Fig. S1. VAMP8 co-localizes with FIP3-endosomes. (A−I) FIP3-GFP (A,D,G) expressing HeLa cells in early (A−C) and late (D−I) telophase were fixed and imaged with anti-VAMP8 (B,E) or anti-VAMP4 (H) antibodies. (C,F,K) show merged images, with yellow representing the degree of overlap between VAMPs and FIP3-GFP. In (C) asterisks mark centrosomes. In (F,I) asterisks mark midbody. Scale bar: 5 m. (J) HeLa cells, transduced with VAMP8-GFP (green) and FIP3-mCherry (red) were incubated with anti-GFP antibody (blue), then fixed and imaged. Left panel shows merged image. Scale bar: 5 m.

Fig. S2. Monitoring FIP3-endosome fusion using VAMP8-pHluorin. (A,B) HeLa cell transduced with VAMP8-pHluorin was imaged by time-lapse microscopy. (A) Image is imaged taken at 0 sec. (B) Image is a projection of time-lapse series. Arrows point to VAMP8-pHluorin fusion events. (C) Fluorescent intensity quantification of individual fusion events. (D−F) HeLa cell transduced with VAMP8-GFP and FIP3-mCherry was fixed and imaged. (F) shows brightfield image of cell. (G−H) VAMP8-pHluorin-expressing HeLa cell was imaged before (G) and after (H) treatment with NH₄Cl.

Fig. S3. Characterization of FIP3/VAMP8 endosome dynamics in early telophase. (A−C) HeLa cell expressing FIP3-mCherry (A) and VAMP8-pHluorin (B). Bright-field image is shown in (C). Scale bar: 5 m. (D,E) HeLa cell expressing VAMP8-pHluorin was imaged by time-lapse microscopy. Shown images are sequential time-lapse images. In (D), arrow points to a VAMP8-pHluorin fusion event. In (E), arrowhead points to VAMP8-pHluorin accumulation just outside the intracellular bridge and arrows are VAMP8-pHluorin-associated endosomes trafficked inside the intracellular bridge. Scale bar: 5 m.

Fig. S4. Characterization of microtubules and organelles in large volume tomographic reconstructions. (A−E) FIP3-GFP-expressing HeLa cells were fixed and stained with anti-GFP antibody, followed with secondary antibodies conjugated with 15 nm immuno-gold beads (marked by arrows). Asterisk marks the midbody matrix. Scale bars: 200 nm. (F−H) Gallery and reconstructions of FIP3-endosomes (F), spherical 53 nm organelles (G), and lytic endosomes (H). (I,J) Gallery of microtubule ends displaying a flared (I) or non-flared (J) conformations.

Fig. S5. Changes in microtubule organization during progression from early to late telophase. (A,B) Reconstructed models of microtubules in late telophase. (A) displays microtubule plus (white spheres) and minus (red spheres) ends. (B) displays microtubules with non-flared (white spheres) and flared (red spheres) ends. Arrowheads points to a site of secondary ingression. Scale bar: 200 nm. (C) RK cells stably expressing tubulin-GFP were imaged using time-lapse microscopy. Arrow marks midbody and arrowheads identify the regions of tubulin-GFP depletion.

Fig. S6. Spastin knockdown delays cytokinesis. (A−C) Mock (A,C), spastin siRNA-treated (C) or blebbistatin-treated (B,C) HeLa cells were analyzed for DNA content using flow cytometry. (C) Quantification of cells in G1, S and G2M phases. (D,E) Mock and spastin siRNA-treated HeLa cells were analyzed for microtubule organization and FIP3 trafficking defects (E) and the percentage of cells in telophase (D). Data shown are the means and standard deviations from three independent experiments. n represents total number of cells analyzed.