Synaptic vesicle mobility and presynaptic F-actin are disrupted in a N-ethylmaleimide-sensitive factor allele of Drosophila

Abstract

N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin.

Figure 1.

FRAP analysis of synaptic vesicle mobility. (a) An image of Syt-GFP within the nerve terminal boutons of a control (elav^3A-Gal4 × UAS-SytGFP) Drosophila larva. Colored shapes indicated the regions from which mean fluorescence intensities were taken and are shown in subsequent panels. (b) Time sequence of the same bouton before (0), immediately after photobleaching (1), 10 s (10), and 60 s (60) after bleaching. The arrow indicates the 12 × 30-pixel region that was bleached. (c) SytGFP expression in a mutant (elav^3A-Gal4::UAS-NSF2^E/Q × UAS-SytGFP) larva shown at the same time intervals as in panel b; the arrow indicates the region that was photobleached. The inset shows the area (purple square) from which mean fluorescence intensity was measured. (d) The time course of changes in fluorescence intensity for the colored regions indicated in panels a and c. The red line represents the region within the bleach zone, and the blue line a region adjacent to the bleach zone; the pink line is the average intensity of the whole target bouton, and the green line is the average intensity of a neighboring bouton. The purple line represents the bleached region in the mutant bouton shown in c. (e) Each colored line represents the mean fluorescence intensity from the cross-section indicated by the white rectangle in panel a, at times 0, 1, 10, 30, and 60 s. Scale bar, (a) 2.5 μm.

Figure 2.

Summary of FRAP analysis. (a) The time course of fluorescence intensity changes. The symbols represent the mean value from each of control and NSF2^E/Q boutons at each time point. Error bars, SEM. Lines are best fit to the one-phase exponential equation y = A(1 − e^(−k/x)) + B. (b) Summary of recovery index values, where the bars represent the mean value and the error bars the SEM for each of the genotypes and conditions indicated. The right axis, % recovery, applies only to the pair of sol-GFP bars. Sample size: n = number of boutons is indicated on each bar; the number of larvae were control, 9; NSF2^E/Q, 6; fixed, 2; control CD8-GFP, 2; NSF2^E/Q CD8-GFP, 2; control sol-GFP, 2; and NSF2^E/Q sol-GFP, 2.

Figure 3.

Effect of swinholide and latrunculin on vesicle mobility. (a) Images of actin-GFP and Syt-GFP before and after application of 5 μM swinholide. The normally punctate distribution of actin is abolished after drug treatment (the scale bar for actin-GFP image is 2 μm), whereas the donut-shaped synaptotagmin signal remains virtually unchanged. This image was taken 40 min after application of swinholide (scale bar for Syt-GFP image is 1.5 μm) (b) Summary of recovery index values in the absence of drug or in the presence of 5 μM swinholide (Swi), 10 μM latrunculin (Lat), or 10 μM jasplakinolide (Jas), where the bars represent the mean value and the error bars the SEM for each of the genotypes and conditions indicated. Sample size (n = number of boutons) is indicated on each bar; the number of larvae were as follows: control no drug, 5; control + Swi, 3; control + Lat, 7; control + Jas, 2; NSF2^E/Q, 5; NSF2^E/Q + Swi, 3; NSF2^E/Q + Lat, 4; and NSF2^E/Q + Jas, 2.

Figure 4.

Altered actin distribution and localization of F-actin in NSF2^E/Q boutons. (a) Images are of actin-GFP distribution in control and NSF2^E/Q nerve terminals. In controls the GFP signal shows a nonuniform punctate appearance, whereas in the NSF2^E/Q nerve terminal it is much more diffuse. (b) Single-slice confocal images of control actin-GFP–expressing nerve terminal labeled with phalloidin reveals that the actin-GFP puncta correspond to filamentous actin. Most of the phalloidin signal in this image is postsynaptic, corresponding to the subsynaptic reticulum of the muscle; however, the presynaptically expressed GFP coincides with phalloidin labeling. (c) Similar image of actin-GFP expressing NSF2^E/Q boutons. This image was selected because it shows the exclusion of phalloidin signal from the actin-GFP signal, marked by the double arrowhead, whereas there are some GFP concentrations that do coincide with phalloidin, marked by the single arrowheads. (d) Colocalization of actin-GFP with synaptotagmin. Control nerve terminals expressing actin-GFP were immunostained with an antibody against Drosophila synaptotagmin. The two signals overlap to a great extent, but there are some areas in which the GFP signal is seen in the absence of Syt labeling. Scale bars, 4 μm.

Figure 5.

Altered actin distribution in an NSF2 loss-of-function allele. (a and b) First instar larval NMJs from Df(3R)urd/+ and Df(3R)urd/NSF2⁵⁵ expressing actin-GFP. Clear concentrations of the actin-GFP signal can be seen in the controls (arrows), but these are absent in the loss-of-function allele. Images were acquired at the same confocal settings. Scale bar, 10 μm. (c and d) A second pair of NMJs shown at higher magnification and double-labeled with the neural marker anti-HRP. Clear actin-GFP puncta are visible (arrows) within the Df(3R)urd/+ NMJs, whereas in the Df(3R)urd/NSF2⁵⁵ boutons the actin-GFP appears diffuse and the puncta are not apparent.

Figure 6.

Presynaptic actin is dynamic. (a) Time-lapse image sequence of actin-GFP expressed in a control nerve terminal. The time elapsed from the beginning of the sequence is indicated in the top left corner. The punctate actin structures indicated by the triangles at time zero have moved laterally by the end of the series, whereas the puncta indicated by the arrow at time 60 increases in brightness and then disappears by time 240. Finally, at time 330 some actin-GFP, at the double triangle, appears to splinter off a larger puncta and move down and out of the bouton. (b) A frequency distribution of the SD of individual pixels over the time series of image

Figure 7.

Actin Fractionation. (a) Larval brain homogenates were examined for actin before and after high-speed centrifugation by SDS-PAGE and Western blotting. This image is an example Western blot showing the input and the supernatant and pellet fractions. The input homogenates from elav-Gal4 × NSF2^WT and elav-Gal4 × NSF2^E/Q samples were equivalent but the amount of actin found in the pellet appears less for the NSF2^E/Q sample. (b) Quantification of the amount of actin in high-speed pellet, expressed as a percentage of the input actin, for each of five trials. In all trials, the black bars represent the elav-Gal4 × NSF2^E/Q result. For trials 1, 2, and 3 the open bars represent data obtained from yw controls, whereas for trials 4 and 5 the hatched bars represent data from elav-Gal4 × UAS-NSF2^WT controls. (c) Pretreatment of larval brain homogenates eliminates actin detected in the pellet fraction. Twenty brains were dissected from elav-Gal4 × UAS-NSF2^WT larvae, 10 were incubated in HL3 alone, and 10 were incubated in HL3 plus 5 μM swinholide followed by centrifugation, SDS-PAGE, and Western blotting. There was no detectable signal in the pellet samples that were pretreated with swinholide, indicating the signal in untreated samples arises from F-actin. This blot is representative of two independent trials with swinholide pretreatment.

Figure 8.

Frap analysis of actin-GFP. (a) Images of control (elav^3A-Gal4 × Actin-GFP) and NSF2^E/Q (elav^3A-Gal4::UAS-NSF2^E/Q × UAS-Actin-GFP) nerve terminals expressing actin-GFP subjected to FRAP. Arrow indicates the region that was photobleached, 0 indicates before photobleaching, 0.5 is immediately after photobleaching, and 10 and 20 are 10 and 20 s after photobleaching. Scale bar, 5 μm. (b) The time course of recovery of actin-GFP signal after photobleaching, where the symbols represent the mean value and the bars the SEM at each time point. The line is the best fit of the one-phase exponential equation y = A(1 − e^(−k/x)) + B to the recovery data points. Scale bar, 2 μm. (c) Summary of the % recovery values obtained for control and NSF2^E/Q boutons, in the absence or presence of 5 μM swinholide. Sample size (n = number of boutons) is indicated on each bar, from three larvae each.