Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis

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.

Structure–function analysis of the cysteine string protein inDrosophila: cysteine string, linker and C terminus

“…The boxes marked 'DCSP1', 'DCSP2', and 'DCSP3' in Fig.·1A comprise those amino acids that are common to all decapeptide spots recognized by the respective antibody. The data demonstrate that mAbs DCSP1 and DCSP2 recognize all cDNA-encoded proteins while mAb DCSP3 binds only to the isoforms encoded by cDNA-1 and cDNA-3 Eberle et al, 1998). We speculate that a hitherto undetected fourth splice variant (cDNA-2*) may code for the second largest isoform recognized in western blots, as indicated in Fig.·1B, which summarizes cDNA structures, approximate isoform sizes, and antibody recognition in western blots.…”

“…This signal matches in size the largest isoform of wild-type homogenates, and in fractionation and deacylation experiments behaves like all four isoforms of wild-type flies (Figs·2, 3, and data not shown). Immunohistochemistry employing mAb DCSP1 on head and body sections of these flies shows an expression pattern of CSP1 both in nervous and non-nervous tissues that is indistinguishable from wild-type stainings (Eberle et al, 1998), with high concentrations of CSP1 in synaptic terminals on muscles and in the neuropil of brain and thoracic ganglia ( Fig.·4 and data not shown). All tested cDna1 transformants rescue the premature death ( Fig.·5A) and the temperaturesensitive paralytic phenotype (Fig.·6) of the null mutant Csp U1 , demonstrating that the 2.3·kb upstream genomic region and the first two introns contain all essential regulatory regions of the Csp gene and that the CSP1 isoform is sufficient to maintain all known functions of the four natural isoforms.…”

“…In Drosophila four different CSP isoforms (denoted as CSP1-CSP4 in Fig.·1) are observed in western blots of head homogenates , while only the largest isoform (CSP1) is detected in non-neural organs (Eberle et al, 1998). No information is presently available on the functional differentiation of the various isoforms or the relevance of their selective distribution in the brain (Eberle et al, 1998;Nie et al, 1999).…”

“…No information is presently available on the functional differentiation of the various isoforms or the relevance of their selective distribution in the brain (Eberle et al, 1998;Nie et al, 1999). Three different cDNAs have been isolated so far (Zinsmaier et al, 1990.…”

DrosophilaABC transporter mutantswhite,brownandscarlethave altered contents and distribution of biogenic amines in the brain

“…These are shown with respect to the immunolocalization of a synaptic vesicle protein, cysteine string protein (CSP) (Zinsmaier et al, 1990;Eberle et al, 1998), for histamine ( Fig. 2).…”