Fig. 3.

FIP3- and VAMP8-containing endosomes fuse with the ICB at the site of the secondary ingression. (A–C) Late telophase HeLa cell expressing FIP3–mCherry and VAMP8–GFP were imaged by time-lapse microscopy. (A) Localization of FIP3–mCherry (left panel) or VAMP8–GFP (right panel) at the beginning of time-lapse analysis. (B,C) Bright field and fluorescence images at the formation (B, marked with asterisk) and expansion (C, marked with asterisks) of the secondary ingression. Scale bar: 5 μm. (D–F) Late telophase cells expressing FIP3–mCherry (E) and VAMP8–pHluorin (F) were imaged by time-lapse microscopy. Only cells with forming secondary ingression were chosen for analysis. (D) Bright field images of the same cell during the initiation (left image) and expansion (middle and right images) of the secondary ingression. Asterisks mark the midbody; arrows mark leading edges of expanding secondary ingression; LE (leading edge) and C (control) show the areas quantified. (G) Quantification of the ratio between VAMP8–pHluorin and FIP3–mCherry fluorescence in cells with the secondary ingression. In each image, two sections within the ICB (each 2 μm long) were quantified (for example, see D). One section is at the leading edge of expanding secondary ingression (LE), and the second control section (C) is on the same side of the ICB close to the cell body. Data shown are individual ratios derived from three cells with secondary ingression at different time points. Lines mark the mean (1.56 for LE and 1.18 for C); n is the number of cells analyzed.