A

null Selleck Quisinostat allele jkk-1(km2) ( Kawasaki et al., 1999) suppressed the arl-8 phenotype to the same degree as wy733 and wy735 ( Figure 1D), indicating that loss of JKK-1 activity suppresses the arl-8 phenotype. We used the jkk-1(km2) allele for our subsequent analyses. We quantified the suppression of arl-8 by jkk-1. Compared to the arl-8 single mutants, SNB-1::YFP is redistributed into more distal axonal regions in arl-8; jkk-1 double mutants ( Figures 1I and 1J). The size of the proximal SNB-1::YFP puncta (0–25 μm from commissure) is also significantly reduced in the double mutants ( Figure 1J). Furthermore, while the arl-8 mutants exhibited reduced number of presynaptic SNB-1::YFP puncta, this defect is abolished in the arl-8; jkk-1 double mutants ( Figure 1K). Similar results were obtained with two additional SV proteins, RAB-3 ( Figures S2A–S2D) and SNG-1/synaptogyrin (data not shown). Therefore, jkk-1 mutations partially and strongly suppressed multiple aspects of the arl-8 mutant phenotype in DA9. MAP kinases (MAPKs) act in cascades in which each MAPK is activated via phosphorylation by a MAPK kinase (MAPKK), which is in turn activated by a MAPKK kinase (MAPKKK) (Davis, 2000). JKK-1 was shown to be a specific upstream activator of c-Jun N-terminal kinase (JNK)-1, a homolog of mammalian JNK3 (Kawasaki et al.,

1999). A null mutation in jnk-1, gk7, caused the same degree of suppression of the arl-8 phenotype as jkk-1(km2) ( Figures 1E and 1I–1K). Moreover, the degree learn more of suppression in arl-8, jnk-1; jkk-1 triple mutants is indistinguishable

from that in either double mutants ( Figures 1F and 1I–1K), indicating that jkk-1 and jnk-1 function in the same pathway. jkk-1 and jnk-1 mutants were previously shown to partially mislocalize SNB-1::GFP to the dendrite in the DD motoneurons ( Byrd et al., 2001). We found that Urease the jkk-1 and jnk-1 single mutants appeared grossly normal in SV protein localization in DA9 ( Figures 1G and 1H) and did not show mislocalization of SV proteins to the DA9 dendrite (data not shown). However, these mutants did exhibit subtle but significant decreases in SNB-1::YFP puncta size ( Figure 1J) and increases in puncta number ( Figure 1K), suggesting that JNK also promotes SV clustering in wild-type animals. To determine whether JNK functionally interacts with arl-8 broadly in the C. elegans nervous system, we examined several other neuron types, including the cholinergic motoneuron DB7, the GABAergic DD motoneurons and the thermosensory neuron AFD, all of which have a proximal axonal region devoid of presynapses and form en passant presynapses in the distal axon ( Hallam and Jin, 1998; Klassen and Shen, 2007; Hellman and Shen, 2011).