Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration.

Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1L(del)). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1L(del) flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1L(del) flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency.

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.

Transmembrane agrin regulates dendritic filopodia and synapse formation in mature hippocampal neuron cultures.

The transmembrane isoform of agrin (Tm-agrin) is the predominant form expressed in the brain but its putative roles in brain development are not well understood. Recent reports have implicated Tm-agrin in the formation and stabilization of filopodia on neurites of immature central and peripheral neurons in culture. In maturing central neurons, dendritic filopodia are believed to facilitate synapse formation. In the present study we have investigated the role of Tm-agrin in regulation of dendritic filopodia and synaptogenesis in maturing cultures of rat hippocampal neurons. We did this by infecting the neurons with an RNAi lentivirus to deplete endogenous agrin during the developmental period when filopodia density on the dendritic arbor was high, and synapse formation was rapid. We found that dendritic filopodia density was markedly reduced, as was synapse density along dendrites. Moreover, synapse formation was more sharply reduced on dendrites of infected neurons contacted by uninfected axons than on uninfected dendrites contacted by infected axons. The results are consistent with a physiological role for Tm-agrin in the maturation of hippocampal neurons involving positive regulation of dendritic filopodia and consequent promotion of synaptogenesis, but also suggest a role for axonal agrin in synaptogenesis.

Long-term fate of neural precursor cells following transplantation into developing and adult CNS.

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.

Long-term fate of neural precursor cells following transplantation into developing and adult CNS.

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.

Synapse-forming axons and recombinant agrin induce microprocess formation on myotubes.

We examined cell-surface behavior at nerve-muscle contacts during synaptogenesis in cocultures of rat ventral spinal cord (VSC) neurons and myotubes. Developing synapses in 1-d-old cocultures were identified by the presence of axon-induced acetylcholine receptor (AChR) aggregation. Identified regions were then examined by transmission and scanning electron microscopy. The myotube surface near contacts with axons that induced AChR aggregation typically displayed ruffles, microvilli, and filopodia (microprocesses), indicating motility of the myotube surface. At some of these contact sites microprocesses were wrapped around the axon, resulting in the partial or total "submersion" of the axon within the myotube contours. Sites of myotube contact with somata and dendrites of the same neurons showed much less evidence of motility and surface interaction than sites of contact with axons. Moreover, the distance between opposed membranes of axons and myotubes was smaller than between dendrites or somata and myotubes, suggesting stronger adhesion of axons. These results suggest polarized expression of molecules involved in the induction of microprocess formation and adhesion in developing VSC neurons. We therefore tested the ability of agrin, which is preferentially secreted by axons, to induce microprocess formation in myotubes. Addition of recombinant C-terminal agrin to culture medium resulted in formation of microprocesses within 3 hr. Myotubes transfected with full-length rat agrin constructs displayed numerous filopodia, as revealed by fluorescence microscopy. The results suggest that the induction of muscle cell surface motility may be linked to the signaling processes that trigger the initial formation of the neuromuscular junction.

Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro.

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

Integrins stimulate phosphorylation of neurofilament NF-M subunit KSP repeats through activation of extracellular regulated-kinases (Erk1/Erk2) in cultured motoneurons and transfected NIH 3T3 cells.

Integrin-mediated interactions of cells with components of the extracellular matrix (ECM) regulate cell survival, cell proliferation, cell differentiation and cell migration through activation of multiple intracellular signal transduction pathways. In this study, we have demonstrated that integrin-matrix interactions promote KSP tail-domain phosphorylation of neurofilament medium molecular weight subunits (NF-M) in cultured rat spinal cord motoneurons and NF-M transfected NIH 3T3 cells. We found that laminin and fibronectin induce NF-M tail-domain phosphorylation in motoneurons and NIH 3T3 cells transfected with NF-M, respectively. This phosphorylation was selectively inhibited by PD98059, a specific MEK1 inhibitor. This suggests that laminin and fibronectin-induced MEK1 activation and the downstream targets Erk1 and Erk2 are involved in NF-M KSP tail-domain phosphorylation. This pathway appears to represent one of the mechanisms whereby integrin-extracellular matrix interactions are involved in phosphorylation of the NF-M KSP tail domain.

Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture.

Agrin secreted by motor neurons is a critical signal for postsynaptic differentiation at the developing neuromuscular junction. We used cultures of chick ventral spinal cord neurons with rat myotubes and immunofluorescence with species-specific antibodies to determine the distribution of agrin secreted by neurons and compare it to the distribution of agrin secreted by myotubes. In addition, we determined the distribution of agrin secreted by isolated chick ventral spinal cord neurons and rat motor neurons grown on a substrate that binds agrin. In cocultures, neuronal agrin was concentrated along axons at sites of axon-induced acetylcholine receptor (AChR) aggregation and was found at every such synaptic site, consistent with its role in synaptogenesis. Smaller amounts of agrin were found on dendrites and cell bodies and rarely were associated with AChR aggregation. Muscle agrin, recognized by an antibody against rat agrin, was found at nonsynaptic sites of AChR aggregation but was not detected at synaptic sites, in contrast to neuronal agrin. In cultures of isolated chick neurons or rat motor neurons, agrin was deposited relatively uniformly around axons and dendrites during the first 2-3 days in culture. In older cultures, agrin immunoreactivity was markedly more intense around axons than dendrites, indicating that motor neurons possess an intrinsic, developmentally regulated program to target agrin secretion to axons.

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: refinements and applications.

Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve-muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better-defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon-induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon-induced AChR aggregation on muscle cell species, by the use of chick-rat chimeric co-cultures. (5) We provide evidence for the role of alternatively-spliced agrin isoforms in synapse formation by using single cell RT-PCR with neurons collected from co-cultures after observation of axon-induced AChR aggregation. Microsc. Res. Tech. 49:26-37, 2000. Published 2000 Wiley-Liss, Inc.

ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles.

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein-endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)-which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH(2) terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils.

Tropomodulin is a tropomyosin-binding protein that terminates "pointed-end" actin filament polymerization. To test the hypothesis that regulation of tropomodulin:actin filament stoichiometry is critical for maintenance of actin filament length, tropomodulin levels were altered in cells by infection with recombinant adenoviral expression vectors, which produce either sense or antisense tropomodulin mRNA. Neonatal rat cardiomyocytes were infected, and sarcomeric actin filament organization was examined. Confocal microscopy indicated that overexpression of tropomodulin protein shortened actin filaments and caused myofibril degeneration. In contrast, decreased tropomodulin content resulted in the formation of abnormally long actin filament bundles. Despite changes in myofibril structure caused by altered tropomodulin expression, total protein turnover of the cardiomyocytes was unaffected. Biochemical analyses of infected cardiomyocytes indicated that changes in actin distribution, rather than altered actin content, accounted for myofibril reorganization. Ultrastructural analysis showed thin-filament disarray and revealed the presence of leptomeres after tropomodulin overexpression. Tropomodulin-mediated effects constitute a novel mechanism to control actin filaments, and our findings demonstrate that regulated tropomodulin expression is necessary to maintain stabilized actin filament structures in cardiac muscle cells.

Intercellular communication that mediates formation of the neuromuscular junction.

Reciprocal signals between the motor axon and myofiber induce structural and functional differentiation in the developing neuromuscular junction (NMJ). Elevation of presynaptic acetylcholine (ACh) release on nerve-muscle contact and the correlated increase in axonal-free calcium are triggered by unidentified membrane molecules. Restriction of axon growth to the developing NMJ and formation of active zones for ACh release in the presynaptic terminal may be induced by molecules in the synaptic basal lamina, such as S-laminin, heparin binding growth factors, and agrin. Acetylcholine receptor (AChR) synthesis by muscle cells may be increased by calcitonin gene-related peptide (CGRP), ascorbic acid, and AChR-inducing activity (ARIA)/heregulin, which is the best-established regulator. Heparin binding growth factors, proteases, adhesion molecules, and agrin all may be involved in the induction of AChR redistribution to form postsynaptic-like aggregates. However, the strongest case has been made for agrin's involvement. "Knockout" experiments have implicated agrin as a primary anterograde signal for postsynaptic differentiation and muscle-specific kinase (MuSK), as a putative agrin receptor. It is likely that both presynaptic and postsynaptic differentiation are induced by multiple molecular signals. Future research should reveal the physiological roles of different molecules, their interactions, and the identity of other molecular participants.

Chicken skeletal muscle tropomodulin: novel localization and characterization.

Tropomodulin is a 40.6-kDa isoform-specific tropomyosin-binding protein which inhibits actin filament elongation from the slow-growing (pointed) end and localizes at or near the pointed ends of thin filaments in rat skeletal muscle. Immunofluorescent localization using affinity-purified anti-tropomodulin antibodies in avian myofibril preparations demonstrates novel immunoreactivity at the Z-disc in addition to the previously reported localization at the periphery of I-Z-I brushes where actin filaments terminate. Identical results were obtained using antibody preparations generated against either bacterially expressed tropomodulin or human erythrocyte tropomodulin. Chicken muscle preparations contain Mr 43000 polypeptides which bind antibodies generated against tropomodulin in Western blot analysis, as well as 125I-labeled tropomyosin in blot overlays. Tropomodulin mRNA expression in adult muscle was confirmed by RNase protection assays, and the sequence of our tropomodulin cDNA amplified from chicken muscle mRNA preparations by polymerase chain reaction closely matches clones selected by chicken muscle cDNA library screening. The novel immunolocalization we report raises new possibilities for the role of tropomodulin in the organization of avian skeletal muscle at the Z-disc. We conclude that tropomodulin is likely to be important in striated muscle biology as a structural component in the Z-disc region which participates in the process of thin filament organization and assembly.

Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons.

We used a novel mammalian coculture system to study ACh receptor (AChR) redistribution and synaptic structure at nerve-muscle contacts. Ventral spinal cord (VSC) neurons were plated on cultures containing extensive myotubes but few fibroblasts. Neurite-induced redistribution of AChRs occurred within 6 hr after plating neurons and was maximal between 36-48 hr. This AChR redistribution appeared in two patterns: (1) AChR density at sites directly apposed to the neurite where neurites crossed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induced aggregates whereas the colocalization of laminin was variable. Electron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing synapses and, in older cultures, features of more mature synaptic structure. Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neurofilament subunits or for microtubule associated protein 2 (MAP2), respectively. Axons showed a 10-fold higher induction of AChR aggregation than did dendrites. Thus, at least one essential signaling molecule necessary for the induction of AChR aggregation at sites of interaction with muscle appears to be expressed in a polarized fashion in developing VSC neurons.

Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle.

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.

Biogenesis of transverse tubules in skeletal muscle in vitro.

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.

Localization of actin, beta-spectrin, 43 x 10(3) Mr and 58 x 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes.

I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4-6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 x 10(3) Mr and 58 x 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.