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. 2019 Mar 22;10:1331. doi: 10.1038/s41467-019-09164-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — The APP/GB1a complex co-localizes with CSTN and JIP proteins in axons. a Scheme depicting the BiFC principle²⁷. Complex formation of APP-VN with GB1a-VC reconstitutes Venus fluorescence and leads to BiFC. GB1b-VC serves as a negative control. Representative confocal images show hippocampal neurons (DIV10) expressing APP-VN together with GB1a-VC or GB1b-VC. BiFC is observed in axons and dendrites for GB1a-VC. Microtubule-associated protein Map2 identifies dendrites; mCherry served as a volume marker. Neurons were imaged 7 h post-transfection²⁷. Scale bar 10 μm. b Higher magnification of axons and dendrites of hippocampal neurons transfected with APP-VN and GB1a-VC. The BiFC complex (Venus) partly co-localizes with piccolo (magenta) in axons. The BiFC complex is also present along dendritic shafts but excluded from spines (PSD-95, magenta). Scale bar 5 μm. c Partial co-localization (white, arrowheads) of the BiFC complex (green) with FLAG-CSTN-1, FLAG-CSTN-3, FLAG-JIP-1b, and FLAG-JIP-3 (magenta) and endogenous kinesin light-chain 1 (KLC1) (blue) in the axons of neurons. Scale bar 5 μm. d Quantification of the co-localization of the BiFC complex with FLAG-CSTN-1, FLAG-CSTN-3, FLAG-JIP-1b, and FLAG-JIP-3. The n numbers of neurons analyzed are indicated. e Scheme illustrating that APP together with interacting JIP and CSTN proteins link the GB1a/APP complex in cargo vesicles to axonal kinesin-1 motors. The neural adaptor protein X11-like (X11L) connects APP to CSTN-1²². f Time-lapse images of a well-separated APP-VN/GB1a-VC complex trafficking anterogradely in axons (acquisition times in seconds). White arrowheads mark a fluorescent APP-VN/GB1a-VC complex. A kymograph shows the entire time-lapse recording (right). Scale bars 25 μm. Data are presented as mean ± s.e.m. g Top: Analysis showing the percentage of mobile and non-mobile vesicles per axon within 5 min in hippocampal neurons expressing APPmCherry or the BiFC complex. Bottom: Number of vesicles moving antero-gradely and retrogradely per axons within 5 min. Data are presented in a min to max-box and whisker plot, with whiskers representing the smallest and largest values, the boxes representing the 25–75% percentile and the middle line representing the median. P > 0.05, one-way ANOVA. Source data are provided as a Source Data file