Laser Confocal scanning microscopy of the surface membrane/T-tubular system and the sarcoplasmic reticulum in insect striated muscle stained with DiIC18(3)

The structure of the surface membrane/transverse tubular (T-tubular) system and of the sarcoplasmic reticular (SR) of the labial adductor muscle of the honey bee (Apis mellifera) was examined by laser confocal scanning microscopy, after staining with the fluorescent membrane probe DiIC₁₈(3). The following components of the surface membrane/T-tubular system were visualized: transverse tubular networks that are located in the A-band close to the A–I junction and form dyads with the SR, longitudinal tubules that link the T-tubular networks within and between sarcomeres, and surface invaginations of larger diameter that contain tracheoles. The well developed SR forms a dense network of branching and anastomosing tubules in the A-band. A few tubular elements in the interfibrillar space in the 1-band link the SR of adjacent sarcomeres. This study demonstrates the advantages of the laser confocal microscope and lipophilic fluorescent dyes for studying the 3-D structure of cellular membrane systems.     

Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis.     

Imaging of changes in sarcoplasmic reticulum [Ca(2+)] using Oregon Green BAPTA 5N and confocal laser scanning microscopy

We describe experiments in which the low affinity indicator Oregon Green BAPTA 5N was used to record the spatially resolved changes in [Ca(2+)] from intracellular stores in rat gastric myocytes. Cells were loaded with the membrane permeant form of the indicator and imaged using a confocal scanning laser microscope. In doubly stained cells the Oregon Green signal colocalized with BIODIPY 558/568 Brefeldin A, a label for the endo/sarcoplasmic reticulum (SR) and Golgi apparatus. Oregon Green BAPTA 5N was calibrated in gastric myocytes, giving an in situ K(d) of 90 microM. The resting free [Ca(2+)] within the SR averaged 65 microM. A reversible decrease in Oregon Green fluorescence was observed on bath application of Inositol triphosphate (IP(3)) (10 microM) to permeabilized cells. Similar changes were also observed when cyclopiazonic acid (5 microM) was applied to intact myocytes, again with recovery of store [Ca(2+)] following drug washout. Identical patterns of Ca(2+) depletion were seen when caffeine (1 microM) and carbachol (10 microM) were applied sequentially to the same cells, suggesting that activation of ryanodine and IP(3)-sensitive channels can result in the release of Ca(2+) from the same regions of the SR.     

Variation in the lipid composition of rabbit muscle sarcoplasmic reticulum membrane with muscle type

The compositions of sarcoplasmic reticulum (SR) membranes from rabbit caudofemoralis, tibialus, and soleus muscles (fast, mixed, and slow twitch, respectively) were analyzed. Compared to caudofemoralis (fast twitch) SR, soleus (slow twitch) SR contained a significantly greater percentage of cholesterol, phosphatidylinositol, and sphingomyelin and a lesser percentage of phosphatidylcholine. Correlations between properties reported for the SR isolated from different muscle types and our analyses of the compositions are discussed. We suggest that the greater cholesterol content and the greater sphingomyelin to phosphatidylcholine ratio present in soleus SR contribute to decreased bilayer fluidity and, hence, decreased Ca2+-ATPase activity.     

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

 We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. Accepted: 28 August 1996     

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.     

Tandem scanning confocal microscopy (TSCM) of normal and ischemic living kidneys

Tandem Scanning Confocal Microscopy (TSCM) allows one to section optically into and record real-time images of living organs and tissues in a noninvasive fashion. In this paper, we will present some initial TSCM observations of subcapsular nephrons in the living, intact kidneys of Munich-Wistar rats and evaluate the nephron's responses to temporary ischemia and to intravenous infusion of mannitol. The rats were anesthetized with Inactin and a laparotomy performed to expose the kidneys. Using a TSCM equipped with a 20 x water-immersion objective, we optically sectioned through the intact kidney capsule and recorded real-time images of living subcapsular glomeruli and uriniferous tubules. The proximal tubule brush border was highly reflective and allowed us to distinguish between the first and second segments of the proximal tubules as well as the distal tubules. Cellular elements of the blood could be seen passing rapidly through peritubular capillaries and individual glomerular capillary loops. With fluorescent filters in place, intravenously injected carboxyfluorescein was seen to pass through the glomerular capillary loops and then progressively through the different segments of the uriniferous tubules. Ligation of the renal artery resulted in rapid swelling of proximal tubule cells into the tubular lumens, loss of reflectiveness of the microvillous brush borders, and closure of the peritubular capillary spaces. Upon release of the ligature, the proximal tubule lumens again became patent, often opening up abruptly and in a zipper-like fashion down the length of the tubules. Increasing the glomerular filtration rate by intravenous infusion of mannitol resulted in increases in tubular luminal and perimeter dimensions. Mannitol also acted as an effective impermeant osmotic agent and prevented most of the cellular swelling which was otherwise seen in response to renal ischemia.     

Pacemaker activity in the insect (T. molitor) heart: role of the sarcoplasmic reticulum

The electrophysiological properties of the myogenic cardiac cells of insects have been analyzed, but the mechanisms that regulate the pacemaker activity have not been elucidated yet. In mammalian pacemaker cells, different types of membrane ion channels seem to be sequentially activated, perhaps in a cooperative fashion with the current generated by Ca(2+) extrusion mediated by the electrogenic Na(+)/Ca(2+) exchanger, which is sustained by the diastolic sarcoplasmic reticulum (SR) Ca(2+) release. The objective of the present work was to investigate the role of the SR function on the basal beating rate (BR), and BR modulation by extracellular Ca(2+) concentration ([Ca(2+)](o)) and neurotransmitters in the in situ dorsal vessel (heart) of the mealworm beetle Tenebrio molitor. The main observations were as follows: 1) basal BR was reduced by 50% by inhibition of SR function, but not affected by perfusion with CsCl or ZD7288; 2) spontaneous activity was abolished by Cd(2+); 3) a robust positive chronotropic response could be elicited to serotonin (5-HT), but not to norepinephrine or carbamylcholine; 4) SR inhibition abolished the sustained chronotropic stimulation by [Ca(2+)](o) elevation and by 5-HT, while the latter was unaffected by CsCl. It is concluded that, in T. molitor heart, BR is markedly, but not exclusively, dependent on the SR function, and that BR control and modulation by both [Ca(2+)](o) and 5-HT requires a functional SR.     

Three-dimensional electron microscopy of the sarcoplasmic reticulum and T-system in embryonic chick skeletal muscle cells in vitro

Summary The formation of the sarcoplasmic reticulum (SR) and the transverse tubular system (T-system) in embryonic chick skeletal muscle cells in vitro was studied by either the critical point drying-physical rupturing or physical rupturing-freeze drying together with rotary shadowing. In these cells, two membranous systems were observed. One was composed of flattened sacs which were either isolated or were connected to each other with slender processes to form mostly longitudinally oriented strands. Initially, these sacs had small granules at their surface and were found mainly under the sarcolemma. Later, they became smooth at their surface, extending throughout the cytoplasm to form irregular and dense networks. At later phases, the networks tended to be disposed at right angle to nascent myofibrils, exhibiting a characteristic honeycomb appearance. From the similarities in thin section images, they were identified as developing SR.The other membranous system were tubules with many enlargements. They were frequently associated with coated vesicles which appeared to take part in the formation, elongation, and anastomosing of developing tubules. These tubules could be impregnated with a tannic acid-glutaraldehyde-potassium ferrocyanide complex and, thus, were identified as T-tubules.Abbreviations CPD critical-point drying - ES exoplasmic surface of the sarcolemma - FD freeze-drying - PR physical rupture - PS protoplasmic surface of the sarcolemma - SR sarcoplasmic reticulum - TAGPF tannic acid-glutaraldehyde-potassium ferrocyanide - T-system transverse tubular system     

Erythrosin B (USFD&C RED 3) inhibits calcium transport and atpase activity of muscle sarcoplasmic reticulum

Erythrosin B (USFD&;C RED 3) inhibits the transport of calcium ions into isolated rabbit muscle sarcoplasmic reticulum vesicles with an IC₅₀ of ～ 0.5 μM and inhibits the Ca²⁺Mg²⁺ ATPase activity with an IC₅₀ of ～ 1 μM. The dye also binds to this tissue with an apparent K_d of ～ 300 nM. Other iodinated and brominated fluorescein analogs and blue dextran also inhibit ATPase activity and displace bound dye, suggesting that erythrosin may bind to a site near to but not identical with the nucleotide site. The dye should prove to be a useful probe for transport and ATPase activity.     

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.     

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane

Summary Two-dimensional crystalline arrays of Ca²⁺-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na₃VO₄ in a Ca²⁺-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C₂(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca²⁺-ATPase, while negative potential disrupts preformed Ca²⁺-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca²⁺-ATPase.     

Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the effect of ionol. II. Physico- chemical properties of the sarcoplasmic reticulum membrane

Physico-chemical parameters of membranes of skeletal muscles' sarcoplasmic reticulum in antioxidant insufficiency, which was modelled by excluding alpha-tocopherol from the animals ration, and after treatment with phenol antioxidant ionol were studied. It was shown that activation of lipid peroxidation in vitamin E insufficiency results in a significant lowering of microviscosity of lipid bilayer membranes of sarcoplasmic reticulum. Using polarography significant changes in membrane protein conformation were revealed, which were characterized by lowering of integrity and by disorganization of protein globules. Treatment of animals with antioxidant insufficiency with ionol led to certain normalization of changes of physico-chemical characteristics of the learned membrane structures caused by lipid peroxidation.     

The musculature of three species of gastrotrichs surveyed with confocal laser scanning microscopy (CLSM)

The muscular system of gastrotrichs consists of circular, longitudinal and helicoidal bands that when analysed with confocal laser scanning microscopy, provide new insights into their functional organization and phylogenetic importance. We therefore undertook a comparative study of the muscle organization in three species of Gastrotricha from the orders Macrodasyida (Paradasys sp., Lepidodasyidae; Turbanella sp., Turbanellidae) and Chaetonotida (Polymerurus nodicaudus, Chaetonotidae). The general muscle organization of the marine interstitial macrodasyidans, Paradasys and Turbanella, not only confirms earlier observation on other species but also adds new details concerning the organization and number of helicoidal, longitudinal and other muscle bands (e.g. semicircular band). The freshwater, epibenthic–epiphytic chaetonotid, Polymerurus nodicaudus, has a similar muscular organization to other species of Chaetonotidae, especially species of Chaetonotus, Halichaetonotus and Lepidodermella. Perhaps unique to Polymerurus is the combined presence of an unbranched Rückenhautmuskel (also in Halichaetonotus and Lepidodermella) and a specialized dorsoventral caudal muscle, which flank the splanchnic component of the longitudinal muscles (only in Chaetonotus and Lepidodermella). This combination, together with the presence of splanchnic dorsoventral muscles, known only in Xenotrichulidae, implies a unique phylogenetic position for Polymerurus, and indicates a potential basal position of this taxon among the Chaetonotidae studied so far (i.e. Aspidiophorus, Chaetonotus, Halichaetonotus and Lepidodermella).