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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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Increased levels of pJNK did not cause lysosome accumulation in jip3nl7.(A) Induction of caJNK3-EGFP at 4 dpf increased the level of pJNK immunofluorescence (middle) in a subset of axon terminals but did not lead to lysosome accumulation as compared to control (B). Scale bars = 10 µm. (C, D) This result was confirmed by Lysotracker red labeling. Surrounding, non-caJNK3-EGFP positive axons show similar numbers, size and density of lysosomes both 4 hours and 13 hours after induction of caJNK3. The pLL nerve was visualized by phase contrast optics and is outlined. Arrowhead indicates axonal swellings caused by high levels of activated JNK. HC denotes neuromast hair cells that strongly label with Lysotracker red. (E) Whole embryo expression of Jip3 and Jip3ΔJNK by mRNA injection partially suppressed the accumulation of lysosomes in jip3nl7 mutant axon terminals at 3 dpf as assayed by expression of Lamp1-mTangerine in pLL neurons. Wildtype – Lamp1-mTangerine positive small puncta only; Mild – small puncta and aggregates visible; Severe - few to no small puncta apparent and large aggregations of Lamp1-mTangerine. (F–I) Injection of 10 pg of a DNA construct encoding Jip3ΔJNK-mCherry rescued lysosome accumulation in jip3nl7 axon terminals. Larvae that expressed Jip3ΔJNK-mCherry (red) in pLL axons and carried the neurod:EGFP transgene were first imaged live (F,H) to identify expressing axon terminals. They were then individually fixed, stained for pJNK (pseudo-colored magenta) and Lamp1 (white), and subsequently the same axon terminals were reimaged (G,I). Arrowheads point to axon terminals in wildtype (F,G; NM1) and jip3nl7 (H,I; NM5) that express Jip3ΔJNK-mCherry (red) at 5 dpf. Arrows point to axon terminals in the same NMs that did not express this construct. Note that expression of Jip3ΔJNK-mCherry in jip3nl7 completely rescued lysosome accumulation (yellow arrowheads in I″) but failed to rescue high levels of pJNK (yellow arrowheads in I′).
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pgen-1003303-g007: Increased levels of pJNK did not cause lysosome accumulation in jip3nl7.(A) Induction of caJNK3-EGFP at 4 dpf increased the level of pJNK immunofluorescence (middle) in a subset of axon terminals but did not lead to lysosome accumulation as compared to control (B). Scale bars = 10 µm. (C, D) This result was confirmed by Lysotracker red labeling. Surrounding, non-caJNK3-EGFP positive axons show similar numbers, size and density of lysosomes both 4 hours and 13 hours after induction of caJNK3. The pLL nerve was visualized by phase contrast optics and is outlined. Arrowhead indicates axonal swellings caused by high levels of activated JNK. HC denotes neuromast hair cells that strongly label with Lysotracker red. (E) Whole embryo expression of Jip3 and Jip3ΔJNK by mRNA injection partially suppressed the accumulation of lysosomes in jip3nl7 mutant axon terminals at 3 dpf as assayed by expression of Lamp1-mTangerine in pLL neurons. Wildtype – Lamp1-mTangerine positive small puncta only; Mild – small puncta and aggregates visible; Severe - few to no small puncta apparent and large aggregations of Lamp1-mTangerine. (F–I) Injection of 10 pg of a DNA construct encoding Jip3ΔJNK-mCherry rescued lysosome accumulation in jip3nl7 axon terminals. Larvae that expressed Jip3ΔJNK-mCherry (red) in pLL axons and carried the neurod:EGFP transgene were first imaged live (F,H) to identify expressing axon terminals. They were then individually fixed, stained for pJNK (pseudo-colored magenta) and Lamp1 (white), and subsequently the same axon terminals were reimaged (G,I). Arrowheads point to axon terminals in wildtype (F,G; NM1) and jip3nl7 (H,I; NM5) that express Jip3ΔJNK-mCherry (red) at 5 dpf. Arrows point to axon terminals in the same NMs that did not express this construct. Note that expression of Jip3ΔJNK-mCherry in jip3nl7 completely rescued lysosome accumulation (yellow arrowheads in I″) but failed to rescue high levels of pJNK (yellow arrowheads in I′).

Mentions: Our data demonstrated that lysosomes accumulate in jip3nl7 mutant axon terminals (see Figure 2) and elevated pJNK levels cause axon terminal swellings (see Figure 6). Next, we asked whether elevated pJNK could cause lysosomal accumulation. To test this, we used the approach described above to conditionally expressed caJNK3 at 4 dpf in wildtype larvae. Larvae expressing caJNK3 in pLL neurons were immunolabeled with an anti-Lamp1 antibody and axon terminals were imaged. This analysis demonstrated that elevation of pJNK levels did not increase Lamp1 levels above controls (Figure 7A, 7B). Importantly, lysosome number and dynamics appeared normal in the presence of activated JNK, as Lysotracker red vital dye labeling was similar between caJNK3 expressing axons and non-expressing neighboring axons (Figure 7C, 7D).


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

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

Increased levels of pJNK did not cause lysosome accumulation in jip3nl7.(A) Induction of caJNK3-EGFP at 4 dpf increased the level of pJNK immunofluorescence (middle) in a subset of axon terminals but did not lead to lysosome accumulation as compared to control (B). Scale bars = 10 µm. (C, D) This result was confirmed by Lysotracker red labeling. Surrounding, non-caJNK3-EGFP positive axons show similar numbers, size and density of lysosomes both 4 hours and 13 hours after induction of caJNK3. The pLL nerve was visualized by phase contrast optics and is outlined. Arrowhead indicates axonal swellings caused by high levels of activated JNK. HC denotes neuromast hair cells that strongly label with Lysotracker red. (E) Whole embryo expression of Jip3 and Jip3ΔJNK by mRNA injection partially suppressed the accumulation of lysosomes in jip3nl7 mutant axon terminals at 3 dpf as assayed by expression of Lamp1-mTangerine in pLL neurons. Wildtype – Lamp1-mTangerine positive small puncta only; Mild – small puncta and aggregates visible; Severe - few to no small puncta apparent and large aggregations of Lamp1-mTangerine. (F–I) Injection of 10 pg of a DNA construct encoding Jip3ΔJNK-mCherry rescued lysosome accumulation in jip3nl7 axon terminals. Larvae that expressed Jip3ΔJNK-mCherry (red) in pLL axons and carried the neurod:EGFP transgene were first imaged live (F,H) to identify expressing axon terminals. They were then individually fixed, stained for pJNK (pseudo-colored magenta) and Lamp1 (white), and subsequently the same axon terminals were reimaged (G,I). Arrowheads point to axon terminals in wildtype (F,G; NM1) and jip3nl7 (H,I; NM5) that express Jip3ΔJNK-mCherry (red) at 5 dpf. Arrows point to axon terminals in the same NMs that did not express this construct. Note that expression of Jip3ΔJNK-mCherry in jip3nl7 completely rescued lysosome accumulation (yellow arrowheads in I″) but failed to rescue high levels of pJNK (yellow arrowheads in I′).
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Related In: Results  -  Collection

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pgen-1003303-g007: Increased levels of pJNK did not cause lysosome accumulation in jip3nl7.(A) Induction of caJNK3-EGFP at 4 dpf increased the level of pJNK immunofluorescence (middle) in a subset of axon terminals but did not lead to lysosome accumulation as compared to control (B). Scale bars = 10 µm. (C, D) This result was confirmed by Lysotracker red labeling. Surrounding, non-caJNK3-EGFP positive axons show similar numbers, size and density of lysosomes both 4 hours and 13 hours after induction of caJNK3. The pLL nerve was visualized by phase contrast optics and is outlined. Arrowhead indicates axonal swellings caused by high levels of activated JNK. HC denotes neuromast hair cells that strongly label with Lysotracker red. (E) Whole embryo expression of Jip3 and Jip3ΔJNK by mRNA injection partially suppressed the accumulation of lysosomes in jip3nl7 mutant axon terminals at 3 dpf as assayed by expression of Lamp1-mTangerine in pLL neurons. Wildtype – Lamp1-mTangerine positive small puncta only; Mild – small puncta and aggregates visible; Severe - few to no small puncta apparent and large aggregations of Lamp1-mTangerine. (F–I) Injection of 10 pg of a DNA construct encoding Jip3ΔJNK-mCherry rescued lysosome accumulation in jip3nl7 axon terminals. Larvae that expressed Jip3ΔJNK-mCherry (red) in pLL axons and carried the neurod:EGFP transgene were first imaged live (F,H) to identify expressing axon terminals. They were then individually fixed, stained for pJNK (pseudo-colored magenta) and Lamp1 (white), and subsequently the same axon terminals were reimaged (G,I). Arrowheads point to axon terminals in wildtype (F,G; NM1) and jip3nl7 (H,I; NM5) that express Jip3ΔJNK-mCherry (red) at 5 dpf. Arrows point to axon terminals in the same NMs that did not express this construct. Note that expression of Jip3ΔJNK-mCherry in jip3nl7 completely rescued lysosome accumulation (yellow arrowheads in I″) but failed to rescue high levels of pJNK (yellow arrowheads in I′).
Mentions: Our data demonstrated that lysosomes accumulate in jip3nl7 mutant axon terminals (see Figure 2) and elevated pJNK levels cause axon terminal swellings (see Figure 6). Next, we asked whether elevated pJNK could cause lysosomal accumulation. To test this, we used the approach described above to conditionally expressed caJNK3 at 4 dpf in wildtype larvae. Larvae expressing caJNK3 in pLL neurons were immunolabeled with an anti-Lamp1 antibody and axon terminals were imaged. This analysis demonstrated that elevation of pJNK levels did not increase Lamp1 levels above controls (Figure 7A, 7B). Importantly, lysosome number and dynamics appeared normal in the presence of activated JNK, as Lysotracker red vital dye labeling was similar between caJNK3 expressing axons and non-expressing neighboring axons (Figure 7C, 7D).

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