Fig 2: 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

Fig 3: Nesprin-1-alpha2 pulls down kinesin light-chain from mouse C2C12 myotube extracts, only if the STAR domain (with LEWD) is present. Extracts of mouse C2C12myotubes (Input) and the component of the myotube extract pulled-down by wild type N1α2 (Wt) or STAR deletion N1α2 (Star del) were probed with (a) KLC (63–90) mAb or (b) mAb against GST tag as loading control. Whole blots are shown; black vertical lines separate the blue markers (taken with visible light) from the western blot (by chemiluminescence). In blot (a), the markers were visible in the chemiluminescent exposure, so a separate visible-light exposure was not needed. Black arrows indicate bands of expected size.

Fig 4: Nesprin-1-alpha2 pulls down kinesin light-chain from human myotube extracts, only if the STAR domain (with LEWD) is present. No pull-down of emerin or lamin A/C was detected. Extracts of human myotubes (Input) and the component of the myotube extract pulled-down by wild type N1α2 (Wt) or STAR deletion N1α2 (Star del) were probed with (a) KLC1 (rabbit pAb, GeneTex, GTX114510), (b) KLC1 (mouse mAb, Santa Cruz, sc-515792), (c) MANNEM1, emerin mAb, (d) MANNLAC1, Lamin A/C mAb or (e) mAb against GST tag as loading control. Whole blots are shown; black vertical lines separate the blue markers (taken with visible light) from the western blot (by chemiluminescence). In some blots (b–d), the markers were visible in the chemiluminescent exposure, so a separate visible-light exposure was not needed. Black arrows indicate bands of expected size.