Cytoplasmic actin in postsynaptic structures at the neuromuscular junction.

Abstract

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...

Desmin and Actin Filaments in Membrane-Cytoskeletal Preparations of the Electric Tissue of Electrophorus electricus, L.

“…Actin has been identified in central and peripheral synapses (KELLEY and COTMAN, 1978;HALL et al, 1981) and it appeared to be associated with purified preparations of postsynaptic membrane from the electric tissue of the torpedo (GYSIN et al, 1984;PORTER and FROEHNER, 1985). Although actin is present in the electrocytes of the torpedo (WALKER et al, 1985;KORDELI et al, 1986) and in those of E. electricus, L (AYRES SA et al, 1991), no actin filaments were detected within the cytoplasm in the former case (KORDELI et al, 1986).…”

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

“…In particular, an additional meshwork of thin, yet unidentified, filaments was observed in the rat beyond the cytoplasmic surface of the junctional folds (33). In addition to molecules immunologically related to the Torpedo 43-kD protein (23, 24), several cytoskelctal components such as a cytoplasmic form of actin (30), talin (60), a-actinin, filamin, vinculin (7), and a 51,000-D protein related to intermediate-sized filaments (12) have been detected by immunoflu0rescence at the neuromuscular junction. Most of these proteins persisted after denervation and were de facto attributed to the postsynaptic domain.…”

Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.

“…Myotome cells were isolated from stage [20][21][22] Xenopus laevis embryos and cultured on coverglass squares according to published methods (30). To induce the formation of new AChR clusters, we treated the cells with polyornithine-coated latex spheres (4.5 or 10 tzm) as previously described (31, 32).…”