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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 failed to accumulate distal to injury.(A) Schematic and time-line of the injury model experiment and fluorescent intensity quantification. pLL nerve (identified using the neurod:EGFP transgene) was severed using finely pulled glass capillaries. DIC image of a representative injury illustrates peripheral tissue remained mostly intact. Three hours post-injury, larvae were fixed and stained for GFP (to identify the nerve) and either pJNK or tJNK. Thirty µm areas immediately proximal or distal to the injury were imaged and the mean fluorescent intensity of pJNK or tJNK was determined in summed projected stacks through the nerve only in areas that overlapped with GFP expression (outlined by dotted lines in B–I). Background mean fluorescent intensity was determined in adjacent tissue. (B–I) Proximal and distal nerve (dotted outline) adjacent to site of injury (dashed line) in wildtype and jip3nl7 larvae immunolabled for pJNK (B–E) and tJNK (F–I). (J) Levels of pJNK were decreased distal to nerve injury in jip3nl7 but proximal levels were comparable to wildtype (ANOVA, post-hoc contrasts; *-p<0.05). (K) tJNK levels trended towards a decrease proximal to the site of injury in jip3nl7 (ANOVA, post-hoc contrasts; p<0.1790) but were not different in the retrograde pool, distal to axonal severing.
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pgen-1003303-g004: pJNK failed to accumulate distal to injury.(A) Schematic and time-line of the injury model experiment and fluorescent intensity quantification. pLL nerve (identified using the neurod:EGFP transgene) was severed using finely pulled glass capillaries. DIC image of a representative injury illustrates peripheral tissue remained mostly intact. Three hours post-injury, larvae were fixed and stained for GFP (to identify the nerve) and either pJNK or tJNK. Thirty µm areas immediately proximal or distal to the injury were imaged and the mean fluorescent intensity of pJNK or tJNK was determined in summed projected stacks through the nerve only in areas that overlapped with GFP expression (outlined by dotted lines in B–I). Background mean fluorescent intensity was determined in adjacent tissue. (B–I) Proximal and distal nerve (dotted outline) adjacent to site of injury (dashed line) in wildtype and jip3nl7 larvae immunolabled for pJNK (B–E) and tJNK (F–I). (J) Levels of pJNK were decreased distal to nerve injury in jip3nl7 but proximal levels were comparable to wildtype (ANOVA, post-hoc contrasts; *-p<0.05). (K) tJNK levels trended towards a decrease proximal to the site of injury in jip3nl7 (ANOVA, post-hoc contrasts; p<0.1790) but were not different in the retrograde pool, distal to axonal severing.

Mentions: Given the ability of Jip3 to bind components of the retrograde motor and pJNK [14], [15], we reasoned that Jip3 might directly mediate pJNK retrograde transport/clearance from axon terminals by attaching this active kinase to the dynein motor complex. To determine if Jip3 has a specific role in pJNK transport, we used two complimentary approaches. First, we developed an axon injury model for use in the zebrafish pLL nerve to indirectly assay pJNK transport, similar to a protocol previously used in mouse sciatic nerve (Figure 4A; see Materials and Methods for procedure details; [15]). Following injury, cargos that are transported in the anterograde direction will accumulate proximal to the injury site, whereas retrograde cargos will accumulate distal to the injury site. Severing the pLL nerve between NM2 and NM3 at 5 dpf resulted in accumulation of pJNK in the pLL nerve proximal and distal to the site of injury in wildtype larvae by 3 hours post-injury. In contrast, pJNK failed to accumulate distal to the site of injury in jip3nl7 mutants (Figure 4B–4E, 4J), indicating failed retrograde pJNK transport in mutant axons. Total JNK levels were not significantly different proximal or distal to injury site in jip3nl7 mutants (Figure 4F–4I, 4K), though there was a strong trend towards decreased levels of the tJNK anterograde pool (proximal to the injury site) in jip3nl7 mutants. This data supports the hypothesis that loss of Jip3 inhibits pJNK retrograde transport, which would lead to accumulations of this kinase in axon terminals.


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 failed to accumulate distal to injury.(A) Schematic and time-line of the injury model experiment and fluorescent intensity quantification. pLL nerve (identified using the neurod:EGFP transgene) was severed using finely pulled glass capillaries. DIC image of a representative injury illustrates peripheral tissue remained mostly intact. Three hours post-injury, larvae were fixed and stained for GFP (to identify the nerve) and either pJNK or tJNK. Thirty µm areas immediately proximal or distal to the injury were imaged and the mean fluorescent intensity of pJNK or tJNK was determined in summed projected stacks through the nerve only in areas that overlapped with GFP expression (outlined by dotted lines in B–I). Background mean fluorescent intensity was determined in adjacent tissue. (B–I) Proximal and distal nerve (dotted outline) adjacent to site of injury (dashed line) in wildtype and jip3nl7 larvae immunolabled for pJNK (B–E) and tJNK (F–I). (J) Levels of pJNK were decreased distal to nerve injury in jip3nl7 but proximal levels were comparable to wildtype (ANOVA, post-hoc contrasts; *-p<0.05). (K) tJNK levels trended towards a decrease proximal to the site of injury in jip3nl7 (ANOVA, post-hoc contrasts; p<0.1790) but were not different in the retrograde pool, distal to axonal severing.
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Related In: Results  -  Collection

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pgen-1003303-g004: pJNK failed to accumulate distal to injury.(A) Schematic and time-line of the injury model experiment and fluorescent intensity quantification. pLL nerve (identified using the neurod:EGFP transgene) was severed using finely pulled glass capillaries. DIC image of a representative injury illustrates peripheral tissue remained mostly intact. Three hours post-injury, larvae were fixed and stained for GFP (to identify the nerve) and either pJNK or tJNK. Thirty µm areas immediately proximal or distal to the injury were imaged and the mean fluorescent intensity of pJNK or tJNK was determined in summed projected stacks through the nerve only in areas that overlapped with GFP expression (outlined by dotted lines in B–I). Background mean fluorescent intensity was determined in adjacent tissue. (B–I) Proximal and distal nerve (dotted outline) adjacent to site of injury (dashed line) in wildtype and jip3nl7 larvae immunolabled for pJNK (B–E) and tJNK (F–I). (J) Levels of pJNK were decreased distal to nerve injury in jip3nl7 but proximal levels were comparable to wildtype (ANOVA, post-hoc contrasts; *-p<0.05). (K) tJNK levels trended towards a decrease proximal to the site of injury in jip3nl7 (ANOVA, post-hoc contrasts; p<0.1790) but were not different in the retrograde pool, distal to axonal severing.
Mentions: Given the ability of Jip3 to bind components of the retrograde motor and pJNK [14], [15], we reasoned that Jip3 might directly mediate pJNK retrograde transport/clearance from axon terminals by attaching this active kinase to the dynein motor complex. To determine if Jip3 has a specific role in pJNK transport, we used two complimentary approaches. First, we developed an axon injury model for use in the zebrafish pLL nerve to indirectly assay pJNK transport, similar to a protocol previously used in mouse sciatic nerve (Figure 4A; see Materials and Methods for procedure details; [15]). Following injury, cargos that are transported in the anterograde direction will accumulate proximal to the injury site, whereas retrograde cargos will accumulate distal to the injury site. Severing the pLL nerve between NM2 and NM3 at 5 dpf resulted in accumulation of pJNK in the pLL nerve proximal and distal to the site of injury in wildtype larvae by 3 hours post-injury. In contrast, pJNK failed to accumulate distal to the site of injury in jip3nl7 mutants (Figure 4B–4E, 4J), indicating failed retrograde pJNK transport in mutant axons. Total JNK levels were not significantly different proximal or distal to injury site in jip3nl7 mutants (Figure 4F–4I, 4K), though there was a strong trend towards decreased levels of the tJNK anterograde pool (proximal to the injury site) in jip3nl7 mutants. This data supports the hypothesis that loss of Jip3 inhibits pJNK retrograde transport, which would lead to accumulations of this kinase in axon terminals.

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