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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01172

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Myopathy mutations in -skeletal-muscle actin cause a range of molecular defects

Céline F. Costa^1,*, Heidi Rommelaere², Davy Waterschoot², Kamaljit K. Sethi¹, Kristen J. Nowak^3,4,, Nigel G. Laing^3,4, Christophe Ampe² and Laura M. Machesky^1,

¹ School of Biosciences, Division of Molecular Cell Biology, University of Birmingham, Birmingham B15 2TT, UK
² Department of Biochemistry, Ghent University and Flanders Interuniversity Institute for Biotechnology (VIB09), B-9052 Gent, Belgium
³ Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Australian Neuromuscular Research Institute, Nedlands
⁴ Centre for Medical Research, West Australian Institute for Medical Research, QEII Medical Centre, Nedlands, WA 6009, Australia

View larger version (98K): [in a new window]   Fig. 1. Location of ACTA1 mutations on the three-dimensional representation of the actin molecule. The 19 mutations selected for this study, encompassing 17 residues, are located on the four subdomains of the actin molecule (labeled I-IV). They lead to different severities of disease. The structure was taken from PDB code 1ATN and displayed by NetViewerLite (Accessory Software).

View larger version (64K): [in a new window]   Fig. 2. Behavior of actin mutants on native gels. The 35S-labeled products of the in vitro translation assays were run on native polyacrylamide gels with or without ATP and imaged using autoradiography. On the native gel with ATP, G182D stuck to CAP and both mutants L94P and E259V remained bound to CCT and prefoldin, and showed an absence of folded actin. The 16 other mutants were folded correctly, as indicated by an abundance of folded actin that was similar to the wild type. On the gel without ATP, G15R, N115S, V163L and G182D showed an increase in binding to CAP. Representative gels shown from three experiments performed.

View larger version (44K): [in a new window]   Fig. 3. Interaction between -skeletal-muscle actin and the different mutants with thymosin ß4, profilin-IIA, DNase I and vitamin-D-binding protein (DBP). 35S-Labeled -skeletal-muscle actin and mutants were produced by in vitro translation, run on native gels after mixing with thymosin ß4, profilin IIa, DNase I or DBP and subjected to autoradiography. Wild-type -skeletal-muscle actin shifted up (profilin, DNase I, DBP) or down (thymosin ß4) in complex with ABPs compared with -skeletal-muscle actin alone. Most of the mutants interacted with the different ABPs (Table 2). R183C and G15R are shown as examples of those that behaved like the wild type; the others are not shown but are listed in Table 2. The control lanes in each case represent the mutant actin run in the absence of added ABP. The mutant I64N was only partially shifted when DNAse I was added (asterisk). The mutants L94P and E259V remained associated with CCT (arrow) and prefoldin and the released product (arrowhead) showed little (L94P) or no (E259V) binding to ABPs. The mutants G182D and V163L, which stuck to CAP, did not shift with thymosin ß4 (up arrow), which might reflect the relatively low affinity of actin for thymosin and competition with CAP. Both mutants shifted with DBP and DNase I, however, indicating proper folding. The gel shown is a representative of three experiments with similar results.

View larger version (32K): [in a new window]   Fig. 4. Co-polymerization assays of wild type -skeletal-muscle actin and mutants. Samples of an in vitro transcription-translation reaction and -actin from rabbit skeletal muscle were mixed, induced to polymerize and pelleted. The amounts of 35S-labeled actin in aggregates (first pellet, before polymerization and without carrier actin), supernatants (did not pellet under any conditions) and final pellets (F-actin, after two rounds of polymerization) were calculated. Mutants with more than 50% of the 35S-labeled actin in filaments were considered to be `wild type'. These include H40Y, M132V, V163L, R183C and D286G. G15R had only 40% polymerization and was considered to be `borderline'. Mutants with less than 50% of 35S-labeled actin in filaments were considered to be impaired in co-polymerization: I64N, N115S, I136M, G182D, R183G, Q263L, G268C, G268R, N280K, I357L, V370F.

View larger version (99K): [in a new window]   Fig. 5. Expression of wild-type and category-II (CAP-associated) mutants in NIH3T3 fibroblasts. Wild-type actin (A; scale bar, 25 µm) was transfected as a control and was incorporated into stress fibers, ruffles and lamellipodia, and generally co-localized with the endogenous filamentous actin, as visualized with phalloidin (A',A''). The mutant G182D showed a dotted pattern in the cytoplasm (B) and co-localized with the endogenous actin in stress fibers and lamellipodia (B',B''). G15R was partially incorporated into the stress fibers and lamellipodia (C). In some cells, the mutant protein formed aggregates that stained with phalloidin (C,C'', arrow). (A''-C'') Red corresponds to the Myc staining and green to the phalloidin staining. (A'',C'') Nuclei were stained with Hoechst and appear blue.

View larger version (61K): [in a new window]   Fig. 6. Expression of category III (co-polymerization defect) and category IV (no biochemical defect) mutants in NIH3T3 fibroblasts. I64N incorporated into stress fibers, localized with the endogenous actin and was observed in aggregates and long rods in the cytoplasm, which did not stain with phalloidin (A; scale bar, 25 µm). D286G incorporated into stress fibers in some cells (B) but their structures were wavy (B, inset). D286G also formed aggregates in the cytoplasm that did not stain with phalloidin (C). (A-C) Red indicates Myc staining and green phalloidin staining. (A) The nuclei were stained with Hoechst and appear blue.

View larger version (126K): [in a new window]   Fig. 7. Rod formation by expression of V163L (category II), H40Y (category IV) and I357L (category III) in NIH3T3 fibroblasts. V163L did not localize to normal actin structures but recruited endogenous actin to aberrant structures (A-A'',B-B'', arrows). In the nucleus, it formed aggregate star structures (A-A''; scale bar, 25 µm). Some aggregates were stained with phalloidin but did not detectably contain the mutant actin (B-B'' arrows). H40Y did not localize to normal actin structures but formed rods and aggregates in the nucleus and cytoplasm (C-C'' and not shown). I357L (D-D'') incorporated into stress fibers and lamellipodia and also formed cytoplasmic aggregates, which did not localize with endogenous actin. (A'-D') Red indicates Myc staining and green phalloidin staining. The nuclei were stained with Hoechst and appear blue.

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