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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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pJNK levels were elevated in jip3nl7 axon terminals.(A–H) Immunolabeling for active JNK (pJNK; red in merge; white in single channel) in proximal (NM1) and distal (NM3) neuromasts at 2 and 5 dpf. pJnk levels were elevated in all axon terminals in jip3nl7 mutants (A–I; arrowheads). (I,J) Mean fluorescent intensity (background subtracted; see Materials and Methods for details) of pJNK and total JNK (tJNK) labeling in NM1 axon terminals and the pLL ganglion (pLLg) at 5 dpf. (ANOVA, post-hoc contrasts; *-p<0.001). Scale bars = 10 µm.
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pgen-1003303-g003: pJNK levels were elevated in jip3nl7 axon terminals.(A–H) Immunolabeling for active JNK (pJNK; red in merge; white in single channel) in proximal (NM1) and distal (NM3) neuromasts at 2 and 5 dpf. pJnk levels were elevated in all axon terminals in jip3nl7 mutants (A–I; arrowheads). (I,J) Mean fluorescent intensity (background subtracted; see Materials and Methods for details) of pJNK and total JNK (tJNK) labeling in NM1 axon terminals and the pLL ganglion (pLLg) at 5 dpf. (ANOVA, post-hoc contrasts; *-p<0.001). Scale bars = 10 µm.

Mentions: Jip3 has been shown to interact with components of the Kinesin-1 motor to regulate anterograde transport [11]–[13], but a role for Jip3 in retrograde transport has not been described previously. Therefore, we next sought to address how Jip3 functioned to regulate retrograde axonal transport. Jip3 was originally identified as a JNK-interacting protein and has been shown to facilitate JNK activation in vitro[14]. Thus, we would predict that loss of Jip3 would lead to decreased JNK activation. As JNK activity can impact numerous intracellular processes that could potentially affect axonal transport machinery [32], [33], we assayed levels and localization of active JNK (pJNK) using pan-pJNK immunolabeling. Surprisingly, instead of a decrease, we found elevated levels of pJNK in the mutant axon terminals innervating all NMs from 2 dpf onward (Figure 3A–3I and data not shown; see Materials and Methods for an explanation of fluorescent intensity measurement). In contrast, total JNK (tJNK) levels in jip3nl7 were comparable to controls (Figure 3J and Figure S4A–S4D). Western blot analysis of whole embryo extracts revealed no increase in overall tJNK or pJNK levels in jip3nl7 (Figure S4E, S4F), pointing to a change in localization of pJNK rather than overall JNK expression or activity.


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

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

pJNK levels were elevated in jip3nl7 axon terminals.(A–H) Immunolabeling for active JNK (pJNK; red in merge; white in single channel) in proximal (NM1) and distal (NM3) neuromasts at 2 and 5 dpf. pJnk levels were elevated in all axon terminals in jip3nl7 mutants (A–I; arrowheads). (I,J) Mean fluorescent intensity (background subtracted; see Materials and Methods for details) of pJNK and total JNK (tJNK) labeling in NM1 axon terminals and the pLL ganglion (pLLg) at 5 dpf. (ANOVA, post-hoc contrasts; *-p<0.001). Scale bars = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585007&req=5

pgen-1003303-g003: pJNK levels were elevated in jip3nl7 axon terminals.(A–H) Immunolabeling for active JNK (pJNK; red in merge; white in single channel) in proximal (NM1) and distal (NM3) neuromasts at 2 and 5 dpf. pJnk levels were elevated in all axon terminals in jip3nl7 mutants (A–I; arrowheads). (I,J) Mean fluorescent intensity (background subtracted; see Materials and Methods for details) of pJNK and total JNK (tJNK) labeling in NM1 axon terminals and the pLL ganglion (pLLg) at 5 dpf. (ANOVA, post-hoc contrasts; *-p<0.001). Scale bars = 10 µm.
Mentions: Jip3 has been shown to interact with components of the Kinesin-1 motor to regulate anterograde transport [11]–[13], but a role for Jip3 in retrograde transport has not been described previously. Therefore, we next sought to address how Jip3 functioned to regulate retrograde axonal transport. Jip3 was originally identified as a JNK-interacting protein and has been shown to facilitate JNK activation in vitro[14]. Thus, we would predict that loss of Jip3 would lead to decreased JNK activation. As JNK activity can impact numerous intracellular processes that could potentially affect axonal transport machinery [32], [33], we assayed levels and localization of active JNK (pJNK) using pan-pJNK immunolabeling. Surprisingly, instead of a decrease, we found elevated levels of pJNK in the mutant axon terminals innervating all NMs from 2 dpf onward (Figure 3A–3I and data not shown; see Materials and Methods for an explanation of fluorescent intensity measurement). In contrast, total JNK (tJNK) levels in jip3nl7 were comparable to controls (Figure 3J and Figure S4A–S4D). Western blot analysis of whole embryo extracts revealed no increase in overall tJNK or pJNK levels in jip3nl7 (Figure S4E, S4F), pointing to a change in localization of pJNK rather than overall JNK expression or activity.

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