Indeed, time-lapse imaging revealed that unc-104(gf) significantly reduced the capture probability in the axon shaft in both wild-type and arl-8 mutant animals ( Figure 7K). Interestingly, unlike the jkk-1 and syd-2 mutations, unc-104(gf) did Sotrastaurin not affect the dissociation rate ( Figure 7L). Similarly, for RAB-3 clusters at the mature synapses, unc-104(gf) significantly reduced the capture probability ( Figure 7M), with no

significant effect on the dissociation rate ( Figure 7N). Thus, control of UNC-104/KIF1A motor activity probably represents a mechanism that regulates STV capture. Consistent with this hypothesis, unc-104(gf) single mutants exhibited decreased presynaptic SNB-1::YFP puncta size ( Figures 7D and 7O). Together, these findings suggest that both STV exit from and entry into stable clusters are subject to molecular regulation. While the exit process is regulated by ARL-8, the JNK-1 AZD5363 pathway, and SYD-2, the entry process is controlled by ARL-8 and

UNC-104/KIF1A. The fact that the abnormal distribution of presynaptic components in arl-8 mutants can be partially suppressed by either an increase in dissociation caused by the jkk-1 and syd-2 mutations or a decrease in capture efficiency caused by the unc-104(gf) mutation argues that it is the balance between the entry and exit processes that controls the position and size of presynaptic protein clusters. This model predicts that perturbation the of both STV entry and exit may produce a stronger phenotype compared to manipulation of a single factor. Indeed, unc-104(gf); jkk-1 double mutants,

in which capture is decreased and dissociation increased, showed enhanced reduction in the size of presynaptic SNB-1::YFP puncta compared to either single mutants ( Figures 7A, 7D, 7E, and 7O), supporting the notion that unc-104 and jkk-1 function in parallel to modulate STV clustering. Conversely, we built double mutants between two partial loss-of-function alleles, arl-8(tm2388) and unc-104(lf) ( Figure S7A), in which capture is increased and dissociation decreased. While the single mutants showed much weaker phenotypes than their respective strong loss-of-function mutants ( Figures 7G, 7H, and 7P), the double mutants showed a strongly enhanced phenotype, with large RAB-3 puncta forming in the ventral axon and commissural regions ( Figures 7I and 7P). The downstream functions of small G proteins are mediated by effector proteins that bind specifically to the GTP-bound form of the G proteins (Donaldson and Jackson, 2011). The strong genetic interaction between arl-8 and unc-104 led us to test whether UNC-104/KIF1A is an ARL-8 effector. We first performed affinity chromatography with glutathione S-transferase (GST)-tagged human ARL8A and various GFP-tagged human KIF1A fragments ( Figure 8A; CC1-FHA-CC2, 430–694 aa; CC3-UDR, 694–1,209 aa; CC3, 694–775 aa; and UDR, 776–1,209 aa).

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