DISTRIBUTION AND ORIENTATION OF RHODAMINE-PHALLOIDIN BOUND TO THIN-FILAMENTS IN SKELETAL AND CARDIAC MYOFIBRILS

Phalloidin staining of muscle does not reflect the known disposition o f sarcomeric thin filaments. Quantitative image analysis and steady-st ate fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isol ated skeletal myofibrils labeled while in rigor reveals fluorescence t hat is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phal loidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-mu m dark areas on ei ther side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether lon g-term storage in glycerol changes the binding of phalloidin to thin f ilaments in myofibrils, minimally permeabilized (briefly skinned) myof ibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments w as randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extra ction. In relaxed skeletal myofibrils with sarcomere lengths greater t han 3.0 mu m, staining was nearly uniform all along the actin filament s. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bi nd rhodamine phalloidin uniformly Our results support the hypothesis t hat nebulin can block the binding of phalloidin to actin in skeletal m yofibrils and nebulette can block phalloidin binding to cardiac thin f ilaments. (C) 1997 Wiley-Liss, Inc.