Abstract
Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants (jip3nl7) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3nl7, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3nl7, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein.
Highlights
Active transport of proteins and organelles between the neuronal cell body and axon terminals is necessary for the formation and maintenance of functional neural circuits
As Jun N-terminal Kinase (JNK)-interacting protein 3 (Jip3) has been shown to interact with members of the anterograde and retrograde motor complexes [11,12,13,22,23] and interruptions in transport have been associated with axon swellings like those observed in jip3nl7 [24,25], we focused our investigations on the potential function of Jip3 in axonal transport
We found that increased pJNK levels by expression of constitutively active form of JNK3 (caJNK3) correlated with the presence of axon terminal swellings (Figure 6F)
Summary
Active transport of proteins and organelles between the neuronal cell body and axon terminals is necessary for the formation and maintenance of functional neural circuits. Anterograde (to axon terminals) and retrograde (to the cell body) transport rely on motor proteins of the Kinesin and Dynein families respectively. These motors use the energy of ATP hydrolysis to walk along microtubule tracks, carrying cargo to its proper destination. It is tempting to speculate that the use of distinct adapter proteins may confer specificity to motorcargo interactions in the dynein motor system. Despite their importance for the understanding of dynein-based cargo transport, the identity of specific dynein cargo adapters is dramatically lacking [5]
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