ABSTRACT
The "cysteine string protein" (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a "J" domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the "tall cells" of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.
Subject(s)
Drosophila melanogaster/genetics , Insect Proteins/genetics , Membrane Proteins/genetics , Age Factors , Animals , Blotting, Western , Chaperonins/chemistry , Chaperonins/genetics , Cloning, Molecular , Electroretinography , Exocytosis/physiology , Female , Gene Expression Regulation, Developmental , HSP40 Heat-Shock Proteins , Insect Proteins/chemistry , Larva/chemistry , Larva/physiology , Male , Membrane Proteins/analysis , Molecular Sequence Data , Mutagenesis, Site-Directed/physiology , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/genetics , Nervous System/chemistry , Nervous System/growth & development , Phenotype , Presynaptic Terminals/chemistry , Sequence Analysis, DNA , Sequence Homology, Amino Acid , TemperatureABSTRACT
Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.
Subject(s)
Drosophila/physiology , Membrane Proteins , Mutation , Nerve Tissue Proteins/genetics , Synapses/physiology , Animals , Calcium/pharmacology , Drosophila/genetics , Evoked Potentials/drug effects , Gene Deletion , HSP40 Heat-Shock Proteins , Larva/physiology , Nerve Tissue Proteins/physiology , Neuromuscular Junction/physiology , Synaptic Membranes/physiology , Synaptic Transmission , TemperatureABSTRACT
Multimeric complexes of synaptic vesicle and terminal membrane proteins are important components of the neurotransmitter release mechanism. The csp gene of Drosophila encodes proteins homologous to synaptic vesicle proteins in Torpedo. Monoclonal antibodies demonstrate different distributions of isoforms at distinct subsets of terminals. Deletion of the csp gene in Drosophila causes a temperature-sensitive block of synaptic transmission, followed by paralysis and premature death.
