KIF5B knockdown abolishes the enrichment of lysosomes on detyrosinated microtubules and impacts lysosome–autophagosome fusion. (A–D) Conventional epifluorescence images of cells stably expressing LAMP-2 mCherry, transiently transfected with scrambled siRNA (A), KIF3A siRNA (B), KIF1B siRNA (C), and KIF5B siRNA (D), followed by immunofluorescence for detyrosinated microtubules (magenta). Lysosomes (green) in scrambled siRNA–transfected cells are distributed throughout the cell including the perinuclear region and periphery, whereas lysosomes in cells transfected with KIF3A, KIF1B, and KIF5B siRNA cluster at the perinuclear region with very few lysosomes at the cell periphery. (E) Percentage of lysosomes associated with detyrosinated microtubules normalized to the overall percentage of detyrosinated microtubules, quantified from super-resolution images. The bars indicate the median, and the error bar is the SD for three different cells analyzed in each case. The total number of lysosomes analyzed for scrambled siRNA, n = 355 (on Detyro = 223, others = 132); KIF3A siRNA, n = 332 (on Detyro = 201, others = 131); KIF1B siRNA, n = 396 (on Detyro = 291, others = 105); and KIF5B siRNA, n = 312 (on Detyro = 124, others = 188). Bar, 2 µm. (F–I) Conventional epifluorescence images of cells transiently expressing LC3B-GFP-RFP, cotransfected with scrambled siRNA (F), KIF3A siRNA (G), KIF1B siRNA (H), and KIF5B siRNA (I). (J) Quantification of the percentage of autophagosomes and autolysosomes for 10 different cells in each case. The asterisk indicates the statistical significance for a two-tailed two-sample t test (*, P < 0.05). OE, overexpressing KIF5B.