The Multiple Functions of Cysteine-String Protein Analyzed atDrosophilaNerve Terminals

Abstract

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) of csp null-mutant Drosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by an in vivo structure/function analysis. Expression of mutant CSP lacking the J domain at csp null-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca 2ϩ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca 2ϩ levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca 2ϩ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca 2ϩ levels. We detected no effects of csp mutations on individual presynaptic Ca 2ϩ signals triggered by action potentials, suggesting that presynaptic Ca 2ϩ entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca 2ϩ levels at rest and during stimulation.

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

“…These loss-of-function white-eyed mutants lack filled pigment granules in the eye but were thought otherwise to be normal. Our subsequent (Bronk et al, 2005), western blot (Zinsmaier et al, 1990) DCSP, Drosophila cysteine string protein; KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; PFA, paraformaldehyde. white, brown and scarlet have reduced brain biogenic amines findings clearly revealed that white is not normal, however, either for histamine or other biogenic amines.…”

MARCKS Regulation of Mucin Secretion by Airway Epithelium in Vitro

“…Members of the CSP family are involved in exocytosis, and they bind to secretory vesicle membranes (e.g., synaptic vesicles, chromaffin granules, zymogen granules) via their hydrophobic side chains of palmitoyl (6,7,38). CSP was first discovered in retina and brain of Drosophila as a neuronal-specific protein (39) and, since then, wide expression of several CSP isoforms has been shown in both neuronal and nonneuronal tissues. Airway and mammary epithelial cells have been reported previously to express CSP (9, 21).…”

Peptide-Induced Modulation of Synaptic Transmission and Escape Response inDrosophilaRequires Two G-Protein-Coupled Receptors

“…The value for the dissociation constant (K d ) was taken as 865 nM following previous work by Macleod et al (2002) and Bronk et al (2005). Briefly, the maximum (R max ) and minimum (R min ) fluorescence values for the preparation in the experimental chamber are obtained and used to calculate the K d of the indicator (Grynkiewicz et al 1985).…”