The distribution of membrane-glycogen complexes in the orbital surface layer of rabbit superior rectus

The distribution of membrane-glycogen complexes along the length of individual muscle fibers was compared among three fiber populations in the orbital surface layer of rabbit superior rectus. These three populations were (a) 61 singly innervated fibers (SIFs), (b) 10 multiply innervated fibers of relatively constant 10 μm diameter (10 μm MIFs), and (c) 22 multiply innervated fibers which are of about 5 μm diameter toward the middle of their length and of about 15 μm diameter toward their proximal and distal segments (5–15 μn MIFs). The orbital surface layer was sampled by electron microscopy at 68 sequential locations. Membrane-glycogen complexes were not seen in any of the 1738 samples of the SIFs. In the MIFs, such complexes were observed in 14% of the 1541 samples. However, both the 10 μm MIFs and 5–15 μm MIFs displayed a preferential concentration of membrane-glycogen complexes toward their distal fiber portions, and such complexes were seen in about 50% of the MIF samples near the beginning of the muscle's distal third. In the distal portion of 5–15 μm MIFs, there was a direct relationship between their increasing fiber diameter and their increasing frequency of occurrence of membrane-glycogen complexes.     

The elasticity of single titin molecules using a two-bead optical tweezers assay

Titin is responsible for the passive elasticity of the muscle sarcomere. The mechanical properties of skeletal and cardiac muscle titin were characterized in single molecules using a novel dual optical tweezers assay. Antibody pairs were attached to beads and used to select the whole molecule, I-band, A-band, a tandem-immunoglobulin (Ig) segment, and the PEVK region. A construct from the PEVK region expressing >25% of the full-length skeletal muscle isoform was chemically conjugated to beads and similarly characterized. By elucidating the elasticity of the different regions, we showed directly for the first time, to our knowledge, that two entropic components act in series in the skeletal muscle titin I-band (confirming previous speculations), one associated with tandem-immunoglobulin domains and the other with the PEVK region, with persistence lengths of 2.9 nm and 0.76 nm, respectively (150 mM ionic strength, 22 degrees C). Novel findings were: the persistence length of the PEVK component rose (0.4-2.7 nm) with an increase in ionic strength (15-300 mM) and fell (3.0-0.3 nm) with a temperature increase (10-60 degrees C); stress-relaxation in 10-12-nm steps was observed in the PEVK construct and hysteresis in the native PEVK region. The region may not be a pure random coil, as previously thought, but contains structured elements, possibly with hydrophobic interactions.     

Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.     

Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

Mitochondrial Activity in Orobanche cernua from Different Regions of the Scape and as Influenced by Host

Orobanche cernua, a holoparasite, was harvested from different hosts, namely, Solanum melongena, Petunia hybrida, Lycopersicum esculentum, Solanum nigrum and Datura metel. Mitochondrial particles were isolated and they were evaluated in terms of the marker enzyme, cytochrome c oxidase, and protein in the mitochondrial fraction. Protein levels in whole homogenate and mitochondrial fraction of parasite growing on different host plants did not vary significantly, whereas the recovery of protein (% of whole homogenate) in the mitochondrial fraction of parasite growing on D. metel was higher. Cytochrome c oxidase activity in parasites growing on the three host plants varied, being highest when the parasite grew on S. melongena, followed sequentially by that on P. hybrida and D. metel. Protein in whole homogenate and mitochondrial fraction was significantly lower, 25–36% and 15–33%, respectively, in distal region when compared with the proximal region. Similarly, cytochrome oxidase and respiratory activity was significantly lower, 23–34% and 18–23%, respectively, in the distal region of the parasite. In addition, variations in cytochrome oxidase and respiratory activity in the proximal and distal regions of the parasite growing on different hosts was also significant. Results indicated that mitochondria in haustoria‐bearing proximal region of Orobanche scape play a special role to meet the metabolic demand of the parasite.     

Steered molecular dynamics studies of titin I1 domain unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.     

Localization of the parallel elastic components in frog skinned muscle fibers studied by the dissociation of the A- and I-bands.

Localization of the parallel elastic components (PECs) in skinned muscle fibers was investigated by analyzing the change of the resting tension, which accompanies the dissociation of the A- and I-bands. The A-band was dissociated from both ends by increasing the concentration of KCl under relaxing conditions (0.09-0.54 M KCl, 4.0 mM MgATP, 1.0 mM Mg2+, 4.0 mM EGTA, pH 6.0-9.0, 20 degrees C). At sarcomere lengths greater than or equal to 3.5 microns, the length of the A-band was estimated by comparing the intensity of the first-order optical diffraction line with the results of model calculations. These results were supported by differential-interference microscopy and sodium dodecyl sulfate gel electrophoresis. It was shown that the resting tension decreased nearly in proportion to the residual length of the A-band. At sarcomere lengths less than or equal to 4.0 microns, the resting tension after the dissociation of the A-band was lowered to less than 10% of the initial value. On the other hand, at sarcomere lengths greater than or equal to 5.0 microns the resting tension after the dissociation of the A-band still showed approximately 35% of the initial value and did not change even after the I-band was dissociated by a solution containing KI. From these results, we propose that most of the PECs contributing to resting tension bind almost uniformly to the A-band and there are also PECs connecting Z-lines.     

Clustering of cyclic-nucleotide-gated channels in olfactory cilia

Olfactory cilia contain the known components of olfactory signal transduction, including a high density of cyclic-nucleotide-gated (CNG) channels. CNG channels play an important role in mediating odor detection. The channels are activated by cAMP, which is formed by a G-protein-coupled transduction cascade. Frog olfactory cilia are 25-200 microm in length, so the spatial distribution of CNG channels along the length should be important in determining the sensitivity of odor detection. We have recorded from excised cilia and modeled diffusion of cAMP into a cilium to determine the spatial distribution of the CNG channels along the ciliary length. The proximal segment, which in frog is the first 20% of the cilium, appears to express a small fraction of the CNG channels, whereas the distal segment contains the majority, mostly clustered in one region.     

Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure of the sarcoplasmic reticulum (SR) in the red, white and intermediate striated muscle fibers of the extensor digitorum longus muscle of the rat was examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of the A-I junction. Numerous slender sarcotubules, originating from the A-band side terminal cisternae, extend obliquely or longitudinally and form oval or irregular shaped networks of various sizes in front of the A-band, then become continuous with the tiny mesh (fenestrated collar) in front of the H-band. The A-and H-band SR appears as a single sheet of anastomotic tubules. Numerous sarcotubules, originating from the I-band side terminal cisternae, extend in threedimensional directions and form a multilayered network over the I-band and Z-line regions. At the I-band level, paired transversely oriented mitochondria partly embrace the myofibril. The I-band SR network is poorly developed in the narrow space between the paired mitochondria, but is well developed in places devoid of these mitochondria.The three-dimensional structure of the SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a much smaller total volume of SR than the mitochondria-poor white fiber. Moreover, the volume of SR of the intermediate fiber is intermediate between the two.     

The jumping mechanism of Xenopsylla cheopis. II. The fine structure of the jumping muscle.

The ultrastructure of the trochanteral depressor muscle of the oriental rat flea is described. It is shown to be similar to that of the tubular leg muscles of other insects except in the volume and arrangement of the sarcoplasmic reticulum. The sarcoplasmic reticulum occupies approximately 18% of the volume of the muscle fibres. It is in three configurations:a regular array of parallel tubules opposite the A-band, a collar of sacculi involved in the formation of the dyads at the edge of the A-band and a loosely woven arrangement of tubules around the I-band. This tripartite arrangement of the sarcoplasmic reticulum and its large surface area is discussed in relation to the action of the muscle as the main propulsive muscle in the jump of the flea.     

Hormonal effects on mitochondrial respiration: potential role of endogenous lipolytic activities

Hormonal effects on heart mitochondrial metabolism are investigated by comparing respiratory rates, Ca2+ uptake capacity, and lipolytic activities of mitochondria isolated from control rats to those of mitochondria isolated from thyroparathyroidectomized animals. Two biochemically and morphologically distinct populations of heart mitochondria are prepared--one derived from the region of the cell directly beneath the sarcolemma (subsarcolemmal mitochondria), the other originally between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria isolated from normal rat cardiac tissue have both lower respiratory rates and Ca2+ uptake capacity than do interfibrillar mitochondria. However, when these mitochondrial populations are isolated from hearts from thyroparathyroidectomized rats, there is a selective increase in the maximal ability of the subsarcolemmal mitochondria to accumulate Ca2+, which is accompanied by a proportionate increase in their maximal respiratory rates. Neither Ca2+ uptake capacity nor respiratory rates are similarly increased in the interfibrillar mitochondria. Cytochrome contents and mitochondrial protein recoveries are not significantly changed in either of these mitochondrial preparations. The relationship between these selective increases in respiratory properties of the subsarcolemmal mitochondria to endogenous lipolytic activities is also investigated. It was previously demonstrated that, in the absence of Ca2+, both the rate and extent of formation of free fatty acids from endogenous phospholipids is greater in subsarcolemmal than interfibrillar mitochondria (J. W. Palmer et al. (1981) Arch. Biochem. Biophys. 211, 674-682). In this study it is shown that lipolysis is also more sustained in the subsarcolemmal mitochondria when Ca2+ is added. In the subsarcolemmal mitochondria isolated from thyroparathyroidectomized rats, however, the rates of release of stearic acid and oleic acid are reduced in both the presence and absence of Ca2+. In the presence of added Ca2+, the rate of release of arachidonic acid is also decreased compared to control subsarcolemmal mitochondria, suggesting that the expressed activity of Ca2+-activated phospholipase A2 is lower in those mitochondria isolated from the thyroparathyroidectomized animals, in which respiratory rates and Ca2+ uptake capacity are increased.     

The ommatidia of Arca noae: a three-tier structure with a central light-guiding element for the receptor cell

The compound eyes of ark clams appear to function as an optical system to trigger shell closure against predators. We have analyzed the structure of the ommatidia of Arca noae by thin section electron microscopy and serial sectioning, Concanavalin A-gold labeling and acid phosphatase cytochemistry. Our results demonstrate that the ommatidia are a three-tier structure composed of a central single receptor cell, surrounded and covered by proximal pigment cells followed by rows of distal pigment cells. The receptor cells of Arca noae have no lens and the disks of their receptive segment are derived from sensory cilia. The distal mitochondrial segment in the cytoplasm between the nucleus and the receptive segment is surrounded by a mass of Concanavalin A-reactive glycogen particles. Although both, proximal and distal pigment cells have numerous microvilli, only those of the proximal pigment cells form a well-aligned brush border. The microvilli of the latter are ≈9-11?μm long and have a diameter of ≈70-80?nm. Numerous microlamellar bodies cover them. The microlamellar bodies are stored in acid phosphatase-negative secretory granules of the pigment granule-free apical cytoplasm of proximal pigment cells before their secretion. Observation of living compound eyes indicated that the apex of proximal pigment cells transmitted significantly more light than the surrounding distal pigment cells. Hence, the regular geometry of the brush border seems to be a light-guiding structure for receptor cells similar to an optical fiber.     

Morphometry of the galliform cecum: A comparison between Gambel's quail and the domestic fowl

Summary Tissues from the proximal, middle, and distal regions of the ceca of Gambel's quail and domestic fowl were examined by scanning and transmission electron microscopy. Cellular and subcellular structures, including epithelial cell height, mitochondrial volume fraction, microvillous surface area, proportion of goblet cells, and junctional complex characteristics, were quantified by a variety of stereologic procedures and other measurement techniques. The mucosal surface of quail cecum shows a much more highly developed pattern of villous ridges and flat areas than that of fowl cecum. The fowl has significantly greater cell heights than the quail in all cecal regions. The mitochondrial volume fraction does not differ significantly with species or region, but mitochondria are concentrated on the apical side of the nucleus. In both species, the proximal cecal region has the greatest microvillous surface area. All 3 components of junctional complexes, including zonula occludens, zonula adhaerens, and macula adhaerens, are quantified. When all factors are considered, the quail cecum appears to have morphological characteristics consistent with a greater potential capacity for absorption than the fowl cecum.     

Effect of cold environment on skeletal muscle mitochondria in growing rats

Summary Growing rats (4 weeks old) were kept for 3 weeks at 11° C and 24° C respectively. The cold-adapted animals showed a significantly higher oxygen consumption (64%). Volume density of subsarcolemmal and interfibrillar mitochondria as well as volume density of fat droplets were estimated in M. soleus and the diaphragm of both groups. In cold-adapted animals, the total volume of mitochondria was significantly increased by 24% in diaphragm and 37% in M. soleus. The volume of subsarcolemmal mitochondria was almost doubled in each muscle, but the volume of interfibrillar mitochondria did not change significantly. The surface of the inner mitochondrial membranes per unit volume of mitochondrion in M. soleus was significantly increased both in interfibrillar and subsarcolemmal mitochondria, whereas the surface of the outer mitochondrial membranes per unit volume of mitochondrion was increased only in the subsarcolemmal mitochondria. The volume of fat droplets in the diaphragm and M. soleus of cold adapted animals increased significantly by 62% and 150% respectively.     

Magnesium secretion by the distal segment of the Malpighian tubules of the black field cricket Teleogryllus oceanicus

The short distal segment of unstimulated Teleogryllus Malpighian tubules secreted hyperosmotic fluid containing primarily Mg (125mmoll(-1)), Cl (242mmoll(-1)) and Na (43mmoll(-1)). Remarkably, the volume secreted by the distal segment in unit time was independent of segment length, i.e. the volume was constant regardless of the length of the segment. Magnesium was secreted at a rate of 75.5pmolmin(-1)mm(-1); the highest rate recorded for any epithelium. Low concentrations of K (20mmoll(-1)) were present but almost no P or S. Ca (2.5mmoll(-1)) concentration was higher than in the main segment. The short distal segment secreted 100% of the Mg, 54% of the Cl and 23% of the Na secreted by the whole tubule. The main segment secreted fluid containing primarily K (199mmoll(-1)), Cl (149mmoll(-1)), Na (104mmoll(-1)) and P (48mmoll(-1)) with very low concentrations of Ca (1mmoll(-1)) and S. The main segment appeared to reabsorb a small fraction of the Mg secreted by the distal segment. The fluid secreted by the whole tubule was isosmotic and alkaline, approximately pH8.     

The retina of the phalangid,Opilio ravennae,with particular reference to arhabdomeric cells

Summary The retina of the phalangid, Opilio ravennae, consists of retinula cells with distal rhabdomeres, arhabdomeric cells, and sheath cells. The receptive segment of retinula cells shows a clear separation into a Proximal rhabdom, organized into distinct rhabdom units formed by three or four retinula cells, and a Distal rhabdom, consisting of an uniterrupted layer of contiguous rhabdomeres. One of the cells comprising a retinula unit, the so-called distal retinula cell (DRC), has two or three branches that pass laterally alongside the rhabdom, thereby separating the two or three principal retinula cells of a unit. The two morphologically distinct layers of the receptive segment differ with respect to the cellular origin of rhabdomeral microvilli: DRC-branches contribute very few microvilli to the proximal rhabdom and develop extremely large rhabdomeres in the distal rhabdom only, causing the rhabdom units to fuse. Principal retinula cells, on the other hand, comprise the majority of microvilli of the proximal rhabdom, but their rhabdomeres diminish in the distal rhabdom. It is argued that proximal and distal rhabdoms serve different functions in relation to the intensity of incident light.In animals fixed 4 h after sunset, pigment granules retreat from the distal two thirds of the receptive segment. A comparison of retinae of day- and night-adapted animals shows that there is a slight (approximately 15%) increase in the cross-sectional area of rhabdomeral microvilli in dark-adapted animals, which in volume corresponds to the loss of pigment granules from the receptive segment. The length of the receptive segment as well as the pattern and shape of rhabdom units, however, remain unchanged.Each retinula unit is associated with one arhabdomeric cell. Their cell bodies are located close to those of retinula cells, but are much smaller and do not contain pigment granules. The most remarkable feature is a long, slender distal dendrite that extends up to the base of the fused rhabdom where it increases in diameter and develops a number of lateral processes interdigitating with microvilli of the rhabdom. The most distal dendrite portion extends through the center of the fused rhabdom and has again a smooth outline. All dendrites end in the distal third of the proximal rhabdom and are never present in the layer of the contiguous distal rhabdom. Arhabdomeric cells are of essentially the same morphology in day- and night-adapted animals. They are interpreted as photoinsensitive secondary neurons involved in visual information-processing that channel current collected from retinula cells of the proximal rhabdom along the optic nerve. A comparison is made with morphological equivalents of these cells in other chelicerate species.     

Persistence length of titin from rabbit skeletal muscles measured with scattering and microrheology techniques

The persistence length of titin from rabbit skeletal muscles was measured using a combination of static and dynamic light scattering, and neutron small angle scattering. Values of persistence length in the range 9-16 nm were found for titin-II, which corresponds to mainly physiologically inelastic A-band part of the protein, and for a proteolytic fragment with 100-nm contour length from the physiologically elastic I-band part. The ratio of the hydrodynamic radius to the static radius of gyration indicates that the proteins obey Gaussian statistics typical of a flexible polymer in a -solvent. Furthermore, measurements of the flexibility as a function of temperature demonstrate that titin-II and the I-band titin fragment experience a similar denaturation process; unfolding begins at 318 K and proceeds in two stages: an initial gradual 50% change in persistence length is followed by a sharp unwinding transition at 338 K. Complementary microrheology (video particle tracking) measurements indicate that the viscoelasticity in dilute solution behaves according to the Flory/Fox model, providing a value of the radius of gyration for titin-II (63 +/- 1 nm) in agreement with static light scattering and small angle neutron scattering results.     

Rat soleus muscle ultrastructure after hindlimb suspension

The aim of the present investigation was to determine, by quantitative electron microscopy, the effects of a 5-wk tail-suspension period on rat soleus muscle ultrastructure. A marked decline (-60%) in muscle mass occurred. The mean fiber cross-sectional area decreased to a greater extent (-75%) than the capillary-to-fiber ratio (-37%), leading to a higher capillary density (+148%) after hypokinesia. The total mitochondrial volume density remained unchanged, whereas the volume density of myofibrils was slightly but significantly reduced (-6%). A shift from subsarcolemmal to interfibrillar mitochondria occurred. Interfibrillar mitochondrial volume density was highest near the fiber border and decreased toward the fiber center. An increase in volume density of satellite cells suggested muscle regenerative events. Soleus atrophy with tail suspension greatly decreases the muscular volume but leaves the ultrastructural composition of muscle fibers relatively unaffected.     

Molecular dissection of N2B cardiac titin's extensibility

Titin is a giant filamentous polypeptide of multidomain construction spanning between the Z- and M-lines of the cardiac muscle sarcomere. Extension of the I-band segment of titin gives rise to a force that underlies part of the diastolic force of cardiac muscle. Titin's force arises from its extensible I-band region, which consists of two main segment types: serially linked immunoglobulin-like domains (tandem Ig segments) interrupted with a proline (P)-, glutamate (E)-, valine (V)-, and lysine (K)-rich segment called PEVK segment. In addition to these segments, the extensible region of cardiac titin also contains a unique 572-residue sequence that is part of the cardiac-specific N2B element. In this work, immunoelectron microscopy was used to study the molecular origin of the in vivo extensibility of the I-band region of cardiac titin. The extensibility of the tandem Ig segments, the PEVK segment, and that of the unique N2B sequence were studied, using novel antibodies against Ig domains that flank these segments. Results show that only the tandem Igs extend at sarcomere lengths (SLs) below approximately 2.0 microm, and that, at longer SLs, the PEVK and the unique sequence extend as well. At the longest SLs that may be reached under physiological conditions ( approximately 2.3 microm), the PEVK segment length is approximately 50 nm whereas the unique N2B sequence is approximately 80 nm long. Thus, the unique sequence provides additional extensibility to cardiac titins and this may eliminate the necessity for unfolding of Ig domains under physiological conditions. In summary, this work provides direct evidence that the three main molecular subdomains of N2B titin are all extensible and that their contribution to extensibility decreases in the order of tandem Igs, unique N2B sequence, and PEVK segment.