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

Figure 8

Intracellular sorting of utrophin fragments by expression of eukaryotic vectors in COS7 cells (EUT11, EUT12, and EUT21, all carrying the FLAG-tag at the C-terminus). Cells in the vertical row at the left are all stained with the anti-Flag M2 mAB ((a), (b), (c), (d), (e), (f)). Rhodamine-phalloidin staining for actin displays stress fibres and a dense cortical actin cytoskeleton along the cell membranes in the COS7 cells spreading on the substratum ((a), (c), (e) at the right). Anti-UT11 recognises full-length utrophin and N-utrophin giving intensive staining in and around the nucleus that results from the heterologously expressed EUT11 plus the endogenous utrophin ((b) at the right). Anti-UT31 (spectrin 14–16 in the rod) stains COS7 cells diffusely throughout the cytoplasm without marking any defined actin structures ((d), (f) at the right). EUT21 (first two spectrin domains) exclusively concentrates in the nuclei ((e), (f)). EUT12 (actin-binding domain alone) precisely traces the actin microfilaments and the cortical cytoskeletal structures ((c), (d)). For further explanation see the text.