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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 interaction with JNK was necessary for pJNK clearance and the prevention of axon swellings.(A–C) Axon terminal swellings and pJNK accumulation were rescued by Jip3 but not by Jip3ΔJNK. Axonal swellings were visualized live by neurod:EGFP transgene expression; following live imaging, pJNK was assayed by individual larva immunolabeling at 4 dpf. White arrowheads mark axon terminals expressing the DNA constructs. Yellow arrow points to a swelling in an axon not expressing Jip3-mCherry. Red arrowhead denotes an underlying pJNK positive cell not expressing Jip3-mCherry. (D) Ratio of axon terminal swellings in each class (mild = small swellings, severe = large swellings) show rescue of axonal swellings by full-length Jip3 but not Jip3ΔJNK. DNA indicates the rescue construct injected; Jip3 = full-length Jip3-mCherry; ΔJNK = Jip3ΔJNK-mCherry; Con = uninjected control. Embryo genotype, determined by control axon terminal morphology, is indicated below each bar. (E) Ratio of pJNK levels in Jip3 or Jip3ΔJNK expressing axon terminals to those not expressing the rescue construct at 4 dpf. Jip3, but not Jip3ΔJNK, suppressed increased pJNK levels in jip3nl7 (Wilcoxon rank-sum; *-p<0.01). (F) Induction of constitutively active JNK3 tagged with EGFP (caJNK3; green) for 15 hours at 4 dpf increased the level of pJNK immunofluorescence concomitant with the induction of swellings shown by both the caJNK3-EGFP fill and Tag1 immunolabeling of neuronal membranes. Arrowhead points to a caJNK3-EGFP expressing axon. Yellow arrow indicates an axon terminal in the same NM not expressing this construct. (G) Axon terminal swellings were absent in axon terminals expressing an inactive form of the same construct (caJNK3-IA), indicating that JNK activation was necessary to induce swellings. (H,I) Efficacy of both caJNK3 and caJNK3-IA were assayed by Western blot analysis of phospho-cJun, a downstream target of active JNK. While whole embryo overexpression of caJNK3-EGFP by RNA injection induced elevated levels of phospho-cJun at 24 hpf (H), similar expression of the inactive form of caJNK3-EGFP (caJNK3-IA) failed to induce a similar increase (I). Scale bars = 10 µm.
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pgen-1003303-g006: Jip3 interaction with JNK was necessary for pJNK clearance and the prevention of axon swellings.(A–C) Axon terminal swellings and pJNK accumulation were rescued by Jip3 but not by Jip3ΔJNK. Axonal swellings were visualized live by neurod:EGFP transgene expression; following live imaging, pJNK was assayed by individual larva immunolabeling at 4 dpf. White arrowheads mark axon terminals expressing the DNA constructs. Yellow arrow points to a swelling in an axon not expressing Jip3-mCherry. Red arrowhead denotes an underlying pJNK positive cell not expressing Jip3-mCherry. (D) Ratio of axon terminal swellings in each class (mild = small swellings, severe = large swellings) show rescue of axonal swellings by full-length Jip3 but not Jip3ΔJNK. DNA indicates the rescue construct injected; Jip3 = full-length Jip3-mCherry; ΔJNK = Jip3ΔJNK-mCherry; Con = uninjected control. Embryo genotype, determined by control axon terminal morphology, is indicated below each bar. (E) Ratio of pJNK levels in Jip3 or Jip3ΔJNK expressing axon terminals to those not expressing the rescue construct at 4 dpf. Jip3, but not Jip3ΔJNK, suppressed increased pJNK levels in jip3nl7 (Wilcoxon rank-sum; *-p<0.01). (F) Induction of constitutively active JNK3 tagged with EGFP (caJNK3; green) for 15 hours at 4 dpf increased the level of pJNK immunofluorescence concomitant with the induction of swellings shown by both the caJNK3-EGFP fill and Tag1 immunolabeling of neuronal membranes. Arrowhead points to a caJNK3-EGFP expressing axon. Yellow arrow indicates an axon terminal in the same NM not expressing this construct. (G) Axon terminal swellings were absent in axon terminals expressing an inactive form of the same construct (caJNK3-IA), indicating that JNK activation was necessary to induce swellings. (H,I) Efficacy of both caJNK3 and caJNK3-IA were assayed by Western blot analysis of phospho-cJun, a downstream target of active JNK. While whole embryo overexpression of caJNK3-EGFP by RNA injection induced elevated levels of phospho-cJun at 24 hpf (H), similar expression of the inactive form of caJNK3-EGFP (caJNK3-IA) failed to induce a similar increase (I). Scale bars = 10 µm.

Mentions: Next, we addressed whether the direct interaction between Jip3 and JNK was necessary for retrograde pJNK transport by asking whether the pJNK accumulation in jip3nl7 could be rescued with a Jip3 variant that lacked the JNK binding domain (Jip3ΔJNK: amino acids 202–214; [32]). DNA constructs were injected into zygotes to mosaically express Jip3-mCherry or Jip3ΔJNK-mCherry in individual pLL ganglion neurons. At 4 dpf, axon terminals expressing the respective fusions were imaged live and scored for axon morphology before larvae were individually immunolabeled for pJNK and the same axon terminals were re-imaged. As each NM is innervated by 2 axons and this innervation is segregated in space [34], we could use the non-expressing half of the NM to identify which larvae were jip3nl7 mutants as well as utilize it as a normalizing factor for the quantification of pJNK immunofluorescence. Though full-length Jip3 rescued axon terminal swellings and the accumulation of pJNK, Jip3ΔJNK was unable to rescue either phenotype (Figure 6A–6E). Importantly, expression of Jip3ΔJNK by mRNA injection rescued axon length, providing evidence that deletion of this region did not result in protein instability or failed processing, and pointing to a JNK-independent mechanism for Jip3's role in axon outgrowth (Figure S7). In summary, these data show that direct interaction between Jip3 and JNK is necessary for pJNK retrograde transport and also revealed a correlation between the accumulation of pJNK due to loss of Jip3-JNK interaction and the generation of axon terminal swellings.


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 interaction with JNK was necessary for pJNK clearance and the prevention of axon swellings.(A–C) Axon terminal swellings and pJNK accumulation were rescued by Jip3 but not by Jip3ΔJNK. Axonal swellings were visualized live by neurod:EGFP transgene expression; following live imaging, pJNK was assayed by individual larva immunolabeling at 4 dpf. White arrowheads mark axon terminals expressing the DNA constructs. Yellow arrow points to a swelling in an axon not expressing Jip3-mCherry. Red arrowhead denotes an underlying pJNK positive cell not expressing Jip3-mCherry. (D) Ratio of axon terminal swellings in each class (mild = small swellings, severe = large swellings) show rescue of axonal swellings by full-length Jip3 but not Jip3ΔJNK. DNA indicates the rescue construct injected; Jip3 = full-length Jip3-mCherry; ΔJNK = Jip3ΔJNK-mCherry; Con = uninjected control. Embryo genotype, determined by control axon terminal morphology, is indicated below each bar. (E) Ratio of pJNK levels in Jip3 or Jip3ΔJNK expressing axon terminals to those not expressing the rescue construct at 4 dpf. Jip3, but not Jip3ΔJNK, suppressed increased pJNK levels in jip3nl7 (Wilcoxon rank-sum; *-p<0.01). (F) Induction of constitutively active JNK3 tagged with EGFP (caJNK3; green) for 15 hours at 4 dpf increased the level of pJNK immunofluorescence concomitant with the induction of swellings shown by both the caJNK3-EGFP fill and Tag1 immunolabeling of neuronal membranes. Arrowhead points to a caJNK3-EGFP expressing axon. Yellow arrow indicates an axon terminal in the same NM not expressing this construct. (G) Axon terminal swellings were absent in axon terminals expressing an inactive form of the same construct (caJNK3-IA), indicating that JNK activation was necessary to induce swellings. (H,I) Efficacy of both caJNK3 and caJNK3-IA were assayed by Western blot analysis of phospho-cJun, a downstream target of active JNK. While whole embryo overexpression of caJNK3-EGFP by RNA injection induced elevated levels of phospho-cJun at 24 hpf (H), similar expression of the inactive form of caJNK3-EGFP (caJNK3-IA) failed to induce a similar increase (I). Scale bars = 10 µm.
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Related In: Results  -  Collection

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pgen-1003303-g006: Jip3 interaction with JNK was necessary for pJNK clearance and the prevention of axon swellings.(A–C) Axon terminal swellings and pJNK accumulation were rescued by Jip3 but not by Jip3ΔJNK. Axonal swellings were visualized live by neurod:EGFP transgene expression; following live imaging, pJNK was assayed by individual larva immunolabeling at 4 dpf. White arrowheads mark axon terminals expressing the DNA constructs. Yellow arrow points to a swelling in an axon not expressing Jip3-mCherry. Red arrowhead denotes an underlying pJNK positive cell not expressing Jip3-mCherry. (D) Ratio of axon terminal swellings in each class (mild = small swellings, severe = large swellings) show rescue of axonal swellings by full-length Jip3 but not Jip3ΔJNK. DNA indicates the rescue construct injected; Jip3 = full-length Jip3-mCherry; ΔJNK = Jip3ΔJNK-mCherry; Con = uninjected control. Embryo genotype, determined by control axon terminal morphology, is indicated below each bar. (E) Ratio of pJNK levels in Jip3 or Jip3ΔJNK expressing axon terminals to those not expressing the rescue construct at 4 dpf. Jip3, but not Jip3ΔJNK, suppressed increased pJNK levels in jip3nl7 (Wilcoxon rank-sum; *-p<0.01). (F) Induction of constitutively active JNK3 tagged with EGFP (caJNK3; green) for 15 hours at 4 dpf increased the level of pJNK immunofluorescence concomitant with the induction of swellings shown by both the caJNK3-EGFP fill and Tag1 immunolabeling of neuronal membranes. Arrowhead points to a caJNK3-EGFP expressing axon. Yellow arrow indicates an axon terminal in the same NM not expressing this construct. (G) Axon terminal swellings were absent in axon terminals expressing an inactive form of the same construct (caJNK3-IA), indicating that JNK activation was necessary to induce swellings. (H,I) Efficacy of both caJNK3 and caJNK3-IA were assayed by Western blot analysis of phospho-cJun, a downstream target of active JNK. While whole embryo overexpression of caJNK3-EGFP by RNA injection induced elevated levels of phospho-cJun at 24 hpf (H), similar expression of the inactive form of caJNK3-EGFP (caJNK3-IA) failed to induce a similar increase (I). Scale bars = 10 µm.
Mentions: Next, we addressed whether the direct interaction between Jip3 and JNK was necessary for retrograde pJNK transport by asking whether the pJNK accumulation in jip3nl7 could be rescued with a Jip3 variant that lacked the JNK binding domain (Jip3ΔJNK: amino acids 202–214; [32]). DNA constructs were injected into zygotes to mosaically express Jip3-mCherry or Jip3ΔJNK-mCherry in individual pLL ganglion neurons. At 4 dpf, axon terminals expressing the respective fusions were imaged live and scored for axon morphology before larvae were individually immunolabeled for pJNK and the same axon terminals were re-imaged. As each NM is innervated by 2 axons and this innervation is segregated in space [34], we could use the non-expressing half of the NM to identify which larvae were jip3nl7 mutants as well as utilize it as a normalizing factor for the quantification of pJNK immunofluorescence. Though full-length Jip3 rescued axon terminal swellings and the accumulation of pJNK, Jip3ΔJNK was unable to rescue either phenotype (Figure 6A–6E). Importantly, expression of Jip3ΔJNK by mRNA injection rescued axon length, providing evidence that deletion of this region did not result in protein instability or failed processing, and pointing to a JNK-independent mechanism for Jip3's role in axon outgrowth (Figure S7). In summary, these data show that direct interaction between Jip3 and JNK is necessary for pJNK retrograde transport and also revealed a correlation between the accumulation of pJNK due to loss of Jip3-JNK interaction and the generation of axon terminal swellings.

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