The multiple functions of cysteine-string protein analyzed at Drosophila nerve terminals.
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 Ca2+ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+ 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 Ca2+ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+ levels. We detected no effects of csp mutations on individual presynaptic Ca2+ signals triggered by action potentials, suggesting that presynaptic Ca2+ 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 Ca2+ levels at rest and during stimulation.
Bronk
Peter
P
Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, Arizona 85721-0077, USA.
Nie
Zhiping
Z
Klose
Markus K
MK
Dawson-Scully
Ken
K
Zhang
Jinhui
J
Robertson
R Meldrum
RM
Atwood
Harold L
HL
Zinsmaier
Konrad E
KE
D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D005075:Biological Evolution; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D003952:Diagnostic Imaging / Q000379:methods; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D009469:Neuromuscular Junction / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D017354:Point Mutation / Q000502:physiology*; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors
D029721:Drosophila Proteins; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; C088460:cysteine string protein; D006735:Horseradish Peroxidase; D002118:Calcium
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