Research Article

Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

Figure 9

Intracellular sorting of utrophin fragments by expression of eukaryotic vectors in myoblast and myotube primary cultures (EUT11, EUT12, and EUT21, all carrying the FLAG-tag at the C-terminus). In each picture in the left vertical row ((a)–(f)), one transfected cell (green) is present stained with anti-FLAG for the corresponding utrophin fragment. Staining with rhodamine-phalloidin for actin reveals a variable number of red cells in each picture ((b)–(e) in the right vertical row) indicating that the transfected cells often fuse with nontransfected cells when forming myotubes. EUT11 and EUT21 solidly stain nucleus and cytoplasm in single spindle-shaped myoblasts. EUT12 (actin-binding domain) sharply stains actin microfilaments ((c) and (d)) superimposable to the red pattern of actin stain ((c) and (d) at the right). EUT12 also concentrates in regions where actin may attach to the substratum (d). EUT11 distributes over the cytoplasm in a diffuse and punctate manner (b) leaving the myotube nuclei unstained. In contrast, EUT21 is almost exclusively confined to the cell nuclei. For further explanations, see the Methods section and the text.
904547.fig.009