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JNK-interacting protein 3 mediates the retrograde transport of activated c-Jun N-terminal kinase and lysosomes.

Drerup CM, Nechiporuk AV - PLoS Genet. (2013)

Bottom Line: Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3(nl7) , 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA.

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 (jip3(nl7) ) 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 jip3(nl7) , 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 jip3(nl7) , 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.

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Retrograde JNK3 transport frequency was decreased in jip3nl7 mutants.(A) Immunolabeling for pJNK in an axon expressing JNK3-mEos showed a high degree of colocalization (arrowheads) indicating that a large percentage of axonal JNK3-mEos is activated. (B,C) Representative stills from a live imaging session showing axonal transport of JNK3-mEos in a pLL axon of a wildtype (B) and jip3nl7 mutant (C) at 2 dpf (see Videos S6 and S7). Pink arrowhead denotes anterograde movement, yellow retrograde movement. (D,E) Kymographs generated from these imaging sessions. (F) Number of retrograde JNK3-mEos puncta (corrected for size of analyzed region and time of imaging session) was decreased in jip3nl7 (ANOVA, post-hoc contrasts; *-p<0.05). Distance of individual retrograde movement bouts (G) and velocity (H) were unaltered in jip3nl7. Anterograde transport distance was decreased (*-p<0.05; Ant = anterograde; Ret = retrograde). Scale bars = 10 µm.
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pgen-1003303-g005: Retrograde JNK3 transport frequency was decreased in jip3nl7 mutants.(A) Immunolabeling for pJNK in an axon expressing JNK3-mEos showed a high degree of colocalization (arrowheads) indicating that a large percentage of axonal JNK3-mEos is activated. (B,C) Representative stills from a live imaging session showing axonal transport of JNK3-mEos in a pLL axon of a wildtype (B) and jip3nl7 mutant (C) at 2 dpf (see Videos S6 and S7). Pink arrowhead denotes anterograde movement, yellow retrograde movement. (D,E) Kymographs generated from these imaging sessions. (F) Number of retrograde JNK3-mEos puncta (corrected for size of analyzed region and time of imaging session) was decreased in jip3nl7 (ANOVA, post-hoc contrasts; *-p<0.05). Distance of individual retrograde movement bouts (G) and velocity (H) were unaltered in jip3nl7. Anterograde transport distance was decreased (*-p<0.05; Ant = anterograde; Ret = retrograde). Scale bars = 10 µm.

Mentions: Finally, we used our in vivo live imaging to concretely determine if retrograde JNK transport was impaired in jip3nl7 mutant pLL axons using transient expression of JNK3 tagged with mEos. We chose to use JNK3 for our in vivo analysis because Jip3 has been shown to bind most strongly to the JNK3 homolog [14], and jnk3 is strongly expressed in the zebrafish nervous system (Figure S6A, S6B). Phospho-JNK immunolabeling of embryos expressing JNK3-mEos driven by the 5kbneurod promoter in pLL axons demonstrated that a large portion of JNK3-mEos positive vesicles carried the active form of this kinase (Figure 5A). Live imaging experiments revealed JNK3-mEos positive puncta traveled bidirectionally in wildtype and jip3nl7 mutants at 2 dpf (Figure 5B, 5C; Videos S6 and S7). Using kymograph analysis (Figure 5D, 5E), we found a decrease in the number of JNK3-mEos positive puncta moving in the retrograde direction at 2 dpf in jip3nl7 mutants (Figure 5F; wildtype:2.99±0.48 vs. jip3nl7:1.15±0.58 particles/100 µm*min, p≤0.05; Wilcoxon rank-sum) while retrograde movement distance and velocity were largely unchanged (Figure 5G, 5H). Taken together with the results from our injury model, these data confirmed that the frequency of retrograde pJNK transport was hindered in jip3nl7 mutants.


JNK-interacting protein 3 mediates the retrograde transport of activated c-Jun N-terminal kinase and lysosomes.

Drerup CM, Nechiporuk AV - PLoS Genet. (2013)

Retrograde JNK3 transport frequency was decreased in jip3nl7 mutants.(A) Immunolabeling for pJNK in an axon expressing JNK3-mEos showed a high degree of colocalization (arrowheads) indicating that a large percentage of axonal JNK3-mEos is activated. (B,C) Representative stills from a live imaging session showing axonal transport of JNK3-mEos in a pLL axon of a wildtype (B) and jip3nl7 mutant (C) at 2 dpf (see Videos S6 and S7). Pink arrowhead denotes anterograde movement, yellow retrograde movement. (D,E) Kymographs generated from these imaging sessions. (F) Number of retrograde JNK3-mEos puncta (corrected for size of analyzed region and time of imaging session) was decreased in jip3nl7 (ANOVA, post-hoc contrasts; *-p<0.05). Distance of individual retrograde movement bouts (G) and velocity (H) were unaltered in jip3nl7. Anterograde transport distance was decreased (*-p<0.05; Ant = anterograde; Ret = retrograde). Scale bars = 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585007&req=5

pgen-1003303-g005: Retrograde JNK3 transport frequency was decreased in jip3nl7 mutants.(A) Immunolabeling for pJNK in an axon expressing JNK3-mEos showed a high degree of colocalization (arrowheads) indicating that a large percentage of axonal JNK3-mEos is activated. (B,C) Representative stills from a live imaging session showing axonal transport of JNK3-mEos in a pLL axon of a wildtype (B) and jip3nl7 mutant (C) at 2 dpf (see Videos S6 and S7). Pink arrowhead denotes anterograde movement, yellow retrograde movement. (D,E) Kymographs generated from these imaging sessions. (F) Number of retrograde JNK3-mEos puncta (corrected for size of analyzed region and time of imaging session) was decreased in jip3nl7 (ANOVA, post-hoc contrasts; *-p<0.05). Distance of individual retrograde movement bouts (G) and velocity (H) were unaltered in jip3nl7. Anterograde transport distance was decreased (*-p<0.05; Ant = anterograde; Ret = retrograde). Scale bars = 10 µm.
Mentions: Finally, we used our in vivo live imaging to concretely determine if retrograde JNK transport was impaired in jip3nl7 mutant pLL axons using transient expression of JNK3 tagged with mEos. We chose to use JNK3 for our in vivo analysis because Jip3 has been shown to bind most strongly to the JNK3 homolog [14], and jnk3 is strongly expressed in the zebrafish nervous system (Figure S6A, S6B). Phospho-JNK immunolabeling of embryos expressing JNK3-mEos driven by the 5kbneurod promoter in pLL axons demonstrated that a large portion of JNK3-mEos positive vesicles carried the active form of this kinase (Figure 5A). Live imaging experiments revealed JNK3-mEos positive puncta traveled bidirectionally in wildtype and jip3nl7 mutants at 2 dpf (Figure 5B, 5C; Videos S6 and S7). Using kymograph analysis (Figure 5D, 5E), we found a decrease in the number of JNK3-mEos positive puncta moving in the retrograde direction at 2 dpf in jip3nl7 mutants (Figure 5F; wildtype:2.99±0.48 vs. jip3nl7:1.15±0.58 particles/100 µm*min, p≤0.05; Wilcoxon rank-sum) while retrograde movement distance and velocity were largely unchanged (Figure 5G, 5H). Taken together with the results from our injury model, these data confirmed that the frequency of retrograde pJNK transport was hindered in jip3nl7 mutants.

Bottom Line: Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3(nl7) , 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA.

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 (jip3(nl7) ) 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 jip3(nl7) , 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 jip3(nl7) , 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.

Show MeSH
Related in: MedlinePlus