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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, an actively transported protein, was necessary for axon extension and the prevention of axon terminal swellings.(A) Schematic of a larval zebrafish illustrating the basic anatomy of the primary posterior lateral line (pLL) system. Neuromasts (NMs; terminal NM cluster-ter) are innervated by the pLL nerve (green), which emanates from the pLL ganglion (pLLg). (B) Wildtype neurod:EGFP transgenic at 5 dpf with the pLLg and pLL nerve (pLLn) indicated. (B′,B″) Panels illustrate pLL axon terminals that innervate NM3 (B′) and the distal end of the pLL nerve including the axon terminals at the terminal NM cluster (ter; B″; red arrowheads point to axon terminals). (C) jip3nl7 mutants displayed truncated pLL nerves and distal pLL nerve thinning (C″) as well as swollen axon terminals in all NMs (NM3 shown in C′). Scale bars B and C = 100 µm. Scale bars in B′, B″, C′ and C″ = 10 µm. (D, E) Long central nervous system axons of the reticulospinal tract (arrowhead) and pLL efferent axons (arrow), visualized by the phox2b:EGFP transgenic reporter, were also truncated in jip3nl7 mutants. End of trunk indicated by the asterisk. (F) Schematic of the zebrafish Jip3 protein showing conserved structural and binding domains. The red arrowhead indicates the location of the jip3nl7 mutation, which generates a premature stop codon at amino acid 184. (G,H) In situ hybridization analysis revealed that jip3 was expressed in the central and peripheral nervous systems at 2 dpf in wildtype but was lost in jip3nl7. (I) Schematic of the paradigm designed to image axon transport in the pLL nerve. (J) Transient expression of Jip3-mCherry in 1 neuron of the pLL ganglion at 30 hpf. (K) Jip3-mCherry was localized to a growth cone of an extending axon at 30 hpf. The pLL ganglion and nerve were visualized by expression of the neurod:EGFP transgene. (L) Jip3-mCherry is actively transported in pLL axons (Video S1). Arrowhead (pink) and arrow (yellow) indicate anterograde and retrograde particle movement respectively. (M) Kymograph of time-lapse imaging in J. Scale bars in J–L = 10 µm.
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pgen-1003303-g001: Jip3, an actively transported protein, was necessary for axon extension and the prevention of axon terminal swellings.(A) Schematic of a larval zebrafish illustrating the basic anatomy of the primary posterior lateral line (pLL) system. Neuromasts (NMs; terminal NM cluster-ter) are innervated by the pLL nerve (green), which emanates from the pLL ganglion (pLLg). (B) Wildtype neurod:EGFP transgenic at 5 dpf with the pLLg and pLL nerve (pLLn) indicated. (B′,B″) Panels illustrate pLL axon terminals that innervate NM3 (B′) and the distal end of the pLL nerve including the axon terminals at the terminal NM cluster (ter; B″; red arrowheads point to axon terminals). (C) jip3nl7 mutants displayed truncated pLL nerves and distal pLL nerve thinning (C″) as well as swollen axon terminals in all NMs (NM3 shown in C′). Scale bars B and C = 100 µm. Scale bars in B′, B″, C′ and C″ = 10 µm. (D, E) Long central nervous system axons of the reticulospinal tract (arrowhead) and pLL efferent axons (arrow), visualized by the phox2b:EGFP transgenic reporter, were also truncated in jip3nl7 mutants. End of trunk indicated by the asterisk. (F) Schematic of the zebrafish Jip3 protein showing conserved structural and binding domains. The red arrowhead indicates the location of the jip3nl7 mutation, which generates a premature stop codon at amino acid 184. (G,H) In situ hybridization analysis revealed that jip3 was expressed in the central and peripheral nervous systems at 2 dpf in wildtype but was lost in jip3nl7. (I) Schematic of the paradigm designed to image axon transport in the pLL nerve. (J) Transient expression of Jip3-mCherry in 1 neuron of the pLL ganglion at 30 hpf. (K) Jip3-mCherry was localized to a growth cone of an extending axon at 30 hpf. The pLL ganglion and nerve were visualized by expression of the neurod:EGFP transgene. (L) Jip3-mCherry is actively transported in pLL axons (Video S1). Arrowhead (pink) and arrow (yellow) indicate anterograde and retrograde particle movement respectively. (M) Kymograph of time-lapse imaging in J. Scale bars in J–L = 10 µm.

Mentions: jip3nl7 was isolated in a forward genetics screen for which we utilized the TgBAC(neurod:EGFP)nl1 transgenic zebrafish (hereafter referred to as neurod:EGFP; [16]). This transgenic strain expresses an EGFP reporter in the central and peripheral nervous systems, including the posterior lateral line (pLL) ganglion and the long sensory axons emanating from it (Figure 1A, 1B; for screen details consult the Materials and Methods). We focused our screen on the long sensory axons of the pLL because of their planar character and superficial localization. These axons originate from the pLL ganglion, located just posterior to the ear, and extend along the trunk, branching to innervate mechanosensory hair cells that reside within surface sensory organs called neuromasts (NMs; axon terminals innervating NM3 and terminal NMs are shown in Figure 1B′ and 1B″ respectively). Initial pLL nerve extension and NM formation is complete by 2 dpf (days post-fertilization), and by 5 dpf a functional neural circuit has developed between NM hair cells and afferent pLL axons [17]. The recessive jip3nl7 mutant (Figure 1C) was isolated because it displayed truncation of pLL axons (incomplete penetrance; Figure 1C″) and swollen axon terminals innervating all trunk NMs (penetrance 100%; NM3 in Figure 1C′ and data not shown). To determine if long central nervous system axons were also affected by loss of Jip3, we analyzed axons of the reticulospinal tract as well as the efferent axons that project from the CNS to innervate the pLL NMs by crossing the jip3nl7 mutation into the TgBAC(phox2b:EGFP)w37 transgenic line [18]. Similar to pLL afferents, both reticulospinal tract and pLL efferent axons were truncated in jip3nl7 mutants (Figure 1D, 1E). jip3nl7 mutants were homozygous viable and the pLL axonal phenotype did not have a maternal component, as progeny derived from homozygous crosses displayed identical phenotypes to that of progeny derived from heterozygous crosses (data not shown).


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, an actively transported protein, was necessary for axon extension and the prevention of axon terminal swellings.(A) Schematic of a larval zebrafish illustrating the basic anatomy of the primary posterior lateral line (pLL) system. Neuromasts (NMs; terminal NM cluster-ter) are innervated by the pLL nerve (green), which emanates from the pLL ganglion (pLLg). (B) Wildtype neurod:EGFP transgenic at 5 dpf with the pLLg and pLL nerve (pLLn) indicated. (B′,B″) Panels illustrate pLL axon terminals that innervate NM3 (B′) and the distal end of the pLL nerve including the axon terminals at the terminal NM cluster (ter; B″; red arrowheads point to axon terminals). (C) jip3nl7 mutants displayed truncated pLL nerves and distal pLL nerve thinning (C″) as well as swollen axon terminals in all NMs (NM3 shown in C′). Scale bars B and C = 100 µm. Scale bars in B′, B″, C′ and C″ = 10 µm. (D, E) Long central nervous system axons of the reticulospinal tract (arrowhead) and pLL efferent axons (arrow), visualized by the phox2b:EGFP transgenic reporter, were also truncated in jip3nl7 mutants. End of trunk indicated by the asterisk. (F) Schematic of the zebrafish Jip3 protein showing conserved structural and binding domains. The red arrowhead indicates the location of the jip3nl7 mutation, which generates a premature stop codon at amino acid 184. (G,H) In situ hybridization analysis revealed that jip3 was expressed in the central and peripheral nervous systems at 2 dpf in wildtype but was lost in jip3nl7. (I) Schematic of the paradigm designed to image axon transport in the pLL nerve. (J) Transient expression of Jip3-mCherry in 1 neuron of the pLL ganglion at 30 hpf. (K) Jip3-mCherry was localized to a growth cone of an extending axon at 30 hpf. The pLL ganglion and nerve were visualized by expression of the neurod:EGFP transgene. (L) Jip3-mCherry is actively transported in pLL axons (Video S1). Arrowhead (pink) and arrow (yellow) indicate anterograde and retrograde particle movement respectively. (M) Kymograph of time-lapse imaging in J. Scale bars in J–L = 10 µm.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1003303-g001: Jip3, an actively transported protein, was necessary for axon extension and the prevention of axon terminal swellings.(A) Schematic of a larval zebrafish illustrating the basic anatomy of the primary posterior lateral line (pLL) system. Neuromasts (NMs; terminal NM cluster-ter) are innervated by the pLL nerve (green), which emanates from the pLL ganglion (pLLg). (B) Wildtype neurod:EGFP transgenic at 5 dpf with the pLLg and pLL nerve (pLLn) indicated. (B′,B″) Panels illustrate pLL axon terminals that innervate NM3 (B′) and the distal end of the pLL nerve including the axon terminals at the terminal NM cluster (ter; B″; red arrowheads point to axon terminals). (C) jip3nl7 mutants displayed truncated pLL nerves and distal pLL nerve thinning (C″) as well as swollen axon terminals in all NMs (NM3 shown in C′). Scale bars B and C = 100 µm. Scale bars in B′, B″, C′ and C″ = 10 µm. (D, E) Long central nervous system axons of the reticulospinal tract (arrowhead) and pLL efferent axons (arrow), visualized by the phox2b:EGFP transgenic reporter, were also truncated in jip3nl7 mutants. End of trunk indicated by the asterisk. (F) Schematic of the zebrafish Jip3 protein showing conserved structural and binding domains. The red arrowhead indicates the location of the jip3nl7 mutation, which generates a premature stop codon at amino acid 184. (G,H) In situ hybridization analysis revealed that jip3 was expressed in the central and peripheral nervous systems at 2 dpf in wildtype but was lost in jip3nl7. (I) Schematic of the paradigm designed to image axon transport in the pLL nerve. (J) Transient expression of Jip3-mCherry in 1 neuron of the pLL ganglion at 30 hpf. (K) Jip3-mCherry was localized to a growth cone of an extending axon at 30 hpf. The pLL ganglion and nerve were visualized by expression of the neurod:EGFP transgene. (L) Jip3-mCherry is actively transported in pLL axons (Video S1). Arrowhead (pink) and arrow (yellow) indicate anterograde and retrograde particle movement respectively. (M) Kymograph of time-lapse imaging in J. Scale bars in J–L = 10 µm.
Mentions: jip3nl7 was isolated in a forward genetics screen for which we utilized the TgBAC(neurod:EGFP)nl1 transgenic zebrafish (hereafter referred to as neurod:EGFP; [16]). This transgenic strain expresses an EGFP reporter in the central and peripheral nervous systems, including the posterior lateral line (pLL) ganglion and the long sensory axons emanating from it (Figure 1A, 1B; for screen details consult the Materials and Methods). We focused our screen on the long sensory axons of the pLL because of their planar character and superficial localization. These axons originate from the pLL ganglion, located just posterior to the ear, and extend along the trunk, branching to innervate mechanosensory hair cells that reside within surface sensory organs called neuromasts (NMs; axon terminals innervating NM3 and terminal NMs are shown in Figure 1B′ and 1B″ respectively). Initial pLL nerve extension and NM formation is complete by 2 dpf (days post-fertilization), and by 5 dpf a functional neural circuit has developed between NM hair cells and afferent pLL axons [17]. The recessive jip3nl7 mutant (Figure 1C) was isolated because it displayed truncation of pLL axons (incomplete penetrance; Figure 1C″) and swollen axon terminals innervating all trunk NMs (penetrance 100%; NM3 in Figure 1C′ and data not shown). To determine if long central nervous system axons were also affected by loss of Jip3, we analyzed axons of the reticulospinal tract as well as the efferent axons that project from the CNS to innervate the pLL NMs by crossing the jip3nl7 mutation into the TgBAC(phox2b:EGFP)w37 transgenic line [18]. Similar to pLL afferents, both reticulospinal tract and pLL efferent axons were truncated in jip3nl7 mutants (Figure 1D, 1E). jip3nl7 mutants were homozygous viable and the pLL axonal phenotype did not have a maternal component, as progeny derived from homozygous crosses displayed identical phenotypes to that of progeny derived from heterozygous crosses (data not shown).

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