Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study

Abstract

A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.

Propagation in the transverse tubular system and voltage dependence of calcium release in normal and mdx mouse muscle fibres

“…The normal appearance of the sarcomeres can be appreciated in the DIC images of normal and mdx fibres after the enzymatic dissociation process. The di‐8 ANEPPS fluorescence images from both normal and mdx fibres showed a double row of T‐tubules per sarcomere, a distinctive feature of mammalian skeletal muscle fibres that has been documented previously (Revel, 1962; Dulhunty, 1989; Krolenko et al 1995; Franzini‐Armstrong et al 1998; Lannergren et al 1999). Importantly, there are no major T‐system structural differences between normal and mdx fibres.…”

“…In the magnified views in Fig. 1 and , it can be observed that the T‐tubules are organized in a double row per sarcomere pattern, a well‐known feature of mammalian skeletal muscle fibres that has been reported with electron microscopy (Franzini‐Armstrong et al 1988; Dulhunty, 1989) and with confocal fluorescence microscopy (Krolenko et al 1995; Lannergren et al 1999).…”

Three-dimensional reconstruction and analysis of the tubular system of vertebrate skeletal muscle

“…The latter property underscores the role of the t-system in compartmentalizing its luminal solutes that would otherwise affect its excitability. The t-system is also a dynamic structure, changing its volume or vacuolating with osmotic stress or other factors (Krolenko et al, 1995;Krolenko and Lucy, 2001;Launikonis and Stephenson, 2002a;Launikonis and Stephenson, 2004).…”

External Cl^--Dependent Formation of Watery Vacuoles by Long-Term Hypotonic Shock in 3T3-L1 Cells

“…Vacuoles in cells are formed by radiation injury, virustransformation and toxin injury [17][18][19]. Osmotic stress with glycerol also induces vacuolization of tubular system in muscle cells [20]. Vesicle membranes pass Cl -through ion channels [21,22], and Cl -is related to the regulation of water movements [23][24][25].…”