Fig. 6: Kinesin-1 regulates early endosome dynamics.
a Confocal microscopy of BMDCs from WT or cKOKif5b mice stained with anti-tubulin and anti-Kif5b. Bars: 5 μm. The indicated box is shown at a higher magnification in the inset. Bars: 2 μm. b Confocal microscopy of WT BMDCs stained with anti-EEA1 and anti-Kif5b. The intersection between the two stainings is shown in white. The indicated boxes are shown at higher magnification in the insets. Bars: 2 μm. c A schematic representation of early endosome WGA labelling. d WT and cKOKif5b BMDCs were labelled with WGA-488 and stained with anti-EEA1. Bars: 2 μm (left panel). Quantification of the colocalization between WGA-488 and EEA1-555 (right panel; WT blue histogram, cKOKif5b red histogram; n = 7 cells per condition). All images of single cells are representative of >100 cells observed in three independent experiments (a–d). e Spinning disk video microscopy was performed on WT and cKOKif5b BMDCs labelled with WGA-488. Representative series of images are shown every 25 s. Bars: 2 μm. Images are representative of four independent experiments. See also Supplementary Movies 1 and 2. f The number of tubulations unable to detach per cell during the 5 min acquisition. (WT blue circles, cKOKif5b red squares; n = 20 cells per condition). Statistical analysis: ***P < 0.0001 in a two-tailed unpaired Student’s t test. g The number of WGA-488-positive vesicular structures >1 μm in size was measured in WT (blue line) and cKOKif5b (red line) BMDCs during the 5 min acquisition (n = 13 cells per condition). Statistical analysis: ***P < 0.0001 in a two-way ANOVA and Sidak test’s correction for multiple comparison. h Schematic representation of the WGA labelling protocol (upper panel). Spinning disk video microscopy was performed on WT and cKOKif5b BMDCs labelled with WGA-555 and WGA-488. Representative series of images observed in three independent experiments are shown at the indicated time periods (lower panel). Bars: 2 μm. i n = 10 cells per condition. Statistical analysis of the experiment in (WT blue histogram, cKOKif5b red histogram) (h); ***P < 0.0001 in a two-way ANOVA and Sidak test’s correction for multiple comparison. d–i Graphs show mean ± S.E.M.
