Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death.
This phenotype is abolished in a mutated version of CHMP6-N designed to prevent CHMP6-N binding to its ESCRT-II partner.Of interest, deleting the first 10 amino acids from CHMP6-N does not interfere with its arrival at the intercellular bridge but almost completely abolishes the abscission failure phenotype.Our work advances the mechanistic understanding of ESCRT-mediated membrane fission in cells and introduces an easily applicable tool for upstream inhibition of the ESCRT pathway in live mammalian cells.
Affiliation: Department of Life Sciences and the National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
- Cell Death/genetics*
- Endosomal Sorting Complexes Required for Transport/genetics/metabolism*
- Madin Darby Canine Kidney Cells
- Membrane Fusion/genetics
- Protein Binding
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Figure 1: The ESCRT-III component CHMP6 localizes to the intercellular bridge during cytokinesis. (A) Schematic model of the late intercellular bridge. The dark zone is located at the center of the intercellular bridge. Bridge cleavage occurs at narrow constriction sites located peripherally on both sides of the dark zone. (B) Percentage of abscission failure in MDCK cells overexpressing ESCRT-II and CHMP6 components. Cells were cotransfected with GFP/mCherry-VPS25, GFP/mCherry-VPS36, GFP/mCherry-VPS22, or mCherry-CHMP6 together with mCherry/GFP-tubulin, respectively, and were imaged for 3–4 h using a confocal spinning disk. CHMP6 (n = 49), VPS22 (n = 60), VPS25 (n = 25), VPS36 (n = 82), and control cells (expressing either GFP/mCherry-tubulin or mCherry/GFP empty plasmids; n = 44). The rate of abscission failure in MDCK cells overexpressing CHMP6 was not significantly different from that in control cells (χ12 test, p = 0.068), whereas the rates of abscission failure in MDCK cells overexpressing VPS22, VPS25, or VPS36 were significantly higher than in control cells (**χ12 test, p < 0.01; ***χ12 test, p < 0.001). (C) Live-cell imaging of MDCK cells undergoing cytokinesis reveals acute recruitment of CHMP6 to the intercellular bridge. Cells expressing low levels of mCherry-CHMP6 together with GFP-tubulin were imaged using a spinning-disk confocal microscope at 7-min intervals. Shown are maximum-intensity projections of different time points during cytokinesis from a representative cell. Top, overlay of CHMP6 (green) and microtubules (red); bottom, CHMP6 signal alone. Time (indicated in minutes) is relative to abscission (Supplemental Video S1; n = 10; bar, 2 μm). Arrows indicate the position of the first abscission site. (D) Spatial organization of CHMP6, in early (top) and late (bottom) intercellular bridges. MDCK cells expressing Flag-CHMP6 were fixed, stained with anti–α-tubulin and anti-Flag antibodies, and imaged by SIM. Early and late bridges were categorized based on the diameter of the intercellular bridge at the constriction site (see Materials and Methods). Each panel shows (from left to right) a three-dimensional (3D) reconstruction of an overlay of CHMP6 (green) and tubulin (white; bar, 2 μm); a zoomed-in, 3D rendered image of the protein structure alone (bar, 1 μm); a zoomed-in, 3D rendered image of the protein structure rotated 90° (bar, 1 μm); and a schematic model for CHMP6 organization at the intercellular bridge based on SIM measurements. Zoomed-in and rotated images of late bridges are of the structure labeled by a solid arrow in the full 3D reconstructed image. The model for late bridges refers to the structure indicated by a dashed arrow in the full 3D reconstructed image, as this is a more advanced time point in the process. In early intercellular bridges, CHMP6 concentrates in two rings located on the rims of the dark zone. The rings are 0.47 ± 0.11 μm apart (n = 17). The dark zone is 0.69 ± 0.12 μm (n = 36) wide. The diameter of the rings is 1.11 ± 0.19 μm (n = 37). In late intercellular bridges, the CHMP6 structure elongates asymmetrically, peripherally to the center of the bridge (solid arrow), forming a series of cortical rings with decreasing diameters (ring diameters in zoomed-in image: 900, 500, and 450 nm). Finally, CHMP6 is located in two separate pools: one on the rims of the dark zone, and one that colocalizes with the site of microtubule constriction (dashed arrow; see also E). (E) The peripheral CHMP6 pool is located at the site of microtubule constriction. Plot shows tubulin (gray) and CHMP6 (green) line intensity profiles along the intercellular bridge (indicated by a blue arrow in D).
Mammalian cell division ends with abscission—the cleavage of a thin intercellular membrane bridge connecting two daughter cells at the end of cytokinesis (Figure 1A). Recently the endosomal sorting complexes required for transport (ESCRT) membrane fission machinery (composed of five different subfamilies: ESCRT-0, -I, -II, and -III and VPS4) has been shown to mediate cytokinetic abscission (Carlton and Martin-Serrano, 2007; Morita et al., 2007; Elia et al., 2011, 2013; Guizetti et al., 2011). During cytokinesis, components of the ESCRT machinery assemble into cortical rings on the membrane of the midbody dark zone, an electron-dense structure at the center of the bridge (Figure 1A). Abscission is accompanied by rearrangement of ESCRT-III components to the abscission sites, located ∼1 μm away from either side of the center of the bridge (Figure 1A; Elia et al., 2011, 2012, 2013; Guizetti et al., 2011). Although these studies identified the ESCRT complex as the molecular machinery that drives abscission, many of the mechanistic steps that lead to ESCRT-mediated abscission and their regulation remained unresolved.