Antibodies Against Cysteine String Proteins Inhibit Evoked Neurotransmitter Release at Xenopus Neuromuscular Junctions

Abstract

Cysteine string proteins (CSPs) are evolutionarily conserved proteins that are associated with synaptic vesicles and other regulated secretory organelles. To investigate the role of CSPs in vertebrate neuromuscular transmission, we introduced anti-CSP antibodies into the cell bodies of Xenopus spinal motor neurons that form synapses with embryonic muscle cells in culture. These antibodies produced a rapid (within 3-6 min), and in most cases complete, inhibition of stimulus-dependent neurotransmitter secretion. However, spontaneous neurotransmitter release was stable (both in frequency and amplitude) throughout the period of antibody exposure. Several control experiments validated the specificity of the anti-CSP antibody effects. First, the anti-CSP antibody actions were not mimicked either by antibodies against another synaptic vesicle protein SV2, or by nonspecific immunoglobins. Second, heat treatment of the anti-CSP antibodies eliminated their effect on evoked secretion. Third, immunoblot experiments showed that the anti-CSP and anti-SV2 antibodies were highly selective for their respective antigens in these Xenopus cultures. We conclude from these results that CSPs are vital constituents of the pathway for regulated neurotransmitter release in vertebrates. Moreover, the selective inhibition of evoked, but not spontaneous transmitter release by anti-CSP antibodies indicates that there is a fundamental difference in the machinery that mediates these secretory processes.

Interaction between Constitutively Expressed Heat Shock Protein, Hsc 70, and Cysteine String Protein Is Important for Cortical Granule Exocytosis in Xenopus Oocytes

“…3) suggests that this pool of csp may also reach the plasma membrane via ongoing constitutive exocytosis. Several prior investigations have noted that perturbation of csp function in the regulated pathway does not affect spontaneous neurotransmitter release at nerve endings (16,44) or the basal secretory process in adrenal or pancreatic cells (22)(23)(24)47). These results support the hypothesis that csps evolved to perform a function important for the regulated secretory pathway.…”

“…The supernatant was mixed with 5ϫ concentrated Laemmli sample buffer and resolved on a SDS-polyacrylamide gel for immunoblot analysis as before (7,26,31). An antibody specific for the amino terminus of csp (N-end antibody) was obtained by covalently conjugating recombinant Xenopus csp 1-112 to Pierce's aminolink resin (following the manufacturer's instructions) and adsorbing csp antiserum (16) to this matrix, washing with phosphate-buffered saline, and eluting the bound antibody using 0.1 M glycine-HCl (pH 3.0). After adjusting the pH to 7.5 with 1 M Tris base, the antibody capable of binding the carboxyl terminus of csp was removed by passing the N-end antibody through an aminolink resin to which the recombinant carboxyl end of Xenopus csp (residues 137-197) was coupled.…”

“…Csp was originally identified as a novel synaptic antigen in Drosophila (11), and several subsequent investigations indicated that csp might be important as a modulator of presynaptic Ca 2ϩ channels (12)(13)(14)(15)(16)(17). However, it also became evident that regulation of presynaptic Ca 2ϩ channels was not the sole function of csp (8 -10).…”

Cysteine String Protein Interacts with and Modulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator

“…In neurons, Csp is associated predominantly with synaptic vesicles (19), and in secretory cells it is found in large dense core or secretory granules (23,24). Studies of membrane dye labeling (25) and neurotransmitter release (26,27) indicated that Csp knockouts exhibit defective neurotransmitter exocytosis; presynaptic endocytosis and vesicle recycling appear to be intact. Studies in neuroendocrine (28) and endocrine cells (24) have confirmed that altered Csp expression elicits an impaired secretory phenotype.…”

A Role for Myosin 1e in Cortical Granule Exocytosis in Xenopus Oocytes

“…22) or a 1:1000 dilution of cortical granule lectin antiserum (a kind gift from Dr. J. Hedrick, University of California, Davis). The extract of Xenopus brain that served as a control in Fig.…”