These results indicate that JNK continues

to impact presynaptic pattern after its initial establishment. To understand where JNK might function within the neuron, we further examined the subcellular localization of JKK-1 and JNK-1 in DA9 using functional GFP fusion proteins. Consistent selleckchem with a presynaptic function, both JKK-1::GFP and JNK-1::GFP are highly enriched at the presynaptic terminals and colocalize with mCherry::RAB-3 (Figures S4B–S4J and S4K–S4S). This presynaptic enrichment requires the anterograde motor for STVs, UNC-104/KIF1A (Hall and Hedgecock, 1991); JKK-1::GFP and JNK-1::GFP are absent from the presynaptic terminals and accumulate in the cell body and dendrite in unc-104 mutants (data not shown). Axonal transport is critical for the establishment and maintenance of presynapses (Hirokawa et al., 2010). Alterations in the cell-wide distribution of presynaptic components could originate from changes in their axonal transport dynamics. We previously showed that presynaptic protein mislocalization in arl-8 mutants probably results from premature clustering of STVs during trafficking ( Klassen et al., 2010). To further investigate the role of the JNK pathway in regulating STV clustering during transport, we performed time-lapse

imaging of STVs labeled with GFP::RAB-3 in vivo. Within the proximal axon of wild-type DA9, small mobile and stationary GFP::RAB-3 puncta can be visualized with a high-sensitivity charge-coupled device camera, evident Epacadostat as diagonal and vertical lines in the kymographs, respectively, with the mobile puncta representing trafficking STVs, which pause frequently en route and form stationary puncta ( Klassen

et al., 2010; Figures 3A and 3B). arl-8 mutants did not exhibit changes in the directionality or velocity of STV movements ( Klassen et al., tuclazepam 2010). However, we observed significant increases in the number and fluorescence intensity of stationary puncta ( Figures 3C, 3F, and 3G). Meanwhile, there was a decrease in the number of moving events ( Figure 3H). The jkk-1 mutation strongly alleviated the STV trafficking abnormalities in arl-8 mutants; the number of stationary puncta en route and their fluorescence intensity were significantly reduced in the arl-8; jkk-1 double mutants ( Figures 3D, 3F, and 3G). Accordingly, there was a significant increase in the number of moving events ( Figure 3H). The jkk-1 single mutants showed no significant difference from wild-type animals ( Figures 3E–3H). The coexistence of stable and motile puncta is consistent with previous findings that STVs undergo intermittent moving and stationary phases en route to the presynaptic terminals (Ahmari et al., 2000; Sabo et al., 2006). The transitions between these two states might serve as regulated switches to control the trafficking and aggregation of STVs.