In a genetic screen for suppressors of
goa-1 (Gao) null mutants we recovered 3 alleles of
unc-16, which encodes the C. elegans JIP3 ortholog. A recent pioneering study showed that impairing UNC-16 function increases the early endosome content of axons (Brown et al., 2009). Null mutations in UNC-108 (Rab2) also suppress
goa-1 null mutants and have early endosome - related membrane trafficking defects (Chun et al., 2008; Mangahas et al., 2008; Edwards et al. WM2009 abstract). To determine if
unc-16 and
unc-108 mutants have similar neuronal membrane trafficking defects, we examined the trafficking of the tagged pro-neuropeptide NLP-21-Venus in
unc-16 mutant neurons. In
unc-108 null mutants, early endosomes remove the Venus tag from immature dense core vesicles after pro-neuropeptide processing resulting in reduced levels of the Venus tag in neuronal somas (59% of wild type) and axons (33% of wild type). Although
unc-16 mutants also had reduced NLP-21-Venus levels in cell somas (29% of wild type) and axons (59% of wild type), the dissimilar ratios of cell soma-to-axonal Venus tag compared to
unc-108 mutants suggested different membrane trafficking defects. Further underscoring the differences between
unc-108 and
unc-16 mutants, we found that
unc-16 mutants have lysosomes in their axons, which we never observed in wild type or
unc-108 mutant axons. Lysosomes are the end-stage organelle of a pathway extending from early and recycling endosomes through late endosomes to lysosomes and also involving Golgi-derived vesicles. To determine the extent to which components of the endo-lysosomal system accumulate in
unc-16 mutant axons, we imaged the following markers expressed from integrated arrays in cholinergic neuronal cell somas and axons: Golgi (AMAN-2-Venus), early endosomes (YFP-RAB-5 and RFP-SYN-13), recycling endosomes (CFP-RAB-11), late endosomes (CFP-RAB-7), and lysosomes (CTNS-1-RFP). We found that the entire endo-lysosomal pathway accumulates at high levels in
unc-16 mutant cholinergic axons (5 - 7 fold higher than wild type for Golgi and early endosome markers, 1.8 - fold higher for recycling endosomes, 4-fold higher for late endosomes, and infinitely higher for lysosomes, which are not observed in wild type). In
unc-16 mutant cell somas, only the RAB-5 early endosome marker was strongly depleted (decreased to 23% of wild type), while the cell soma levels of the other markers ranged from 60% - 86% of wild type. We plan to use time-lapse imaging to determine if lysosomes aberrantly enter
unc-16 mutant axons or if they form within axons.