Minus end–directed organelle redistribution using moss-derived kinesin-14. (A) Fixed-cell imaging of recycling endosomes (middle) tagged with FKBP-mCherry-RAB11 and the dynein adaptor FRB-BICDN-HA (top) in COS-7 cells, with or without rapalog (100 nM, 1-h treatment). (B) Moss-derived kinesin-14 VIb constructs. Addition of GCN4 leucine zipper creates a dimer of dimers, effectively creating a tetrameric motor. (C) Fixed-cell imaging of kinesin-14 VIb constructs in COS-7 cells. (D) Similar to A, but with cells expressing dimeric FRB-GFP–ppKin14-VIb. (E) Assay: early endosomes were tagged with iLID-mCherry-RAB5. Blue light illumination induced unfolding of iLID and binding to SSPB-GFP–ppKin14-VIb or SSPB-GFP-GCN4–ppKin14-VIb, stimulating anterograde transport (gray). (F) Quantification of live-cell imaging of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS cells treated with nocodazole (10 µM) to depolymerize microtubules. Cells were illuminated continuously for 30 s with 100 µW cm⁻² of blue light, followed by 150 s of darkness. Graph shows normalized intensity of opto-kinesin on individual endosomes. (G) Live-cell imaging and quantifications (H–K) of iLID-mCherry-RAB5 in U2OS cells expressing the indicated constructs before or after 10 min of illumination. Quantifications in H–K show peripheral enrichment index (H) or perinuclear enrichment index (I) of iLID-mCherry-RAB5. Plots in J and K show per-cell change in peripheral (J) or perinuclear (K) enrichment index after 10 min. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean of control cells. Boxed numbers show the percentage of responsive cells for each dataset (peripheral or perinuclear enrichment greater than mean + two times the SD of control). Graphs represent mean and 95% confidence intervals of at least 15 cells from a total of at least three experiments. Blue box indicates 470-nm illumination. Scale bars are 10 µm.