Abstract
Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.
Introduction
Skeletal muscle membrane repair became a topic of intense research interest when it was implicated in the pathogenesis of muscular dystrophy. Mutations in the dysferlin gene were identified as a new cause of muscular dystrophy (Bashir et al., 1998; Liu et al., 1998), and dysferlin-deficient mouse myofibers were shown to demonstrate defective calcium-dependent membrane repair (Bansal et al., 2003). Dysferlin is a member of the ferlin family of vesicle fusion proteins characterized by the rare feature of six or seven tandem C2 domains (Lek et al., 2012), motifs associated with calcium-regulated lipid or protein binding. Thus, dysferlin emerged as a key mediator of calcium-activated vesicle-mediated membrane repair. Pathologies relating to defective vesicle fusion link ferlin animal models (Washington and Ward, 2006; Covian-Nares et al., 2010), with mutations in human otoferlin causing a form of inherited human deafness resulting from defective synaptic vesicle fusion in the cochlea (Roux et al., 2006; Dulon et al., 2009; Johnson and Chapman, 2010).
Recently, it has been demonstrated that dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53, also called TRIM72) (Cai et al., 2009b). MG53 bears the characteristic structural motifs of a TRIM-domain E3-ubiquitin ligase and rapidly accumulates at sites of membrane damage (Cai et al., 2009b). MG53 knock-out mice display a mild, progressive muscular dystrophy that is also characterized by defective membrane resealing of skeletal myofibers (Cai et al., 2009a). The substrate of the MG53 E3 ligase is unknown; however, MG53 is proposed to participate in calcium-independent stages of membrane resealing via formation of a disulphide-linked protein scaffold to which membrane repair components bind (Cai et al., 2009a), such as dysferlin.
Although it has long been established that damaged cells require calcium to survive a membrane injury (Steinhardt et al., 1994), molecular roles for calcium in the resealing response are unclear. There are three families of calcium-binding proteins with proposed roles in membrane repair: dysferlin (Bansal et al., 2003), annexins (McNeil et al., 2006; Bouter et al., 2011), and calpains (Mellgren et al., 2009). We developed a novel ballistics assay to study the acute responses of human skeletal myotubes to a membrane injury. Using rapid fixation, we “freeze-frame” injury repair, and have used confocal and super-resolution 3D-structured illumination microscopy (3D-SIM) to reconstruct the spatial and temporal assembly of endogenously expressed muscle membrane repair proteins. Our results unify discrete calcium-dependent roles for activated calpains and dysferlin that function upstream of recruited annexins. Moreover, we propose the calcium dependence of membrane repair involves unique interplay between cellular signaling pathways activated both by calcium entry via the membrane lesion, and by L-type voltage-gated calcium channels (VGCCs) in response to the persistent depolarization induced by a membrane breach. Together, these signaling pathways initiate a specialized response to membrane injury.
Materials and Methods
Primary myoblast culture
Human muscle biopsy samples were minced into 1–2 mm pieces and transferred into a scored, collagen-coated T25 with 1 ml of growth media containing 20% FCS, 10% amniomax, 1:200 gentamycin, 1:1 DMEM:Ham's F12 (Invitrogen). Flasks were sealed to retain humidity and supplemented with 2 ml fresh growth media after 48 h. Myoblast outgrowth was typically observed 48–72 h after plating. Expanded cultures were trypsinized, and myoblast-rich cultures derived through fluorescent cell sorting for anti-NCAMPE-CY7 labeling (mouse anti-human CD56, BD Biosciences). Myoblasts were induced to differentiate by culture in differentiation media (1:1 DMEM:Ham's F12, 3% horse serum, 1:200 gentamycin) for 4–5 d.
Control myoblasts.
Biopsies (male and female) subjected to malignant hypothermia testing with normal in vitro contracture results.
Patient myoblasts.
Patient myoblasts were as follows: Dysferlinopathy 1 (female), L344P, and splice acceptor site preceding exon 49; Dysferlinopathy 2 and Dysferlinopathy 3 are male siblings, Q1061_A1062insAE and R1586X; sarcoglycanopathy homozygous R77C (male); caveolinopathy E33K (female). Ethical approval for this research project was provided by the Children's Hospital at Westmead (10/CHW/45).
Ballistics-induced membrane damage
Ballistics-induced injury was performed using a Bio-Rad Helios Gene Gun with bullet cartridges prepared using silica microparticles (4 μm diameter, Sigma). Myotubes on Thermanox coverslips and differentiated for 4–6 d were shot at 300 psi in a 24-well containing 200 μl PBS (Invitrogen) in a laminar flow hood. Immediately after discharging the gun, 200 μl PBS was immediately added to the well to refresh the fluid expelled by the helium blast.
Minus calcium conditions.
Coverslips were preincubated in 500 μl calcium-free PBS containing 10 mm EGTA for 30 s, then shot in 200 μl calcium-free PBS.
Trypan permeability.
To assess membrane permeability of shot myotubes, trypan blue was added to shot and unshot myotubes at 2, 10, 30, 60, and 120 s after injury. Shot myotubes were shown to exclude trypan blue at 120 s after injury in the presence of calcium, but not in calcium-free buffer, consistent with impaired resealing in −Ca conditions.
Calcium-titration.
Calcium-free PBS was mixed at the appropriate ratio with calcium-containing PBS (0.9 μm). Addition of calcium chloride was used for concentrations >0.9 μm. All solutions used were equalized to room temperature (23°C–26°C).
Calcium-channel inhibitors.
A total of 100 mm stocks of cadmium-, nickel-, and gadolinium-chloride (Sigma) were diluted in PBS to a final concentration of 800 μm. Stocks of diltiazem (50 mm in MQ), verapamil (50 mm in MQ), and nifedipine (50 mm in DMSO) were freshly prepared and diluted in PBS just before the experiment. Differentiated human myotubes were preincubated with diltiazem (50 μm), nifedipine (100 μm), or verapamil (100 μm) for 1 h in culture media before injury, preincubated for 30 s in PBS containing the same inhibitor, then shot in PBS containing the inhibitor.
Immunocytochemistry
For the ballistics time course, the T2 sec sample was obtained by flooding the shot well with 500 μl ice-cold fixative (3% PFA in PBS containing 20% sucrose, pH 7). For other time points, shot coverslips were picked up using a bent needle and forceps and transferred onto droplets of ice-cold fixative on a Parafilm-covered glass plate resting on ice. After fixation, coverslips were washed (3 dips in room temperature PBS baths), permeabilized with PBS plus 0.15% saponin for 10 min, washed, devitellinized with room temperature methanol/acetone 1:1 for 4 min, washed, blocked (PBS containing 2% BSA) for 30 min, then incubated in primary antibody diluted in block overnight in the cold room. Coverslips were washed, reblocked for 15 min, incubated with secondary antibody diluted in block for 2 h, washed and mounted on a 22 × 50 mm glass coverslips with a droplet of Fluorsave (Calbiochem) or Vectashield (Vector Laboratories) mounting reagents.
Antibodies
Western blot.
Western blot included the following: Hamlet-1 (Novocastra, 1:500), Romeo-1 (Epitomics, 1:500), anti-sarcomeric actin 5C5 (Sigma, 1:1000), and anti-GAPDH (Millipore Bioscience Research Reagents, 1:2000).
Immunocytochemistry.
Immunocytochemistry included the following: Hamlet-1 (1:25), Hamlet-2 (Novocastra, 1:25), Romeo-1 (1:200), SAB2100636 (Sigma, 1:200), MG53 rabbit polyclonal (gift from Prof. Jianjie Ma, 1:200), annexin A1 (BD Biosciences, 1:100), annexin A2 (BD Biosciences, 1:100), AHNAK (Sapphire Bioscience, 1:250), caveolin-3 (BD Biosciences, 1:500), syntaxin-4 (Synaptic Systems, 1:250), Munc-18 (Edwards Scientific, 1:250), dystrophin (Novocastra, NCL-DYS3, 1:50), annexin V (Sapphire Bioscience, 1:50), LAMP-1 (Hybridoma Bank, H4A3, 1:500), GM130 (BD Biosciences, 1:100), VAMP-4 (Synaptic Systems, 1:200), and goat anti-rabbitAlexa594, goat anti-rabbitAlexa555, and donkey anti-mouseAlexa488 (Invitrogen, 1:200).
Scrape injury and cell harvesting
Cells grown in 6-well plates were washed once with 2 ml ice-cold PBS containing freshly added protease inhibitor mixture (1:500, Sigma-Aldrich, P8340), then solubilized in 250 μl RIPA (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mm EDTA, 1:500 protease inhibitor mixture) for 30 min on ice with gentle rocking. Samples were collected and centrifuged for 1 min at 13,000 × g to pellet cell debris. Supernatant was transferred to a fresh tube and snap frozen at −80°C. Cells grown in 6-well plates were washed once with room temperature PBS (24°C-26°C), scraped with a rubber policeman in 500 μl PBS, and pelleted by centrifugation for 5 min at 300 × g. Supernatant was removed and cell pellets were resuspended in RIPA, rotated at 4°C for 30 min, then centrifuged for 1 min at 13,000 × g to pellet cell debris. Supernatant was transferred to a fresh tube and snap frozen at −80°C.
SDS-PAGE and Western blotting
RIPA supernatants were reconstituted to 1 × SDS loading buffer (1% SDS 5.5% glycerol, 55 mm DTT, bromophenol blue). A total of 10 μg total protein was separated on Nu-PAGE 4–12% gradient gels, transferred to PVDF membranes, and probed with antibodies diluted in block (PBS plus 0.1% Tween, 5% skim milk) as previously described (Waddell et al., 2011).
Microscopy
Confocal.
Images were captures using a Leica SP2 or SP5 scanning confocal microscope with a 63× oil objective.
3D-SIM.
Imaging was performed using a DeltaVision OMX V3 3D-Structured Illumination Microscopy System (OMX 3D-SIM, Applied Precision) as previously described (Riglar et al., 2011). Raw 3-phase images were reconstructed as previously described (Gustafsson et al., 2008; Schermelleh et al., 2008). Reconstructed images were rendered in 3D, with interpolation, using IMARIS v. 7.4 (Bitplane Scientific). Colocalization correlation was performed according to Costes et al. (2004), using IMARIS software. Correct alignment of the individual fluorescent channels was validated using Tetraspec beads (Invitrogen) before image capture.
Results
Our study exploited the advantage of rapid ballistics injury to resolve the acute response of human skeletal myotubes to membrane injury, with the specific goal of determining the interplay between the calcium-independent injury response proposed for MG53 (Cai et al., 2009a), with calcium-dependent elements of membrane repair proposed for dysferlin (Bansal et al., 2003). Using trypan blue exclusion as a marker of membrane permeability, we established ballistics conditions that created membrane lesions that were repairable within 2 min (data not shown, see Materials and Methods). Immunolabeling of coverslips damaged via ballistics at 10 s after injury reveals that both dysferlin and MG53 show rapid, calcium-dependent stages of injury recruitment (Fig. 1a, 10 s after injury). 3D-SIM reveals that, in the presence of physiological calcium, dysferlin and MG53 compartments interdigitate to form a concentrated lattice, specifically bound to exposed lipids encircling ballistics lesions (Fig. 1a, left, +Ca). When extracellular calcium is chelated by EGTA (30 s) and myotubes are shot in calcium-free buffer, dysferlin and MG53 still show enrichment to injury sites but appear as diffuse halos surrounding ballistics lesions (Fig. 1a, right). Moreover, rotation of −Ca lesions in the x-z plane reveals dysferlin and MG53 occupy distinct spatial planes, with dysferlin-rich vesicular compartments lying on the cytoplasmic face of accumulated MG53 on the plasma membrane (Fig. 1a, right, bottom rows). Using only brief chelation with EGTA (10 s vs 30 s), we captured intermediates of dysferlin and MG53 merging, with small rafts of aligned but unfused dysferlin-rich vesicles on the cytoplasmic face of MG53 clusters at the plasma membrane (Fig. 1a, middle). By titrating levels of extracellular calcium, we show that ≥200 μm [Ca]EC provides the critical calcium concentration required to activate fusion of dysferlin and MG53 compartments, and their refinement into a concentrated lattice specifically bound to the exposed lipids at the edge of injury sites (Fig. 1b).
We occasionally observed ballistics lesions positive for MG53 but negative for dysferlin (Fig. 1c). At these sites, MG53 labeling remained as a diffuse halo, similar to −Ca conditions. Furthermore, distal to injury sites, and in −Ca conditions, we observed little or no colocalization of dysferlin and MG53 (Fig. 1a,d; Pearson coefficient <0.4). Collectively, our results imply that MG53 and dysferlin have separate routes of injury-activated recruitment. Therefore, dysferlin is not required to trigger MG53 injury recruitment but is associated with a calcium-dependent phase of MG53 accumulation at injury sites.
We next studied the temporal sequence of injury-activated recruitment of MG53 and dysferlin, with that of annexin-A1, a phospholipid-binding protein also implicated in membrane repair (McNeil et al., 2006). We have previously shown that annexin-A1, together with dysferlin and MG53, colabel damaged longitudinal tubules in overstretched human muscle (Waddell et al., 2011). MG53 showed diffuse enrichment at injury sites at 2 s after injury (Fig. 2a), before robust detection of dysferlin. By 10 s after injury, MG53 and dysferlin intensely labeled the circumference of the ballistics lesions. In contrast, enrichment of vesicular compartments labeled by annexin-A1 only became evident at 20–30 s post-injury, but did not show specificity for the lesion site, instead showing amorphous labeling peripheral to the membrane lesions (Fig. 2b). The concentrated lattice formed by dysferlin and MG53 encircling injury sites at early time points expands and spreads throughout the injury site during repair (Fig. 2a, 30–90 s after injury). At 90 s after injury, lesions appear as “filled patches” that appear as a network of invaginating membranes (Fig. 2a, 90 s), often characterized by a dominant arc of labeled membrane (Fig. 2b, 90 s; see Fig. 4, 90 s). 3D-SIM of two lesions at 90 s after injury represent the two outcomes observed at this time point, an unfilled lesion with an expansive dysferlin and MG53 lattice surrounding the injury site (Fig. 2c, top), and a filled lesion with the characteristic arc of strongly labeled dysferlin and MG53 among a bed of interdigitated lattice (Fig. 2c, bottom).
Repair of ballistics injury in human skeletal myotubes showed calcium-dependent expansion and contraction phases of membrane resealing (Fig. 2d), as previously reported using an in vivo model of membrane injury within a developing Drosophila embryo (Abreu-Blanco et al., 2011). We did not observe injury-enriched labeling for a panel of additional dysferlin-interacting or muscular dystrophy proteins, or endomembrane or vesicle markers (Fig. 3). In contrast to results by Reddy et al. (2001.) in fibroblasts, lysosomal markers were not enriched at sites of membrane injury in human myotubes, although we occasionally observed evidence for lysosomal exocytosis at sites distal to the membrane injury (Fig. 3).
A calpain-cleaved C-terminal fragment of dysferlin, mini-dysferlinC72, has a specialized role in membrane repair
Our ballistics assay also revealed that dysferlin may be detected at sites of injury with antibodies recognizing a C-terminal epitope (Hamlet-1), but not N-terminal epitopes (i.e., Romeo, Hamlet-2, SAB2100636) (Fig. 4), raising the possibility of a truncated dysferlin isoform. We used Western analysis to look for a “mini-dysferlin” and observed a ∼72 kDa C-terminal dysferlin isoform in myotubes shot in the presence of calcium but absent in myotubes shot without extracellular calcium and in unshot myotubes (Fig. 5a). To obtain greater quantities of protein for biochemical studies, we induced mechanical injury to the myotube plasma membrane by scraping cells from the tissue culture substratum with a rubber policeman, and similarly observed calcium-dependent formation of a ∼72 kDa C-terminal dysferlin fragment with scrape injury (Fig. 5b), herein referred to as mini-dysferlinC72. Injury-specific formation of mini-dysferlinC72 was not observed in primary myotubes from three dysferlinopathy patients (Fig. 5c, D1–3), each possessing at least one allele with an intact 3′ region of their DYSF gene. These results negate the likelihood of an alternate splice isoform and suggest mini-dysferlinC72 is derived from full-length dysferlin.
Closely correlating with our ballistics immunostaining results, robust detection of mini-dysferlinC72 by Western blot required an activating concentration of extracellular calcium ≥200 μm (Fig. 5d, Hamlet-1) and was accompanied by detection of the corresponding N-terminal ∼170 kDa cleavage product (Fig. 5d, Romeo-1). With results showing injury-dependent and calcium-dependent dysferlin cleavage, we explored a role for calpains and showed that pretreatment of human skeletal myotubes with the calpain inhibitor calpeptin significantly attenuated formation of mini-dysferlinC72 by Western blot with scrape injury (Fig. 5e) and reduced levels of recruited dysferlin in myotubes damaged by ballistics (Fig. 5g). The levels of cleaved mini-dysferlinC72 relative to full-length dysferlin did not vary with myogenic differentiation, suggesting that capacity to cleave dysferlin in response to injury is not dependent upon the myogenic maturity of the sample (Fig. 5e). Through refinement of the calpeptin treatment regimen, we determined maximal inhibition of injury-activated dysferlin cleavage with ≥30 μm calpeptin using 3 h preincubation and refreshment of a third of the media 30 min before injury (Fig. 5f). To further support a role for calpains in the injury-specific cleavage of dysferlin, we performed scrape injury experiments in PBS buffered from pH 5.5–8.5, and demonstrated that maximum levels of cleaved mini-dysferlinC72 occur at neutral pH 7.5 (Fig. 5h).
Collectively, our results indicate that dysferlin is a substrate of activated calpains, releasing a C-terminal fragment with a specific role in membrane repair. Molecular weight calculations predict that mini-dysferlinC72 includes the two most ancestrally conserved C2 domains (Lek et al., 2010), with structural parallels to the classical mediators of vesicle fusion, the synaptotagmins.
Injury-recruitment of dysferlin and MG53 is blocked by cadmium and attenuated by L-type voltage-gated channel antagonists
Given that otoferlin (Ramakrishnan et al., 2009) and sea-urchin ferlin (Covian-Nares et al., 2010) are regulated by specific VGCCs, we explored a role for VGCC in the regulation of dysferlin in membrane repair. First, we used broad nonspecific blockers of different classes of VGCC and showed cadmium (Cd; blocks L-, P-, and R-types), but not nickel (Ni; blocks P/Q-, R-, and T-types) or gadolinium (Gd; blocks stretch-activated channels), blocked recruitment of MG53 and dysferlin to sites of membrane injury, and detection of mini-dysferlinC72 by Western blot (Fig. 6a). Cadmium is a classical inhibitor of L-type channels, although it may exert off-target effects with access to the cellular cytoplasm after membrane injury. Therefore, we examined the effects of specific L-type channel antagonists and showed that diltiazem, nifedipine, and verapamil blocked or attenuated injury-induced recruitment of dysferlin and MG53 (Fig. 6b). None of the specific L-type antagonists blocked formation of cleaved mini-dysferlinC72 detected by Western blot (Fig. 6e); rather, they appeared to induce an uncoordinated vesicle fusion response, with “clumps” of mini-dysferlinC72 and MG53 at the lesion circumference (Fig. 6b,c). Super-resolution 3D-SIM shows distinct spatial separation of the dysferlin and MG53 in the presence of L-type VGCC antagonists, with dysferlin (green) lying on the cytoplasmic face of MG53 (red) membrane compartments (Fig. 6c), similar to low-calcium conditions (Fig. 1a). In the case of verapamil treatment, we also observed formation of a population of abnormally large vesicles containing cleaved mini-dysferlinC72 and MG53 (i.e., positive for C-terminal Hamlet-1, but negative for N-terminal Romeo-1) (mean diameter 0.8 μm, range 0.3–3.7 μm; Fig. 6d). Our results suggest that, despite promiscuous calcium entry through wound sites, VGCCs play a role in coordinating the regulated fusion of mini-dysferlinC72 and MG53 compartments for membrane repair.
Discussion
Collectively, our results implicate two cooperative calcium signaling cascades for muscle membrane repair, separately activated by calcium entry through membrane lesions, as well as specific calcium signaling via L-type VGCC. We refine the muscle membrane repair paradigm to propose the following working model (Fig. 7). Membrane injury causes promiscuous influx of calcium at sites of membrane injury, local activation of calpains, and strong and persistent depolarization of L-type VGCC. Dysferlin is cleaved by activated calpains, releasing a C-terminal fragment, mini-dysferlinC72. Mini-dysferlinC72-rich cytoplasmic vesicles are rapidly recruited to sites of membrane injury and fuse with MG53 decorated plasma membrane compartments in a process coordinated by L-type VGCC. Mini-dysferlinC72 undergoes calcium-dependent protein or phospholipid binding via its C2 domains, initiating a calcium-dependent phase of MG53 injury recruitment. The role of MG53 at sites of membrane injury may relate to its functions as a phosphatidylserine binding protein and a ubiquitin ligase; MG53 may be initially recruited via calcium-independent mechanisms to rapidly ubiquitinate target receptors/channels (currently unknown) damaged by the membrane injury to target them for recycling/repair or degradation.
3D-SIM provides the first evidence that mini-dysferlinC72 is delivered to sites of membrane injury in cytoplasmic vesicles that align and fuse with plasma membrane domains enriched with MG53 in a process coordinated by L-type VGCCs, a role consistent with VGCC-regulated vesicle fusion of mammalian otoferlin (Ramakrishnan et al., 2009) and sea-urchin ferlin (Covian-Nares et al., 2010). Interestingly, a role for L-type channels in response to acute membrane injury of transected spinal cord axons has been reported (Nehrt et al., 2007), suggesting that L-type channels may mediate cellular signaling in response to acute membrane injuries in other tissues.
Annexin-A1 did not specifically label injury sites, and showed different temporal activation and biodistribution in injured myotubes, suggesting that annexin-A1 may participate in a separate arm of the membrane repair response. Repaired lesions were often characterized by a dominant arc of mini-dysferlinC72 and MG53 among an interdigitated lattice. This pattern of labeling is consistent with a repair model whereby mini-dysferlinC72 and MG53 initially bind the exposed edge of the damage site, then infiltrate and stabilize the surrounding bilayer as it expands to reseal the membrane injury. The membrane arc represents the edges of the original lesion that are elongated, aligned, and pushed centrally as new membrane is added to the lesion surrounds to facilitate resealing of the membrane breach.
A specific role for mini-dysferlinC72 in membrane repair is consistent with studies described by Roostalu and Strahle (2012), who developed an elegant in vivo model of muscle membrane damage in a developing zebrafish embryo and showed that C-terminal fragments of ectopically expressed human dysferlin showed injury recruitment, but N-terminal fragments did not. Furthermore, biologically derived mini-dysferlinC72 bears similarity to a truncated dysferlin identified in a mildly affected dysferlinopathy patient with a genomic deletion within the DYSF gene (Krahn et al., 2010). Remarkably, this patient-derived mini-dysferlin also bears the last two C2 domains and transmembrane domain and was shown to functionally restore defective membrane repair when expressed in dysferlin-deficient mouse muscle fibers (Krahn et al., 2010).
Our discovery that activated calpains generate mini-dysferlinC72 with a specialized role in membrane repair reveals a novel function for calpains in the cellular response to membrane injury and is consistent with studies by Mellgren et al. (2009) that establish a requirement for m- or μ-calpain, but not calpain-3, for muscle membrane repair. m-Calpain and μ-calpain are named based on their activating calcium concentrations in the millimolar or micromolar range, respectively (Cong et al., 1989; Goll et al., 2003). In our study, 200 μm extracellular calcium was the critical concentration required for both biochemical detection of cleaved mini-dysferlinC72 (Fig. 5) and for tight refinement of dysferlin and MG53 into concentrated rings encircling sites of membrane injury by immunolabeling (Fig. 1). However, this calcium concentration does not clearly implicate one calpain over another and may also reflect a critical extracellular calcium concentration for conductance and signaling by L-type calcium channels to recruit or activate vesicle fusion.
Analysis of the biodistribution (Murphy et al., 2006), autolysis (Murphy et al., 2006), and damage-induced activity (Gailly et al., 2007) of calpains in single muscle fibers reveals a population of membrane-associated preactivated μ-calpain previously proposed as ideally positioned to rapidly mediate a role in membrane resealing (for review, see Lamb, 2007). In −Ca conditions, a cleaved mini-dysferlinC72 (positive for Hamlet-1 but negative for the N-terminal Romeo-1 epitope) shows a diffuse halo of enrichment within 10 seconds of a membrane injury. However, it is important to consider that ballistics will also structurally damage intracellular calcium stores and induce local elevations in intracellular calcium that may be sufficient for calpain activation. Further studies are required to define roles of μ-calpain and m-calpain in dysferlin cleavage and membrane repair, to refine feasible scenarios that include potential roles for both isoforms; for example, preactivated μ-calpain may cleave dysferlin, whereas activated m-calpain may remodel the cytoskeleton associated with the expansion and contraction stages of membrane repair (Fig. 2) (Abreu-Blanco et al., 2011).
Collectively, our results confirm that muscle cells use a mechanism similar to synaptic exocytosis for membrane repair (Steinhardt et al., 1994). The structural similarity between synaptotagmins and the predicted structure of cleaved mini-dysferlinC72 cannot be overlooked, and suggests functional parallels between protein- or phospholipid-binding roles of mini-dysferlinC72 in membrane repair and that of synaptotagmin for secretory exocytosis. Importantly, our results suggest that cellular functions of dysferlin, and perhaps other mammalian ferlins, may be diversified via enzymatic cleavage to release different C2 domain-containing fragments with specialized functions. Our results shed new light on the established paradigm of calcium-dependent membrane repair, highly relevant to disease pathogenesis in dysferlinopathy, and perhaps also relevant to other pathologies characterized by calcium signaling with membrane injury.
Footnotes
This work was supported by the Australian National Health and Medical Research Council Project Grant 570744 to S.T.C. and K.N.N., Australian Postgraduate Award to A.L., Fellowship 571905 to C.B.W., the Jain Foundation (S.T.C.), the NSW Muscular Dystrophy Association (S.T.C., K.N.N.), and the Brain Foundation (S.T.C., K.N.N.). We thank Dr Neil Street (Department of Anaesthesia, Children's Hospital at Westmead) for his assistance with patient and control biopsy samples for primary culture; Dr. X. M. Wang in the Flow Cytometry Core Facility at Westmead Millennium Institute (supported by the Australian National Health and Medical Research Council and Cancer Institute New South Wales) for performing flow cytometry; Prof. Jianjie Ma and Dr. Noah Weisleder (UMNDJ, Piscataway, New Jersey) for the polyclonal anti-MG53 antibody; Bodson designs (Sydney, Australia) for the graphic design for Figure 7; and our patients and their families.
The authors declare no competing financial interests.
- Correspondence should be addressed to Dr. Sandra T. Cooper, Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia. sandra.cooper{at}sydney.edu.au