BACKGROUND
transport and assembly of synaptic vesicles has been the subject of several studies.
vesicles and their components are transported along axon microtubules to the nerve terminal,  where they participate in synaptic physiology, undergoing a cycle of exo- and endocytosis.
however, vesicle traffic within terminal boutons is not well understood although recent advances in this area have been made  <cit> .
classically, vesicles were believed to be relatively stationary until released  <cit> .
however, more recent studies provided evidence for a mobile vesicle pool  <cit>  best described by a caged-diffusion model  <cit>  and differential vesicle mobility in the reserve and recycling pool has been suggested within the frog motor nerve terminals  <cit> .
additionally, nunes et al.
<cit>  observed dynamic vesicles in the drosophila melanogaster bouton, and dynamic vesicles have been reported at ribbon synapses in lizards  <cit> .
vesicle movement may result from diffusion or directed transport.
actin polymerization in the nerve terminal may promote vesicle movement through a listeria comet mechanism  <cit>  or it may act as a substrate for myosin motors to shuttle vesicles.
in light of a previous screen from our lab identifying nonmuscle myosin ii  as a candidate molecule important in neuromuscular junction  development  <cit> , we have focused on determining a neuromuscular function for this actin-based myosin motor.
nmmii is present in the nervous system of xenopus, mouse, rat and chicken  <cit> , and in the cns of drosophila  <cit> .
nmmii can both crosslink f-actin and has been shown to transport vesicles on f-actin  <cit> .
this study was undertaken to determine whether nmmii contributes to synaptic vesicle mobility.
we used the genetic model system drosophila melanogaster to manipulate nmmii expression, pharmacology to inhibit nmmii activity, and the optically accessible neuromuscular synapse of third instar larvae to investigate vesicle dynamics.
we show that nmmii is concentrated pre- and postsynaptically and importantly, we report that unstimulated synaptic vesicle mobility exhibited a dependence on nmmii expression.
these results report the first evidence for nmmii having a function in synaptic vesicle dynamics at the drosophila nmj.
RESULTS
nonmuscle myosin ii is localized pre- and postsynaptically at the nmj
although nmmii has been previously found in the drosophila cns  <cit> , it has not been previously localized to the nmj.
therefore we used immunocytochemistry to investigate the localization of nmmii at the nmj .
first, we demonstrated the presence of nmmii by staining the nmj with anti-nmmii and found a robust signal labelling the nerve terminal, which co-localized to a high degree with anti-hrp, a general marker of insect neural membrane.
we did notice however that some of the nmmii staining was found outside the boutons defined by the anti-hrp signal suggesting that some of the nmmii signal has a postsynaptic origin.
to further investigate this possibility we double labelled the nmj with the post-synaptic marker, discs large and anti-nmmii.
we found colocalization between discs large and nmmii, indicating nmmii is found post-synaptically.
to determine whether nmmii is also found presynaptically, we expressed uasziprnai in the muscle using the muscle specific driver 24bgal <dig>  this dramatically reduces nmmii in the muscle, revealing presynaptic nmmii, which colocalizes with the specific neural marker anti-hrp.
together these results demonstrate the presence of nmmii at the drosophila nmj.
expression level of nonmuscle myosin ii in zipper alleles
in order to study nmmii we manipulated its expression level using genetic tools.
in drosophila, nmmii is encoded by the zipper  gene.
we used the heterozygous loss-of-function zip <dig> allele , overexpressed zip with the zip transgene, zipgs <dig> , and expressed a zip knockdown construct, ziprnai .
before proceeding with using these reagents in experiments we confirmed the different zip tools were functioning as expected by western blot analysis of protein prepared from larval brain extracts .
nmmii expression in elav3agal <dig>  larvae were used as a control and normalized to  <dig> .
rnai knockdown of nmmii significantly reduced the nmmii level to 28%  of the control.
the heterozygous loss-of-function allele zip1/cyo reduced nmmii expression to 57%  of the control while overexpression of nmmii increased protein levels by 95% compared  to elav3agal <dig>  .
statistical analysis revealed significant variation among the groups , while post hoc newman-keuls comparison between groups showed that each group was different from the other  with the exception that zip1/cyo was not found to be statistically different from the knockdown sample despite the 50% difference in average protein levels.
inhibition of myosin with ml- <dig> results in a dose-dependent decrease in vesicle mobility
nmmii has been previously shown to function in nmj development  <cit> .
for this reason, we investigated whether nmmii affected vesicle dynamics at the drosophila nmj.
to assess the effects of nmmii on vesicle dynamics, synaptic vesicle mobility was measured using frap under conditions of altered myosin activity.
to visualize synaptic vesicles in vivo, the recombinant synaptotagmin-gfp construct, elavc155gal4; uas-sytgfp, was used to drive expression of synaptotagmin-gfp in the nervous system.
synaptotagmin-gfp has previously been shown to mark synaptic vesicles  <cit> .
to begin our examination of myosin activity at the synapse we used ml- <dig>  an inhibitor of myosin light chain kinase , and tested synaptic vesicle mobility under different doses of this myosin inhibitor .
application of ml- <dig> to elavc155gal4; uas-sytgfp/y larvae resulted in a dose-dependent reduction in the mobility of synaptic vesicles compared to vesicle dynamics in a saline + dmso control .
vesicle mobility in this saline + dmso treated control  did not significantly differ from the saline only control  used here in the subsequent frap experiments.
a significant decrease in vesicle mobility was observed with  <dig> μm ml- <dig>  which resulted in the most dramatic reduction of vesicle mobility  while  <dig> μm ml- <dig> resulted in slightly, but significantly less vesicle mobility  compared to the control.
a dose of  <dig> μm ml- <dig>  had no effect on vesicle dynamics .
together this indicates a dose-dependent response of vesicle mobility to myosin inhibition.
ml- <dig> is a general myosin inhibitor while -blebbistatin is known to be selective towards inhibiting nmmii  <cit> .
however -blebbistatin is autofluorescent under blue light and is quickly deactivated upon exposure to blue light  <cit>  thus limiting our ability to use this drug with our frap conditions.
we did however obtain nmmii alleles to determine their affect on vesicle mobility.
<dig> equation: frap = 1-a-b  <cit>  where a, b = constants that represent the apparent bleached fraction, k <dig>  k <dig> = rates, t = time
nonmuscle myosin ii contributes to synaptic vesicle dynamics
in order to clarify whether nmmii was contributing to the inhibition of vesicle dynamics by ml- <dig>  we genetically combined elavc155gal4;uas-sytgfp with the nmmii alleles; zip <dig>  uasziprnai and zipgs <dig>  this allowed us to visualize the affect on vesicle dynamics when nmmii expression was altered .
when nmmii levels are knocked down using elavc155gal4;uas-sytgfp/+; uasziprnai/+  the rate of vesicle movement is significantly reduced compared to the elavc155gal4;uas-sytgfp/y  control.
surprisingly, vesicle dynamics were also reduced  when nmmii is overexpressed using elavc155gal4;uas-sytgfp/+; zipgs50077/+.
interestingly, the heterozygous loss-of-function nmmii allele, zip1/+ resulted in enhanced mobility of vesicles  .
although the bleach depth was not significantly different between the heterozygous loss-of-function allele, the rnai knockdown sample and control samples, the o/e samples did show significantly lower bleach depth .
we therefore further analysed the frap recoveries to ensure differences in bleach depth did not account for our results.
to refine our analysis, we calculated the recovery index of the frap curve  <cit>  which accounts for differences in bleach depth between genotypes.
these calculations led to the same conclusions: the heterozygous loss-of-function allele of nmmii increased the recovery index, while both the rnai knockdown and overexpression of nmmii reduced the recovery index .
overall these results indicate that nmmii contributes to vesicle mobility in a manner which is highly dependent on the expression level of nmmii .
↑ = increased relative to control, ↓ = decreased relative to control, ↓↓ = further decreased relative to control
discussion
this report has identified the presence of nmmii in the presynaptic terminal and indicates a function for nmmii in synaptic vesicle mobility at the nmj of drosophila melanogaster.
nmmii has been implicated in synaptic transmission in rats  <cit> , but has not previously been shown at the nmj of drosophila and this is the first evidence of nmmii having a function in synaptic vesicle mobility.
using drosophila, as a genetically malleable tool, and the confocal imaging technique, frap, we were able to quantify the effect of nmmii on vesicle mobility.
frap revealed that nmmii plays a complex role in vesicle dynamics and begins to clarify our knowledge of how synaptic vesicles may be available for release upon stimulation.
vesicle mobility is affected by a complex interaction with nonmuscle myosin ii
immunocytological staining first identified that nmmii is found both pre- and postsynaptically in the drosophila nmj.
abolishment of postsynaptic nmmii expression through rnai confirmed expression of nmmii presynaptically.
with nmmii present in the presynaptic terminal, this suggests a possible function for nmmii in trafficking vesicles within the bouton.
to determine whether nmmii impacts vesicle dynamics, we carried out in vivo imaging techniques to visualize synaptic vesicle mobility.
we found an intriguing complex interaction between the expression level of nmmii and the dynamics of vesicle mobility.
inhibiting mlck reduced vesicle mobility, consistent with jordan et al.
<cit> .
more specifically, increasing either nmmii expression by 95% or reducing nmmii expression to 28% reduced vesicle mobility, while moderately reducing nmmii to 57% enhanced vesicle mobility.
a limitation of the present study is an estimate of nmmii activity or levels specifically at nerve terminal boutons in the various mutant strains used.
the postsynaptic presence of nmmii makes immunohistochemical techniques for measuring presynaptic nmmii difficult.
we estimated neuronal nmmii levels using western blots of larval brains.
this demonstrated that nmmii levels were up- and down-regulated significantly by the genotypes used.
the 50% reduction in protein level from the heterozygous loss-of-function to the rnai knockdown strains did not exhibit a statistical difference.
however, we did find substantially different effects on vesicle mobility suggesting that the apparent difference in expression levels is functionally significant.
thus it appears that tight regulation of nmmii expression is essential in maintaining appropriate vesicle dynamics: a small reduction in nmmii levels enhances mobility whereas too much or too little impairs mobility.
together this supports a role for nmmii in normal synaptic vesicle mobility at the drosophila nmj.
while these findings suggest a role for nmmii in synaptic vesicle mobility, it does not exclude the possibility that other myosin motors are also involved.
in support of this, the general myosin inhibitor, ml- <dig>  reduced synaptic vesicle mobility in a dose dependent manner with no stimulation of vesicle mobility at low concentrations.
in addition, other myosin motors have been shown to associate with synaptic vesicles and be involved in synaptic transmission.
for example, myosin v can bind to a myosin receptor found on a subpopulation of high density vesicles  <cit>  and is found to be associated with vesicles isolated from chick brain  <cit> , but was not found to alter hippocampal synaptic transmission in mice  <cit> .
however, myosin ii has been associated with normal synaptic transmission.
in cultured rat superior cervical ganglion neurones, myosin iib was found to inhibit synaptic transmission  <cit> .
a reduction in synaptic transmission upon inhibition of myosin ii was also observed in rat cholinergic synapses  <cit> , while mlck was found to be involved in maintaining repetitive synaptic transmission  <cit> .
myosin ii has also been shown to be involved in vesicle mobility in other systems.
nonmuscle myosin ii has been shown to transport vesicles on actin filaments in clam oocytes  <cit>  and nonmuscle myosin ii has been shown to contribute to vesicle transport from the golgi to the endoplasmic reticulum  <cit> .
thus, while the present work identifies a function for nmmii in synaptic vesicle mobility, the precise mechanisms of myosin motors in synaptic transmission and the precise role of nmmii as a vesicle motor remains to be clarified.
since nmmii interacts with actin, it will also be important to investigate the affects of nmmii on actin stability and dynamics in the nmj and to determine whether the affects of nmmii on vesicle mobility translate into affects on synaptic transmission.
CONCLUSIONS
our results show that nmmii is found presynaptically at the drosophila nmj and plays a functional role at the nmj.
we report, for the first time, a function for nmmii in normal synaptic vesicle mobility in the unstimulated neuron, which is dependent on the expression level of nmmii.
further experimentation to address the function of nmmii at the nmj, through electrophysiological assays, manipulating nmmii activity by its' kinases and phosphatases and measuring actin activity, is required to more clearly define the precise role of nmmii at the drosophila nmj.

 transport and assembly of synaptic vesicles has been the subject of several studies. vesicles and their components are transported along axon microtubules to the nerve terminal, where they participate in synaptic physiology, undergoing a cycle of exo- and endocytosis. however, vesicle traffic within terminal boutons is not well understood although recent advances in this area have been made. classically, vesicles were believed to be relatively stationary until released. however, more recent studies provided evidence for a mobile vesicle pool best described by a caged-diffusion model and differential vesicle mobility in the reserve and recycling pool has been suggested within the frog motor nerve terminals. additionally, nunes et al. observed dynamic vesicles in the drosophila melanogaster bouton, and dynamic vesicles have been reported at ribbon synapses in lizards. vesicle movement may result from diffusion or directed transport. actin polymerization in the nerve terminal may promote vesicle movement through a listeria comet mechanism or it may act as a substrate for myosin motors to shuttle vesicles. in light of a previous screen from our lab identifying nonmuscle myosin ii as a candidate molecule important in neuromuscular junction development, we have focused on determining a neuromuscular function for this actin-based myosin motor. nmmii is present in the nervous system of xenopus, mouse, rat and chicken, and in the cns of drosophila. nmmii can both crosslink f-actin and has been shown to transport vesicles on f-actin. this study was undertaken to determine whether nmmii contributes to synaptic vesicle mobility. we used the genetic model system drosophila melanogaster to manipulate nmmii expression, pharmacology to inhibit nmmii activity, and the optically accessible neuromuscular synapse of third instar larvae to investigate vesicle dynamics. we show that nmmii is concentrated pre- and postsynaptically and importantly, we report that unstimulated synaptic vesicle mobility exhibited a dependence on nmmii expression. these results report the first evidence for nmmii having a function in synaptic vesicle dynamics at the drosophila nmj.  nonmuscle myosin ii is localized pre- and postsynaptically at the nmj although nmmii has been previously found in the drosophila cns, it has not been previously localized to the nmj. therefore we used immunocytochemistry to investigate the localization of nmmii at the nmj. first, we demonstrated the presence of nmmii by staining the nmj with anti-nmmii and found a robust signal labelling the nerve terminal, which co-localized to a high degree with anti-hrp, a general marker of insect neural membrane. we did notice however that some of the nmmii staining was found outside the boutons defined by the anti-hrp signal suggesting that some of the nmmii signal has a postsynaptic origin. to further investigate this possibility we double labelled the nmj with the post-synaptic marker, discs large and anti-nmmii. we found colocalization between discs large and nmmii, indicating nmmii is found post-synaptically. to determine whether nmmii is also found presynaptically, we expressed uasziprnai in the muscle using the muscle specific driver 24bgal this dramatically reduces nmmii in the muscle, revealing presynaptic nmmii, which colocalizes with the specific neural marker anti-hrp. together these results demonstrate the presence of nmmii at the drosophila nmj. expression level of nonmuscle myosin ii in zipper alleles in order to study nmmii we manipulated its expression level using genetic tools. in drosophila, nmmii is encoded by the zipper gene. we used the heterozygous loss-of-function zip allele, overexpressed zip with the zip transgene, zipgs, and expressed a zip knockdown construct, ziprnai. before proceeding with using these reagents in experiments we confirmed the different zip tools were functioning as expected by western blot analysis of protein prepared from larval brain extracts. nmmii expression in elav3agal larvae were used as a control and normalized to. rnai knockdown of nmmii significantly reduced the nmmii level to 28% of the control. the heterozygous loss-of-function allele zip1/cyo reduced nmmii expression to 57% of the control while overexpression of nmmii increased protein levels by 95% compared to elav3agal. statistical analysis revealed significant variation among the groups, while post hoc newman-keuls comparison between groups showed that each group was different from the other with the exception that zip1/cyo was not found to be statistically different from the knockdown sample despite the 50% difference in average protein levels. inhibition of myosin with ml- results in a dose-dependent decrease in vesicle mobility nmmii has been previously shown to function in nmj development. for this reason, we investigated whether nmmii affected vesicle dynamics at the drosophila nmj. to assess the effects of nmmii on vesicle dynamics, synaptic vesicle mobility was measured using frap under conditions of altered myosin activity. to visualize synaptic vesicles in vivo, the recombinant synaptotagmin-gfp construct, elavc155gal4; uas-sytgfp, was used to drive expression of synaptotagmin-gfp in the nervous system. synaptotagmin-gfp has previously been shown to mark synaptic vesicles. to begin our examination of myosin activity at the synapse we used ml- an inhibitor of myosin light chain kinase, and tested synaptic vesicle mobility under different doses of this myosin inhibitor. application of ml- to elavc155gal4; uas-sytgfp/y larvae resulted in a dose-dependent reduction in the mobility of synaptic vesicles compared to vesicle dynamics in a saline + dmso control. vesicle mobility in this saline + dmso treated control did not significantly differ from the saline only control used here in the subsequent frap experiments. a significant decrease in vesicle mobility was observed with μm ml- which resulted in the most dramatic reduction of vesicle mobility while μm ml- resulted in slightly, but significantly less vesicle mobility compared to the control. a dose of μm ml- had no effect on vesicle dynamics. together this indicates a dose-dependent response of vesicle mobility to myosin inhibition. ml- is a general myosin inhibitor while -blebbistatin is known to be selective towards inhibiting nmmii. however -blebbistatin is autofluorescent under blue light and is quickly deactivated upon exposure to blue light thus limiting our ability to use this drug with our frap conditions. we did however obtain nmmii alleles to determine their affect on vesicle mobility. equation: frap = 1-a-b where a, b = constants that represent the apparent bleached fraction, k k = rates, t = time nonmuscle myosin ii contributes to synaptic vesicle dynamics in order to clarify whether nmmii was contributing to the inhibition of vesicle dynamics by ml- we genetically combined elavc155gal4;uas-sytgfp with the nmmii alleles; zip uasziprnai and zipgs this allowed us to visualize the affect on vesicle dynamics when nmmii expression was altered. when nmmii levels are knocked down using elavc155gal4;uas-sytgfp/+; uasziprnai/+ the rate of vesicle movement is significantly reduced compared to the elavc155gal4;uas-sytgfp/y control. surprisingly, vesicle dynamics were also reduced when nmmii is overexpressed using elavc155gal4;uas-sytgfp/+; zipgs50077/+. interestingly, the heterozygous loss-of-function nmmii allele, zip1/+ resulted in enhanced mobility of vesicles. although the bleach depth was not significantly different between the heterozygous loss-of-function allele, the rnai knockdown sample and control samples, the o/e samples did show significantly lower bleach depth. we therefore further analysed the frap recoveries to ensure differences in bleach depth did not account for our results. to refine our analysis, we calculated the recovery index of the frap curve which accounts for differences in bleach depth between genotypes. these calculations led to the same conclusions: the heterozygous loss-of-function allele of nmmii increased the recovery index, while both the rnai knockdown and overexpression of nmmii reduced the recovery index. overall these results indicate that nmmii contributes to vesicle mobility in a manner which is highly dependent on the expression level of nmmii. ↑ = increased relative to control, ↓ = decreased relative to control, ↓↓ = further decreased relative to control discussion this report has identified the presence of nmmii in the presynaptic terminal and indicates a function for nmmii in synaptic vesicle mobility at the nmj of drosophila melanogaster. nmmii has been implicated in synaptic transmission in rats, but has not previously been shown at the nmj of drosophila and this is the first evidence of nmmii having a function in synaptic vesicle mobility. using drosophila, as a genetically malleable tool, and the confocal imaging technique, frap, we were able to quantify the effect of nmmii on vesicle mobility. frap revealed that nmmii plays a complex role in vesicle dynamics and begins to clarify our knowledge of how synaptic vesicles may be available for release upon stimulation. vesicle mobility is affected by a complex interaction with nonmuscle myosin ii immunocytological staining first identified that nmmii is found both pre- and postsynaptically in the drosophila nmj. abolishment of postsynaptic nmmii expression through rnai confirmed expression of nmmii presynaptically. with nmmii present in the presynaptic terminal, this suggests a possible function for nmmii in trafficking vesicles within the bouton. to determine whether nmmii impacts vesicle dynamics, we carried out in vivo imaging techniques to visualize synaptic vesicle mobility. we found an intriguing complex interaction between the expression level of nmmii and the dynamics of vesicle mobility. inhibiting mlck reduced vesicle mobility, consistent with jordan et al.. more specifically, increasing either nmmii expression by 95% or reducing nmmii expression to 28% reduced vesicle mobility, while moderately reducing nmmii to 57% enhanced vesicle mobility. a limitation of the present study is an estimate of nmmii activity or levels specifically at nerve terminal boutons in the various mutant strains used. the postsynaptic presence of nmmii makes immunohistochemical techniques for measuring presynaptic nmmii difficult. we estimated neuronal nmmii levels using western blots of larval brains. this demonstrated that nmmii levels were up- and down-regulated significantly by the genotypes used. the 50% reduction in protein level from the heterozygous loss-of-function to the rnai knockdown strains did not exhibit a statistical difference. however, we did find substantially different effects on vesicle mobility suggesting that the apparent difference in expression levels is functionally significant. thus it appears that tight regulation of nmmii expression is essential in maintaining appropriate vesicle dynamics: a small reduction in nmmii levels enhances mobility whereas too much or too little impairs mobility. together this supports a role for nmmii in normal synaptic vesicle mobility at the drosophila nmj. while these findings suggest a role for nmmii in synaptic vesicle mobility, it does not exclude the possibility that other myosin motors are also involved. in support of this, the general myosin inhibitor, ml- reduced synaptic vesicle mobility in a dose dependent manner with no stimulation of vesicle mobility at low concentrations. in addition, other myosin motors have been shown to associate with synaptic vesicles and be involved in synaptic transmission. for example, myosin v can bind to a myosin receptor found on a subpopulation of high density vesicles and is found to be associated with vesicles isolated from chick brain, but was not found to alter hippocampal synaptic transmission in mice. however, myosin ii has been associated with normal synaptic transmission. in cultured rat superior cervical ganglion neurones, myosin iib was found to inhibit synaptic transmission. a reduction in synaptic transmission upon inhibition of myosin ii was also observed in rat cholinergic synapses, while mlck was found to be involved in maintaining repetitive synaptic transmission. myosin ii has also been shown to be involved in vesicle mobility in other systems. nonmuscle myosin ii has been shown to transport vesicles on actin filaments in clam oocytes and nonmuscle myosin ii has been shown to contribute to vesicle transport from the golgi to the endoplasmic reticulum. thus, while the present work identifies a function for nmmii in synaptic vesicle mobility, the precise mechanisms of myosin motors in synaptic transmission and the precise role of nmmii as a vesicle motor remains to be clarified. since nmmii interacts with actin, it will also be important to investigate the affects of nmmii on actin stability and dynamics in the nmj and to determine whether the affects of nmmii on vesicle mobility translate into affects on synaptic transmission.  our results show that nmmii is found presynaptically at the drosophila nmj and plays a functional role at the nmj. we report, for the first time, a function for nmmii in normal synaptic vesicle mobility in the unstimulated neuron, which is dependent on the expression level of nmmii. further experimentation to address the function of nmmii at the nmj, through electrophysiological assays, manipulating nmmii activity by its' kinases and phosphatases and measuring actin activity, is required to more clearly define the precise role of nmmii at the drosophila nmj. 
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. in the present study we have identified nonmuscle myosin ii as a candidate molecule important for synaptic vesicle traffic within drosophila larval neuromuscular boutons. 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. frap analysis showed that vesicle dynamics were highly dependent on the expression level of nonmuscle myosin ii. 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. the actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear.
 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.  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.  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. 