Dysferlin Interacts with Annexins A1 and A2 and Mediates Sarcolemmal Wound-healing
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m307247200
ISSN1083-351X
AutoresNiall J. Lennon, Alvin T. Kho, Brian J. Bacskai, Sarah L. Perlmutter, Bradley T. Hyman, Robert H. Brown,
Tópico(s)RNA Research and Splicing
ResumoMutations in the dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes. Mutations in the dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes. Mutations in the dysferlin gene DYSF cause limb girdle muscular dystrophy (LGMD) 1The abbreviations used are: LGMDlimb girdle muscular dystrophyPBSphosphate-buffered salineFLIMfluorescence lifetime imaging microscopyDAPI4′,6-diamidino-2-phenylindole.1The abbreviations used are: LGMDlimb girdle muscular dystrophyPBSphosphate-buffered salineFLIMfluorescence lifetime imaging microscopyDAPI4′,6-diamidino-2-phenylindole. type 2B and Miyoshi myopathy (1.Liu J. Aoki M. Illa I. Wu C. Fardeau M. Angelini C. Serrano C. Urtizberea J.A. Hentati F. Hamida M.B. Bohlega S. Culper E.J. Amato A.A. Bossie K. Oeltjen J. Bejaoui K. McKenna-Yasek D. Hosler B.A. Schurr E. 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Genet. 1999; 8: 871-877Crossref PubMed Scopus (161) Google Scholar) that shows homology to the Caenorhabditis elegans FER1 protein thought to mediate fusion of intracellular vesicles to the sperm plasma membrane (6.Achanzar W.E. Ward S. J. Cell Sci. 1997; 110: 1073-1081Crossref PubMed Google Scholar). Dysferlin has a single transmembrane domain at its C terminus and six C2 domains along the length of the cytoplasmic domain (7.Aoki M. Liu J. Richard I. Bashir R. Britton S. Keers S.M. Oeltjen J. Brown H.E. Marchand S. Bourg N. Beley C. McKenna-Yasek D. Arahata K. Bohlega S. Cupler E. Illa I. Majneh I. Barohn R.J. Urtizberea J.A. Fardeau M. Amato A. Angelini C. Bushby K. Beckmann J.S. Brown Jr., R.H. Neurology. 2001; 57: 271-278Crossref PubMed Scopus (117) Google Scholar). Recent studies have illustrated Ca2+-dependent binding of the first C2 domain of dysferlin to phospholipid; a mutation that causes muscular dystrophy negatively affects this binding (8.Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Dysferlinopathic muscle tissue reveals an accumulation of vesicles at the plasma membrane (9.Selcen D. Stilling G. Engel A.G. Neurology. 2001; 56: 1472-1481Crossref PubMed Scopus (132) Google Scholar, 10.Bansal D. Miyake K. Vogel S.S. Groh S. Chen C.C. Williamson R. McNeil P.L. Campbell K.P. Nature. 2003; 423: 168-172Crossref PubMed Scopus (748) Google Scholar). We have demonstrated previously a weak interaction between dysferlin and caveolin-3, a skeletal and cardiac muscle protein that organizes lipid and protein components of caveolae (11.Matsuda C. Hayashi Y.K. Ogawa M. Aoki M. Murayama K. Nishino I. Nonaka I. Arahata K. Brown Jr., R.H. Hum. Mol. Genet. 2001; 10: 1761-1766Crossref PubMed Scopus (199) Google Scholar).To elucidate the function of dysferlin, we examined gene expression patterns in normal and dysferlin-deficient mice at different ages and in different muscle compartments. We have employed a novel analysis algorithm, Relevance Networks (12.Butte A.J. Tamayo P. Slonim D. Golub T.R. Kohane I.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12182-12186Crossref PubMed Scopus (463) Google Scholar), that correlates relative gene expression levels in high-density microarray samples. The algorithm identified a novel cluster of genes whose relative expression levels are highly correlated in all dysferlinopathic samples. Examination of this network prompted us to investigate further the roles of annexin A1 and annexin A2 in the dysferlinopathies.Annexins are widely expressed Ca2+- and phospholipid-binding proteins that are implicated in membrane trafficking, transmembrane channel activity, inhibition of phospholipase A2 and cell-matrix interactions (13.Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1022) Google Scholar). The functions of many of the annexins are not clear. However, annexins A1 and A2 have been shown to aggregate intracellular vesicles and lipid rafts in a Ca2+-dependent manner at the cytosolic surface of plasma membranes in many cell types (14.Babiychuk E.B. Draeger A. J. Cell Biol. 2000; 150: 1113-1124Crossref PubMed Scopus (226) Google Scholar, 15.Lambert O. Gerke V. Bader M.F. Porte F. Brisson A. J. Mol. Biol. 1997; 272: 42-55Crossref PubMed Scopus (95) Google Scholar). Annexin A1 mediates this aggregation by forming a heterotetramer with S100A11, and annexin A2 has been postulated to have a similar relationship with S100A10 (p11) (16.Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1610) Google Scholar).This investigation provides the first evidence for a Ca2+-dependent interaction between dysferlin and annexins A1 and A2. After a membrane injury, there is disruption of dysferlin binding to annexin A1, Ca2+-dependent vesicle aggregation, and fusion with the surface membrane. We show that this membrane repair process is severely upset in dysferlinopathic myotubes. These findings confirm the disrupted membrane healing seen in dysferlin knockout mice (10.Bansal D. Miyake K. Vogel S.S. Groh S. Chen C.C. Williamson R. McNeil P.L. Campbell K.P. Nature. 2003; 423: 168-172Crossref PubMed Scopus (748) Google Scholar) and extend that work by suggesting possible interacting partners for dysferlin. We propose a central role for dysferlin in "patch" fusion events that compose a novel wound healing model in skeletal muscle sarcolemma.MATERIALS AND METHODSMicroarray Sample Preparations—For the SJL/J and SWR/J samples, 10 animals of each species were killed at 6 weeks and at 8 months of age. Each group of 10 mice was divided into two groups of 5 mice each. RNA was extracted from one quadriceps and one gastrocnemius from each mouse in the group, and a pooled RNA sample from each group was prepared for hybridization to the Affymetrix murine MGU74Av2 arrays (Affymetrix, Santa Clara, CA), as described previously in detail (17.Haslett J.N. Sanoudou D. Kho A.T. Bennett R.R. Greenberg S.A. Kohane I.S. Beggs A.H. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15000-15005Crossref PubMed Scopus (283) Google Scholar). The remaining muscles were retained for subsequent immunohistochemical and Western blot analysis.Affymetrix software was used to calculate the relative expression signal of each gene from the average difference of intensities between matching and mismatched probe-pairs designed to hybridize a particular sequence. Although we obtained gene expression data using various parameters such as age and muscle group, the distinction used for analysis of relevance networks in this study was whether the samples were normal (SWR/J) or dysferlin-deficient (SJL/J).Relevance Networks—Relevance networks were generated (18.Butte A.J. Ye J. Haring H.U. Stumvoll M. White M.F. Kohane I.S. Pac. Symp. Biocomput. 2001; 6: 6-17Google Scholar) by using the RelNet software developed by Atul Butte of Children's Hospital Bioinformatics Program, Boston. 2Available from www.chip.org/relnet/. Raw data from microarrays run with samples from the gastrocnemius and quadriceps of 6-week- and 8-month-old SWR/J and SJL/J were uploaded to the RelNet software. The RelNet algorithm comprehensively compares all gene probe sets with each other in a pair-wise manner and generates networks of highly correlated genes. Networks were initially generated encompassing all samples with a correlation threshold of 0.95 and subsequently from control and dysferlin-deficient samples separately with a correlation threshold of 0.965.Co-immunoprecipitation—SWR/J protein homogenate (1 mg) was resuspended in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 12 mm deoxycholic acid). Immunoprecipitations with the polyclonal annexin antibodies were performed using Biomag protein A magnetic beads (Qiagen, Valencia, CA). Immunoprecipitation experiments using the monoclonal dysferlin antibody, NCL-Hamlet, were performed using the Catch and Release kit (Upstate Biotechnology, Lake Placid, NY).Cell Culture—Primary myoblast cells were cultured from 2-day-old SWR/J and SJL/J pups as described previously (19.Bischoff R. Heintz C. Dev. Dyn. 1994; 201: 41-54Crossref PubMed Scopus (149) Google Scholar) in medium containing 20% fetal bovine serum and 2% chick embryo extract in Dulbecco's modified Eagle's medium at 37 °C in 5% CO2. Cells were induced to differentiate and fuse at 30–50% confluency by switching to serum-deprived medium (2.5% horse serum in Dulbecco's modified Eagle's medium).Tissue Section Immunocytochemistry—Eight-micron-thick tissue sections from SWR/J and SJL/J quadriceps were fixed for 4 min with ice-cold methanol/acetone (1:1) and preincubated for 30 min with phosphate buffered saline containing 10% (v/v) goat serum prior to staining with primary antibodies using established methods. The primary antibodies were applied in three double-staining combinations as indicated. Images were collected with an Eclipse E800 M microscope (Nikon, Mellville, NY) and Spot RT Software (Sterling heights, MI).Fluorescence Lifetime Imaging Microscopy (FLIM)—SWR/J myotubes were preincubated for 10 min at 37 °C in PBS containing either 1.8 mm CaCl2 or 10 mm EGTA. Cells were injured by dragging a scalpel blade twice across the surface of the dish, in the presence of blue dextran (Molecular Probes). Cells were fixed in PBS containing 4% paraformaldehyde for 15 min and blocked in PBS containing 10% goat serum for 30 min prior to incubation with primary antibodies diluted in PBS containing 0.5% Triton-X (v/v) for 60 min at 37 °C. Baseline decay times for FLIM were gathered as described (20.Bacskai B.J. Skoch J. Hickey G.A. Allen R. Hymen B.T. J. Biomed. Optics. 2003; 8: 368-375Crossref PubMed Scopus (140) Google Scholar) from injured and non-injured cells by labeling the dysferlin in the cell with a 488-nm fluorescein fluorophore attached to either a primary antibody label or a fluorescent secondary antibody. Decay times were measured with a commercial multiphoton microscope (Radiance 2000, Bio-Rad) with attached Ti:Saphire laser (Tsunami, Spectra Physics) and a fast microchannel plate detector (Hamamatsu, Bridgewater, NJ) connected to high-speed time correlated single photon counting acquisition hardware (SPC-830; Becker & Hickl, Berlin); data was analyzed using SPCImage software (Becker & Hickl). To investigate the interaction of two proteins, the cells were incubated with both the donor 488-nm-labeled dysferlin antibody and a 594-nm (annexins A1 and A6) or 568-nm (annexin A2) labeled acceptor antibody attached to the protein of interest.Scrape Wounding and Expression of Lamp-1—SWR/J and SJL/J myotubes were preincubated for 10 min at 37 °C in PBS containing either 1.8 mm CaCl2 or 2 mm EGTA. Cells were injured by dragging a scalpel blade twice across the surface of the dish in the presence of Texas Red dextran (Molecular Probes). Dishes were transferred to ice and cells were fixed in PBS containing 4% paraformaldehyde for 15 min. They were blocked in PBS containing 10% goat serum for 30 min prior to incubation with anti-Lamp-1 (1D4B) diluted in PBS for 60 min at 37 °C. Cells were then incubated with Alexa 488-goat anti-mouse antibody (Molecular Probes) for 30 min at room temperature and with DAPI (Sigma) for 5 min. For each of the triplicate dishes, myotubes were counted according to positive DAPI, Texas Red-dextran, and anti-Lamp-1 surface staining.Microinjury and Time-lapse Fluorescence Microscopy—SWR/J and SJL/J myotubes in 35-mm culture dishes were loaded with 2 μm Indo1-AM for 20 min in Dulbecco's modified Eagle's medium with 2.5% horse serum. During imaging, myotubes were incubated in Hank's buffered saline containing 1.8 mm CaCl2. Myotubes were individually wounded by disruption of the sarcolemmal surface using a finely pulled-glass capillary attached to a micromanipulator. Regions of interest were located at or near the sites of injury, at points distant from the sites of injury, or on adjacent, non-injured cells using the Lasersharp software (Bio-Rad). The ratio of the Indo1-AM fluorescence at 405 nm to that at 485 nm was determined within each region of interest for each time point of the experiment. Images were collected through a 40× objective at time-lapse intervals of 1 s using a laser scanning confocal microscope (Radiance 2000, Bio-Rad).RESULTSRelevance Networks Reveal Functionally Related Genes in Dysferlinopathy—We first used high density oligonucleotide microarrays to compare the gene expression profiles of control muscle (SWR/J) to that of muscle from the mouse model of dysferlinopathy, the SJL/J strain (data available as supplementary Table I). The SJL/J strain is an established model of dysferlin deficiency that occurs as a result of a splice-site mutation that removes part of the C2-E domain of the dysferlin protein, resulting in a greatly reduced expression of the protein (21.Vafiadaki E. Reis A. Keers S. Harrison R. Anderson L.V. Raffelsberger T. Ivanova S. Hoger H. Bittner R.E. Bushby K. Bashir R. NeuroReport. 2001; 12: 625-629Crossref PubMed Scopus (51) Google Scholar). As a control, we used the closely related SWR/J strain whose dysferlin levels are normal."Unsupervised" data analysis identifies relationships between genes in a data set. One such analysis algorithm is that used to generate relevance networks (12.Butte A.J. Tamayo P. Slonim D. Golub T.R. Kohane I.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12182-12186Crossref PubMed Scopus (463) Google Scholar, 22.Butte A. Nat. Rev. Drug Discovery. 2002; 1: 951-960Crossref PubMed Scopus (401) Google Scholar). By using this system, genes that are highly correlated over many data sets are organized into networks, with each node representing a gene and the links between nodes representing correlations in relative expressions levels between genes. Applying the relevance network algorithm to all microarrays used in the study revealed a cluster of muscle contractile genes whose expression was correlated regardless of age or species (Table Ia). This network confirms the ability of this approach to extract functionally relevant information from the raw expression data.Table IRelevance network analysis of microarray data Open table in a new tab To determine whether there exists some group of genes whose expression levels are similarly affected by the disruption of dysferlin expression, we generated relevance networks from both the control and dysferlinopathic arrays separately. One such network is shown in Table Ib. This network, which is specific to the dysferlinopathic samples, contains genes involved in many of the processes seen in muscular atrophy. To investigate whether members of this network were functionally important in dysferlinopathy, we considered each of the members for potential functional interactions with dysferlin. The annexins (Table Ib, nodes 22, 23, and 41) were of particular interest because they have been shown to bind phospholipid in a calcium-dependant manner.Dysferlin Binds to Annexins A1 and A2 in Vitro—The altered expression of dysferlin in the SJL/J samples was confirmed by Western analysis (Fig. 1a). Immunoprecipitation experiments demonstrated that both annexins A1 and A2 co-precipitate with dysferlin from muscle homogenates but not with each other (Fig. 1b). Similarly, dysferlin co-precipitates with both annexins. By contrast, neither dysferlin nor the annexins co-immunoprecipitate with dystrophin from muscle homogenates.Dysferlin Co-localizes with Annexins A1 and A2 at the Sarcolemma—Co-localization studies on tissue sections of mouse skeletal muscle show that both annexins A1 and A2 co-localize with dysferlin at the plasma membrane in normal skeletal muscle sections (Fig. 2, a and b); some annexin A1 is also possibly evident in cytoplasm. The expression patterns of both annexins A1 and A2 appear abnormal at the membrane of dysferlin-deficient (SJL/J) muscle (Fig. 2, a and b). Normal sarcolemmal distribution of dystrophin in SJL/J mice confirms that the abnormal localization of annexin A2 is not an artifact of an already disrupted membrane (Fig. 2c). Although no dysferlin is evident on Western analysis of SJL/J muscle homogenates (Fig. 1a), some expression of this protein is visible on immunofluorescent staining of tissue sections (Fig. 2, a and b) because of the higher sensitivity of this technique.Fig. 2Dysferlin co-localizes with annexins A1 and A2. Shown are representative images of normal and SJL/J muscle tissue sections (quadriceps) co-stained for either annexin A1 (A1) (a) or annexin A2 (A2) (b) and dysferlin, or co-stained for annexin A2 (A2) and dystrophin (c).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Injury State and Ca2+Modulate the Binding of Annexins to Dysferlin—Dysferlin contains six calcium-responsive C2 domains; binding of calcium to at least one of these domains (C2A) is essential for the binding of dysferlin to phospholipid (8.Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Both annexins A1 and A2 are capable of binding to and aggregating phospholipids in a Ca2+-dependent manner (15.Lambert O. Gerke V. Bader M.F. Porte F. Brisson A. J. Mol. Biol. 1997; 272: 42-55Crossref PubMed Scopus (95) Google Scholar, 16.Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1610) Google Scholar). The aggregation and fusion of intracellular vesicles at the plasma membrane in response to a membrane injury event has been demonstrated in several non-muscle cell types (normal rat kidney, 3T3, L6E9, and Chinese hamster ovary) and has been shown to be Ca2+-dependent (23.Reddy A. Caler E.V. Andrews N.W. Cell. 2001; 106: 157-169Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). To investigate whether the interaction of dysferlin and the annexins is affected by the injury state of the sarcolemma, we performed immunofluorescent staining followed by fluorescence lifetime imaging microscopy on injured or intact SWR/J myotubes in the presence or absence of Ca2+ (Fig. 3). FLIM measures the decay half-life of fluorescent molecules immunologically attached to individual proteins. A shortening of the lifetime of a donor fluorophore in the presence of an acceptor indicates fluorescent resonance energy transfer between them. FLIM allows sensitive measurement of protein-protein interactions on a spatial scale of <10 nm (20.Bacskai B.J. Skoch J. Hickey G.A. Allen R. Hymen B.T. J. Biomed. Optics. 2003; 8: 368-375Crossref PubMed Scopus (140) Google Scholar).Fig. 3[Ca2+] and injury state affect interaction between dysferlin and annexins A1 and A2.a, representative images from both the injured and non-injured population of SWR/J muscle cells that have been stained with a 488-nm fluorophore bound to the dysferlin antibody (green) and a 594-nm fluorophore attached to the annexin A2 (A2) antibody (red). Blue dextran was used as an indicator of injury. b–d, by using the decay half-life of the dysferlin-associated 488-nm fluorophore alone as a baseline (left hand column of each graph), the effect on that half-life of labeling annexins A1, A2, or A6 (A1, A2, or A6) with a known acceptor 568-nm fluorophore (annexin A1 and A6) or 594-nm fluorophore (annexin A2) was assayed in the presence or absence of Ca2+ in both uninjured and injured myotubes. *, p < 0.0001; **, p < 0.00001; ***, p < 0.000001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A scalpel blade was used to injure SWR/J myotubes in culture in the presence of Ca2+ or EGTA. Injured cells were identified by the presence of membrane impermeable blue dextran (Fig. 3a). The fluorescence lifetime of the immunofluorescent label on the dysferlin antibody was measured alone or in the presence of a fluorescently labeled annexin antibody (Fig. 3, b–d). The faster decay time of the fluorophore attached to dysferlin in the presence of one attached to annexin A1 indicates that annexin A1 associates with dysferlin in non-injured cultured myotubes in the presence of Ca2+ and that a disruption of the membrane destroys this association (Fig. 3b). In uninjured myotubes, independent of Ca2+, a 568-nm fluorophore attached to annexin A1 significantly reduced the decay half-life of the 488-nm fluorophore attached to dysferlin from a baseline of 2.2 ns ± 0.28 ns to 1.8 ns ± 0.07 ns. In injured myotubes, there is no significant FLIM-detectable association between dysferlin and annexin A1. Annexin A1 did not associate with dysferlin when intracellular Ca2+ was depleted by pre-incubation with EGTA (Fig 3b).Annexin A2 is shown to associate with dysferlin in both non-injured and injured cells in the presence of Ca2+ (Fig. 3c). In these experiments, the directly labeled antibody attached to dysferlin has a baseline fluorescence lifetime of 2.3 + 0.27 ns. In the presence of Ca2+, this is significantly reduced to 1.6 ± 0.07 ns in intact myotubes and 1.5 ± 0.40 ns in the injured myotubes. No reduction is seen in the absence of Ca2+.Annexin A6 did not demonstrate any significant interaction with dysferlin in cultured myotubes (Fig. 3d). These data combined suggest that annexins A1 and A2 may have significant but functionally distinct interactions with dysferlin, and this interaction may be mediated through the unique N-terminal domains of annexins A1 and A2 and not a conserved domain that would also be present on annexin A6. It is also possible that dysferlin and the annexins might form a complex with other as yet unidentified proteins to mediate their functions. The baseline decay times shown were unaffected by injury state or Ca2+ concentration.Intracellular Vesicles Fuse with the Sarcolemma Post-injury— Reddy et al. (23.Reddy A. Caler E.V. Andrews N.W. Cell. 2001; 106: 157-169Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar) have reported that Ca2+-regulated exocytosis of lysosomes follows plasma membrane injury in several cell types. To determine whether the same is true in skeletal muscle, we looked for the presence of the lumenal domain of the lysosomal protein Lamp-1 on the surface of cultured myotubes post-injury. Injured myotubes were positively identified by uptake of the membrane-impermeable dye Texas Red dextran from the media during the injury event. Myotube membranes were not permeabilized during the immunofluorescent staining procedure, allowing the selective identification of surface-expressed protein. In the presence of Ca2+, sarcolemmal expression of Lamp-1 was detected on 86% of dextran-positive SWR/J (dysferlin-positive myotubes, line arrows) but not on uninjured cells (Fig. 4, a and d, open block arrows). Chelation of Ca2+ with EGTA in the SWR/J dishes significantly (p < 0.004) reduces the amount of surface Lamp-1 seen post-injury with only 48% of dextran-positive cells with detectable Lamp-1 (Fig. 4, b and d). We found that, in the presence of calcium, 60% of cultured SJL/J myotubes (dysferlin-negative) stained positive for Lamp-1 expression at the sarcolemma post-injury (Fig. 4, c and d), indicating a significant (p < 0.04) reduction in the number of membrane repair events in these cells compared with control. The Ca2+-independent repair process in these cells however remained active, with 41% of injured cells expressing Lamp-1 in dishes preincubated with EGTA (Fig. 4d). This Lamp-1 detection level is not significantly different from that of normal (SWR/J) cells in the absence of Ca2+.Fig. 4Surface expression of Lamp-1 after injury is reduced in dysferlin-deficient myotubes.a–c, cultured SWR/J (a, b) or SJL/J (c) myotubes were injured by scraping culture dish twice with scalpel blade in the presence of Texas Red dextran and either Ca2+ or EGTA as indicated. The myotubes were fixed and immunofluorescently stained for the surface expression of the lumenal lysosomal marker Lamp-1. Injured cells (solid line arrows) were positively identified by their uptake of the membrane-impermeable Texas-Red dextran (uninjured cells are marked with open block arrows). Myotubes were counter-stained with the nuclear marker, DAPI. d, the number of dextran-positive cells expressing Lamp-1 on their surface was quantified visually and plotted as a percentage of the total number of wounded cells. N, total number of wounded cells counted; *, p < 0.004; **, p < 0.04.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Dysferlinopathic Sarcolemma Has Significantly Reduced Healing Ability—To determine whether a disruption of dysferlin expression has an effect on the efficiency of sarcolemmal repair, SWR/J and SJL/J myotubes were loaded with the fluorescent Ca2+ indicator, Indo1-AM, and changes in [Ca2+]i were monitored while the sarcolemma was disrupted with a micropipette. Several cells from each preparation of cultured myotubes were recorded (Fig. 5, a and b), and triplicate preparations were used to determine mean recovery times (Fig. 5d). SWR/J cells displayed a transient elevation of [Ca2+]i in response to membrane disruption that returned to normal levels within a few seconds (Fig. 5a, red traces). This return to normal Ca2+ levels was significantly delayed in myotubes cultured from SJL/J mice (Fig. 5a, green trace). Concurrent measurements taken in uninjured cells show that no photobleaching of the Ca2+ indicator occurred for the duration of the experiments (Fig. 5b). Note that traces from injured myotubes (Fig. 5a) have been scaled to standardize baseline Indo1-AM ratios and times of injury for easier comparison, whereas traces from uninjured cells (Fig. 5b) have not been scaled to demonstrate the similarities in baseline ratios between separate dishes. From the individual traces, mean values for the recovery in each species were determined (Fig. 5c), and mean recovery half-times (time to recover to half maximal ratio) were calculated (Fig. 5d). Normal myotubes (SWR/J) have a mean recovery half-time of 4.7 ± 1.5 s. SJL/J myotubes have a mean recovery half-time of 16 ± 2.7 s, which is significantly longer than normal (p < 0.006). Also, measurements taken from injured SJL/J myotubes at regions distant from the sites of injury (traces not shown) indicate normal calcium sequestration and clearance in these cells, as the recovery half-time for these regions is 8 ± 1.7 s (SJL/J Away).Fig. 5Dysferlin is required for efficient rapid resealing of microinjury wounds.a and b, Indo1-AM-loaded SWR/J (red) and SJL/J (green) myotubes were subjected to time-lapse fluorescence microscopy, while a micropipette was used to injure the cell membrane. The 405–485 nm emission ratio of Indo1-AM was measured each second at the site of inj
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