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Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport.

Chiba K, Araseki M, Nozawa K, Furukori K, Araki Y, Matsushima T, Nakaya T, Hata S, Saito Y, Uchida S, Okada Y, Nairn AC, Davis RJ, Yamamoto T, Kinjo M, Taru H, Suzuki T - Mol. Biol. Cell (2014)

Bottom Line: In JIP1-deficient neurons, we find that both the fast velocity (∼2.7 μm/s) and high frequency (66%) of anterograde transport of APP cargo are impaired to a reduced velocity (∼1.83 μm/s) and a lower frequency (45%).Furthermore, efficient APP axonal transport is not influenced by phosphorylation of APP at Thr-668, a site known to be phosphorylated by JNK.Our quantitative analysis indicates that enhanced fast-velocity and efficient high-frequency APP anterograde transport observed in neurons are mediated by novel roles of JIP1b.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.

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Interaction of the N-terminal half of KLC1 with JIP1b. (A) Structure of N-terminal HA- or C-terminal EGFP-tagged mouse KLC1 and N-terminal FLAG-tagged mouse JIP1b proteins. Numbers represent amino acid positions. The shaded box indicates the coiled-coil/heptad repeats region, and ovals indicate TPR motifs in KLC1. JIP1bΔC11 is the truncated protein lacking 11 C-terminal amino acids (697YTCPTEDIYLE707). JBD, JNK-binding motif; SH3, Src homology domain 3; PI, phosphotyrosine interaction or phosphotyrosine binding (PTB) domain. Summary of KLC1 interactions with JIP1b is shown on the right, classified as binding (+), weak binding (±), and nonbinding (–) in a coimmunoprecipitation assay. N.D., not determined. (B, C) Coimmuno­precipitation of KLC1 and JIP1b. The IPs and lysate samples were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies. Numbers shown at the left indicate molecular weight standards (kilodaltons). Numbers shown below indicate lane numbers. Protein bands at ∼60 and 35 kDa in the IP lanes shown in this and later figures are derived from the antibody used.
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Figure 3: Interaction of the N-terminal half of KLC1 with JIP1b. (A) Structure of N-terminal HA- or C-terminal EGFP-tagged mouse KLC1 and N-terminal FLAG-tagged mouse JIP1b proteins. Numbers represent amino acid positions. The shaded box indicates the coiled-coil/heptad repeats region, and ovals indicate TPR motifs in KLC1. JIP1bΔC11 is the truncated protein lacking 11 C-terminal amino acids (697YTCPTEDIYLE707). JBD, JNK-binding motif; SH3, Src homology domain 3; PI, phosphotyrosine interaction or phosphotyrosine binding (PTB) domain. Summary of KLC1 interactions with JIP1b is shown on the right, classified as binding (+), weak binding (±), and nonbinding (–) in a coimmunoprecipitation assay. N.D., not determined. (B, C) Coimmuno­precipitation of KLC1 and JIP1b. The IPs and lysate samples were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies. Numbers shown at the left indicate molecular weight standards (kilodaltons). Numbers shown below indicate lane numbers. Protein bands at ∼60 and 35 kDa in the IP lanes shown in this and later figures are derived from the antibody used.

Mentions: The role of JIP1b in efficient anterograde transport of APP cargo was examined by detailed analysis of the interaction of JIP1b and KLC1. First, we expressed four region-deleted KLC1 proteins with N-terminal hemagglutinin (HA) or C-terminal EGFP tags in COS7 cells and examined their binding to N-terminal FLAG-JIP1b or FLAG-JIP1bΔC11, the latter lacking the 11 known KLC-binding C-terminal amino acids (schematic protein structures are shown in Figure 3A).


Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport.

Chiba K, Araseki M, Nozawa K, Furukori K, Araki Y, Matsushima T, Nakaya T, Hata S, Saito Y, Uchida S, Okada Y, Nairn AC, Davis RJ, Yamamoto T, Kinjo M, Taru H, Suzuki T - Mol. Biol. Cell (2014)

Interaction of the N-terminal half of KLC1 with JIP1b. (A) Structure of N-terminal HA- or C-terminal EGFP-tagged mouse KLC1 and N-terminal FLAG-tagged mouse JIP1b proteins. Numbers represent amino acid positions. The shaded box indicates the coiled-coil/heptad repeats region, and ovals indicate TPR motifs in KLC1. JIP1bΔC11 is the truncated protein lacking 11 C-terminal amino acids (697YTCPTEDIYLE707). JBD, JNK-binding motif; SH3, Src homology domain 3; PI, phosphotyrosine interaction or phosphotyrosine binding (PTB) domain. Summary of KLC1 interactions with JIP1b is shown on the right, classified as binding (+), weak binding (±), and nonbinding (–) in a coimmunoprecipitation assay. N.D., not determined. (B, C) Coimmuno­precipitation of KLC1 and JIP1b. The IPs and lysate samples were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies. Numbers shown at the left indicate molecular weight standards (kilodaltons). Numbers shown below indicate lane numbers. Protein bands at ∼60 and 35 kDa in the IP lanes shown in this and later figures are derived from the antibody used.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230617&req=5

Figure 3: Interaction of the N-terminal half of KLC1 with JIP1b. (A) Structure of N-terminal HA- or C-terminal EGFP-tagged mouse KLC1 and N-terminal FLAG-tagged mouse JIP1b proteins. Numbers represent amino acid positions. The shaded box indicates the coiled-coil/heptad repeats region, and ovals indicate TPR motifs in KLC1. JIP1bΔC11 is the truncated protein lacking 11 C-terminal amino acids (697YTCPTEDIYLE707). JBD, JNK-binding motif; SH3, Src homology domain 3; PI, phosphotyrosine interaction or phosphotyrosine binding (PTB) domain. Summary of KLC1 interactions with JIP1b is shown on the right, classified as binding (+), weak binding (±), and nonbinding (–) in a coimmunoprecipitation assay. N.D., not determined. (B, C) Coimmuno­precipitation of KLC1 and JIP1b. The IPs and lysate samples were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies. Numbers shown at the left indicate molecular weight standards (kilodaltons). Numbers shown below indicate lane numbers. Protein bands at ∼60 and 35 kDa in the IP lanes shown in this and later figures are derived from the antibody used.
Mentions: The role of JIP1b in efficient anterograde transport of APP cargo was examined by detailed analysis of the interaction of JIP1b and KLC1. First, we expressed four region-deleted KLC1 proteins with N-terminal hemagglutinin (HA) or C-terminal EGFP tags in COS7 cells and examined their binding to N-terminal FLAG-JIP1b or FLAG-JIP1bΔC11, the latter lacking the 11 known KLC-binding C-terminal amino acids (schematic protein structures are shown in Figure 3A).

Bottom Line: In JIP1-deficient neurons, we find that both the fast velocity (∼2.7 μm/s) and high frequency (66%) of anterograde transport of APP cargo are impaired to a reduced velocity (∼1.83 μm/s) and a lower frequency (45%).Furthermore, efficient APP axonal transport is not influenced by phosphorylation of APP at Thr-668, a site known to be phosphorylated by JNK.Our quantitative analysis indicates that enhanced fast-velocity and efficient high-frequency APP anterograde transport observed in neurons are mediated by novel roles of JIP1b.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.

Show MeSH