Fig. 5.

Effects of skip residue deletions on myosin incorporation into sarcomeres. (A) Cardiomyocytes electroporated with WT and mutant GFP-tagged skip residue deletion constructs (ΔS) were imaged by confocal microscopy 96 h later. (Scale bar, 10 μm.) (B) Cardiomyocytes were cotransfected with mutant GFP- and WT mCherry-tagged constructs as indicated. Cells were imaged by confocal microscopy 96 h later. The two boxes in the WT and ΔS4 merge panels show the high magnification view of the sarcomeric I band and H zone (the latter corresponding to the bare zone) and the lack of colocalization between the mutant GFP- and the WT mCherry-tagged myosins. (Scale bar, 5 μm.) (C) Linescan analysis showing the relative intensity across the sarcomere of WT GFP and mCherry (Left) and ΔS4 GFP- and WT mCherry- tagged myosins (Right). Cells from three independent transfections were imaged and a total of 420 sarcomeres for each graph analyzed; data were obtained by averaging the two fluorescence signals. x axis: pixel distance (0.086 μm per pixel); y axis: fluorescence intensity. The location of the I-band and H-zone are reported. (D) Colocalization of ΔS4 GFP construct with the endogenous myosin, and time course incorporation into the sarcomeres. (Ab-F59): cardiomyocytes were transfected with WT or ΔS4 GFP-tagged myosin constructs; 96 h later cells were fixed and stained with F59 antimyosin primary antibody that recognizes only the myosin head domain, and the Alexa Fluor 568 secondary antibody with orange-red emission color. All panels are overlays of GFP and mCherry fluorescence signals. (36 h, 48 h): cardiomyocytes cotranfected with ΔS4 GFP- and WT mCherry-tagged myosin rod constructs were imaged by confocal microscopy 36 and 48 h later. (Scale bar, 5 μm.)