Caveolae promote successful abscission by controlling intercellular bridge tension during cytokinesis

Abstract

During cytokinesis, the intercellular bridge (ICB) connecting the daughter cells experiences pulling forces, which delay abscission by preventing the assembly of the ESCRT scission machinery. Abscission is thus triggered by tension release, but how ICB tension is controlled is unknown. Here, we report that caveolae, which are known to regulate membrane tension upon mechanical stress in interphase cells, are located at the midbody, at the abscission site, and at the ICB/cell interface in dividing cells. Functionally, the loss of caveolae delays ESCRT-III recruitment during cytokinesis and impairs abscission. This is the consequence of a twofold increase of ICB tension measured by laser ablation, associated with a local increase in myosin II activity at the ICB/cell interface. We thus propose that caveolae buffer membrane tension and limit contractibility at the ICB to promote ESCRT-III assembly and cytokinetic abscission. Together, this work reveals an unexpected connection between caveolae and the ESCRT machinery and the first role of caveolae in cell division.

Fig. 1.. Caveolae are present at the midbody and at the EPs during cytokinesis.

(A) Purified midbody remnants from HeLa cells that stably express MKLP1-GFP were immunostained for endogenous Cavin1. Scale bars, 2 μm. (B) Left: Localization of endogenous Cavin1 in HeLa cells stained with α-tubulin + acetylated tubulin (hereafter “Tubulin”), RacGAP1, AuroraB, and 4′,6-diamidino-2-phenylindole (DAPI) at the indicated stages of cytokinesis. Scale bars, 10 μm (general views) and 1 μm (insets of the boxed regions). Top right: Percentage of the indicated structures positive for endogenous Cavin1. Mean ± SD, n > 20 cells per condition. N = 3 independent experiments. (C) Scanning EM pictures of an ICB (left) and midbody at different magnifications (successive insets). Red arrows point to membrane invaginations. Scale bars, 10 μm (left), 500 nm (middle), and 100 nm (right). (D) TEM pictures of a midbody, with inset showing caveolae (red arrow). Scale bars, 10 μm (left) and 100 nm (inset). (E) Left and middle: Two examples of endogenous staining for Cavin1, together with Tubulin and DAPI, in late bridges. Insets show different categories of Cavin1 localization at EPs. Scale bars, 10 μm. Right: Percentage of late bridges with/without secondary constriction where Cavin1 is accumulated, present or absent at EPs, as indicated. Mean ± SD, n > 30 cells per condition. N = 3 independent experiments. One-sided Student’s t tests. NS, nonsignificant (P > 0.05). (F) TEM pictures of an EP region, with the ICB highlighted with red lines and caveolae marked with red arrows. The inset shows caveolae (red arrows) connected to the plasma membrane (PM) and facing the extracellular space (asterisks). Scale bars, 10 μm and 100 nm (inset).

Fig. 2.. Cavin1 and Cav1 dynamically colocalize at the midbody, the EPs, and the abscission site.

(A) Top: Snapshots of a spinning disk confocal microscopy movie of cells coexpressing Cavin1-GFP and Cav1-RFP and incubated with SiR-tubulin. Squares: EPs. Brackets: Midbodies. Facing arrowheads: Abscission site. Time 0 indicates the time of abscission. Note the progressive disappearance of Cavin1/Cav1 in EP regions as cells progress to abscission. Scale bars, 5 μm. Bottom: Percentage of Cavin1-GFP dots colocalizing with Cav1-RFP dots at the furrow, midbody, and EPs. Mean ± SD, n = 6 movies of dividing cells. (B) Intensity of Cavin1-GFP staining as a function of time at EP1 and EP2 in n = 10 cells throughout cytokinesis (see representative video in fig. S3A). The brightest EP was defined as EP1, and the values of EP1 (green) and EP2 (magenta) were normalized to the maximum intensity observed in EP1 during cytokinesis for each cell analyzed. All movies were registered with respect to the time of abscission (t = 0). Mean ± SD, n = 10 movies. (C) Top: Endogenous staining for Cav1 and Cavin1 in cells stably expressing CHMP4B-GFP, with a zoom centered on the ICB. Arrow: CHMP4B-GFP polymerizing cone. Arrowhead: Abscission site (AS). Note the colocalization of Cavin1/Cav1 at the ICB/midbody and at the abscission site. Bottom: Intensity profiles between * and ** along the bridge of the merged picture. Scale bars, 1 μm. (D) Snapshots of a spinning disk confocal microscopy movie of a cell expressing Cavin1-mApple and CHMP4B-GFP incubated with SiR-tubulin. Arrow: CHMP4B-GFP polymerizing cone. Note that the green Cavin1 dot at the tip of the CHMP4B cone disappears while the cone constricts the microtubules at the future abscission site. Scale bars, 2 μm.

Fig. 3.. Depletion of Cavin1 impairs ESCRT-III localization and abscission.

(A) Lysates of parental HeLa, Control KO, and Cavin1 KO cells were blotted for Cavin1, Cav1, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (loading control). (B) TEM micrographs of ultrathin sections of Control KO and Cavin1 KO cells in interphase (left; scale bars, 100 nm) and in cytokinesis (right; scale bars, 500 and 100 nm for insets). Red arrows: Caveolae profiles observed only in Control KO cells. See fig. S4 (C and D) for full fields of view and quantifications. (C) Percentage of bi-/multinucleated cells in indicated cells. Mean ± SD, n > 500 cells per condition. N = 3 independent experiments. (D) Time from the start of furrow ingression to furrow/bridge regression in the Cavin1 KO cells that became binucleated (n = 32 cells). (E) Left: Cumulative distribution of the fraction of cells that completed abscission, measured by phase-contrast time-lapse microscopy in indicated cells. Kolmogorov-Smirnov tests. Right: Mean abscission times (min) ± SD in indicated cells, with n > 60 cells per condition. N = 3 independent experiments. (F) Proportion of late cytokinetic bridges with endogenous CHMP4B localized both at the midbody and at the abscission site (see representative image) in indicated cells. Mean ± SD, n > 30 cells per condition. N = 3 independent experiments. Scale bar, 2 μm. (G) Top: Control KO and Cavin1 KO cells stably expressing CHMP4B-GFP were recorded by spinning disk confocal time-lapse microscopy. t1: Time from cleavage furrow ingression (t = 0 min) to CHMP4B-GFP recruitment at the midbody; t2: Time from CHMP4B-GFP at the midbody to CHMP4B-GFP at midbody + abscission site. Scale bars, 5 μm. Bottom: t1 and t2 (min) ± SD. n > 23 cells. N = 4 independent experiments. Two-sided (C, E, and F) and unpaired (G) Student’s t tests. NS, nonsignificant (P > 0.05).

Fig. 4.. Caveolae limit acto-myosin II at the EPs and control ICB tension during cytokinesis.

(A) Scheme illustrating the laser ablation experiment. (B) Left: Snapshots of ICBs from Control KO and Cavin1 KO cells, before (t0) and after the laser ablation (t0 + 1 s) with corresponding kymographs (red arrows mark the moment of ablation). Dotted line: Position of the laser cut. Scale bars, 2 μm. Right: Retraction speed (micrometers per second) of midbodies in Control KO and Cavin1 KO ablated bridges. Horizontal bar: Mean, n = 23 to 24 ICBs from N = 3 independent experiments. Unpaired Student’s t tests. (C) Top: Representative images of the three categories of endogenous F-actin staining (fluorescent phalloidin) accumulation in late ICBs: none at EPs, at one EP, and at both EPs. Scale bars, 5 μm. Bottom: Percentage of late bridges for each F-actin category. Mean ± SD, n > 20 cells per condition. N = 3 independent experiments. (D) Top: Representative images of the three categories of endogenous pMRLC staining accumulation in late ICBs: none at EPs, at one EP, and at both EPs. Scale bars, 5 μm. Bottom: Percentage of late bridges for each pMRLC category. Mean ± SD, n > 30 cells per condition. N = 3 independent experiments. (E) Left: Endogenous pMRLC and Tubulin staining in a Cavin1-NeonGreen (NG) stable cell line, with a zoom centered on the ICB. Scale bars, 2 μm. Right: Intensity profiles of the EP1 and EP2 regions. (F) Pixel intensity of pMRLC (y axis) and Cavin1-NG (x axis) at EP1 and EP2 from (E). Box a: High pMRLC/low Cavin1 intensity pixels. Box b: High Cavin1/low pMRLC intensity pixels. In (C) and (D), paired Student’s t tests. NS, nonsignificant (P > 0.05). a.u., arbitrary units.

Fig. 5.. Increased ICB tension upon Cavin1 depletion is responsible for the cytokinetic defects.

(A) Quantification of pMRLC localization at the EPs as in Fig. 4D (same representative pictures; scale bars, 5 μm) in indicated cells, treated or not with the ROCK inhibitor Y27632. Mean ± SD, n > 30 cells per condition. N = 3 independent experiments. (B) Retraction speed (micrometers per second) of midbodies in Control KO and Cavin1 KO ablated bridges, treated or not with the ROCK inhibitor Y27632. Horizontal bar: Mean, n > 12 ICBs from N = 3 independent experiments. (C) Endogenous CHMP4B localization was quantified as in Fig. 3F (same representative picture; scale bar, 2 μm) in indicated cells, treated or not with the ROCK inhibitor Y27632 (no synchronization). Mean ± SD, n > 30 cells per condition. N = 3 independent experiments. (D) Left: Cumulative distribution of the fraction of cells that completed abscission, measured by phase-contrast time-lapse microscopy in indicated cells, treated or not with the ROCK inhibitor Y27632. Right: Mean abscission times (min) ± SD in indicated cells, with n > 60 cells per condition. N = 4 independent experiments. (E) Left: Cumulative distribution of the fraction of cells that completed abscission, measured by phase-contrast time-lapse microscopy in Control KO and Cavin1 KO cells, plated at low versus high confluency (see Materials and Methods). Middle: Mean abscission times (min) ± SD in indicated cells, with n > 24 cells per condition. N = 3 independent experiments. Right: Representative pictures of cells in low versus high confluency. Phase contrast (gray) and SiR-tubulin (red) are overlaid. Scale bars, 5 μm. Paired (A, C, D, and E) and unpaired (B) Student’s t tests. Kolmogorov-Smirnov tests for cumulative curves (D and E). NS, nonsignificant (P > 0.05).

Fig. 6.. Model: Caveolae buffer ICB tension by limiting the F-actin/myosin II–dependent contractility and membrane tension, thereby promoting ESCRT-III polymerization and successful abscission.

(A) Cells with caveolae (WT cells). Box: Proposed mechanism for the control of abscission by caveolae. (B) Cells without caveolae (e.g., Cavin1 KO cells).