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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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Jip3 scaffolds lysosomes to DLIC for retrograde transport.(A,B) Stills from a wildtype imaging session at 3 dpf in which Lamp1-EGFP (A) and Jip3-mCherry (B) co-transport was analyzed (Video S10). Pink and yellow arrowheads point to two retrograde Jip3/Lamp1 positive cargos. (C,D) Kymographs generated from this imaging session for individual cargos. (E) Schematized kymograph of co-transport. Yellow lines denote Jip3-positive lysosomes moving in the retrograde direction. (F,G) mTangerine-DLIC expression in a wildtype (F) and jip3nl7 mutant (G) NM1 axon terminal at 3 dpf. (H,I) Stills from analysis of Lamp1 (H) and DLIC (I) co-transport at 3 dpf in a wildtype (Video S11). Green arrow-anterograde co-labeled puncta. Yellow arrowhead-DLIC positive lysosome undergoing retrograde transport. (J) The ratio of DLIC positive lysosomes moving in the retrograde direction was significantly decreased in jip3nl7 mutants (ANOVA, *-p<0.05; Anterograde-Ant; Retrograde-Ret). (K–M) Kymographs from this imaging session and schematized kymograph depicting co-labeled anterograde lysosomes in green and retrograde in yellow.
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pgen-1003303-g008: Jip3 scaffolds lysosomes to DLIC for retrograde transport.(A,B) Stills from a wildtype imaging session at 3 dpf in which Lamp1-EGFP (A) and Jip3-mCherry (B) co-transport was analyzed (Video S10). Pink and yellow arrowheads point to two retrograde Jip3/Lamp1 positive cargos. (C,D) Kymographs generated from this imaging session for individual cargos. (E) Schematized kymograph of co-transport. Yellow lines denote Jip3-positive lysosomes moving in the retrograde direction. (F,G) mTangerine-DLIC expression in a wildtype (F) and jip3nl7 mutant (G) NM1 axon terminal at 3 dpf. (H,I) Stills from analysis of Lamp1 (H) and DLIC (I) co-transport at 3 dpf in a wildtype (Video S11). Green arrow-anterograde co-labeled puncta. Yellow arrowhead-DLIC positive lysosome undergoing retrograde transport. (J) The ratio of DLIC positive lysosomes moving in the retrograde direction was significantly decreased in jip3nl7 mutants (ANOVA, *-p<0.05; Anterograde-Ant; Retrograde-Ret). (K–M) Kymographs from this imaging session and schematized kymograph depicting co-labeled anterograde lysosomes in green and retrograde in yellow.

Mentions: In cultured cells, DLIC, a dynein accessory protein, functions in dynein-dependent lysosome transport [30]. As Jip3 has been shown to interact with DLIC [22], we hypothesized that Jip3 might serve as an adapter for lysosome-DLIC attachment during retrograde lysosome transport in axons. To ascertain whether Jip3 co-localized with moving lysosomes and could function in such a direct role, we performed sequential imaging of axons expressing both Jip3-mCherry and Lamp1-EGFP cargos at 2 and 3 dpf. Co-transport analysis revealed that Jip3 is present on lysosomes moving in the retrograde direction at both time-points (Figure 8A–8E; Video S10). Interestingly, the percentage of lysosomes that were transported in the retrograde direction labeled with Jip3 was higher at 3 dpf than at 2 dpf (2 dpf: 15%±3.8%, N = 5 vs. 3 dpf: 37%±4.2%, N = 4). This may indicate a differential reliance on Jip3 for the transport of this organelle beyond 2 dpf, leading to the decrease in lysosome retrograde transport frequency only after 2 dpf in jip3nl7 (see Figure 2).


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

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

Jip3 scaffolds lysosomes to DLIC for retrograde transport.(A,B) Stills from a wildtype imaging session at 3 dpf in which Lamp1-EGFP (A) and Jip3-mCherry (B) co-transport was analyzed (Video S10). Pink and yellow arrowheads point to two retrograde Jip3/Lamp1 positive cargos. (C,D) Kymographs generated from this imaging session for individual cargos. (E) Schematized kymograph of co-transport. Yellow lines denote Jip3-positive lysosomes moving in the retrograde direction. (F,G) mTangerine-DLIC expression in a wildtype (F) and jip3nl7 mutant (G) NM1 axon terminal at 3 dpf. (H,I) Stills from analysis of Lamp1 (H) and DLIC (I) co-transport at 3 dpf in a wildtype (Video S11). Green arrow-anterograde co-labeled puncta. Yellow arrowhead-DLIC positive lysosome undergoing retrograde transport. (J) The ratio of DLIC positive lysosomes moving in the retrograde direction was significantly decreased in jip3nl7 mutants (ANOVA, *-p<0.05; Anterograde-Ant; Retrograde-Ret). (K–M) Kymographs from this imaging session and schematized kymograph depicting co-labeled anterograde lysosomes in green and retrograde in yellow.
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pgen-1003303-g008: Jip3 scaffolds lysosomes to DLIC for retrograde transport.(A,B) Stills from a wildtype imaging session at 3 dpf in which Lamp1-EGFP (A) and Jip3-mCherry (B) co-transport was analyzed (Video S10). Pink and yellow arrowheads point to two retrograde Jip3/Lamp1 positive cargos. (C,D) Kymographs generated from this imaging session for individual cargos. (E) Schematized kymograph of co-transport. Yellow lines denote Jip3-positive lysosomes moving in the retrograde direction. (F,G) mTangerine-DLIC expression in a wildtype (F) and jip3nl7 mutant (G) NM1 axon terminal at 3 dpf. (H,I) Stills from analysis of Lamp1 (H) and DLIC (I) co-transport at 3 dpf in a wildtype (Video S11). Green arrow-anterograde co-labeled puncta. Yellow arrowhead-DLIC positive lysosome undergoing retrograde transport. (J) The ratio of DLIC positive lysosomes moving in the retrograde direction was significantly decreased in jip3nl7 mutants (ANOVA, *-p<0.05; Anterograde-Ant; Retrograde-Ret). (K–M) Kymographs from this imaging session and schematized kymograph depicting co-labeled anterograde lysosomes in green and retrograde in yellow.
Mentions: In cultured cells, DLIC, a dynein accessory protein, functions in dynein-dependent lysosome transport [30]. As Jip3 has been shown to interact with DLIC [22], we hypothesized that Jip3 might serve as an adapter for lysosome-DLIC attachment during retrograde lysosome transport in axons. To ascertain whether Jip3 co-localized with moving lysosomes and could function in such a direct role, we performed sequential imaging of axons expressing both Jip3-mCherry and Lamp1-EGFP cargos at 2 and 3 dpf. Co-transport analysis revealed that Jip3 is present on lysosomes moving in the retrograde direction at both time-points (Figure 8A–8E; Video S10). Interestingly, the percentage of lysosomes that were transported in the retrograde direction labeled with Jip3 was higher at 3 dpf than at 2 dpf (2 dpf: 15%±3.8%, N = 5 vs. 3 dpf: 37%±4.2%, N = 4). This may indicate a differential reliance on Jip3 for the transport of this organelle beyond 2 dpf, leading to the decrease in lysosome retrograde transport frequency only after 2 dpf in jip3nl7 (see Figure 2).

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