Fig 2: KIF3 is essential for the long-range anterograde transport of REs driven by KIF13A.(a–c) Confocal live-cell microscopy of RAB11A-GFP labeled recycling endosomes in control (a), KIF3A-KD (b) and BLOC1S1-KD (c) cells (see also Figure 6—videos 1 and 2). Magnified insets (of boxed areas) of consecutive time-lapse images (image/2 s) showed that, compared to control cells, the occurrence of backward movement of recycling endosomes following paused anterograde transport is significantly increased in KIF3A-KD and BLOC1S1-KD cells. White arrows labeled 1 and 2 indicate representative recycling endosomes in these cells. The arrows were drawn perpendicularly to the direction of motion and were reversed when moving in the opposite direction. A trajectory diagram of the representative recycling endosomes is shown on the left bottom. Time stamps are in the format of minutes: seconds. (d) Histogram showing the occurrence of forward and backward motion of recycling endosomes following paused anterograde transport in control (n = 50) and KIF3A-KD (n = 124) cells. (e, f) Confocal live-cell microscopy of KIF13A-GFP tubules in control and KIF3A-KD cells (see Figure 6—video 3). Magnified insets (of boxed areas) of consecutive time-lapse images (image/2 s) showed impaired long-range transport of KIF13A tubules in KIF3A-KD cells. White arrows labeled 1 and 2 indicate tips of representative KIF13A-positive tubules in control (e) and KIF3A-KD (f) cells. The arrows were drawn perpendicularly to the direction of motion and were reversed when moving in the opposite direction. A trajectory diagram of the representative KIF13A tubule tips is shown on the left bottom. Time stamps are in the format of min:s. Scale bars in all pictures, 10 µm. See also Figure 6—figure supplement 1, Figure 6—video 1, 2, 3, 4 and 5.

Fig 3: Analysis of endosomal Rab RNAi screen and the localization of Rabs to REs AIFM images of HeLa cells to study the localization of KIF13A‐YFP to REs.BPCR analysis of Rab‐knockdown HeLa cells to measure the knockdown efficiency of each shRNA as indicated. DNA band intensities were quantified and indicated on the gels.CSubcellular fractionation of HeLa cells to study the localization of different Rabs (red box) with respect to STX13 or AP‐1.D, EIFM analysis of HeLa cells that were transfected with different constructs as indicated.FLive cell imaging of mCherry‐Rab22A with respect to GFP‐Rab9A in HeLa cells. Arrowheads point to the Rab22A‐positive tubular structures. Magnified view of insets (at 0, 16, 40 s) are shown separately. Scale bars: 10 μm.GIFM images of Rab22A sh, BLOC‐1 sh and control sh HeLa cells that were transfected with indicated constructs.HGraphs represent the measurement of corrected total cell fluorescence (CTCF) in HeLa cells of Fig 2A. n = 6–8 cells. Average CTCF values (AU: arbitrary units) and their respective fold changes (mean ± SEM) are indicated.Data information: In (A), arrowheads and arrows point to the KIF13A‐positive tubular REs and E/SEs, respectively. In (D, E, G), arrowheads point to the STX13‐ or KIF13A‐positive tubular REs or E/SEs. Scale bars: 10 μm.

Fig 4: BLOS1 acts as an adaptor protein of KIF3 complex in the regulation of RE anterograde transport.(a) Immunofluorescence staining of LDLR in KIF3B-FLAG expressing Hep G2 cells. Merged and single labeling images of magnified insets of boxed areas are shown on right. (b) Puncta distribution of LDLR and TfR in KIF3B-R expressing Hep G2 cells. Merged and single labeling images of magnified insets of boxed area are shown on right. (c) Confocal live-cell microscopy of RAB11A-GFP labeled recycling endosomes in KIF3B-R expressing and non-transfected Hep G2 cells. Magnified insets (of box 1 and 2 in the same time points) of consecutive time-lapse images (image/1 s) showed that recycling endosomes in control cells had active motion, while this movement was impaired in KIF3B-R expressing cells (see Figure 5—video 1). White arrows indicate representative recycling endosomes in control and KIF3B-R-expressing cells. Time stamps are in the format of minutes: seconds. (d) Retention of BLOS1 on microtubules caused by co-expressed KIF3A, KIF3B and KIF3C rigor mutants, but not KIF5B or KIF13A rigor mutants in Hep G2 cells. Merged and single labeling images of magnified insets of boxed areas are shown on right. (e) Co-expression of BLOS1 with both KIF3A and KIF3B redistributed KIF3A/B to BLOS1-positive puncta in Hep G2 cells. Merged and single labeling images of magnified insets of boxed area are shown on right. (f, g) Similar retention of KAP3 on microtubules caused by KIF3B-R (f) and redistribution of KIF3A/B resulting from the co-expression of KAP3 (g) in Hep G2 cells. Merged and single labeling images of magnified insets of boxed area are shown on right. (h) Immunoblot of LDLR in KIF3A stable knockdown Hep G2 cells (KIF3A-KD cells). Scale bars in all pictures, 10 µm. See also Figure 5—video 1 and 2, Figure 5—source data 1. Figure 5—source data 1.KIF3A distributes evenly in primary hepatocytes.Representative confocal images of non-puncta and even distribution pattern of overexpressed KIF3A-Scarlet (red) in mouse primary hepatocytes. Bar = 10 µm.

Fig 5: A) Schematic representation of KIF13A domain structure. The longest monomeric human isoform contains one motor domain (MD), one forkhead-associated (FHA) domain, five coiled-coil (CC) domains and two low complexity (LC) regions. After the MD, the neck coil (NC) region is merged with the CC1 domain. The proline (P390) residue is present in the NC-CC1 region and highlighted in the image. The length of each domain (according to the SMART tool) is indicated separately. (B) Schematic representation of KIF13A isoforms from the human. The five isoforms (a to e) of human KIF13A majorly differ by having different deletions, as listed in Table 1. The longest monomeric isoform-a is considered a functional form of KIF13A and used for expression studies. The change in amino acid (aa) sequence (red color letters) in the respective isoform is indicated. The isoform-e express only N-terminal 70 aa and is indicated with * after the modified sequence. The images are drawn at the displayed scale bar.