Fig 1: Elongated tubulin dimers increase Kif5B velocity and localize at the microtubule-mitochondrial interface. (A) TIRF microscopy of hippocampal neurons transfected with Kif5b-eGFP constructs, arrows indicate motile kinesin motors. (B) Frequency distribution of KIF5B velocities in DMSO (control) or taxol-treated samples (10 nM, 4 h) shows increased velocities in taxol-treated cells (n = 27 neuronal fibers from three independent experiments; **P < 0.01 Gaussian least squares fit). (C) Hippocampal neurons loaded with mitotracker after incubation with DMSO as control or taxol (10 nM, 4 h). During transport recordings, PFA was added at the moment an anterograde motile mitochondrion was observed (arrows). (D) 2D-STED microscopy was used to record z-stacks on the fixed samples using antibodies for a-tubulin, TOM20 and KIF5B, image stacks were deconvolved in Huygens (SVI), rendered and analyzed in Imaris. (E) Quantification reveals that the number KIF5B spots present on the surface of motile mitochondria (entire surface considered) does not differ between control (1.3 ± 0.6) and taxol-treated axons (1.3 ± 0.6, n = 40 mitochondria from three independent experiments; Mann-Whitney test). (F) Quantification of the KIF5B spots residing specifically in the surfaceMITO-MT overlap, shows an increase due to taxol-treatment (2.4 ± 1.3 vs. 1.8 ± 0.9 in control, n = 40 mitochondria from three independent experiments; *P < 0.05 unpaired Welch two-tailed t test). (G) Hippocampal neurons were transfected with two constructs: TOM20-mCherry-LOV and Kif5b-GFP-ePDZb1. Upon illumination with blue light, the Light-oxygen-voltage (LOV)-domain and ePDZ1 domain heterodimerize, forcing a link between kinesin motors (KIF5B) and mitochondria (TOM20). Axonal processes were selected based on process length and diameter. (H) Examples of kymographs before and after illumination, indicated by the red dotted line in the kymograph. (I) Quantification of the percentage of motile anterograde mitochondria between the dark and illuminated condition in control and taxol cells, and the control and taxol condition in illuminated (LIGHT) state. (J) Quantification of the anterograde transport velocity between the dark and illuminated condition in control and taxol cells, and between the control and taxol condition in illuminated (LIGHT) state. Indicated values are mean ± SD, bar plots represent mean with SEM.