Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization

Abstract

At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and stonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Here, we test possible roles of the stoned proteins in synaptic vesicle internalization via analyses of third instar larval neuromuscular synapses in two Drosophila stoned (stn) mutants, stn(ts) and stn(8P1). Both mutations reduce presynaptic levels of stonedA and stonedB, although stn(ts) has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha-adaptin, and clathrin. In addition, stn(8P1) mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, we implemented new methods to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stn(ts), an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shi(ts) mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stn(ts) is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which stonedA and stonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane.

Fig. 1.

The levels of stonedA and stonedB at presynaptic terminals are altered distinctively instn^ts andstn^8P1 mutants. Shown are confocal micrographs of larval abdominal body wall muscles 6 and 7 stained with antibodies against stonedA and stonedB. StonedA and stonedB are enriched in presynaptic boutons of the wild-type larval NMJ. Instn^ts boutons presynaptic levels of stonedA often appear similar to those in wild type, whereas levels of stonedB are reduced. Instn^8P1 boutons both stonedA and stonedB are not detectable above background staining. Scale bar, 10 μm.

Fig. 2.

Evoked, but not spontaneous, neurotransmitter release is impaired severely instn^8P1. A, Measurements of postsynaptic responses evoked by nerve stimulation shown for stn^8P1 andstn^8P1/Dp.B, EJP amplitudes are tabulated for other relevant genotypes. The reduced EJP phenotype ofstn^8P1 is caused by a defect instoned function because it is not complemented by thestoned lethal allelestn^13–120 and is complemented by astoned duplication. EJP amplitude forstn^ts, shown for comparison with the identical wild-type EJP amplitude, was reported previously in Stimson et al. (1998). In the graph, from left toright, the number of larvae examined was 22, 14, 14, 3, and 13 for the indicated genotype. C, MEJP sizes do not differ between stn^8P1 andstn^8P1/Dp; thus, altered EJPs derive from reduced quantal content. D, E, Amplitude histograms for MEJP sizes in betweenstn^8P1 andstn^8P1/Dp show essentially identical distributions. Significantly, there is no increase in the number of unusually large events instn^8P1.

Fig. 3.

The stn^8P1mutation alters synaptic vesicle number and size. Shown are electron micrographs (EMs) through motor terminals on larval body wall muscles 6 and 7. Top panels,stn^8P1 boutons have a lower density of synaptic vesicles than do controls. Scale bar, 500 nm. Bottom panels, High magnification views of active zones show thatstn^8P1 boutons have abnormally large vesicles rarely observed in controls. Scale bar, 100 nm. Plots show that in stn^8P1 mutants the synaptic vesicle density is reduced significantly, whereas synaptic vesicle size is increased significantly. From left toright, the number of boutons examined was 46, 30, 19, and 17 for the indicated genotype.

Fig. 4.

The stn^8P1mutation causes altered distribution of the synaptic vesicle proteins synaptotagmin (Syt) and cysteine string protein (Csp), but not of endocytosis proteins, clathrin heavy chain, α-adaptin, and dynamin within motor terminals. A, Lower magnification images of a string of boutons double-stained with antibodies against Syt (top) and Csp (bottom). B, High magnification images of single boutons double-stained with a plasma membrane label (anti-HRP, green) and anti-synaptotagmin (red; top two panels) or with anti-HRP (green) and Csp (red; bottom two panels). Syt and Csp, restricted to well defined subdomains within wild type, or controlstn^8P1/Dp terminals, boutons, are distributed diffusely instn^8P1 mutant motor terminals (A). In wild-type and control terminals synaptic vesicle labeling is surrounded by plasma membrane that has been visualized by anti-HRP staining; however, instn^8P1 this labeling overlaps substantially in the bouton periphery with plasma membrane staining (B). The altered distribution patterns that are observed are consistent with the inefficient retrieval of Syt and Csp from the plasma membrane in stn^8P1mutants. Arrows indicate regions between boutons with low Csp immunoreactivity as compared with Syt (visible in thepaired image). C, Images of larval motor terminals double-stained with anti-HRP (green) and each of three endocytosis proteins (red). The distribution of clathrin heavy chain (top), α-adaptin (middle), and dynamin (bottom) is not altered significantly in stn^8P1 andstn^ts mutants. Thus, the altered localization of synaptic vesicle proteins reflects a function ofstoned downstream of mechanisms that are involved in the expression and localization of endocytosis molecules. The boutons shown in B are ∼4 μm in diameter.

Fig. 5.

Stn^tsspecifically enhances the temperature-sensitive paralysis ofshi^ts mutants. The fraction of flies paralyzed in 2 min of exposure to specific temperatures is plotted as a function of temperature. A, B,Stn^ts lowers the temperature of paralysis of two shi^ts mutants,shi^ts4 (A) andshi^ts2 (B). This effect of stn^ts is specific toshi^ts;stn^ts has no effect on the behavior of two other paralytic mutants,para^ts1 (D) andcomt^tp7 (C), which are defective in action potential propagation and synaptic vesicle fusion, respectively.

Fig. 6.

Stn^ts enhances the rate of synaptic vesicle depletion atshi^ts2 synapses, indicating a specific effect on recycling. A1, A2, At a restrictive temperature for shi^ts2 mutants, high frequency stimulation causes synaptic depression, a sign of synaptic vesicle depletion. A1, With 10 Hz stimulation at 30°C, wild-type NMJs can maintain synaptic transmission for several minutes but eventually will show some depression (see B).A2, In contrast,shi^ts2 NMJs treated under the same conditions show a rapid decline in synaptic transmission.B–G, Synaptic depression profiles for wild-type,shi^ts2, andshi^ts2–stn^tsdouble mutants at 28°C (left panels) and 30°C (right panels). B, C, Synaptic depression in shi^ts mutants is temperature-dependent; at 28°Cshi^ts2 has only a marginal effect on the rate of depression, indicating weak inhibition of synaptic vesicle recycling. D, E, In a wild-type backgroundstn^ts shows only a marginal effect on synaptic depression that is not affected by temperature.F, However, in ashi^ts2 mutant backgroundstn^ts causes a significant acceleration of synaptic depression at 28°C. Thus,stn^ts enhances the weak inhibition of synaptic vesicle recycling that is caused byshi^ts2 at 28°C. This effect ofstn^ts is not complemented by the lethal stoned allelestn^13–120 and, thus, is attributable specifically to loss of stoned function.G, At 30°C, whereshi^ts2 strongly inhibits synaptic vesicle recycling, there is no detectable effect ofstn^ts on depression. This observation indicates that stn^ts affects the same synaptic function (vesicle recycling) as shi; thus, when recycling is blocked completely, no further effects ofstn^ts are visible. Five to seven larvae were examined for each genotype, with the exception ofshi^ts2stn^ts/shi^ts2stn^13–120, for which four larvae were examined.

Fig. 7.

Stn^ts slows the rate of synaptic vesicle internalization. A, Our protocol for measuring the rate of synaptic vesicle endocytosis. The NMJ is subjected to 30 Hz stimulation for 30 sec to induce a burst of exocytosis. FM1-43 applied just before stimulation labels the complete exo–endo cycling pool of synaptic vesicles. FM1-43 applied att = 0 and t = 1 min after stimulation labels only those vesicles that have been internalized after these time points. B, A time course of FM1-43 labeling at wild-type and stn^tsboutons. After stimulation the stn^tsboutons appear to take up more dye than wild-type boutons, indicating significantly delayed vesicle internalization. Scale bar, 5 μm.C, Quantitative fluorescence measurements of FM1-43 labeling (normalized to maximal uptake for each genotype, as described in Materials and Methods) show that FM1-43 uptake is prolonged atstn^ts boutons, indicating that synaptic vesicle endocytosis is delayed. This delay is specifically attributable to loss of stoned function, because it is even more pronounced instn^ts/stn^13–120mutants. For each genotype three to seven larvae were examined under each condition. A fresh larva was used for each experiment; thus, one bouton provides a single data point (see Materials and Methods).