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Mechanical Function of Dystrophin in Muscle Cells Carmela Pasternak, Scott Wong, and Elliot L

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Mechanical Function of Dystrophin in Muscle Cells Carmela Pasternak, Scott Wong, and Elliot L View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Mechanical Function of Dystrophin in Muscle Cells Carmela Pasternak, Scott Wong, and Elliot L. Elson Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 Abstract. We have directly measured the contribution sured as the resistance to identation of a small area of of dystrophin to the cortical stiffness of living muscle the cell surface (to a depth of 1 /~m) by a glass probe cells and have demonstrated that lack of dystrophin 1 #m in radius. The stiffness of the membrane skele- causes a substantial reduction in stiffness. The inferred ton was evaluated as the increment of force (mdyne) molecular structure of dystrophin, its preferential lo- per/~m of indentation. Normal myotubes with an aver- calization underlying the cell surface, and the apparent age stiffness value of 1.23 + 0.04 (SE) mdyne//zm fragility of muscle cells which lack this protein sug- were about fourfold stiffer than myotubes cultured gest that dystrophin stabilizes the sarcolemma and pro- from mdx mice (0.34 5:0.014 mdyne/#m). We tects the myofiber from disruption during contraction. verified by immunofluorescence that both normal and Lacking dystrophin, the muscle cells of persons with mdx myotubes, which were at a similar developmental Duchenne muscular dystrophy (DMD) are abnormally stage, expressed sarcomeric myosin, and that dystro- vulnerable. These facts suggest that muscle cells with plain was detected, diffusely distributed, only in nor- dystrophin should be stiffer than similar cells which mal, not in mdx myotubes. These results confirm that lack this protein. We have tested this hypothesis by dystrophin and its associated proteins can reinforce the measuring the local stiffness of the membrane skeleton myotube membrane skeleton by increasing its stiffness of myotubes cultured from mdx mice and normal con- and that dystrophin function and, therefore, the trois. Like humans with DMD mdx mice lack dystro- efficiency of therapeutic restoration of dystrophin can phin due to an x-linked mutation and provide a good be assayed through its mechanical effects on muscle model for the human disease. Deformability was mea- cells. A early step leading to the pathology of Ducherme chanical measurements to test the function of dystrophin re- muscular dystrophy (DMD) 1 is thought to be the stored to cells which originally lack it and thereby to judge disruption of muscle cells deficient in dystrophin at the cellular level the success of therapeutic strategies for (Mokri and Engel, 1975; Weller et al., 1990). Dystrophin's restoration ofdystrophin. The measurements reported in this inferred molecular structure and cellular location suggest paper provide the basis for an assay of dystrophin function that it is an essential link in a chain of interactions which con- in living muscle cells. nects the actin cytoskeleton to the extraceUular matrix (Er- Dystrophin, a large (427 kD) cytoskeletal protein, has vasti and Campbell, 1993a). These structures could stabilize four distinct domains. From the amino terminus these in- the sarcolemma and protect it from disruption during muscle clude an actin-binding domain analogous to that in ot-acti- contraction (Zubrzycka-Gaarn et al., 1988). Hence, normal nin, a large rod-like domain of 24-spectrin-like repeats, muscle ceils should be mechanically stiffer than those which a domain similar to a region ofDictyostelium c~-actinin with lack dystrophin. We have tested and confirmed this hypothe- two Ca~÷-binding sites (which may be nonfunctional in sis using quantitative measurements of the local surface skeletal muscle dystrophin), and, finally, a carboxyl terminal deformability of muscle cells in culture. In addition to domain presumed to interact with the plasma membrane verifying a proposed mechanical function of dystrophin (Koenig et al., 1988; Ervasti and Campbell, 1993a). Dystro- these measurements demonstrate the possibility of using me- phin seems to interact with a group of dystrophin associated proteins at the plasma membrane, most directly with a 59- kD protein on the cytoplasmic surface of the sarcolemma, Address all correspondence to E. L. Elson, Department of Biochemistry which in turn associates with a complex of four transmem- and Molecular Biophysics, Washington University School of Medicine, Box brahe glycoproteins (Ervasti and Campbell, 1993b). Bound 8231,660 South Euclid Avenue, St. Louis, MO 63110. Ph.: (314) 362-3346. to these at the extracellular surface, a large heavily gly- Fax: (314) 362-7183. cosylated protein, ot-dystroglycan, binds to laminin (Ervasti and Campbell, 1993c). Hence, dystrophin links the actin 1. Abbreviations used in this paper: DAG, dystrophin-associated glycopro- cytoskeleton to the extracellular matrix via a complex of tein; DMD, Duchenne muscular dystrophy. membrane-associated proteins. © The Rockefeller University Press, 0021-9525/95/02/355/7 $2.00 The Journal of Cell Biology, Volume 128, Number 3, February 1995 355-361 355 Measurements of the stiffness of the membrane skeleton 20% CPSR2 (Controlled Process Serum Replacement-Type 2; Sigma directly test the proposed mechanical function of dystrophin. Chemical Co.), 1-3% fetal equine serum and 1% glutamine. 3 d later Ara-C (cytosine ~-D-arabinofuranoside; 10 #g/ml; Sigma Chemical Co.) We have compared the deformabilities of myotubes in culture was added for 24 h to suppress fibroblast growth. Myoblasts started to fuse derived from both normal and mdx mice, which lack dystro- within 2-3 d in differentiationmedium and celldeformation measurements phin and provide a suitable model for DMD (Stedman et al., were performed on myotubes incubated in this medium for 5-9 d. 1991; Partridge, 1991). Deformability was measured as local resistance to cellular indentation (Petersen et al., 1982). Measurement of the Stiffness of the Membrane Skeleton Local cellular stiffness was measured as described for lymphocytes (Paster- Materials and Methods nak and Elson, 1985). This stiffness is a measure of the viscoelastic resis- tance of the cell to indentation. Myotubes were grown and differentiated at- Myotube Culture tached to collagen-coated glass coverslips and then were immersed in a 12 ml thermostated chamber mounted on the microscope stage. Measurements Muscle myotubes were cultured from primary satellitecells of normal were made at 37°C in MEM Earrs medium buffered with 20 mM Hepes (C57BL/10ScSn) and dystrophic mdx (C57BL/MDX) mice (Jackson (Washington University Medical School Tissue Cuimm Support Center). Laboratories,Bar Harbor, ME) accordingto publishedprocedures (Franco The surfacesof myombes 8-20 ~m in diameter,and 150-250-/~m long were and Lansman, 1990; DiMario and Strohinan, 1988) with modifications.12 slightlyindented to a depth of 0.7-0.9 #m by a glass stylus 1 #m in radius preparationsusing three to five mice of each kind were used in this study. which moved according to a triangularwaveform with an amplitude of 1.3 Myoblasts were obtainedfrom freshlyisolated hindlimb muscles of 2-8-wk ~m and a speed of 2.6 ~rrds.The styluswas mounted on a glass beam with old mice. We have detected no difference in the properties of myotubes pre- a spring constantof 2.3 mdyne/gm. The empirical stiffnessparameter was pared from mice of various ages. The muscles were minced to a fine con- defined as the resistanceper unit indentationdepth, in unitsof mdyne/~m. sistency and incubated in a proteolytic enzyme solution containing 0.25% The ingoingcurves arc linear(Fig. 2), and so the stiffnessparameter is well Collagenase B (Boehringer Mannheim Biochemicals, Indianapolis, IN) and defined. 0.1% Trypsin (Sigma Chemical Co., St. Louis, MO) in Hank's Ca- and Mg- free saline (Washington University Medical School Tissue Culture Support Center, St. Louis, Me) for 30 rain at 37°C. After digestion and proteolytic Immunofluorescence enzyme inhibition the tissue was pelleted by low speed centrifugetion. The pellet was rcsnspended in Ham's F-10 medium (Washington University Procedures for fixationand labelingof myotubes on glass coverslipswere Medical School Tissue Culture Support Center) and triturated several times adapted from earlierwork (Miller et al., 1985). Briefly,myotubes grown to release satellite ceils. After low speed centrifugetion, the muscle cells on collagen-coated glass coverslips were fixed for 5 rain in 100% ethanol were resuspended in proliferation medium consisting of Ham's F-10 medium at room temperature, washed with PBS and blocked with 2% BSA and 2% supplemented with 20% fetal bovine serum (Hyclone Labs., Logan, UT) horse serum in PBS at 37°C for 1 h. A monoclonal antibody against adult and 5% fetal equine serum (Sigma Chemical Co.). The cells were filtered skeletal myosin heavy chain, F59 (a gift from Frank Stockdale, Stanford through a 125-t~m pore size nylon mesh (Nytex; Tetko, Inc., Monterey University, Stanford, CA), was added in blocking solution for 1 h at 37°C. Park, CA) and prcplated for I-2 h to remove most fibroblasts. Next the cells The coverslip was then rinsed and incubated with a rhodamine-labeled were plated on dishes coated with 0.1% collagen and allowed to proliferate afffinity-purified goat anti-mouse IgG antibody (Orgenon-Teknika-Cappel, for 4-5 d. The cells were then mildly trypsinized (0.02% trypsin for 3 rain) Malvern, PA). The coverslips were rinsed and mounted in 90% glycerol to promote myoblast detachment, washed and plated on collagen coated containing 0.1 M n-propyl gellate (Sigma Chemical Co.) to prevent pho- (0.1%) round (diam = 18 mm) glass coverslips at a density of about 25,000 tobleaching. The procedure for immunolabeling of dystrophin was similar myohlasts per coverslip in proliferation medium. The next day the cells were except that a monoclunal antibody against dystrophin, DYS1 (Novocastra, switched to differentiation medium consisting of Ham's F-10 medium with Newcastle upon Tyne, England) was used. Figure 1. Images of normal and mdx myotubes during measurement of local deformability.
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