Overexpression of cysteine-string proteins in Drosophila reveals interactions with syntaxin

Abstract

Cysteine-string proteins (CSPs) are associated with secretory vesicles and critical for regulated neurotransmitter release and peptide exocytosis. At nerve terminals, CSPs have been implicated in the mediation of neurotransmitter exocytosis by modulating presynaptic calcium channels; however, studies of CSPs in peptidergic secretion suggest a direct role in exocytosis independent of calcium transmembrane fluxes. Here we show that the individual expression of various CSP isoforms in Drosophila similarly rescues the loss of evoked neurotransmitter release at csp null mutant motor nerve terminals, suggesting widely overlapping functions for each isoform. Thus, the structural difference of CSP variants may not explain the opposing putative functions of CSP in neurotransmitter and peptide exocytosis. Consistently, the individual overexpression of each CSP isoform in wild-type Drosophila shows similar effects such as impaired viability and interference with wing and eye development. The dominant effects caused by the overexpression of CSP are suppressed by the simultaneous overexpression of syntaxin-1A but not by the coexpression of SNAP-25. Although overexpression of CSP itself has no apparent effect on the synaptic physiology of larval motor nerve terminals, it fully suppresses the decrease of evoked release induced by the overexpression of syntaxin-1A. A direct protein-protein interaction of CSP with syntaxin is further supported by coimmunoprecipitations of syntaxin with CSP and by protein binding assays using recombinant fusion proteins. Together, the genetic and biochemical interactions of CSP and syntaxin-1A suggest that CSP may chaperone or modulate protein-protein interactions of syntaxin-1A with either calcium channels or other components of the regulatory machinery mediating depolarization-dependent neurotransmitter exocytosis.

Fig. 1.

A, Protein domain structure of Drosophila CSPs. The different protein isoforms CSP1, CSP2, and CSP3 are derived from alternatively spliced RNA transcripts. All proteins share three conserved domains, the J domain (J, residues 19–82), the linker domain (L, residues 83–113), and the cysteine-string domain (C, residues 114–138). CSPs differ in their C-terminal half; CSP1 contains a 21 amino acid insertion termed variable region 1 (V1), which is not present in CSP2 or CSP3. CSP1 and CSP2 share the same C terminus (V2), which differs by seven residues in CSP3 (V3). B, Analysis of CSP overexpression in wild-type driven by the elav promoter. Immunoblot of fly head protein extracts from wild-type control,csp^R1 null mutants and from flies overexpressing CSP1–3 with a elav promoter (elav-csp1,elav-csp2, elav-csp3) or with a csp promoter contained in a genomic csp transgene (csp-promoter). Proteins were resolved by 11% SDS-PAGE, immunoblotted, and stained against CSP with the monoclonal antibody DCSP1 detecting all CSP isoforms. Each lane contains proteins equivalent to one adult head from flies raised at 25°C. elav-csp2 expression shows a prominent band at 32 kDa, elav-csp1 at 33–34 kDa, and elav-csp3 at 36 kDa. Genotypes: elav-csp1 (w¹¹¹⁸, {elav-GAL4}/w¹¹¹⁸; P{UAS-csp1}/+); elav-csp2(w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+); elav-csp3 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp3}/+); csp-promoter(w¹¹¹⁸; P {csp});csp⁻(w¹¹¹⁸; csp^R1).

Fig. 2.

All CSP isoforms individually rescue the loss of evoked release in csp null mutants. A, Representative EJPs are shown for wild-type-like controlwhite, for larvae expressing CSP1 or CSP3 in acsp null mutant genetic background (elav-csp1;csp⁻and elav-csp3; csp⁻), and csp null mutant larvae (csp⁻). EJPs were recorded from muscle 6 in HL-3 solution at 22°C in the presence of 1 mm Ca²⁺. Scales for voltage trace and time sweep are as indicated. B, Mean amplitudes of evoked EJP responses at 22°C with 1 mmexternal Ca²⁺ from wild-type controlwhite, from larvae overexpressing CSP1–3 in a csp minus background (elav-csp1; csp⁻,elav-csp2; csp⁻, elav-csp3; csp⁻), from controls containing a UAS-csp but not a elav-GAL4 transgene in a csp minus background (UAS-csp1; csp⁻, UAS-csp2; csp⁻), and from a csp null mutation (csp⁻). Error bars represent SEM. Genotypes for A and B: white (w¹¹¹⁸), elav-csp1; csp⁻(w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp1}/+; csp^R1), elav-csp2; csp⁻(w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+; csp^R1), elav-csp3; csp⁻(w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp3}/+; csp^R1), UAS-csp1 (w¹¹¹⁸; P{UAS-csp1}/+; csp^R1), and the csp⁻null mutation (w¹¹¹⁸; csp^R1).

Fig. 3.

Neuronal overexpression of individual CSP isoforms has no effect on neurotransmitter release at larval neuromuscular junctions. A, Representative EJPs recorded from larval neuromuscular junctions of wild-type-like control white, larvae overexpressing CSP2 in the nervous system with the elav promoter (elav-csp2), and control larvae containing only the UAS-csp2 or the elav-GAL4 transgene. EJPs were recorded from muscle 6 in HL-3 solution at 22°C in the presence of 1 mmCa²⁺. Scales for voltage trace and time sweep are as indicated. B, Mean EJP amplitudes from wild-type-like control white, larvae overexpressing CSP1–3 with the elav promoter (elav-csp1, elav-csp2,elav-csp3), and control larvae containing either an elav-GAL4 or a UAS-csp transgene in trans (UAS-csp1, UAS-csp2,UAS-csp3, elav-GAL4). Fifteen EJPs were averaged for n larvae. Recordings were in the presence of 1 mm Ca²⁺ at 22°C. Error bars represent SEM. Genotypes for A andB: white (w¹¹¹⁸), elav-csp1 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+), elav-csp2 (w¹¹¹⁸,P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+), elav-csp3 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp3}/+), UAS-csp1 (w¹¹¹⁸; P{UAS-csp1}/+), UAS-csp2 (w¹¹¹⁸; P{UAS-csp2}/+), UAS-csp3 (w¹¹¹⁸; P{UAS-csp3}/+), elav-GAL4 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸).

Fig. 4.

Overexpression of CSP reduces adult life span. Survival curves of adult flies overexpressing CSP1–3 and wild-type controls. Adult elav-csp1 flies die prematurely after 4–5 d, elav-csp3 within 10–25 d, and elav-csp2 within 51 d. Controls exhibit life spans longer than 55 d. Flies were raised at 25°C. Newly emerged flies were collected within 12 hr, kept at 25°C, and dead flies were counted every 12 hr.

Fig. 5.

Wing defects caused by the elav-driven overexpression of individual CSP isoforms. A–L, Light microscopic images of wings from 2- to 5-d-old flies raised at 25°C unless otherwise indicated. Flies overexpressing CSP1–3 in the nervous system by the elav promoter fail to inflate their wings after eclosion such that wings appear “crumpled” (B–D). This phenotype is not observed in flies overexpressing CSP from a csp promoter contained in a genomic csp transgene, csp-csp (F). Reduction of CSP levels by removing one copy of endogenous csp (elav-csp2; csp⁻/+) partially suppresses the crumpled wing overexpression phenotype (G), and abolishment of endogenous csp (elav-csp2; csp⁻) fully suppresses the overexpression phenotype (H). Simultaneous overexpression of syntaxin-1A driven by a heat-shock promoter (elav-csp; hs-syx) partially suppresses the elav-CSP wing phenotype in flies raised at 25°C (K). Higher levels of syntaxin induced by the application of a daily heat-shock during late larval and pupal development fully suppresses the wing phenotype for flies raised otherwise at 25°C (L). Genotypes: (A) wild type; (B) elav-csp2 (w¹¹¹⁸,P{elav-GAL4}/w¹¹¹⁸;P{UAS-csp2}/+); (C) elav-csp1 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸;P{UAS-csp1}/+); (D) elav-csp3 (w¹¹¹⁸,P{elav-GAL4}/w¹¹¹⁸;P{UAS-csp3}/+); (E) elav-GAL4 (w¹¹¹⁸,P{elav-Gal4}); (F) csp-csp (w¹¹¹⁸; P{csp}—a genomic csp transgene expressing all isoforms); (G) elav-csp3; csp⁻/+ (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+; csp^R1/TM3 Sb); (H) elav-csp3; csp⁻(w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+; csp^R1); (I) elav-csp2 raised at 18°C -compare to (B); (J) hs-syx control (w¹¹¹⁸, P{hs-syx}/+; P{UAS-csp2}/+); (K) elav-csp2; hs-syx at 25°C (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{hs-syx}/+; P{UAS-csp2}/+); (L) elav-csp2; hs-syx raised at 25°C and heat-shocked for 1 hr at 37°C per day.

Fig. 6.

Eye defects caused by the elav-driven overexpression of individual CSP isoforms. A–I, Scanning electron microscopic images of adult eyes from 2- to 5-d-old flies raised at 25°C unless otherwise indicated. The elav-GAL4 driven overexpression of all three CSP isoforms in the nervous system disrupts eye development, causing a rough surface of the eye (B–D). Flies overexpressing CSP from a genomic csp gene fragment with the csp promoter (csp-csp) show normal eyes (E). The rough eye phenotype of elav-CSP2 is partially suppressed by the removal of endogenous CSP (F). Simultaneous overexpression of syntaxin-1A (hs-syx; elav-csp2) driven by the heat-shock promoter at 25°C partially suppresses the rough eye phenotype of CSP overexpressing flies (H). Higher levels of syntaxin-1A coexpression induced by the application of a daily heat-shock during late larval development completely suppress the rough eye phenotype (I). Genotypes for (A) wild type; (B) elav-csp1 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp1}/+); (C) elav-csp2 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+); (D) elav-csp3 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp3}/+); (E) csp-csp (w¹¹¹⁸; P{csp} - a genomic csp transgene expressing all isoforms); (F) elav-csp2; csp⁻(w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+; csp^R1); (G) hs-syx control (w¹¹¹⁸, P{hs-syx}/+; P{UAS-csp2}/+); (H) hs-syx; elav-csp2 raised at 25°C (w¹¹¹⁸, P{elav-GAL4/w¹¹¹⁸; P{hs-syx}/+; P{UAS-csp2}/+); (I) hs-syx; elav-csp2 raised at 25°C and heat-shocked for 1 hr at 37°C per day. Scale bar, 100 μm.

Fig. 7.

Overexpression of CSP suppresses the decrease of evoked release induced by the overexpression of syntaxin-1A. Continuous overexpression of syntaxin-1A from the heat-shock promoter at 25°C reduces neurotransmitter release in a similar manner as the induction of syntaxin expression by a heat-shock. This loss of evoked release is suppressed by the simultaneous overexpression of CSP2.A, Representative EJP recordings from larval neuromuscular junctions of wild-type-like control white, larvae overexpressing CSP2 (elav-csp2), larvae overexpressing syntaxin-1A and containing a csp transgene but not an elav transgene (hs-syx; UAS-csp2), and for larvae overexpressing syntaxin-1A and CSP2 (hs-syx; elav-csp2). All larvae were raised at 25°C and recordings are from muscle 6 at 1 mm Ca²⁺ at 25°C. B, Mean amplitudes of evoked EJP responses at 25°C with 1 mmexternal Ca²⁺ from control white, larvae overexpressing CSP2 (elav-csp2), larvae overexpressing syntaxin-1A (hs-syx and hs-syx; UAS-csp2), and larvae overexpressing CSP and syntaxin-1A (hs-syx; elav-csp2). Recordings were as inA. Bars indicate SEM. Genotypes: white (w¹¹¹⁸); elav-csp2 (w¹¹¹⁸, P{elav-GAL4}/w¹¹¹⁸; P{UAS-csp2}/+); hs-syx (w¹¹¹⁸; P{ hs-syx}/+); hs-syx; UAS-csp2 (w¹¹¹⁸; P{ hs-syx}/+; P{UAS-csp2}/+); and hs-syx; elav-csp2 (w¹¹¹⁸, P{elav-GAL4/w¹¹¹⁸; P{hs-syx}/+; P{UAS-csp2}/+).

Fig. 8.

In vitro binding of CSP and syntaxin -1A. A, Coimmunoprecipitation of syntaxin-1A from Drosophila wild-type extracts.Drosophila head protein extracts were solubilized with Triton X-100 and immunoprecipitated with DCSP1 antibodies detecting all CSP isoforms (IP CSP). Control experiments omitted the monoclonal antibody (no Ab), the fly extract (no extract), or protein A-coupled beads (no protein A). Bound proteins were recovered by centrifugation, washed, and resuspended in SDS buffer. Three percent of the soluble fraction (S) of the immunoprecipitation and one-half of the immunoprecipitated pellet fraction (P) were analyzed on separate immunoblots for the presence of CSP and syntaxin-1A (Syx). CSP antibodies immunoprecipitate CSPs and coimmunoprecipitate syntaxin. Controls show neither a CSP- nor a syntaxin-specific signal. B, Similar coimmunoprecipitation of syntaxin-1A from protein extracts of wild-typeDrosophila and csp^X1deletion mutants where CSP is absent shows copurification of syntaxin from wild-type extracts but not from mutant extracts. C,Recombinant protein binding of syntaxin-1A with CSP. Soluble His-CSP was incubated in a 2:1 molar ratio with immobilized GST-syntaxin-1A (GST-Syx), with immobilized GST-synaptotagmin (GST-Syt), or with immobilized GST protein (GST control) for 2 hr at 4°C. The affinity precipitate was recovered by centrifugation, washed, and resuspended in SDS-PAGE buffer. Recombinant His-CSP (1/25) used for the binding assay and all of the affinity precipitate was analyzed by immunoblotting for the presence of recombinant CSP. Note that <4% of total His-CSP binds by GST-syntaxin (GST-Syx + His-CSP) but not to GST-synaptotagmin (GST-Syt + His-CSP) or the GST control.