Limits...
Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death.

Goliand I, Nachmias D, Gershony O, Elia N - Mol. Biol. Cell (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

Show MeSH

Related in: MedlinePlus

Exogenous expression of CHMP6-N-GFP inhibits abscission through specific interaction with the ESCRT-II protein VPS25 and leads to cell death. (A) Schematic view of the CHMP6 constructs used in the study. Highlighted are CHMP6 myristoylation site (zigzag line), VPS25-binding domain (purple), and CHMP6–ESCRT-III interaction motif (green). CHMP6-N is composed of the first 52 aa of CHMP6. Asterisks indicate three point mutations that were introduced in CHMP6-N-mut in order to interfere with its binding to VPS25. All constructs were fused to GFP/mCherry fluorescent tags. (B and C) Dividing MDCK cells expressing CHMP6-N-GFP (A) or CHMP6-N-mut-GFP (C) together with mCherry-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 representative cells. Top, tubulin signal alone (bar, 10 μm); middle, overlay of CHMP6-N-GFP/CHMP6-N-mut-GFP (green) and tubulin (red); bottom, enlargement of the intercellular bridge taken from the overlay images (bar, 5 μm). Of note, CHMP6-N-GFP arrived at the center of the bridge (arrows). Asterisks in B indicate dying cells exhibiting acute cell contraction and membrane blebbing. While exogenous expression of CHMP6-N-GFP delays abscission and increases cell death, exogenous expression of CHMP6-N-mut-GFP (L21R, R27A and D28A) does not affect abscission or cell viability (Supplemental Videos S5 and S6; bars, 5 μm). (D) Percentage of cells that completed abscission successfully and percentage of cell death observed in daughter cells within the time course of the experiments (3–4 h) under the different conditions. Cell death was determined based on changes in cell morphology as observed in the movie sequences (see asterisks in B). CHMP6-N–GFP (n = 40); CHMP6-N-mut-GFP (n = 44); and control cells expressing GFP (n = 44).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230781&req=5

Figure 3: Exogenous expression of CHMP6-N-GFP inhibits abscission through specific interaction with the ESCRT-II protein VPS25 and leads to cell death. (A) Schematic view of the CHMP6 constructs used in the study. Highlighted are CHMP6 myristoylation site (zigzag line), VPS25-binding domain (purple), and CHMP6–ESCRT-III interaction motif (green). CHMP6-N is composed of the first 52 aa of CHMP6. Asterisks indicate three point mutations that were introduced in CHMP6-N-mut in order to interfere with its binding to VPS25. All constructs were fused to GFP/mCherry fluorescent tags. (B and C) Dividing MDCK cells expressing CHMP6-N-GFP (A) or CHMP6-N-mut-GFP (C) together with mCherry-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 representative cells. Top, tubulin signal alone (bar, 10 μm); middle, overlay of CHMP6-N-GFP/CHMP6-N-mut-GFP (green) and tubulin (red); bottom, enlargement of the intercellular bridge taken from the overlay images (bar, 5 μm). Of note, CHMP6-N-GFP arrived at the center of the bridge (arrows). Asterisks in B indicate dying cells exhibiting acute cell contraction and membrane blebbing. While exogenous expression of CHMP6-N-GFP delays abscission and increases cell death, exogenous expression of CHMP6-N-mut-GFP (L21R, R27A and D28A) does not affect abscission or cell viability (Supplemental Videos S5 and S6; bars, 5 μm). (D) Percentage of cells that completed abscission successfully and percentage of cell death observed in daughter cells within the time course of the experiments (3–4 h) under the different conditions. Cell death was determined based on changes in cell morphology as observed in the movie sequences (see asterisks in B). CHMP6-N–GFP (n = 40); CHMP6-N-mut-GFP (n = 44); and control cells expressing GFP (n = 44).

Mentions: The spatiotemporal organization of ESCRT-II and CHMP6 in cytokinetic abscission and its agreement with the spatiotemporal mapping previously described for other ESCRT components in cytokinesis points to the involvement of these proteins in ESCRT-mediated abscission. To substantiate a role for ESCRT-II and CHMP6 in cytokinetic abscission, we designed a tool for specific inhibition of ESCRT-II–CHMP6 interaction in live cells using exogenous expression of the first 52 aa of CHMP6 in cells (CHMP6-N). siRNA depletion of these components was reported to have no effect on cytokinetic abscission (Morita et al., 2007) and was therefore avoided. According to in vitro studies and work in yeast, binding of CHMP6 to the ESCRT-II component VPS25 facilitates ESCRT-III activation and polymerization (Peel et al., 2011; McCullough et al., 2013). CHMP6 binding to ESCRT-II is mediated by direct interactions between the N-terminus of CHMP6 (aa 11–42) and the ESCRT-II component VPS25 (Im et al., 2009), whereas induction of ESCRT-III polymerization is attributed to the C- terminal domains of CHMP6 (Teis et al., 2010; Peel et al., 2011; McCullough et al., 2013; Figure 3A). Therefore, if CHMP6 arrives at the intercellular bridge via interactions with VPS25, then exogenous CHMP6-N is expected to localize to the intercellular bridge, compete with endogenous CHMP6 over binding to VPS25, and inhibit ESCRT-mediated abscission. Indeed, a CHMP6-N-GFP construct arrives to the intercellular bridge in MDCK cells undergoing cytokinesis (Figure 3B, bottom, arrow). Of note, >80% of cells expressing CHMP6-N-GFP failed to complete abscission within the 3–4 h of the experiment (Figure 3, B and D, and Supplemental Video S5). Late ESCRT components arrived to the intercellular bridges of these cells but failed to form the characteristic ring-like structures or to localize to the constriction sites (Supplemental Figure S2). In 40% of the cells, the inability to complete abscission was accompanied by premature death of the daughter cells, characterized by acute cell contraction and membrane blebbing (asterisks in Figure 3, B and D, and Supplemental Video S5). Such morphology was observed in <10% of the daughter cells upon expression of GFP/mCherry-tubulin plasmids alone or with an mCherry/GFP control plasmid (Figure 3D). Similar results were obtained with CHMP6-N-mCherry.


Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death.

Goliand I, Nachmias D, Gershony O, Elia N - Mol. Biol. Cell (2014)

Exogenous expression of CHMP6-N-GFP inhibits abscission through specific interaction with the ESCRT-II protein VPS25 and leads to cell death. (A) Schematic view of the CHMP6 constructs used in the study. Highlighted are CHMP6 myristoylation site (zigzag line), VPS25-binding domain (purple), and CHMP6–ESCRT-III interaction motif (green). CHMP6-N is composed of the first 52 aa of CHMP6. Asterisks indicate three point mutations that were introduced in CHMP6-N-mut in order to interfere with its binding to VPS25. All constructs were fused to GFP/mCherry fluorescent tags. (B and C) Dividing MDCK cells expressing CHMP6-N-GFP (A) or CHMP6-N-mut-GFP (C) together with mCherry-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 representative cells. Top, tubulin signal alone (bar, 10 μm); middle, overlay of CHMP6-N-GFP/CHMP6-N-mut-GFP (green) and tubulin (red); bottom, enlargement of the intercellular bridge taken from the overlay images (bar, 5 μm). Of note, CHMP6-N-GFP arrived at the center of the bridge (arrows). Asterisks in B indicate dying cells exhibiting acute cell contraction and membrane blebbing. While exogenous expression of CHMP6-N-GFP delays abscission and increases cell death, exogenous expression of CHMP6-N-mut-GFP (L21R, R27A and D28A) does not affect abscission or cell viability (Supplemental Videos S5 and S6; bars, 5 μm). (D) Percentage of cells that completed abscission successfully and percentage of cell death observed in daughter cells within the time course of the experiments (3–4 h) under the different conditions. Cell death was determined based on changes in cell morphology as observed in the movie sequences (see asterisks in B). CHMP6-N–GFP (n = 40); CHMP6-N-mut-GFP (n = 44); and control cells expressing GFP (n = 44).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230781&req=5

Figure 3: Exogenous expression of CHMP6-N-GFP inhibits abscission through specific interaction with the ESCRT-II protein VPS25 and leads to cell death. (A) Schematic view of the CHMP6 constructs used in the study. Highlighted are CHMP6 myristoylation site (zigzag line), VPS25-binding domain (purple), and CHMP6–ESCRT-III interaction motif (green). CHMP6-N is composed of the first 52 aa of CHMP6. Asterisks indicate three point mutations that were introduced in CHMP6-N-mut in order to interfere with its binding to VPS25. All constructs were fused to GFP/mCherry fluorescent tags. (B and C) Dividing MDCK cells expressing CHMP6-N-GFP (A) or CHMP6-N-mut-GFP (C) together with mCherry-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 representative cells. Top, tubulin signal alone (bar, 10 μm); middle, overlay of CHMP6-N-GFP/CHMP6-N-mut-GFP (green) and tubulin (red); bottom, enlargement of the intercellular bridge taken from the overlay images (bar, 5 μm). Of note, CHMP6-N-GFP arrived at the center of the bridge (arrows). Asterisks in B indicate dying cells exhibiting acute cell contraction and membrane blebbing. While exogenous expression of CHMP6-N-GFP delays abscission and increases cell death, exogenous expression of CHMP6-N-mut-GFP (L21R, R27A and D28A) does not affect abscission or cell viability (Supplemental Videos S5 and S6; bars, 5 μm). (D) Percentage of cells that completed abscission successfully and percentage of cell death observed in daughter cells within the time course of the experiments (3–4 h) under the different conditions. Cell death was determined based on changes in cell morphology as observed in the movie sequences (see asterisks in B). CHMP6-N–GFP (n = 40); CHMP6-N-mut-GFP (n = 44); and control cells expressing GFP (n = 44).
Mentions: The spatiotemporal organization of ESCRT-II and CHMP6 in cytokinetic abscission and its agreement with the spatiotemporal mapping previously described for other ESCRT components in cytokinesis points to the involvement of these proteins in ESCRT-mediated abscission. To substantiate a role for ESCRT-II and CHMP6 in cytokinetic abscission, we designed a tool for specific inhibition of ESCRT-II–CHMP6 interaction in live cells using exogenous expression of the first 52 aa of CHMP6 in cells (CHMP6-N). siRNA depletion of these components was reported to have no effect on cytokinetic abscission (Morita et al., 2007) and was therefore avoided. According to in vitro studies and work in yeast, binding of CHMP6 to the ESCRT-II component VPS25 facilitates ESCRT-III activation and polymerization (Peel et al., 2011; McCullough et al., 2013). CHMP6 binding to ESCRT-II is mediated by direct interactions between the N-terminus of CHMP6 (aa 11–42) and the ESCRT-II component VPS25 (Im et al., 2009), whereas induction of ESCRT-III polymerization is attributed to the C- terminal domains of CHMP6 (Teis et al., 2010; Peel et al., 2011; McCullough et al., 2013; Figure 3A). Therefore, if CHMP6 arrives at the intercellular bridge via interactions with VPS25, then exogenous CHMP6-N is expected to localize to the intercellular bridge, compete with endogenous CHMP6 over binding to VPS25, and inhibit ESCRT-mediated abscission. Indeed, a CHMP6-N-GFP construct arrives to the intercellular bridge in MDCK cells undergoing cytokinesis (Figure 3B, bottom, arrow). Of note, >80% of cells expressing CHMP6-N-GFP failed to complete abscission within the 3–4 h of the experiment (Figure 3, B and D, and Supplemental Video S5). Late ESCRT components arrived to the intercellular bridges of these cells but failed to form the characteristic ring-like structures or to localize to the constriction sites (Supplemental Figure S2). In 40% of the cells, the inability to complete abscission was accompanied by premature death of the daughter cells, characterized by acute cell contraction and membrane blebbing (asterisks in Figure 3, B and D, and Supplemental Video S5). Such morphology was observed in <10% of the daughter cells upon expression of GFP/mCherry-tubulin plasmids alone or with an mCherry/GFP control plasmid (Figure 3D). Similar results were obtained with CHMP6-N-mCherry.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus