Confocal-microscopic study of skeletal muscle fibre membrane organelles during Zenker's (spreading) necrosis

The changes of T-system and cellular acidic organelles during spreading (Zenker's) necrosis of frog skeletal muscle fibres have been investigated using laser confocal microscopy and several vital fluorescent dyes acridine orange, RH 414, DiOC6(3), rhodamine 123, fluorescein dextran. The formation of numerous vacuoles as a result of local T-system swelling is most characteristic for initial steps of Zenker's necrosis. Vacuoles can attain tens microns in length. They are located both near nuclear poles and between myofibres. Vacuoles maintain connections with the extracellular space up to the moment of contraction knot rejection, and under definite conditions (glycerol influx to fibre) vacuoles are reversible. They deform nuclei and sarcoplasmic reticulum cisternae. Cellular acidic organelles, accumulating acridine orange (lysosomes, late endosomes, Golgi apparatus cisternae) are situated in direct vicinity with normal and vacuolated T-system. The increase in acidic organelles number and size occur during the pathological process development, and tendency to vacuoles clusterization may be seen. Vacuolation of T-system during necrosis is not followed by vacuole content acidification. The role of cellular acidic organelles and of T-system vacuolation in the development of different muscle pathological changes is discussed.     

Confocal microscopy study of membrane organelles of the skeletal muscle fiber in the process of Zenker’s (spreading) necrosis

Using laser confocal microscopy and some vital fluorescent dyes (acridine orange, RH 414, DiOC₆(3), rhodamine 123, fluorescein dextran), changes of the T-system and cellular acidic organelles were studied during spreading (Zenker’s) necrosis of isolated frog skeletal muscle fibers. The most characteristic of the initial stages of development of Zenker’s necrosis is the formation of numerous vacuoles as a result of local T-system swellings. The vacuole length can reach tens of micrometers. They are located both near nuclear poles and between myofibrils. Until the moment of contraction knot separation, the vacuoles preserve their connections with normal T-tubules and under certain conditions (glycerol influx to the fiber) are reversible. The vacuoles deform nuclei and cisternae of the sarcoplasmic reticulum. Acidic cell organelles accumulating acridine orange (lysosomes, late endosomes, trans-Golgi cisternae) are located in the immediate vicinity both of normal and of vacuolated T-tubules. In the course of the development of the pathological process, the size and number of acidic organelles increases and they tend to be clustered. Vacuolation of the T-system during necrosis was not accompanied by vacuole content acidification. At late stages of necrosis, alterations of nuclei and sarcoplasmic reticulum were observed. The role of cellular acidic organelles and of the T-system vacuolation in development of various muscle pathologies is discussed.     

Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.     

Effect of vacuolization on the sodium concentration in an isolated muscle fiber]

Using the Perkin Elmer flame photometer sodium and potassium concentrations have been measured in muscle fibers from the m. ileofibularis of Rana temporaria. After 30 minutes preincubation in the Ringer solution, made hypertonic by the addition of 0.22M glycerol, the muscle fibers were incubated in the normal Ringer solution for 30 min. These fibers showed a vacuolation and an increase in total fiber sodium up to 37.2 mmol/l +/- 5.9 S. E., or 45.8 mmol/kg H2O +/- 7.3 S. E. No significant changes in potassium concentration were observed. Then, the fibers were exposed again to the Ringer solution containing 0.22 M glycerol. This procedure caused the disappearance of vacuoles and decrease in fiber sodium concentration down to 17.7 mmol/l +/- 1.6 S. E., or 21.8 mmol/kg H2O +/- 2.0 S. E. The effect of vacuolation was not blocked by ouabain (1.10(-4) M). It is suggested that the vacuoles have a high NaCl concentration. A model for NaCl and water accumulation in T-tubules is presented.     

The effects of glycerol and urea on the ultrastructure and contractility of fast and slow rat skeletal muscles

The influence of the influx and efflux of glycerol and urea (400 mmol/l) on the amplitude of isometric twitches and the ultrastructure of isolated fast (EDL) and slow (SOL) muscles of young rats was studied. The influx of non-electrolytes was accompanied by a temporary decrease in the twitch tension. The removal of non-electrolytes resulted in a stable reduction of twitches. Both effects were less pronounced in glycerol experiments on slow muscles. The inhibition of twitches after the removal of non-electrolytes was associated with selective alterations of the T-system: swelling, vacuolation, and lysis of T-tubules. Quantitative analysis of the T-system showed that the extent of these changes may vary for different fibres, and the intensity of morphological alteration of the T-system generally correlated with the degree of twitch inhibition. Reloading of muscles with non-electrolytes tended to improve the T-system structure in some fibres and led to a partial restoration of the amplitude of twitches.     

Cellular vacuolation induced by Clostridium perfringens epsilon-toxin

The epsilon-toxin of Clostridium perfringens forms a heptamer in the membranes of Madin-Darby canine kidney cells, leading to cell death. Here, we report that it caused the vacuolation of Madin-Darby canine kidney cells. The toxin induced vacuolation in a dose-dependent and time-dependent manner. The monomer of the toxin formed oligomers on lipid rafts in membranes of the cells. Methyl-β-cyclodextrin and poly(ethylene glycol) 4000 inhibited the vacuolation. Epsilon-toxin was internalized into the cells. Confocal microscopy revealed that the internalized toxin was transported from early endosomes (early endosome antigen 1 staining) to late endosomes and lysosomes (lysosomal-associated membrane protein 2 staining) and then distributed to the membranes of vacuoles. Furthermore, the vacuolation was inhibited by bafilomycin A1, a V-type ATPase inhibitor, and colchicine and nocodazole, microtubule-depolymerizing agents. The early endosomal marker green fluorescent protein-Rab5 and early endosome antigen 1 did not localize to vacuolar membranes. In contrast, the vacuolar membranes were specifically stained by the late endosomal and lysosomal marker green fluorescent protein-Rab7 and lysosomal-associated membrane protein 2. The vacuoles in the toxin-treated cells were stained with LysoTracker Red DND-99, a marker for late endosomes and lysosomes. A dominant negative mutant of Rab7 prevented the vacuolization, whereas a mutant form of Rab5 was less effective. These results demonstrate, for the first time, that: (a) oligomers of epsilon-toxin formed in lipid rafts are endocytosed; and (b) the vacuoles originating from late endosomes and lysosomes are formed by an oligomer of epsilon-toxin.     

Kinetics of functional and morphological changes during decoupling and recoupling induced by glycerol in isolated muscle fibres of the crayfish

Summary The development of ultrastructural changes in the T-system of isolated muscle fibres of the crayfish by the glycerol procedure is described in correlation with the dissociation of excitation-contraction (E-C) coupling as well as with recoupling of the E-C link. The sequence of events in the process of disconnection of the tubules is as follows: dilation of the T-system tubules, disconnection of the constricted tubular segments from the surface membrane and from the T-system vesicle, disappearance of the lumen and its disintegration. The decoupled state is characterised by the presence of round vesicles uniformly distributed in the entire volume of the fibre. The volume of vesicles accounts well for the residual postglycerol volume increase (15%) of the muscle fibres. Functional and structural recovery can be induced by reapplication of glycerol to fibres decoupled and vesiculated with concentrations of glycerol300mmol · l^-1 in crayfish saline. The restitution starts with the organisation of the material of the disintegrated connecting segment of the T-system tubule into small vesicles which coalesce to form the tubule from the vesicular site. At the same time the surface membrane is invaginated toward the vesicle, thus forming the tubule from the surface membrane site. Recovery starts already in the first minute after application of glycerol and is completed within approximately 15min.     

Vacuolation in T-tubules as a model for tubular-vesicular transformations in biomembrane systems

This review outlines the basic properties of T-tubules in skeletal muscle cells, and the factors that govern reversible vacuolation in T-tubules under experimental conditions. Comparable membranous transformations, involving the plasma membrane or occurring intracellularly, in non-muscle cells are then considered. Finally, the mechanisms of similar transformations in various model membrane systems are discussed. In view of the similarities between reversible vacuolation in the T-system and membrane transformations occurring in a variety of non-muscle cells, it is suggested that reversible vacuolation in T-tubules may be regarded as a general model for tubular-vesicular transformations in biomembranes.     

Two Fluorescent Markers Identify the Vacuolar System of Schizophyllum commune

Vacuole-mediated proteolysis is important to sustained growth of filamentous wood-decaying fungi such as Schizophyllum commune. Demonstrating that specific proteases are vacuole associated has been difficult in these organisms due to the lack of specific markers for vacuolar compartments. We used 5-(and 6-)-carboxy-2′, 7′-dichlorofluorescein diacetate (carboxy-DCFDA) and a proprietary vacuolar membrane marker for yeast (MDY-64; Molecular Probes) for in situ fluorescent labeling of the vacuoles of S. commune mycelia grown on microscope slides. MDY-64 labels numerous small vesicles in S. commune mycelia in addition to larger vacuolar structures. In contrast, carboxy-DCFDA apparently is taken up by a subset of the MDY-64-labeled vesicles, accumulating primarily in larger vacuoles. Staining of mycelia with carboxy-DCFDA shows a transition from mostly cytoplasmic fluorescence in apical cells with little vacuolar fluorescence to nearly complete sequestration of the stain in vacuoles of older cells. In penultimate cells, both cytoplasm and vacuolar structures fluoresce. Vacuoles stained with carboxy-DCFDA typically were spherical and ranged in size from 0.4 μm to 3.2 μm in diameter with a mean of 1.8 um. Occasionally, in penultimate cells, tubular structures which stained with carboxy-DCFDA were found. ScPrB, a principal enzyme of nitrogen-limitation induced autolysis in S. commune, copurified in sucrose density gradients with carboxy-DCFDA and acid phosphatase, demonstrating its vacuolar localization. Received: 23 December 1998 / Accepted: 11 January 1999     

Membrane trafficking in guard cells during stomatal movement: Application of an image processing technique

Pairs of guard cells form small pores called stoma in the epidermis, and the reversible swelling and shrinking of these guard cells regulate the stomatal apertures. The well-documented changes in guard cell volume have been associated with their vacuolar structures. To investigate the contribution of the guard cell vacuoles to stomatal movement, the dynamics of these vacuolar structures were recently monitored during stomatal movement in vacuolar-membrane visualized Arabidopsis plants. Calculation of the vacuolar volume and surface area after reconstruction of three-dimensional images revealed a decrease in the vacuolar volume but an increase in the vacuolar surface area upon stomatal closure. These results implied the possible acceleration of membrane trafficking to the vacuole upon stomatal closure and membrane recycling from the vacuole to the plasma membrane upon stomatal opening. To clarify and quantify membrane trafficking during stomatal movement, we describe in this addendum our development of an improved image processing system.Key words: stomata, guard cells, vacuole, membrane traffic, image processing     

Localization of acid organelles in frog skeletal muscle fibers

By means of fluorescent and phase-contrast microscopy the distribution of acid membrane organelles in normal and vacuolated frog skeletal muscle fibers has been studied. The vacuolation of the T-system was produced by loading and subsequent removal of glycerol (80-110 mM), or it appeared as a result of Zenker's necrosis. Acridine orange (AO) was used as a marker for acid intracellular compartments. AO accumulated in granules localized near the nuclear poles (more seldom around the nucleus)' and in the intermyofibrillar spaces. Typically the AO granules make up short longitudinal chains or regular pairs, where the distances between neighboring granules are short-dated to sarcomere lengths. Almost all granules emit in red, but about one third of them simultaneously emit in green, which is characteristic of AO monomers. In the vicinity of necrotic boundary or under the influence of brefeldin A, a green component of fluorescence appears in most granules. Treatment with monensin leads to granule disappearance. Vacuoles accompanying the glycerol treatment or developing of necrosis do not accumulate AO and exert no effect on the localization of AO-granules. The nature of cellular organelles accumulating AO in skeletal muscle fibers is discussed.     

The Role of the Contractile Vacuole in the Osmoregulation of Tetrahymena pyriformis*

The relationship of cell size and contractile vacuole efflux to osmotic stress was studied in Tetrahymena pyriformis strain W, after transfer into fresh solutions iso- or hypoosmotic to the growth medium. Microscopic measurements of the cell and contractile vacuole dimensions, made with an image-sharing ocular at 27 C, allowed the calculation of the cell size and shape and the vacuolar efflux rate which provide a measure of osmoregulation. The contractile vacuole cycles have no homeostatic oscillations. In 0.03–0.10 osmolar solutions, the cell size and shape are constant while the vacuolar efflux rate has an inverse linear dependence upon extracellular osmolarity. Regression analyses indicate that for cells with systole faster than 0.1 sec (the major part of the population), it is only the final diastolic volume of the contractile vacuole that is related to osmotic stress while the frequency of systole is independent of osmotic stress and has a constant period of 7.7 ± 0.2 sec. Therefore, osmotic stress upon Tetrahymena is regulated by a corresponding change in the filling rate of its contractile vacuole to allow an unaltered cell size and shape. Kinetic measurements of vacuoles during diastole fit the model (dV/dt = K_1-K₂A), where (dV/dt) is the vacuolar filling rate and (A) is the vacuolar surface area. This dependence of vacuolar volume upon its surface area may be ascribed either to elastic components of the vacuolar membrane or to an increasing leakiness of this membrane during diastole. Mitochondrial inhibitors were used to observe the energy requirements of vacuolar operation and of intracellular secretion of water.     

CORRELATED MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES ON ISOLATED SINGLE MUSCLE FIBERS : II. The Properties of the Crayfish Transverse Tubular System: Localization of the Sites of Reversible Swelling

Living muscle fibers of crayfish become dark during efflux of Cl^-. This change in appearance is correlated with occurrence of vacuolation in the fixed fibers. The vacuoles begin at and are mainly confined to the terminals of the transverse tubular system (TTS) which are in diadic contact with the sarcoplasmic reticulum (SR). In electron micrographs swellings more than 1 µ in diameter may be seen connected to the sarcolemma or sarcolemmal invaginations by relatively unswollen tubules about 300–500 A wide. Darkening of the living fibers can be reversed by causing an influx of Cl^-. Vacuoles are then absent in the fixed preparations. These findings accord with the conclusion that the membrane of the TTS is anion permselective. Localization of the selectivity to the membrane of the terminals of the TTS strengthens the hypothesis that a channeling of current flow is responsible for initiation of excitation-contraction coupling. During the swelling, and upon its reversal, the area of the membrane of the terminals must change reversibly by about two to four orders of magnitude. The absence of changes in the dimensions of the unit membrane indicates that the expansion of the membrane and its subsequent shrinkage involve reversible incorporation of cytoplasmic material into the membrane phase.     

Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase-dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase-induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30–50% less LY than controls and returned to higher rates of pinocytosis after invertase-induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY-containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY-containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase-induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.     

Quantitative determination of changes induced by NaCl in vacuoles and cellular size of Lycopersicon esculentum root cells

Changes induced by NaCl in the sizes of both cells and vacuolar compartments of tomato seedling roots were determined using morphometric and stereological analysis. Parameters evaluated showed that cellular volume was smaller in roots grown under saline conditions than in control roots. This reduction was greater when NaCl concentration was increased. Similar behaviour can be observed in the vacuolar compartment where high concentrations of NaCl produce a reduction in vacuolar volume although the number of vacuoles per cell does not change under saline conditions.     

The dynamic changes of tonoplasts in guard cells are important for stomatal movement in Vicia faba

Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxy-succinyl-l-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement.     

Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers.

In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule.     

Calcium-Activated K+ Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatal Closure

Stomatal closing requires the efflux of K+ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K+ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca2+ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K+-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca2+ concentration. These vacuolar K+ (VK) channels had a single channel conductance of 70 pS with 100 mM KCI on both sides of the membrane and were highly selective for K+ over NH4+ and Rb+. Na+, Li+, and Cs+ were not measurably permeant. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K+ channels in the initiation and control of K+ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K+-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca2+ over K+ selectivity (permeability ratio for Ca2+ to K+ of ~3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca2+-induced Ca2+ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K+ and Ca2+ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca2+-induced Ca2+ release from higher plant vacuoles is discussed.     

Localization of Mg2+-ATPase in the membranes of the skeletal muscles and myocardium of the frog Rana temporaria

Using differential centrifugation in sucrose density gradient, from muscles of the frog fractions were obtained which contain fragments of sarcolemma, as well as membranes of T-system tubules and sarcoplasmic reticulum. In isolated membrane fractions, studies were made on the activity of cation-stimulated ATPases (Na+, K+-, Ca2+, Mg2+- and Mg2+-ATPases). Enzymic and electrophoretic analyses showed that the highest content of Mg2+-ATPases is typical of the fractions which are located on the surface of 35% sucrose. The data obtained indicate that Mg2+-ATPase is the enzyme which is specific for the membranes of T-system tubules in skeletal muscles of not only birds but amphibians as well. From cardiac muscle of the frog, membrane fraction was isolated which is similar (with respect to its predominant content of Mg2+-ATPase) to the membranes of T-system tubules. It is suggested that the presence of Mg2+-ATPase in these membranes is a common property of phasic striated muscle fibers in all mature vertebrate animals.     

STUDIES OF THE INNER AND OUTER PROTOPLASMIC SURFACES OF LARGE PLANT CELLS : II. MECHANICAL PROPERTIES OF THE VACUOLAR SURFACE

The vacuolar surface of Nitella is covered with a non-aqueous film too thin to be visible as a separate membrane. The motion of the protoplasm may subject this film to a good deal of mechanical disturbance. Apparently this does not rupture the film for no dye escapes into the protoplasm as the result of such disturbance when the vacuolar sap is deeply stained with neutral red or brilliant cresyl blue. When the deeply stained central vacuole breaks up into several smaller vacuoles, leaving the outer protoplasmic surface in its normal position, there is no evidence of the escape of dye into the protoplasm through the film surrounding the vacuole.