Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac myofibrils

Abstract

Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady‐state 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 isolated skeletal myofibrils labeled while in rigor reveals fluorescence that 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, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2‐μm dark areas on either side of the Z line. Extraction of myosin or tropomyosin‐troponin molecules does not change the nonuniform staining. To test whether long‐term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, 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 was randomly oriented in glycerol‐extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 μm, staining was nearly uniform all along the actin filaments. Exogenous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825–833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments. Cell Motil. Cytoskeleton 37:363–377, 1997. © 1997 Wiley‐Liss, Inc.

Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

“…In control cultured skeletal myotubes, phalloidin stained a thin band within the Z-disc and a thicker band at the pointed ends of the thin filaments ( Figure 8B, arrowheads and arrow, respectively), with relatively less staining of the intervening region of the I-band, as described by others (e.g., Bukatina et al, 1984;Wilson et al, 1987;Szczesna and Lehrer, 1993;Ao and Lehrer, 1995;Zhukarev et al, 1997). In cells with reduced nebulin, however, phalloidin stained along the entire length of the thin filament in the I-band.…”

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

“…Myofibrils were isolated from tissue homogenates by an established method (Zhukarev et al, 1997). Muscle strips were homogenised in 10 volumes of icecold buffer (10 mM TES, pH 7.1, 3 mM MgCl 2 and 10 mM EGTA) using an Ultra-Turrax T25 (IKA-Labortechnik, Staufen, Germany) for 3 times 30 s at medium speed.…”

“…For the microscopic evaluation of the quality of the subcellular fractionation procedure, a drop of the myofibril-enriched suspension was positioned on a glass slide and investigated by bright-field microscopy and immunofluorescence staining with an antibody to actin (Zhukarev et al, 1997). The immunofluorescence survey of myosin isoforms in transverse tissue sections from gastrocnemius muscle was carried out according to Doran et al (2008).…”

“…Myofibrils were isolated from rat skeletal muscle homogenates by an established method (Zhukarev et al, 1997;Bassaglia et al, 2005). Fig.…”

“…Besides bright-field microscopy, also dye staining and immuno-decoration was carried out to evaluate the quality of the subcellular fractionation protocol. Both, methylene blue and immunofluorescence staining of isolated myofibrils with an antibody to actin revealed a regular striation pattern in the longitudinal direction of isolated myofibrillar structures (not shown), indicating a successful enrichment of the contractile machinery by this method (Zhukarev et al, 1997). Two protein dye methods were used to determine the expression pattern of the soluble myofibrillar proteome.…”