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Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres
Cell Biology International 30 (2006) 933e939 www.elsevier.com/locate/cellbi
Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres S.A. Krolenko*, S.Ya. Adamyan, T.N. Belyaeva, T.P. Mozhenok Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., 194064 St. Petersburg, Russian Federation Received 7 June 2006; accepted 26 June 2006
Abstract 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 l-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. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Skeletal muscle; Acid organelles; T-system; Vacuolation; Confocal microscopy; Acridine orange
1. Introduction Data on the localization and structure of lysosomes and related acid membrane organelles in skeletal muscle are rather limited (Canonico and Bird, 1969; Bird et al., 1981; Vult von Steyern et al., 1996; Bechet et al., 2005). Recently, however, it has been shown that the spatial organization of the Golgi apparatus, late endosomes, and some other components of membrane transport pathways in striated muscle fibres differs considerably from that in other vertebrate cells in that they are not concentrated exclusively in the perinuclear region but are more or less uniformly dispersed throughout the whole volume of muscle cells (Ralston, 1993; Rahkila et al., 1996; Kaisto et al., 1999; Ralston et al., 1999, 2001; Lu et al., 2001). Another
Abbreviation: AO, acridine orange. * Corresponding author. Tel.: þ7 812 2971866; fax: þ7 812 2970341. E-mail address: [email protected] (S.A. Krolenko).
unique feature of striated muscle cells is the system of transverse tubules (T-system), which comprise deep and regular invaginations of the surface membrane into the cell. Apart from the transmission of electrical signals from the cell surface to the cisternae of sarcoplasmic reticulum, the T-system participates in membrane transport (Rahkila et al., 1996; Kaisto et al., 1999; Brette and Orchard, 2003). The Tsystem is known to be easily vacuolated under the effect of outward fluxes of non-electrolytes and ions during fatigue and in many instances of muscle pathology (Krolenko et al., 1995, 1998; La¨nnergren et al., 1999, 2002; Devlin et al., 2001; Krolenko and Lucy, 2001, 2002; Cooper et al., 2002; Launikonis and Stephenson, 2002). It has been suggested that the vacuoles that accompany denervation, dystrophy, and some other muscle pathological processes are characterized by low-pH and other features of lysosomes (Libelius et al., 1979a,b). The goal of the present work was to evaluate the spatial distribution of acid membrane organelles and their relationships
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.06.017
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with normal and vacuolated transverse tubules. For this purpose, confocal fluorescent scanning microscopy of living muscle fibres was used with several fluorescence probes, such as acridine orange (AO) for acid organelles and lipophilic styryl dye RH 414 for the T-system membranes. AO has been intensively employed for many decades as a vital dye in cell biology (Palmgren, 1991; Schindler et al., 1996; Millot et al., 1997; Clerc and Barenholz, 1998; Zelenin, 1999), while RH 414 has proven to be an effective dye for the evaluation of normal and vacuolated transverse tubules (Krolenko et al., 1995, 1998). 2. Materials and methods 2.1. Isolation of fibres The frogs (Rana temporaria) were killed by decapitation followed by destruction of the spinal cord with a needle. Single fast muscle fibres or small bundles of fibres were isolated from muscle iliofibularis and the experiments were performed in winter/spring months at room temperature (22e25 C). The composition of Ringer solution and the simple chamber used for microscopy have been described earlier (Krolenko et al., 1995, 1998).
4e6 Kalman means were used. The 488 nm line of an argon laser was used for excitation of fibres stained with AO, RH 414 or their combination, and the 488 and 543 nm lines for the combination of RH 414 with Lyso Tracker Red. The laser power did not exceed usually 30% of the maximum value. The fluorescence emissions of muscle fibres stained with AO alone or in combination with RH 414 were simultaneously recorded in green (500e 560 nm) and red (590e660 nm) channels. Due to the metachromatic properties of AO, this procedure permitted evaluation of the monomeric (green fluorescence) and aggregated (red fluorescence) state of the dye. A similar system of registration was used for BODIPY FL C5-ceramide. In experiments with Lyso Tracker Red, the emission was recorded in the red region of 590e660 nm. For more detailed spectral characteristics of various structures of the AOstained fibres, we used a l-scanning system of Leica microscope (regime Mode/xyl) in the 500e660 nm interval with a step size of 10 nm. This scanning was done before or after the two-channel fluorescence recording in the same region of the fibre. The dimensions of analyzed areas were in the order of 1e2 mm2. Confocal image processing was performed using the ordinary program of Leica TCS SL and Image J program. As a standard for the minimal size of AO granules, we used a clustered position of 3 3 pixels, whose intensity exceeded at least three times the surrounding level.
3. Results
2.2. Chemicals and solutions
3.1. AO-staining
The stock solution of AO (Merck) was diluted in normal or glycerol-containing Ringer solution before each experiment to obtain final concentrations of 0.25 mg/ml. Muscle fibres were stained with AO for 40e120 min in a large volume of solution. As a rule, the dye was not removed when the specimens were viewed by confocal microscope. In some experiments, Lyso Tracker Red at a concentration of 50e100 ng/ml and BODIPY FL C5-ceramide at a concentration of 5 mM were used (both dyes from Molecular Probes, Eugene, OR, USA). To evaluate the membranes of normal and vacuolated T-systems we used lipophilic styryl dye RH 414 (Molecular Probes, Eugene, OR, USA) (Krolenko et al., 1995, 1998). The stock solution of RH 414 (1.5 mg/ ml) was diluted in normal or glycerol-containing Ringer solution before each experiment to obtain a final concentration of 15 mg/ml. Fibres were placed in the RH 414 solution 15e30 min before observations and were viewed in the presence of the dye. Carboxylic ionophore monensin (Sigma) was used at a concentration of 30e50 ng/ml.
Intact skeletal muscle fibres stained with AO were characterized by a weak green fluorescence of the whole sarcoplasm and by bright green nuclei (Fig. 1). The green fluorescence intensity of A-band was usually significantly (up to 2e3 times) higher than in other regions of cytosol. AO accumulated in numerous bright spherical granules located near nuclei poles and throughout the whole sarcoplasm (Fig. 1aec). The diameter of the AO granules could vary in the same fibre from about 0.4 up to 1.5 mm. This size variation was confirmed by observing the diameter of the same granules in serial optical sections. The number of granules per square unit of optical sections varied considerably in different fibres as well as in different regions of the same fibre. For example, in the same Z-series the number of granules in individual sections could vary 1.5e2.0 times. Under our conditions, there were, on average, 80 5 granules per 10,000 mm2 of the optical section. This value was the result of measurements in 60 optical sections from 10 fibres.
2.3. Vacuolation of the T-system Vacuolation of the T-system was induced by soaking muscle fibres in Ringer solution with 100 mM glycerol followed by a return to normal Ringer solution. Loading with glycerol lasted for no less than 30 min, and in most experiments started before the transfer of the fibre into the chamber. Glycerolremoval was usually performed under the confocal microscope. It led to the appearance of many vacuoles within several minutes. To induce devacuolation, normal Ringer solution was replaced by Ringer solution containing glycerol. If the fibres were not injured during the first part of the experiment (injury sometimes occurred because of the high sensitivity of vacuolated fibres to mechanical stresses and laser irradiation), this procedure led to a fast disappearance of vacuoles. The presence of AO or RH 414 did not disturb the development of vacuolation and its reversibility.
2.4. Confocal microscopy The specimens were viewed under confocal fluorescent scanning microscope (Leica TCS SL) with HCX PLAPO CS 631.32 oil objective. Our observations were restricted to the superficial layers of the fibres, i.e. not deeper than 10e15 mm from the upper surface, which usually touched the cover glass. Single or serial optical sections (Z-series) parallel to the long axis of the fibre were collected at different stages of the experiment. Each of the Z-series contained 10e20 successive optical sections 0.3e0.8 mm thick. For each image,
3.2. Location of AO granules Confocal microscopy revealed important spatial features in the localization of AO granules in living frog muscle fibres. Several granules were always located near both poles of elongated nuclei. Occasionally, they surrounded nuclei along their whole perimeter. Beyond the nuclear region, the AO granules were arranged in short chains, triplets or pairs oriented parallel to myofibrils. In many cases the distance between neighbouring granules was about the same and was equal to the length of sarcomere (Fig. 1aec). The double staining of muscle fibres with AO and RH 414, despite both of these dyes emitting in the red part of spectrum, easily allowed transverse tubules to be differentiated from AO granules (Fig. 2). The latter in most cases were located near the transverse tubules, except for places of vernier arrangement of cross striation. The AO
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granules were arranged in separate pairs (Fig. 1d) accounting for about 15% of all granules. The granules in pairs could be positioned both in the same and in different sites of the adjacent Z-line. The paired location of AO granules was often observed in the perinuclear region of the muscle cell. In some cases, several AO granules of different sizes were located in close proximity to each other to form cluster-like structures (Fig. 1e). The Z-serial sections showed that in this case the granules preserved their spherical shape and individuality. A direct, tether-like connection between AO granules was revealed only in sporadic cases (Fig. 1f). Most often the cluster structures were observed near the nuclear poles, but sometimes they were located between the myofibrils. A clustering of AO granules was typical of fibres that were comparatively enriched with granules. 3.3. Spectral properties of AO granules AO is a metachromatic dye: in monomeric form it has its fluorescence maximum at 525 nm, while in di- and oligomeric forms it is at 590e630 nm. Separate recording of emission in the green (500e560 nm) and red (590e660 nm) region with merging of both images established the existence of three types of granules according to their optical properties: green, red, and mixed. All three types of AO granules could be located in the same optical section, often adjacent to each other as components of chains, pairs or clusters (Fig. 1). The majority of the granules had mixed optical properties; they were present in all recorded images and accounted for 85 4% of all granules. Analysis of serial optical sections indicated that these structures did not result from the apposition of adjoining separate red and green granules, but rather reflected the optical properties of AO within individual membrane vesicles. Red AO granules, with no corresponding structures in the green channel, were present comparatively rarely. The granules that were revealed only in the green channel were also observed in sporadic cases. As a rule, green AO granules were smaller than red ones; this could be for methodological reasons (difference in intensity of green and red emission) as well as due to the osmotic swelling of membrane vesicles that accumulated high concentrations of AO. Fig. 3 showed spectral properties of different regions of AO-stained muscle fibre as revealed by l-scanning. The fluorescence spectra of the sarcoplasm, nuclei, and green granules coincided with the spectrum of monomeric AO solution (see, Haugland, 2004). The spectrum of red granules had its Fig. 1. AO localization in intact frog muscle fibre. (a)e(c) A typical single individual section demonstrates the general pattern of AO distribution within muscle fibre after 90 min staining with AO (0.25 mg/ml). (a) AO fluorescence (500e560 nm, green channel). (b) AO fluorescence (590e660 nm, red channel). (c) Merged image of green and red channels. Numerous small granules are seen in both green and red channels. AO granules are present in longitudinal rows or pairs in the sarcoplasm and near the nuclei. The nuclei emit intensively in green region of the spectrum. The green cross striation is due to the more intensive staining of A-bands. Its periodicity corresponds to the sarcomere length (2.2 mm). (d)e(f) Merged images illustrating various patterns of localization of AO granules in different muscle fibre preparations. Pairs in (d), clusters in (e), bridges in (f). Bars, 2 mm.
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Fig. 2. Acidic organelles and the T-system of intact muscle fibre. A merged image demonstrates the localization of AO granules relative to the T-tubules in a fibre stained simultaneously with AO (0.25 mg/ml) and RH 414 (20 mg/ ml). The T-tubules are seen as red dotted regular lines crossing the fibre. The green bleared spots near the centre of image are out-of-focus nuclei. The inset shows two pairs of granules adhering to transverse tubules at higher magnification. The sarcomere length is 2.2 mm.
maximum in the region of 620 nm. The mixed granules (Fig. 4) were characterized by a small (10e30 nm) shift of green maximum and by an extension of the long-wave part of spectrum, which could be accompanied by the appearance of an additional red peak.
Fig. 4. Fluorescence emission spectra of AO granules. (a) A merged image of AO granules obtained using both green (500e560 nm) and red (600e680 nm) channels. (b) Image of the same granules obtained at 548 nm. (c) Fluorescence emission spectra of single granules marked by numbers in (b). Bars, 2 mm.
3.5. Lyso Tracker Red and BODIPY FL C5-ceramide staining
3.4. Action of monensin A 30-min treatment with monensin led to the disappearance of most AO granules, while the staining of other cellular structures did not change (not shown). Most resistant to monensin were granules near the nuclear poles. The effect of monensin was reversible if AO was present in solution throughout the experiment. We did not observe any changes in spectral properties of AO granules in the course of monensin treatment.
Fig. 3. Fluorescence emission spectra of different regions in the muscle fibre stained by AO. (1) Nucleus, (2) green granule, (3) red granule, (4) sarcoplasm.
Staining with Lyso Tracker Red, a specific probe for labeling and tracing of acidic organelles in living cells, demonstrated many bright granules similar to the AO granules’ size and localization pattern (Fig. 5). In contrast to AO this dye did not stain any other cellular structures. Treatment
Fig. 5. Lyso Tracker Red localization and the T-system of intact frog muscle fibre. Simultaneous staining by Lyso Tracker Red (100 ng/ml) and RH 414 (20 mg/ml). Registration of fluorescence at 590e660 nm. The sarcomere length is 2.2 mm.
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with monensin led to the disappearance of most granules within 10e30 min. Red emission of BODIPY FL C5-ceramide is considered to be specific for the Golgi apparatus (Haugland, 2004). Preliminary experiments with this probe revealed red granules located near the nuclear poles and in the bulk sarcoplasm. They were less numerous than the granules stained by AO or Lyso Tracker Red. 3.6. AO granules and vacuolation Vacuolationedevacuolation of transverse tubules due to glycerol-removal and loading had no marked effect on the pattern of AO-staining (Fig. 6). The lumens of vacuoles at all stages of vacuolation did not accumulate AO. The vacuole membranes were often in close contact with one or several AO granules (Fig. 6b). At early stages of vacuolation, the distribution of vacuoles and AO granules had many common features: both structures were located near nuclei, formed longitudinal chains between myofibrils, and were often arranged in pairs in the space of one sarcomere (Figs. 2 and 6a). Similar results were obtained using Lyso Tracker Red. As the vacuoles increased in size, they disturbed the alignment pattern of AO granules. 4. Discussion On the basis of their spectral properties, AO granules in our experiments can be divided into three groups: green, red, and mixed green/red. In agreement with the metachromatic properties of AO and current knowledge about mechanisms of AO accumulation in acid compartments (Palmgren, 1991; Schindler et al., 1996; Millot et al., 1997; Clerc and Barenholz, 1998; Zelenin, 1999), it can be concluded that the green granules contain AO at a comparatively low concentration in monomeric form. The ‘‘pure’’ red granules accumulate very high concentrations of AO, and the dye is present in these granules
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mainly in aggregated form. As l-scanning demonstrates, most granules belong to the mixed type and contain monomeric and di- and oligomeric aggregated AO molecules in various proportions. The disappearance of granules under the effect of carboxylic ionophore monensin indicates that AO accumulates in membrane organelles whose lumen has an acid pH. As reviewed by Weisz (2003) acidic organelles include the transGolgi cisternae, and various types of endosomes, lysosomes and secretory granules. In most cells the pH of these organelles ranges from 4.5 to 6.5. The pH decreases gradually along the endocytic pathway starting from early endosome (approximately 6.2e6.3) to sorting endosome (about 6.0 and up to 5.4) and late endosome (5.0e5.8). The most acidic organelles are lysosomes (4.6e5.3). The pH of trans-Golgi cisternae is between 5.9 and 6.3. It should be noted that all this data have been obtained on cells of warm-blooded animals and the variations from these pH ranges have been found in many cases. The degree of AO accumulation in the lumen of acidic vesicles mainly depends on the transmembrane pH gradient. However, some other factors, such as aggregation of the dye, the permeability of the vesicular membrane, internal space and osmotic properties of vesicles may influence accumulation of AO (Palmgren, 1991; Clerc and Barenholz, 1998; Weisz, 2003). Therefore, it is difficult to identify the type of AO-accumulating organelle solely by the spectral characteristics of the granules. As can be judged by the spectral properties of AO granules, various low-pH compartments, lysosome-related organelles and Golgi apparatus are involved in formation of AO granules. The localization of AO granules in frog muscle fibres is very similar to that of Golgi apparatus and late endosomes in fast skeletal muscle fibres of mammals (Ralston, 1993; Ralston et al., 1999, 2001; Lu et al., 2001). Our preliminary data with BODIPY FL C5-ceramide fibre staining indicate that the trans-cisternae of Golgi apparatus
Fig. 6. Acidic organelles and the T-system of vacuolated muscle fibre. (a) Staining with RH 414 (20 mg/ml). Vacuolation was produced by loading and subsequent removal of 100 mM glycerol. The image demonstrates the numerous and variable-sized vacuoles in the sarcoplasm and in the perinuclear region. (b) A merged image demonstrates the localization of AO granules relative to the T-tubules as revealed by simultaneous staining with AO (0.25 mg/ml) and RH 414 (20 mg/ml). The sarcomere length is 2.2 mm.
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are responsible at least for some part of AO granules. It is reasonable to consider that the compartments accumulating the highest amount of AO are lysosomes and lysosome-related organelles. The double staining of muscle fibres with AO or Lyso Tracker Red and with RH 414 (the latter stains only the plasma membrane and membranes of transverse tubules) (Krolenko et al., 1995, 1998) reveals some new features in the localization of acidic organelles in the sarcoplasm. Firstly, close contacts are present between acidic organelles and T-tubules at the level of the I-band. These involve almost all granules located between the myofibrils and many granules near nuclear poles, where the T-system is arranged more loosely than between myofibrils and, as has been recently demonstrated by Voigt and Dauber (2004), is in contact with Golgi cisternae. The close juxtaposition of AO granules and T-tubules is also preserved in vacuolated fibres produced by glycerol efflux. As judged by the RH 414 staining pattern, AO granules are attached to, but do not fuse with, membranes of T-tubules. This location of acid organelles may indicate that the T-system is important in the events of membrane trafficking. Additionally, a juxtaposition of lysosomes and related organelles with the membrane of transverse tubules can facilitate the participation of lysosomes in the repair of surface membrane ruptures in muscle fibres (McNeil and Steinhardt, 1997; Reddy et al., 2001). AO granules are not distributed uniformly through the whole muscle fibre, but are located in distinct areas of the cell: typically near the poles of nuclei and between myofibrils where they are arranged in short parallel rows and pairs. Due to their attachment to transverse tubules, neighbouring granules are usually separated by distances corresponding to the sarcomere length. A similar peculiarity of spatial distribution has been reported by Ralston and coworkers for Golgi elements and late endosomes in mammalian muscle cells (Ralston, 1993; Ralston et al., 1999, 2001; Lu et al., 2001). It seems that this type of spatial organization of different membranous organelles in skeletal muscle has been missed in most electron microscopic investigations (Mobley and Eisenberg, 1975; Franzini-Armstrong, 1996), but the functional significance of this arrangement of acidic organelles is not yet clear. The general patterns of spatial distribution of acidic membrane organelles in skeletal muscle fibres have many common features with the topography of local swellings (vacuoles) of the transverse tubules that accompany glycerol efflux (Krolenko et al., 1995; Krolenko and Lucy, 2001) or fatigue (Gonzalez-Serratos et al., 1978; La¨nnergren et al., 1999, 2002). This is particularly obvious at early stages of vacuole development. Thus, small T-tubule vacuoles are about the same size as AO granules and are located near nuclei and over the whole cytoplasm where they often are arranged in longitudinal chains and pairs restricted by adjacent rows of T-tubules. We consider these common features of distribution of vesicular membrane structures to be the result of a very limited ‘‘free’’ space for them in skeletal muscle fibre. About 80% of the frog muscle fibre volume is filled with contractile apparatus and up to 20% can be occupied by
sarcoplasmic reticulum, mitochondria, and nuclei (Mobley and Eisenberg, 1975). Spaces between the myofilaments in the sarcomere are inaccessible to membrane vesicles. Most ‘‘vacant’’ spaces are located near nuclei and between myofibrils and, in the case of amphibian muscles, especially in the region of I-bands near Z-lines. It is here that various vesicular organelles can be accumulated and the swollen T-tubules can protrude to form vacuoles. This can explain the close association of vacuoles and AO granules observed during glycerol efflux. On the other hand, we never observed accumulation of AO in the lumen of vacuoles. Vacuolation in our experiments is easily and completely reversible, and the vacuoles maintain connections with the extracellular space (Krolenko et al., 1998; Devlin et al., 2001; Krolenko and Lucy, 2001; Cooper et al., 2002). Under such conditions, it is difficult to expect acidification of the vacuolar lumen.
Acknowledgements This work was supported by the Russian Foundation for Basic Research, grant N 04-04-49394 and in part by the Joint Research Center ‘‘Material science and characterization in high technology’’. We thank Drs J.A. Lucy, M.D. Faddejeva, Yu.M. Rosanov and G.I. Shtain for their helpful advice and discussion.
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Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres S.A. Krolenko*, S.Ya. Adamyan, T.N. Belyaeva, T.P. Mozhenok Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., 194064 St. Petersburg, Russian Federation Received 7 June 2006; accepted 26 June 2006
Abstract 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 l-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. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Skeletal muscle; Acid organelles; T-system; Vacuolation; Confocal microscopy; Acridine orange
1. Introduction Data on the localization and structure of lysosomes and related acid membrane organelles in skeletal muscle are rather limited (Canonico and Bird, 1969; Bird et al., 1981; Vult von Steyern et al., 1996; Bechet et al., 2005). Recently, however, it has been shown that the spatial organization of the Golgi apparatus, late endosomes, and some other components of membrane transport pathways in striated muscle fibres differs considerably from that in other vertebrate cells in that they are not concentrated exclusively in the perinuclear region but are more or less uniformly dispersed throughout the whole volume of muscle cells (Ralston, 1993; Rahkila et al., 1996; Kaisto et al., 1999; Ralston et al., 1999, 2001; Lu et al., 2001). Another
Abbreviation: AO, acridine orange. * Corresponding author. Tel.: þ7 812 2971866; fax: þ7 812 2970341. E-mail address: [email protected] (S.A. Krolenko).
unique feature of striated muscle cells is the system of transverse tubules (T-system), which comprise deep and regular invaginations of the surface membrane into the cell. Apart from the transmission of electrical signals from the cell surface to the cisternae of sarcoplasmic reticulum, the T-system participates in membrane transport (Rahkila et al., 1996; Kaisto et al., 1999; Brette and Orchard, 2003). The Tsystem is known to be easily vacuolated under the effect of outward fluxes of non-electrolytes and ions during fatigue and in many instances of muscle pathology (Krolenko et al., 1995, 1998; La¨nnergren et al., 1999, 2002; Devlin et al., 2001; Krolenko and Lucy, 2001, 2002; Cooper et al., 2002; Launikonis and Stephenson, 2002). It has been suggested that the vacuoles that accompany denervation, dystrophy, and some other muscle pathological processes are characterized by low-pH and other features of lysosomes (Libelius et al., 1979a,b). The goal of the present work was to evaluate the spatial distribution of acid membrane organelles and their relationships
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.06.017
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with normal and vacuolated transverse tubules. For this purpose, confocal fluorescent scanning microscopy of living muscle fibres was used with several fluorescence probes, such as acridine orange (AO) for acid organelles and lipophilic styryl dye RH 414 for the T-system membranes. AO has been intensively employed for many decades as a vital dye in cell biology (Palmgren, 1991; Schindler et al., 1996; Millot et al., 1997; Clerc and Barenholz, 1998; Zelenin, 1999), while RH 414 has proven to be an effective dye for the evaluation of normal and vacuolated transverse tubules (Krolenko et al., 1995, 1998). 2. Materials and methods 2.1. Isolation of fibres The frogs (Rana temporaria) were killed by decapitation followed by destruction of the spinal cord with a needle. Single fast muscle fibres or small bundles of fibres were isolated from muscle iliofibularis and the experiments were performed in winter/spring months at room temperature (22e25 C). The composition of Ringer solution and the simple chamber used for microscopy have been described earlier (Krolenko et al., 1995, 1998).
4e6 Kalman means were used. The 488 nm line of an argon laser was used for excitation of fibres stained with AO, RH 414 or their combination, and the 488 and 543 nm lines for the combination of RH 414 with Lyso Tracker Red. The laser power did not exceed usually 30% of the maximum value. The fluorescence emissions of muscle fibres stained with AO alone or in combination with RH 414 were simultaneously recorded in green (500e 560 nm) and red (590e660 nm) channels. Due to the metachromatic properties of AO, this procedure permitted evaluation of the monomeric (green fluorescence) and aggregated (red fluorescence) state of the dye. A similar system of registration was used for BODIPY FL C5-ceramide. In experiments with Lyso Tracker Red, the emission was recorded in the red region of 590e660 nm. For more detailed spectral characteristics of various structures of the AOstained fibres, we used a l-scanning system of Leica microscope (regime Mode/xyl) in the 500e660 nm interval with a step size of 10 nm. This scanning was done before or after the two-channel fluorescence recording in the same region of the fibre. The dimensions of analyzed areas were in the order of 1e2 mm2. Confocal image processing was performed using the ordinary program of Leica TCS SL and Image J program. As a standard for the minimal size of AO granules, we used a clustered position of 3 3 pixels, whose intensity exceeded at least three times the surrounding level.
3. Results
2.2. Chemicals and solutions
3.1. AO-staining
The stock solution of AO (Merck) was diluted in normal or glycerol-containing Ringer solution before each experiment to obtain final concentrations of 0.25 mg/ml. Muscle fibres were stained with AO for 40e120 min in a large volume of solution. As a rule, the dye was not removed when the specimens were viewed by confocal microscope. In some experiments, Lyso Tracker Red at a concentration of 50e100 ng/ml and BODIPY FL C5-ceramide at a concentration of 5 mM were used (both dyes from Molecular Probes, Eugene, OR, USA). To evaluate the membranes of normal and vacuolated T-systems we used lipophilic styryl dye RH 414 (Molecular Probes, Eugene, OR, USA) (Krolenko et al., 1995, 1998). The stock solution of RH 414 (1.5 mg/ ml) was diluted in normal or glycerol-containing Ringer solution before each experiment to obtain a final concentration of 15 mg/ml. Fibres were placed in the RH 414 solution 15e30 min before observations and were viewed in the presence of the dye. Carboxylic ionophore monensin (Sigma) was used at a concentration of 30e50 ng/ml.
Intact skeletal muscle fibres stained with AO were characterized by a weak green fluorescence of the whole sarcoplasm and by bright green nuclei (Fig. 1). The green fluorescence intensity of A-band was usually significantly (up to 2e3 times) higher than in other regions of cytosol. AO accumulated in numerous bright spherical granules located near nuclei poles and throughout the whole sarcoplasm (Fig. 1aec). The diameter of the AO granules could vary in the same fibre from about 0.4 up to 1.5 mm. This size variation was confirmed by observing the diameter of the same granules in serial optical sections. The number of granules per square unit of optical sections varied considerably in different fibres as well as in different regions of the same fibre. For example, in the same Z-series the number of granules in individual sections could vary 1.5e2.0 times. Under our conditions, there were, on average, 80 5 granules per 10,000 mm2 of the optical section. This value was the result of measurements in 60 optical sections from 10 fibres.
2.3. Vacuolation of the T-system Vacuolation of the T-system was induced by soaking muscle fibres in Ringer solution with 100 mM glycerol followed by a return to normal Ringer solution. Loading with glycerol lasted for no less than 30 min, and in most experiments started before the transfer of the fibre into the chamber. Glycerolremoval was usually performed under the confocal microscope. It led to the appearance of many vacuoles within several minutes. To induce devacuolation, normal Ringer solution was replaced by Ringer solution containing glycerol. If the fibres were not injured during the first part of the experiment (injury sometimes occurred because of the high sensitivity of vacuolated fibres to mechanical stresses and laser irradiation), this procedure led to a fast disappearance of vacuoles. The presence of AO or RH 414 did not disturb the development of vacuolation and its reversibility.
2.4. Confocal microscopy The specimens were viewed under confocal fluorescent scanning microscope (Leica TCS SL) with HCX PLAPO CS 631.32 oil objective. Our observations were restricted to the superficial layers of the fibres, i.e. not deeper than 10e15 mm from the upper surface, which usually touched the cover glass. Single or serial optical sections (Z-series) parallel to the long axis of the fibre were collected at different stages of the experiment. Each of the Z-series contained 10e20 successive optical sections 0.3e0.8 mm thick. For each image,
3.2. Location of AO granules Confocal microscopy revealed important spatial features in the localization of AO granules in living frog muscle fibres. Several granules were always located near both poles of elongated nuclei. Occasionally, they surrounded nuclei along their whole perimeter. Beyond the nuclear region, the AO granules were arranged in short chains, triplets or pairs oriented parallel to myofibrils. In many cases the distance between neighbouring granules was about the same and was equal to the length of sarcomere (Fig. 1aec). The double staining of muscle fibres with AO and RH 414, despite both of these dyes emitting in the red part of spectrum, easily allowed transverse tubules to be differentiated from AO granules (Fig. 2). The latter in most cases were located near the transverse tubules, except for places of vernier arrangement of cross striation. The AO
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granules were arranged in separate pairs (Fig. 1d) accounting for about 15% of all granules. The granules in pairs could be positioned both in the same and in different sites of the adjacent Z-line. The paired location of AO granules was often observed in the perinuclear region of the muscle cell. In some cases, several AO granules of different sizes were located in close proximity to each other to form cluster-like structures (Fig. 1e). The Z-serial sections showed that in this case the granules preserved their spherical shape and individuality. A direct, tether-like connection between AO granules was revealed only in sporadic cases (Fig. 1f). Most often the cluster structures were observed near the nuclear poles, but sometimes they were located between the myofibrils. A clustering of AO granules was typical of fibres that were comparatively enriched with granules. 3.3. Spectral properties of AO granules AO is a metachromatic dye: in monomeric form it has its fluorescence maximum at 525 nm, while in di- and oligomeric forms it is at 590e630 nm. Separate recording of emission in the green (500e560 nm) and red (590e660 nm) region with merging of both images established the existence of three types of granules according to their optical properties: green, red, and mixed. All three types of AO granules could be located in the same optical section, often adjacent to each other as components of chains, pairs or clusters (Fig. 1). The majority of the granules had mixed optical properties; they were present in all recorded images and accounted for 85 4% of all granules. Analysis of serial optical sections indicated that these structures did not result from the apposition of adjoining separate red and green granules, but rather reflected the optical properties of AO within individual membrane vesicles. Red AO granules, with no corresponding structures in the green channel, were present comparatively rarely. The granules that were revealed only in the green channel were also observed in sporadic cases. As a rule, green AO granules were smaller than red ones; this could be for methodological reasons (difference in intensity of green and red emission) as well as due to the osmotic swelling of membrane vesicles that accumulated high concentrations of AO. Fig. 3 showed spectral properties of different regions of AO-stained muscle fibre as revealed by l-scanning. The fluorescence spectra of the sarcoplasm, nuclei, and green granules coincided with the spectrum of monomeric AO solution (see, Haugland, 2004). The spectrum of red granules had its Fig. 1. AO localization in intact frog muscle fibre. (a)e(c) A typical single individual section demonstrates the general pattern of AO distribution within muscle fibre after 90 min staining with AO (0.25 mg/ml). (a) AO fluorescence (500e560 nm, green channel). (b) AO fluorescence (590e660 nm, red channel). (c) Merged image of green and red channels. Numerous small granules are seen in both green and red channels. AO granules are present in longitudinal rows or pairs in the sarcoplasm and near the nuclei. The nuclei emit intensively in green region of the spectrum. The green cross striation is due to the more intensive staining of A-bands. Its periodicity corresponds to the sarcomere length (2.2 mm). (d)e(f) Merged images illustrating various patterns of localization of AO granules in different muscle fibre preparations. Pairs in (d), clusters in (e), bridges in (f). Bars, 2 mm.
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Fig. 2. Acidic organelles and the T-system of intact muscle fibre. A merged image demonstrates the localization of AO granules relative to the T-tubules in a fibre stained simultaneously with AO (0.25 mg/ml) and RH 414 (20 mg/ ml). The T-tubules are seen as red dotted regular lines crossing the fibre. The green bleared spots near the centre of image are out-of-focus nuclei. The inset shows two pairs of granules adhering to transverse tubules at higher magnification. The sarcomere length is 2.2 mm.
maximum in the region of 620 nm. The mixed granules (Fig. 4) were characterized by a small (10e30 nm) shift of green maximum and by an extension of the long-wave part of spectrum, which could be accompanied by the appearance of an additional red peak.
Fig. 4. Fluorescence emission spectra of AO granules. (a) A merged image of AO granules obtained using both green (500e560 nm) and red (600e680 nm) channels. (b) Image of the same granules obtained at 548 nm. (c) Fluorescence emission spectra of single granules marked by numbers in (b). Bars, 2 mm.
3.5. Lyso Tracker Red and BODIPY FL C5-ceramide staining
3.4. Action of monensin A 30-min treatment with monensin led to the disappearance of most AO granules, while the staining of other cellular structures did not change (not shown). Most resistant to monensin were granules near the nuclear poles. The effect of monensin was reversible if AO was present in solution throughout the experiment. We did not observe any changes in spectral properties of AO granules in the course of monensin treatment.
Fig. 3. Fluorescence emission spectra of different regions in the muscle fibre stained by AO. (1) Nucleus, (2) green granule, (3) red granule, (4) sarcoplasm.
Staining with Lyso Tracker Red, a specific probe for labeling and tracing of acidic organelles in living cells, demonstrated many bright granules similar to the AO granules’ size and localization pattern (Fig. 5). In contrast to AO this dye did not stain any other cellular structures. Treatment
Fig. 5. Lyso Tracker Red localization and the T-system of intact frog muscle fibre. Simultaneous staining by Lyso Tracker Red (100 ng/ml) and RH 414 (20 mg/ml). Registration of fluorescence at 590e660 nm. The sarcomere length is 2.2 mm.
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with monensin led to the disappearance of most granules within 10e30 min. Red emission of BODIPY FL C5-ceramide is considered to be specific for the Golgi apparatus (Haugland, 2004). Preliminary experiments with this probe revealed red granules located near the nuclear poles and in the bulk sarcoplasm. They were less numerous than the granules stained by AO or Lyso Tracker Red. 3.6. AO granules and vacuolation Vacuolationedevacuolation of transverse tubules due to glycerol-removal and loading had no marked effect on the pattern of AO-staining (Fig. 6). The lumens of vacuoles at all stages of vacuolation did not accumulate AO. The vacuole membranes were often in close contact with one or several AO granules (Fig. 6b). At early stages of vacuolation, the distribution of vacuoles and AO granules had many common features: both structures were located near nuclei, formed longitudinal chains between myofibrils, and were often arranged in pairs in the space of one sarcomere (Figs. 2 and 6a). Similar results were obtained using Lyso Tracker Red. As the vacuoles increased in size, they disturbed the alignment pattern of AO granules. 4. Discussion On the basis of their spectral properties, AO granules in our experiments can be divided into three groups: green, red, and mixed green/red. In agreement with the metachromatic properties of AO and current knowledge about mechanisms of AO accumulation in acid compartments (Palmgren, 1991; Schindler et al., 1996; Millot et al., 1997; Clerc and Barenholz, 1998; Zelenin, 1999), it can be concluded that the green granules contain AO at a comparatively low concentration in monomeric form. The ‘‘pure’’ red granules accumulate very high concentrations of AO, and the dye is present in these granules
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mainly in aggregated form. As l-scanning demonstrates, most granules belong to the mixed type and contain monomeric and di- and oligomeric aggregated AO molecules in various proportions. The disappearance of granules under the effect of carboxylic ionophore monensin indicates that AO accumulates in membrane organelles whose lumen has an acid pH. As reviewed by Weisz (2003) acidic organelles include the transGolgi cisternae, and various types of endosomes, lysosomes and secretory granules. In most cells the pH of these organelles ranges from 4.5 to 6.5. The pH decreases gradually along the endocytic pathway starting from early endosome (approximately 6.2e6.3) to sorting endosome (about 6.0 and up to 5.4) and late endosome (5.0e5.8). The most acidic organelles are lysosomes (4.6e5.3). The pH of trans-Golgi cisternae is between 5.9 and 6.3. It should be noted that all this data have been obtained on cells of warm-blooded animals and the variations from these pH ranges have been found in many cases. The degree of AO accumulation in the lumen of acidic vesicles mainly depends on the transmembrane pH gradient. However, some other factors, such as aggregation of the dye, the permeability of the vesicular membrane, internal space and osmotic properties of vesicles may influence accumulation of AO (Palmgren, 1991; Clerc and Barenholz, 1998; Weisz, 2003). Therefore, it is difficult to identify the type of AO-accumulating organelle solely by the spectral characteristics of the granules. As can be judged by the spectral properties of AO granules, various low-pH compartments, lysosome-related organelles and Golgi apparatus are involved in formation of AO granules. The localization of AO granules in frog muscle fibres is very similar to that of Golgi apparatus and late endosomes in fast skeletal muscle fibres of mammals (Ralston, 1993; Ralston et al., 1999, 2001; Lu et al., 2001). Our preliminary data with BODIPY FL C5-ceramide fibre staining indicate that the trans-cisternae of Golgi apparatus
Fig. 6. Acidic organelles and the T-system of vacuolated muscle fibre. (a) Staining with RH 414 (20 mg/ml). Vacuolation was produced by loading and subsequent removal of 100 mM glycerol. The image demonstrates the numerous and variable-sized vacuoles in the sarcoplasm and in the perinuclear region. (b) A merged image demonstrates the localization of AO granules relative to the T-tubules as revealed by simultaneous staining with AO (0.25 mg/ml) and RH 414 (20 mg/ml). The sarcomere length is 2.2 mm.
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are responsible at least for some part of AO granules. It is reasonable to consider that the compartments accumulating the highest amount of AO are lysosomes and lysosome-related organelles. The double staining of muscle fibres with AO or Lyso Tracker Red and with RH 414 (the latter stains only the plasma membrane and membranes of transverse tubules) (Krolenko et al., 1995, 1998) reveals some new features in the localization of acidic organelles in the sarcoplasm. Firstly, close contacts are present between acidic organelles and T-tubules at the level of the I-band. These involve almost all granules located between the myofibrils and many granules near nuclear poles, where the T-system is arranged more loosely than between myofibrils and, as has been recently demonstrated by Voigt and Dauber (2004), is in contact with Golgi cisternae. The close juxtaposition of AO granules and T-tubules is also preserved in vacuolated fibres produced by glycerol efflux. As judged by the RH 414 staining pattern, AO granules are attached to, but do not fuse with, membranes of T-tubules. This location of acid organelles may indicate that the T-system is important in the events of membrane trafficking. Additionally, a juxtaposition of lysosomes and related organelles with the membrane of transverse tubules can facilitate the participation of lysosomes in the repair of surface membrane ruptures in muscle fibres (McNeil and Steinhardt, 1997; Reddy et al., 2001). AO granules are not distributed uniformly through the whole muscle fibre, but are located in distinct areas of the cell: typically near the poles of nuclei and between myofibrils where they are arranged in short parallel rows and pairs. Due to their attachment to transverse tubules, neighbouring granules are usually separated by distances corresponding to the sarcomere length. A similar peculiarity of spatial distribution has been reported by Ralston and coworkers for Golgi elements and late endosomes in mammalian muscle cells (Ralston, 1993; Ralston et al., 1999, 2001; Lu et al., 2001). It seems that this type of spatial organization of different membranous organelles in skeletal muscle has been missed in most electron microscopic investigations (Mobley and Eisenberg, 1975; Franzini-Armstrong, 1996), but the functional significance of this arrangement of acidic organelles is not yet clear. The general patterns of spatial distribution of acidic membrane organelles in skeletal muscle fibres have many common features with the topography of local swellings (vacuoles) of the transverse tubules that accompany glycerol efflux (Krolenko et al., 1995; Krolenko and Lucy, 2001) or fatigue (Gonzalez-Serratos et al., 1978; La¨nnergren et al., 1999, 2002). This is particularly obvious at early stages of vacuole development. Thus, small T-tubule vacuoles are about the same size as AO granules and are located near nuclei and over the whole cytoplasm where they often are arranged in longitudinal chains and pairs restricted by adjacent rows of T-tubules. We consider these common features of distribution of vesicular membrane structures to be the result of a very limited ‘‘free’’ space for them in skeletal muscle fibre. About 80% of the frog muscle fibre volume is filled with contractile apparatus and up to 20% can be occupied by
sarcoplasmic reticulum, mitochondria, and nuclei (Mobley and Eisenberg, 1975). Spaces between the myofilaments in the sarcomere are inaccessible to membrane vesicles. Most ‘‘vacant’’ spaces are located near nuclei and between myofibrils and, in the case of amphibian muscles, especially in the region of I-bands near Z-lines. It is here that various vesicular organelles can be accumulated and the swollen T-tubules can protrude to form vacuoles. This can explain the close association of vacuoles and AO granules observed during glycerol efflux. On the other hand, we never observed accumulation of AO in the lumen of vacuoles. Vacuolation in our experiments is easily and completely reversible, and the vacuoles maintain connections with the extracellular space (Krolenko et al., 1998; Devlin et al., 2001; Krolenko and Lucy, 2001; Cooper et al., 2002). Under such conditions, it is difficult to expect acidification of the vacuolar lumen.
Acknowledgements This work was supported by the Russian Foundation for Basic Research, grant N 04-04-49394 and in part by the Joint Research Center ‘‘Material science and characterization in high technology’’. We thank Drs J.A. Lucy, M.D. Faddejeva, Yu.M. Rosanov and G.I. Shtain for their helpful advice and discussion.
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