Functional studies of novel ACTN1 variants. (A) Immunofluorescence analysis in PD220 fibroblast cell line transiently transfected according to standard procedures. Both wild-type (wt) or mutant ACTN1 cDNAs were cloned into the pcDNA3.1-Myc tagged expression vector.⁵  The subcellular localization of exogenous α-actinin 1 (green) was examined using c-myc antibodies (9E10; Santa Cruz Biotechnology), whereas the actin filaments were stained with AlexaFluor594 (red) conjugated phalloidin (Invitrogen). Images were obtained with a Nikon C1si confocal microscope, using ×60 Plan Apo objectives. Images were processed for z-projection (maximum intensity), brightness, and contrast regulation, using ImageJ 1.45 (National Institutes of Health). The cells shown are representative of 3 independent experiments. Scale bar = 50 µm. (B) Domain structure of α-actinin and localization of ACTN1 mutations identified in Japanese families (arrowheads)⁵  and in this article (arrows). The p.Arg46Gln mutation was also identified in a French family.⁶  α-actinin is organized in an actin-binding domain (ABD) at the N terminus, 4 spectrin repeats, and a calmodulin-like domain (CaM) at the C terminus. Antiparallel molecules dimerize in rod-like structures with the ABD at each extremity for cross-linking the actin filaments into bundles.