We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction . Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers . Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed . These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction .The postsynaptic membrane at the adult neuromuscular junctions is highly specialized with several characteristic features that distinguish it from the surrounding nonjunctional membrane. The membrane underneath the nerve terminal is elaborately folded and, along its entire contour, has a thin layer of electron-dense material attached to its cytoplasmic face. The underlying cytoplasm is rich in filaments whose identity has, thus far, not been established (10,12,18,30) . At the crests of the folds of the postsynaptic muscle membrane, acetylcholine receptors (AChRs) are densely packed (14): their density in the membrane is several orders of magnitude higher than that found elsewhere on the muscle surface (13,22) . The biochemical basis of these specializations at the neuromuscular junction is unknown . Because of the presence of filaments beneath the postsynaptic membrane, and because contractile proteins have been implicated in the clustering ofintegral membrane proteins in nonmuscle cells (7, 11), we have investigated whether actin is concentrated at or near the postsynaptic surface of the neuromuscular junction. For these experiments we have used a recently characterized antibody with an unusual specificity; it does not bind myofibrillar actin in vertebrate muscles but recognizes certain forms of cytoplasmic actin (26).A preliminary report of these findings has been published (27). MATERIALS AND METHODSAntiactin antibody was elicited in rabbits by immunization with actin purified from Aplysia (mollusc) body-wall muscle. Forimmunocytochemistry theantiactin was purified from serum by adsorption to Sephacryl-200 to which highly purified Aplysia actin had been coupled by the cyanogen bromide method (26) . The fraction of serum that did not bind to the column was used as a control. Bound THE JOURNAL OF CELL BIOLOGY " VOLUME 90 AUGUST 1981 789-792 © The Rockefeller University Press -0021-9525/81/08/0789/04 $1 .00 RAPID COMMUNICATIONS antiactin was eluted with 3 M ammonium thiocyanate and was dialyzed against 0.15 M NaCI at 4°C.Actin, used to block antibody binding to tissue sections, was prepared by differential extraction of Aplysia thin filaments followed by polyacrylamide gel electrophoresis in SDS (24). The protein band corresponding to actin was eluted from the gel with 10 mM Tris-HCl (pH 7.6) containing 50 pct AT...
We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica . We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin . Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form .In addition to its role in muscle contraction, actin is thought to participate in other processes that involve intracellular motility (including fast axonal transport) and to serve as a universal cytoskeletal element in eukaryotic cells (see references 14,35,43). Although greatly conserved during phylogeny, the actin molecule occurs in a variety of forms (24) . In vertebrate muscle, three types have been separated because of differences in charge: a-actin is present in sarcomeres of the skeletal muscle fibril ; ß-and y-actins are found in embryonic and smooth muscle (13,38,45) . Actins have also been isolated from brain and other nonmuscle eukaryotic tissues; these have been called cytoplasmic, and are more closely related to the actins of embryonic muscle than to myofibrillar actin (24) .Immunocytochemical localization has been one of the main tools for obtaining evidence that actin is involved in intracellular motility . The antiactin antibodies previously used, whether raised against actins of smooth muscle or against aactin, have generally reacted with both cytoplasmic and myofibrillar actins (see below) . To determine how actin might be involved in the rapid movement of organelles along axons of identified neurons of the marine mollusc, Aplysia californica, we have raised antibodies in rabbits using Aplysia body wall muscle actin as immunogen . We found that the antibody obtained can be used to localize actin within neurons (reference 29 and Lubit et al ., manuscript in preparation) . We also found that the specificity of the antibody is unlike all previous antiactins because it reacted only with certain forms of cytoplasmic actin and not with myofibrillar actin . This unique THE JOURNAL OF CELL BIOLOGY " VOLUME 86 SEPTEMBER 1980891-897 ©The Rockefeller University Press " 0021-9525/80/09/0891/07 $1 .00 specificity was determined by competitive binding studies and by immunohistochemical examination of vertebrate cells in which the distribution of actin is known . Reactivity of the antibody with cytoplasmic actin was assessed by determining its loc...
Although actin is thought to participate in several tyes of cell motility other than muscle contraction, no direct evidence has linked it to the force-generating mechanism for fast axonal transport. We have obtained evidence for the involvement of actin by microinjecting, into the serotonergic giant cerebral neuron of Aplysia, two preparations that have been shown to depolymerize actin filaments. One is a fraction of rabbit serum containing a heat-labile gamma globulin that affects actin polymerization in a manner similar to that of cytochalasin and several proteins that are thought to regulate the length of actin filaments. The other is bovine pancreatic DNase I which binds to actin stoichiometrically. Both preparations substantially decreased the transport of storage vesicles containing [3H]serotonin. Phalloidin, a toxic fungal peptide that binds to actin filaments but stabilizes rather than depolymerizes them, did not inhibit transport. We have not yet determined whether the inhibition of transport occurs during export of 13H]serotonin from the cell body into the axon or during trans-
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