinity for ADP-actin (Carlier et al., 1997), this would likely result in increased import of monomers of -skeletal actin into the nucleus, where they subsequently polymerise to form aggregates, that are then cross-linked into more complex structures by -actinin. Further studies are necessary to dissect the molecular basis of accumulation of mutant actin inside the nucleus, in particular to determine whether the aggregates are due to decreased export or increased import of the mutant actin and, in the case of increased import, whether this is due to passive diffusion or active transport by a transporter molecule such as cofilin.
It is difficult to imagine that large aggregates inside the nucleus would not affect nuclear function and structure. One possible explanation for the synchronic bending and movement of the intranuclear aggregates (Fig. 2C) is that they are tethered to a nucleoskeleton. Figure 5 shows that the intranuclear aggregates displace the DNA and their appearance correlates with a reduced mitotic index. Lenart et al. (2005) showed that nuclear actin networks are involved in the congression of chromosomes in oocytes and that this process can fail when the actin cytoskeleton is disrupted, however, the existence of nuclear actin networks remains to be demonstrated in other cell types. Alternatively, the effect of intranuclear aggregates on mitotic index could be the result of a general affect on cell metabolism, disruption of transcription or a toxic effect due to the sequestration of normal sarcomeric proteins such as -actinin-2, so that they are no longer available to perform their normal functions. It is likely that these alterations in muscle cell function (rather than structure) contribute to muscle hypotrophy and weakness in IRM, since patients with pure IRM have relatively normal sarcomeric structure and demonstrate very little of the myofibrillar disorganization usually attributed as the cause of weakness in patients with nemaline myopathy and cytoplasmic rods (Hutchinson et al., 2006).
Finally, why do cells accumulate actin inside the nucleus? High levels of nuclear actin are known to interfere with developmental programs and are toxic at the cellular level (Perrimon et al., 1989; Stuven et al., 2003). The sequestration of actin into intranuclear aggregates may be part of a protective response by the cell which minimizes the impact of unincorporated mutant actin in the cytoplasm. Thus, packing of the mutant actin into intranuclear aggregates and their cross-linking by the -actinins may be an effective way for the cell to minimize the impact of the mutation on sarcomeric assembly. However, once formed, the presence of large aggregates within the nucleus can disrupt nuclear morphology and impair normal cell function.
A limitation of our tissue culture model is that our observations are in mononuclear myoblasts whereas mature muscle fibres are multinucleate. This limits extrapolation of our results to determine the effect on whole skeletal muscle in vivo. Nevertheless, our study provides valuable information concerning the time-course and mechanisms underlying intranuclear rod formation and provides indirect evidence that the presence of these structures disrupts the function of the muscle cell, which is likely to impact on muscle bulk and muscle strength in humans with IRM.
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Acknowledgements |
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We thank Dr Beric Henderson (University of Sydney, Sydney, Australia) for his gift of the pRev(NES)-GFP construct. This work was funded by the National Health and Medical Research Council (NHMRC #139039 and #403941) Australia. A.D. is supported by Australian Postgraduate Award (APA) scholarship.
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References |
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TopSummaryIntroductionMaterial and MethodsResultsDiscussionReferences |
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Bohnsack MT, Stuven T, Kuhn C, Cordes VC, Gorlich D. A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes. Nat Cell Biol (2006) 8:257–63.[CrossRef][Web of Science][Medline]
Carlier MF, Laurent V, Santolini J, Melki R, Didry D, Xia GX, et al. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J Cell Biol (1997) 136:1307–22.
Chhabra D, dos Remedios CG. Cofilin, actin and their complex observed in vivo using fluorescence resonance energy transfer. Biophys J (2005) 89:1902–8.[CrossRef][Web of Science][Medline]
Clarke NF, Ilkovski B, Cooper S, Valova VA, Robinson PJ, Nonaka I, et al. The pathogenesis of ACTA1-related congenital fiber type disproportion. Ann Neurology (2007) 61:552–61.[CrossRef][Web of Science][Medline]
Cooper ST, Lo HP, North KN. Single section Western blot: improving the molecular diagnosis of the muscular dystrophies. Neurology (2003) 61:93–7.
Costa CF, Rommelaere H, Waterschoot D, Sethi KK, Nowak KJ, Laing NG, et al. Myopathy mutations in alpha-skeletal-muscle actin cause a range of molecular defects. J Cell Sci (2004) 117:3367–77.
Goebel HH, Anderson JR, Hubner C, Oexle K, Warlo I. Congenital myopathy with excess of thin myofilaments. Neuromuscul Disord (1997a) 7:160–8.[CrossRef][Web of Science][Medline]
Goebel HH, Piirsoo A, Warlo I, Schofer O, Kehr S, Gaude M. Infantile intranuclear rod myopathy. J Child Neurol (1997b) 12:22–30.
Goebel HH, Warlo I. Nemaline myopathy with intranuclear rods–intranuclear rod myopathy. Neuromuscul Disord (1997) 7:13–9.[CrossRef][Web of Science][Medline]
Gonsior SM, Platz S, Buchmeier S, Scheer U, Jockusch BM, Hinssen H. Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody. J Cell Sci (1999) 112:797–809.[Abstract]
Henderson BR, Eleftheriou A. A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. Exp Cell Res (2000) 256:213–24.[CrossRef][Web of Science][Medline]
Hutchinson DO, Charlton A, Laing NG, Ilkovski B, North KN. Autosomal dominant nemaline myopathy with intranuclear rods due to mutation of the skeletal muscle ACTA1 gene: clinical and pathological variability within a kindred. Neuromuscul Disord (2006) 16:113–21.[CrossRef][Web of Science][Medline]
Ilkovski B, Cooper ST, Nowak K, Ryan MM, Yang N, Schnell C, et al. Nemaline myopathy caused by mutations in the muscle alpha-skeletal-actin gene. Am J Hum Genet (2001) 68:1333–43.[CrossRef][Web of Science][Medline]
Ilkovski B, Nowak KJ, Domazetovska A, Maxwell AL, Clement S, Davies KE, et al. Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms. Hum Mol Genet (2004) 13:1727–43.
Kaindl AM, Ruschendorf F, Krause S, Goebel HH, Koehler K, Becker C, et al. Missense mutations of ACTA1 cause dominant congenital myopathy with cores. J Med Genet (2004) 41:842–8.
Laing NG, Clarke NF, Dye DE, Liyanage K, Walker KR, Kobayashi Y, et al. Actin mutations are one cause of congenital fibre type disproportion. Ann Neurol (2004) 56:689–94.[CrossRef][Web of Science][Medline]
Lazarides E. Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells. J Cell Biol (1976) 68:202–19.
Lenart P, Bacher CP, Daigle N, Hand AR, Elis R, Terasaki M, et al. A contractile nuclear actin network drives chromosome congression in oocytes. Nature (2005) 436:812–8.[CrossRef][Medline]
Matsuzaki F, Matsumoto S, Yahara I, Yonezawa N, Nishida E, Sakai H. Cloning and characterization of porcine brain cofilin cDNA. Cofilin contains the nuclear transport signal sequence. J Biol Chem (1988) 263:11564–8.
Minamide LS, Striegl AM, Boyle JA, Meberg PJ, Bamburg JR. Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function. Nat Cell Biol (2000) 2:628–36.[CrossRef][Web of Science][Medline]
Nakagawa T, Engler JA, Sheng M. The dynamic turnover and functional roles of alpha-actinin in dendritic spines. Neuropharmacology (2004) 47:734–45.[CrossRef][Web of Science][Medline]
Nishida E, Iida K, Yonezawa N, Koyasu S, Yahara I, Sakai H. Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells. Proc Natl Acad Sci USA (1987) 84:5262–6.
Nowak KJ, Wattanasirichaigoon D, Goebel HH, Wilce M, Pelin K, Donner K, et al. Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy. Nat Genet (1999) 23:208–12.[CrossRef][Web of Science][Medline]
Pendleton A, Pope B, Weeds A, Koffer A. Latrunculin B or ATP depletion induces cofilin-dependent translocation of actin into nuclei of mast cells. J Biol Chem (2003) 278:14394–400.
Perrimon N, Engstrom L, Mahowald AP. Zygotic lethals with specific maternal effect phenotypes in Drosophila melanogaster. I. Loci on the X chromosome. Genetics (1989) 121:333–52.
Rajfur Z, Roy P, Otey C, Romer L, Jacobson K. Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat Cell Biol (2002) 4:286–93.[CrossRef][Web of Science][Medline]
Sparrow JC, Nowak KJ, Durling HJ, Beggs AH, Wallgren-Pettersson C, Romero N, et al. Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1). Neuromuscul Disord (2003) 13:519–31.[CrossRef][Web of Science][Medline]
Stuven T, Hartmann E, Gorlich D. Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes. EMBO J (2003) 22:5928–40.[CrossRef][Web of Science][Medline]
Wada A, Fukuda M, Mishima M, Nishida E. Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein. EMBO J (1998) 17:1635–41.[CrossRef][Web of Science][Medline]
Young KG, Kothary R. Spectrin repeat proteins in the nucleus. Bioessays (2005) 27:144–52.[CrossRef][Web of Science][Medline]
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