Distribution of filipin-sterol complexes on cultured muscle cells: cell- substratum contact areas associated with acetylcholine receptor clusters

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol- specific antibiotic filipin before fracturing, the pattern of filipin- sterol complex distribution closely followed the pattern of cell- substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze- fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.     

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.     

A freeze-fracture study of afferent and efferent synapses of hair cells in the sensory epithelium of the organ of Corti in the guinea pig

Summary Afferent and efferent synapses of hair cells in the organ of Corti of the guinea pig have been examined in freeze-fracture replicas.Afferent synapse In the inner hair cells, intramembranous particles 10 nm in diameter are aggregated on the ridge on the P-face of the presynaptic membrane directly beneath the synaptic rod. In the outer hair cells, in which the synaptic rod is located in the presynaptic cytoplasm underneath the presynaptic membrane, small aggregations of intramembranous particles 10 nm in diameter can be found on the P-face of the presynaptic membrane corresponding to the site of the presynaptic dense projection. Intramembranous particles 10 nm in diameter are also densely aggregated on the P-face of the postsynaptic membrane of the outer hair cells.Efferent synapse of the outer hair cells Large intramembranous particles 13 nm in diameter are distributed in clusters composed of four to ten particles on the P-face of the presynaptic membrane. In the P-face of the postsynaptic membrane, disc-like aggregations of intramembranous particles 9 nm in diameter are found. The subsynaptic cistern covers the cytoplasmic surface of the postsynaptic membrane of the efferent synapse; it may cover more than one postsynaptic membrane when several efferent synapses are in close proximity to one another.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Insect UV-, and green-photoreceptor membranes studied by the freeze-fracture technique

Summary The membranes of the microvilli of UV- and green-photoreceptors of the ant Myrmecia gulosa have been studied with the freeze-fracture technique. Both inner fracture faces, the cytoplasmic P-face and the extracellular E-face, are covered by globular particles. The P-face particles appear to be randomly distributed, occasionally forming clusters. Their density is about 7,000/m², and their mean diameter is 8.5 nm. The E-face particles, however, are arranged in an ordered square pattern with a center-to-center spacing of 9 nm. The density and distribution of P- and E-face particles are the same in both the UV- and the green-photoreceptor membranes. No differences were found in the ultrastructural organization of photoreceptor membranes after dark or light adaptation. It is suggested that the P-face particles represent rhodopsin molecules.     

Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.     

The ultrastructural effects of β-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features.

Summary The fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of β- 1–40 (β-AP). PC12 cells treated with β-AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of β-AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and β-AP treated cells had two major P-face IMP populations, small-diameter (4–8 nm) IMPs, and large-diameter (≤ 9nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the β-AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the β-AP. These results demonstrate that β-AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.     

Purification of acetylcholine receptors from the muscle of Electrophorus electricus

Muscle from the electric eel Electrophorus electricus contains acetylcholine receptors at 50 times the concentration of normal mammalian muscle and fully one-tenth the concentration of receptors in its electric organ tissue. Receptor is organized much more diffusely over the surface of Electrophorus muscle cells than is the case in normally innervated mammalian skeletal muscle. Receptor was purified from Electrophorus muscle by affinity chromatography on cobra toxin-agarose and found to contain subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from electric organ tissue of Torpedo californica. Receptor purified from Electrophorus muscle appears virtually identical with receptor purified from Electrophorus electric organ tissue.     

Influence of neostigmine treatment on embryonic development of acetylcholine receptors and neuromuscular junctions

The postulated role of the acetylcholine receptor in the formation of neuromuscular synapses during the course of embryonic development was investigated in the superior oblique muscle of white Peking duck embryos. The possibility that the number of receptors could be experimentally lowered by chronic injections of the anticholinesterase agent, neostigmine methylsulfate, was determined using 125I-alpha- bungarotoxin. The total number of acetylcholine receptors on incubation day 12, 2 d subsequent to the onset of treatment, was reducted 45% as compared to saline-treated controls. A similar reduction in total receptor content (49%) was also observed on day 19. Radioautographic preparations showed that clusters of acetylcholine receptors were rare and that the grain density of extrajunctional receptors was also reduced. Hence, chronic treatment with neostigimine during development was observed to exert an effect on both the number and distribution of receptors in the developing superior oblique muscle. These changes occurred in the absence of any apparent effect on muscle differentiation in general. Myoblasts and myotubes were present on day 14 and further differentiated into myofibers by day 18 in both neostigmine and saline-treated muscles. The cytology of the develop;ing muscle cells also appeared normal. This is in contradistinction to the striking morphological changes that take place in adult mammalian and avian muscle after anticholinesterase treatment. More significantly, the decreased total receptor content and sparsity of clusters had no apparent effect on the formation of developing neuromuscular junctions at the electron microscopic level. The frequency of neuromuscular junctions in neostigmine-treated muscles was similar to that of the controls. It is concluded that acetylcholine receptor clusters are not required for the events leading to the morphological formation of neuromuscular junctions during in vivo development.     

A freeze fracture and thin section study of intestinal cell membranes and intercellular junctions of a nematode, Ascaris

The microvillar and lumenal plasma membrane P-face of Ascaris intestinal cells is shown to be covered by relatively large (13 nm) particles at a fairly high density (1000/μm²), while the E-face has virtually none. The P-face of the lateral cell membranes, those separating the cells, have fewer and smaller (8 nm) particles. The intestinal cells are also shown to be connected by an apical complex of smooth septate and tricellular junctions similar to those found between some insect midgut cells. A periodic layer of tannic acid staining material is found on the cytoplasmic sides of the smooth septate junction, and when the intercellular space is filled with lanthanum, smoothly curved, 10 nm wide septal walls can be seen. Below the belt of septate junctions are a large number of gap junctions. These have closely packed arrays of particles on the P-face with some particle aggregates adhering to the closely packed pit arrays on the E-face.     

Chemosensitivity of single smooth muscle cells to acetylcholine, noradrenaline, and histamine in vitro

Electrical responses to acetylcholine, noradrenaline, and histamine were recorded from solitary smooth muscle cells. Iontophoresis of each transmitter elicited three fast responses: a hyperpolarization, a depolarization, or a biphasic hyperpolarization-depolarization. Each transmitter activated a specific receptor since responses were specifically blocked by antagonists, two transmitters elicited different responses in solitary cells, and desensitization of response to one transmitter did not cause desensitization of responses to other transmitters. Responses were due to increased ion conductances since input resistance decreased during responses and reversal potentials were measured for depolarizing responses (-5 mV) and hyperpolarizing responses (-60 mV). Regional differences in transmitter sensitivity were mapped on solitary cells. Biphasic responses were due to simultaneous activation of receptors mediating hyperpolarizing responses and receptors mediating depolarizing responses which were segregated in the cell membrane. Noradrenaline enhanced action potential amplitude by regulation of voltage-dependent ion conductances. Finally, noradrenaline and histamine elicited periodic hyperpolarizing potentials, which may be due to increased intracellular Ca++.     

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.     

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Temporal and spatial expression patterns of a muscarinic acetylcholine receptor (Acr60C) and an octopamine/tyramine receptor (Octyr) were determined inDrosophila melanogaster using quantitative Northern analysis andin situ hybridization to tissue sections. Expression of mRNA encoding both of these G-protein coupled receptors peaks initially in 18 to 21 hour embryos following the formation of the mature larval nervous system. Levels of mRNA then decline during larval stages, rising to a second peak in 3 to 4-day-old pupae after a period of major nervous system reorganization. The muscarinic acetylcholine receptor mRNA is expressed throughout the cortical regions of the central nervous system in adults and embryos. Particularly high levels of expression of Acr60C are observed in cell bodies adjacent to the antennal lobes, suggesting a major role for this muscarinic receptor in the processing of olfactory information. In contrast, the octopamine/tyramine receptor mRNA is distributed diffusely throughout the adult brain, with patches of signal concentrated in the cortex of the dorsal protocerebrum near the mushroom bodies. These patches may represent individual cells expressingOctyr receptors.     

Membrane specializations in the paired spermatozoa of dytiscid water beetles

The paired spermatozoa of the dytiscid beetles Dytiscus marginalis and Hydaticus seminiger were studied by electron microscopy with the aim of examining whether the regions of the cell membrane in the zones of sperm conjugation might differ from other regions and to explore whether these cells had any other specialized domains of the cell membrane that could be recognized by the freeze-fracturing technique. The spermatozoa are conjugated along one side of the sperm head and proximal tail portion, called the ventral side. The cell membrane was seen to contain tightly packed intramembranous particles (IMPs) that were predominantly located in the external membrane face (the E-face). In thin sections the cell membrane had a ladder-like appearance at these regions and a specialized type of glycocalyx seen as a fluffy material containing granules. Other specialized membrane domains could also be recorded: a ribbon of particles in the protoplasmic face (P-face) of the dorsal side of the spermatozoon at the proximal tail portion and regularly arranged particle rows in the P-face of the distal tail portion. These domains corresponded to regions where the glycocalyx is prominent. Both the E-face and the P-face of the cell membrane were seen to contain numerous intramembranous particles, which suggests an active function for both membrane leaflets; this is in contrast to the situation in most cells where the particles are mainly in the P-face. The functions of the intramembranous particles in the specialized domains of the cell membrane remains unknown. Some particles may represent receptors or ion gates, others proteins with a mechanical function.     

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.     

A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech,Hirudo medicinalis

Summary Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.     

Nerve-Muscle Interaction In Vitro : Role of acetylcholine

Nerve and muscle cells from clonal lines interact in vitro, resulting in the association on the muscle surface of an area of increased acetylcholine sensitivity with a site of nerve-muscle contact. This localization of acetylcholine sensitivity on the muscle cell to a site of contact between nerve and muscle was found to occur when acetylcholine receptors on the muscle had been blocked with α-neurotoxin. Localization was also found to occur when the nerve cell had been prevented from releasing acetylcholine. It is concluded that neither the presence of active acetylcholine receptors on the muscle, nor the release of acetylcholine from the nerve, was required for the events leading to the localization of acetylcholine sensitivity in vitro.     

Spatial and temporal organization of TrkB expression in the developing musculature of the mouse esophagus

TrkB expression was investigated immunocytochemically in the developing musculature of mouse esophagus using conventional and confocal laser scanning microscopy. To demonstrate spatial relationships of TrkB immunoreactive cells to striated and smooth muscle fibers we combined TrkB immunocytochemistry with fluorochrome-tagged alpha-bungarotoxin for labeling of nicotinic acetylcholine receptors, and alpha-smooth muscle actin for labeling of smooth muscle cells. At developmental stages E15 to P7, TrkB immunoreactive cells transiently occurred in a transformation zone where striated intermingled with smooth muscle fibers. This transformation zone started in the rostral esophagus at E15, moved caudally, and disappeared between P7 and P10 in the caudal esophagus. The first TrkB-immunoreactive cells appeared in the outer muscle layer at E15. No TrkB-positive cells exhibited acetylcholine receptor clusters or were positive for alpha-smooth muscle actin. A few showed slight alpha-bungarotoxin staining over their entire surface. Taken together, the appearance of TrkB-expressing cells in the transformation zone suggest a role in muscle transdifferentiation. Alternatively, these results, together with recent in vitro data, suggest that TrkB is expressed in a subpopulation of myoblasts in which acetylcholine receptor clustering may be inhibited through a TrkB-mediated pathway.