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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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Lysosomes accumulated in jip3nl7 axon terminal swellings.(A,B) Lysosome density, as assayed by Lamp1 immunolabeling (A,B-red, A′B′-white) was increased in jip3nl7 NM3 axon terminals at 5 dpf (arrowhead). Larvae carried the neurod:EGFP transgene to label pLL axons. (C,D) Stills from imaging sessions of Lamp1-mTangerine transport in the pLL nerve of wildtype (C) and jip3nl7 mutant (D) embryos at 3 dpf (Videos S4 and S5). Dotted lines denote the lower bound of the axons imaged. (E,F) Lamp1-mTangerine accumulated in the axon terminals of jip3nl7 mutants (F) but not wildtype siblings (E) at 3 dpf (NM1 shown for both). (G) The majority of Lamp1-EGFP positive vesicles in axons co-labeled with Lysotracker red, indicating they were lysosomes. Arrows denote a subset of the Lamp1-EGFP/Lysotracker red co-labeled vesicles. Arrowhead points to a small Lamp1 positive vesicle that was not acidified. HC denotes hair cells rich in Lysotracker red positive, acidic vesicles. (H) Retrograde, but not anterograde, lysosome transport frequency was decreased in jip3nl7 mutants at 3 and 5 dpf (Wilcoxon rank-sum; **-p<0.005). Number of embryos analyzed is indicated on the graph for this and all subsequent bar graphs. (I,J) Kymographs of wildtype (I) and jip3nl7 (J) Lamp1-mTangerine transport shown in C and D. (K,L) Neither distance moved in individual bouts (K) nor velocity of movement (L) were altered in jip3nl7 mutants, save a decrease in anterograde transport distance at 3 dpf (Wilcoxon rank-sum; *p<0.05). Scale bars = 10 µm.
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pgen-1003303-g002: Lysosomes accumulated in jip3nl7 axon terminal swellings.(A,B) Lysosome density, as assayed by Lamp1 immunolabeling (A,B-red, A′B′-white) was increased in jip3nl7 NM3 axon terminals at 5 dpf (arrowhead). Larvae carried the neurod:EGFP transgene to label pLL axons. (C,D) Stills from imaging sessions of Lamp1-mTangerine transport in the pLL nerve of wildtype (C) and jip3nl7 mutant (D) embryos at 3 dpf (Videos S4 and S5). Dotted lines denote the lower bound of the axons imaged. (E,F) Lamp1-mTangerine accumulated in the axon terminals of jip3nl7 mutants (F) but not wildtype siblings (E) at 3 dpf (NM1 shown for both). (G) The majority of Lamp1-EGFP positive vesicles in axons co-labeled with Lysotracker red, indicating they were lysosomes. Arrows denote a subset of the Lamp1-EGFP/Lysotracker red co-labeled vesicles. Arrowhead points to a small Lamp1 positive vesicle that was not acidified. HC denotes hair cells rich in Lysotracker red positive, acidic vesicles. (H) Retrograde, but not anterograde, lysosome transport frequency was decreased in jip3nl7 mutants at 3 and 5 dpf (Wilcoxon rank-sum; **-p<0.005). Number of embryos analyzed is indicated on the graph for this and all subsequent bar graphs. (I,J) Kymographs of wildtype (I) and jip3nl7 (J) Lamp1-mTangerine transport shown in C and D. (K,L) Neither distance moved in individual bouts (K) nor velocity of movement (L) were altered in jip3nl7 mutants, save a decrease in anterograde transport distance at 3 dpf (Wilcoxon rank-sum; *p<0.05). Scale bars = 10 µm.

Mentions: Next, we set out to determine the identity of the mCherry labeled retrograde cargo(s) by looking for accumulation of commonly transported retrograde cargos in jip3nl7 axon terminals using immunofluorescence [29], [30]. Neither late endosomes (Rab7-positive) nor autophagosomes (LC3-positive) accumulated in jip3nl7 axon terminals (Figure S3A–S3D). Consistent with a previous study on Jip3's role in anterograde transport of TrkB [13], TrkB levels were decreased in jip3nl7 axon terminals, as assayed by TrkB antibody labeling (Figure S3E, S3F). In contrast, the axon terminal swellings in jip3nl7 were rich in lysosomes that were visualized using two separate markers, Lamp1 (detected by immunofluorescence; Figure 2A, 2B) and Lysotracker red (vital dye; Figure S3G, S3H).


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

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

Lysosomes accumulated in jip3nl7 axon terminal swellings.(A,B) Lysosome density, as assayed by Lamp1 immunolabeling (A,B-red, A′B′-white) was increased in jip3nl7 NM3 axon terminals at 5 dpf (arrowhead). Larvae carried the neurod:EGFP transgene to label pLL axons. (C,D) Stills from imaging sessions of Lamp1-mTangerine transport in the pLL nerve of wildtype (C) and jip3nl7 mutant (D) embryos at 3 dpf (Videos S4 and S5). Dotted lines denote the lower bound of the axons imaged. (E,F) Lamp1-mTangerine accumulated in the axon terminals of jip3nl7 mutants (F) but not wildtype siblings (E) at 3 dpf (NM1 shown for both). (G) The majority of Lamp1-EGFP positive vesicles in axons co-labeled with Lysotracker red, indicating they were lysosomes. Arrows denote a subset of the Lamp1-EGFP/Lysotracker red co-labeled vesicles. Arrowhead points to a small Lamp1 positive vesicle that was not acidified. HC denotes hair cells rich in Lysotracker red positive, acidic vesicles. (H) Retrograde, but not anterograde, lysosome transport frequency was decreased in jip3nl7 mutants at 3 and 5 dpf (Wilcoxon rank-sum; **-p<0.005). Number of embryos analyzed is indicated on the graph for this and all subsequent bar graphs. (I,J) Kymographs of wildtype (I) and jip3nl7 (J) Lamp1-mTangerine transport shown in C and D. (K,L) Neither distance moved in individual bouts (K) nor velocity of movement (L) were altered in jip3nl7 mutants, save a decrease in anterograde transport distance at 3 dpf (Wilcoxon rank-sum; *p<0.05). Scale bars = 10 µm.
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pgen-1003303-g002: Lysosomes accumulated in jip3nl7 axon terminal swellings.(A,B) Lysosome density, as assayed by Lamp1 immunolabeling (A,B-red, A′B′-white) was increased in jip3nl7 NM3 axon terminals at 5 dpf (arrowhead). Larvae carried the neurod:EGFP transgene to label pLL axons. (C,D) Stills from imaging sessions of Lamp1-mTangerine transport in the pLL nerve of wildtype (C) and jip3nl7 mutant (D) embryos at 3 dpf (Videos S4 and S5). Dotted lines denote the lower bound of the axons imaged. (E,F) Lamp1-mTangerine accumulated in the axon terminals of jip3nl7 mutants (F) but not wildtype siblings (E) at 3 dpf (NM1 shown for both). (G) The majority of Lamp1-EGFP positive vesicles in axons co-labeled with Lysotracker red, indicating they were lysosomes. Arrows denote a subset of the Lamp1-EGFP/Lysotracker red co-labeled vesicles. Arrowhead points to a small Lamp1 positive vesicle that was not acidified. HC denotes hair cells rich in Lysotracker red positive, acidic vesicles. (H) Retrograde, but not anterograde, lysosome transport frequency was decreased in jip3nl7 mutants at 3 and 5 dpf (Wilcoxon rank-sum; **-p<0.005). Number of embryos analyzed is indicated on the graph for this and all subsequent bar graphs. (I,J) Kymographs of wildtype (I) and jip3nl7 (J) Lamp1-mTangerine transport shown in C and D. (K,L) Neither distance moved in individual bouts (K) nor velocity of movement (L) were altered in jip3nl7 mutants, save a decrease in anterograde transport distance at 3 dpf (Wilcoxon rank-sum; *p<0.05). Scale bars = 10 µm.
Mentions: Next, we set out to determine the identity of the mCherry labeled retrograde cargo(s) by looking for accumulation of commonly transported retrograde cargos in jip3nl7 axon terminals using immunofluorescence [29], [30]. Neither late endosomes (Rab7-positive) nor autophagosomes (LC3-positive) accumulated in jip3nl7 axon terminals (Figure S3A–S3D). Consistent with a previous study on Jip3's role in anterograde transport of TrkB [13], TrkB levels were decreased in jip3nl7 axon terminals, as assayed by TrkB antibody labeling (Figure S3E, S3F). In contrast, the axon terminal swellings in jip3nl7 were rich in lysosomes that were visualized using two separate markers, Lamp1 (detected by immunofluorescence; Figure 2A, 2B) and Lysotracker red (vital dye; Figure S3G, S3H).

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