Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction

Abstract

Background: Although the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear.

Results: In the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II.

Conclusion: Our results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

Figure 1

NMMII is localized pre- and postsynaptically at the NMJ. α-NMMII (red) was found to colocalize with the neural marker α-HRP (green). α-NMMII staining is observed which did not colocalize with α-HRP (merge) suggesting NMMII is also present postsynaptically (top row). The postsynaptic marker α-Dlg (green) is observed to colocalize (merge) with α-NMMII (red) suggesting NMMII is present postsynaptically (second row). To confirm presynaptic expression of NMMII, UASzipRNAi was expressed postsynaptically using 24BGal4 (bottom row). This eliminated postsynaptic expression of NMMII. Immunocytochemistry revealed that while postsynaptic staining of NMMII had been eliminated, NMMII was still present presynaptically as seen with the colocalization with α-HRP (merge). No postsynaptic NMMII is visible in the merged image. The top and bottom rows are shown as confocal stacks, while the middle two rows are shown as single confocal slices.

Figure 2

Quantification of NMMII alleles. Western blot of NMMII alleles a, Knockdown (K/D) of NMMII in the nervous system reduced NMMII expression to 28% (n = 5, P < 0.05) whereas the heterozygous loss-of-function zip¹/CyO (Het) reduced NMMII expression to 57% (n = 4, P < 0.05) compared to the elav^3AGal4 control (ct). Overexpression of zip^GS50077 (O/E) in the nervous system increased NMMII expression by 95% (n = 4, P < 0.05) compared to the elav^3AGal4 control (ct). α-β-Tubulin was used as the loading control. Relative NMMII proteins levels were quantified from the western blot analysis. Each band was first normalized to the level of β-tubulin (used as a loading control, not shown) and then the mutant genotypes were normalized to elav^3AGal4 protein levels, which were set to 1. The bars indicate the mean level obtained from 4-5 samples, bars are SEM. Overexpression (O/E), knockdown (K/D), heterozygous loss-of-function (Het).

Figure 3

FRAP indicating the mobility of vesicles in response to application of the Myosin inhibitor, ML-9. a, Acquired images for the recovery of fluorescently labelled vesicles using elav^C155Gal4;UAS-sytGFP/Y after application of ML-9. Images are shown immediately before bleaching (8 sec), immediately after photobleaching (10 sec) and at 16, 48 and 120 sec. Bleached areas are indicated by white arrows. A reduction in recovery is observed with increasing doses of ML-9. b, A significant dose-dependent response was observed for inhibition with ML-9 between the doses of 100 μM ML-9 and 10 μM ML-9 as compared to 0 μM ML-9. 10 μM ML-9 did not significantly reduced vesicle mobility. Error is represented as the 95% confident interval of the curve. FRAP recoveries were fit with double exponential curves and nonlinear regression was used to test for statistical differences. Sample sizes and significance were as follows: 0 μM ML-9 (n = 20), 10 uM ML-9 (n = 22, p > 0.05), 50 μM ML-9 (n = 20, p < 0.05), 100 μM ML-9 (n = 20, p < 0.05).

Figure 4

FRAP recovery curves for vesicle mobility when NMMII expression is altered. a, Acquired images for the recovery of the heterozygous loss-of-function, elav^C155Gal4;UAS-sytGFP/+; zip¹/+ (Het), the control, elav^C155Gal4;UAS-sytGFP/Y (ct), the RNAi knockdown, elav^C155Gal4;UAS-sytGFP/+;UASzipRNAi/+ (K/D) and the overexpression, elav^C155Gal4;UAS-sytGFP/+; zip^GS50077/+ (O/E) of NMMII immediately before bleaching (8 sec), immediately after photobleaching (10 sec) and at 12, 16, 48 and 120 sec post-bleaching. Bleached areas are indicated with white arrows. At 16 sec the bleached region for Het is no longer visible; however, it is still clearly visible in control (ct). At 120 sec, the bleached region is no longer visible in the control (ct), but is still visible for both K/D and O/E. b, Vesicle mobility is affected by the expression level of NMMII. FRAP curves reveal that the heterozygous NMMII loss-of-function (Het) significantly enhanced vesicle mobility as compared to the control (ct) while both knockdown (K/D) and overexpression (O/E) of NMMII significantly reduced vesicle mobility. FRAP recoveries were fit with double exponential curves. Nonlinear regression was used to test for statistical differences; elav^C155Gal4;UAS-sytGFP/Y (n = 26), elav^C155Gal4;UAS-sytGFP/+; zip¹/+ (n = 24, p < 0.05), elav^C155Gal4;UAS-sytGFP/+;UASzipRNAi/+ (n = 22, p < 0.05), elav^C155Gal4;UAS-sytGFP/+; zip^GS50077/+ (n = 20, p < 0.05).