Figure 7. KIF3 and BLOS1 function at specific microtubule intersections.

(a) Schematic of tip of one KIF13A-GFP tubule encounters another KIF13A-GFP tubule. (b) Histogram showing the occurrence of extension and retraction action when tips of KIF13A tubule encounter other KIF13A tubules in control (n = 64) and KIF3A-KD (n = 100) cells. (c) Super-resolution microscopy of the BLOS1-GFP puncta (green) on microtubule tracks (red, labeled by Tubulin Tracker Deep Red) in Hep G2 cells. Merged and single labeling images of magnified insets of boxed area are shown on right. Arrows indicate BLOS1 puncta that locate near microtubule intersections. (d) Frequency of reversed movement after pausing at specific microtubule intersection was increased in both BLOC1S1-KD and KIF3A-KD cells as compared to control cells. Data are presented as Mean ± SEM, n = 5 cells. Two-tailed t test, ***p<0.001. Scale bars in all pictures, 10 μm. See also Figure 7—figure supplement 1, Figure 7—videos 1, 2 and 3.

Figure 7—figure supplement 1. BLOS1 also showed puncta pattern in primary hepatocytes and locates near microtubule intersections.

(a) Overexpressed BLOS1-GFP (green) showed both tubular and puncta distribution in primary hepatocytes. (b) A proportion of BLOS1 puncta (green) locate near the intersections of microtubules (labeled by Tubulin-tracker). Representative BLOS1 puncta were indicated by arrows. Magnified insets of boxed areas are shown on the left bottom left corner of each figure. Scale bars in all pictures, 10 μm.

Figure 7—video 1. 3D-reconstruction of super-resolution Z-stack images of BLOS1-HA (green) and alpha-tubulin (red).

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Z-stacks images were acquired every 170 nm for a total of 50 slices.

Figure 7—video 2. Live-cell imaging of recycling endosomes (indicated by RAB11A-GFP) moving on microtubule tracks (stained by Tubulin Tracker Deep Red probe) showing the passage of recycling endosomes after pausing at specific microtubule intersections.

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Arrows indicate intersections where recycling endosomes (marked with magenta circles) pause and then go through. Images were acquired with Zeiss LSM 880 using the Airyscan module with line mode, and the time interval between every two frames was 0.32 s.

Figure 7—video 3. Live-cell imaging of recycling endosomes (indicated by RAB11A-GFP) moving on microtubule tracks (stained by Tubulin Tracker Deep Red probe) showing the reversed movement of recycling endosomes at specific microtubule intersections.

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Arrows indicate intersections where recycling endosomes (marked with magenta circles) pause and then reversed their moving directions. Images were acquired with Zeiss LSM 880 using the Airyscan module with line mode, and the time interval between every two frames was 0.32 s.