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. 2013 Aug 5;202(3):495–508. doi: 10.1083/jcb.201302078

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© 2013 Fu and Holzbaur

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 7. — Mutations of the JNK phosphorylation site S421 in JIP1 alter KHC activation in vitro and APP directionality in neurons. (A) Mutations at JIP1-S421 alter KHC-tail–binding ability. COS7 cells were cotransfected with GFP-KHC-tail and wild-type or mutant myc-JIP1 and immunoprecipitated with anti-myc antibody. (B) Representative kymographs of KHC-Halo motility show weak activation by myc-JIP1-S421A and enhanced activation by myc-JIP1-S421D in in vitro motility assays. 100 total frames (∼33 s) are shown. (C) KHC-Halo run frequencies in vitro decrease in the presence of JIP1-S421A and increase in the presence of JIP1-S421D. (C–E) Data from three independent experiments (n = 48–101 microtubules and n = 18–254 runs) are shown and statistical comparisons were made versus the wild-type JIP1 condition. (D) KHC-Halo run length is decreased with addition of JIP1-S421A. (E) KHC-Halo speed is increased with addition of JIP1-S421D. (F) Representative kymographs of APP-YFP motility in DRGs transfected with siRNA targeted to mouse JIP1 and rescued with a bicistronic construct coexpressing human wild-type or mutant JIP1 as well as the fluorescent transfection marker BFP. (G) DRGs expressing JIP1-S421D have increased percentages of anterograde APP vesicles, whereas DRGs expressing JIP1-S421A have increased percentages of retrograde APP vesicles. (G–I) Data from three independent experiments (n = 7–9 neurons and n = 78–224 runs) are shown with statistical comparisons made against the wild-type rescue condition. (H) APP-positive vesicles in DRGs expressing JIP1-S421A have decreased anterograde run length, whereas those expressing JIP1-S421D have decreased retrograde run length. (I) No significant differences are observed in APP speeds in DRGs expressing JIP1-S421 phosphomutants. Error bars show the mean ± SEM; *, P < 0.05; **, P < 0.01.