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

Deletions in CHMP6-N suggest a role for the first 10 aa in CHMP6-N–induced inhibition of ESCRT-driven abscission. (A) Schematic view of the CHMP6-N deletion constructs used in the study. VPS25-binding domain is depicted in purple. All constructs were fused to GFP/mCherry fluorescent tags on their C-terminal. (B–D) MDCK cells expressing CHMP6-N-11-52–GFP (B), CHMP6-N-11-42–GFP (C), or CHMP6-N-1-42–GFP (D) together with mCherry-tubulin were imaged during cytokinesis using a spinning-disk confocal microscope at 7-min intervals. Each panel shows a maximum-intensity projection of different time points taken from a representative movie sequence (Supplemental Videos S7–S9 respectively). Top, tubulin signal alone (bars, 10 μm); middle, overlay of the CHMP6-N construct (green) and tubulin (red); bottom, enlargement of the intercellular bridge from the overlay images (bars, 5 μm). All constructs localize to the center of the intercellular bridge during abscission (arrows). Of note, whereas exogenous expression of either CHMP6-N-11-52–GFP or CHMP6-N-11-42–GFP has only a mild effect on abscission and has no effect on cell viability, expression of CHMP6-N-1-42–GFP delays abscission and increases cell death. Asterisks indicate dying cells exhibiting acute cell contraction and membrane blebbing. (E) Percentage of cells that completed abscission successfully and percentage of daughter cell death observed within the time course of the experiments (3–4 h) upon expression of the different constructs. Cell death was determined based on cell morphology (see asterisks in D). CHMP6-N-11-52–GFP (n = 43); CHMP6-N-11-42–GFP (n = 49); and CHMP6-N-1-42–GFP (n = 40).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230781&req=5

Figure 4: Deletions in CHMP6-N suggest a role for the first 10 aa in CHMP6-N–induced inhibition of ESCRT-driven abscission. (A) Schematic view of the CHMP6-N deletion constructs used in the study. VPS25-binding domain is depicted in purple. All constructs were fused to GFP/mCherry fluorescent tags on their C-terminal. (B–D) MDCK cells expressing CHMP6-N-11-52–GFP (B), CHMP6-N-11-42–GFP (C), or CHMP6-N-1-42–GFP (D) together with mCherry-tubulin were imaged during cytokinesis using a spinning-disk confocal microscope at 7-min intervals. Each panel shows a maximum-intensity projection of different time points taken from a representative movie sequence (Supplemental Videos S7–S9 respectively). Top, tubulin signal alone (bars, 10 μm); middle, overlay of the CHMP6-N construct (green) and tubulin (red); bottom, enlargement of the intercellular bridge from the overlay images (bars, 5 μm). All constructs localize to the center of the intercellular bridge during abscission (arrows). Of note, whereas exogenous expression of either CHMP6-N-11-52–GFP or CHMP6-N-11-42–GFP has only a mild effect on abscission and has no effect on cell viability, expression of CHMP6-N-1-42–GFP delays abscission and increases cell death. Asterisks indicate dying cells exhibiting acute cell contraction and membrane blebbing. (E) Percentage of cells that completed abscission successfully and percentage of daughter cell death observed within the time course of the experiments (3–4 h) upon expression of the different constructs. Cell death was determined based on cell morphology (see asterisks in D). CHMP6-N-11-52–GFP (n = 43); CHMP6-N-11-42–GFP (n = 49); and CHMP6-N-1-42–GFP (n = 40).

Mentions: To test whether binding of CHMP6-N to VPS25 is sufficient for inhibiting abscission, we generated a series of CHMP6-N truncations without perturbing the VPS25-CHMP6 interaction domain (aa 11–42; Im et al., 2009). Three additional CHMP6-N truncations were conjugated to GFP: CHMP6-N-11-52, CHMP6-N-1-42, and CHMP6-N-11-42 (Figure 4A). All three constructs efficiently localize to the intercellular bridge of dividing MDCK cells (Figure 4, B–D, bottom, arrows, and Supplemental Videos S7–S9), confirming that CHMP6-N localization to the intercellular bridge is mediated through binding to VPS25. Of interest, constructs lacking the first 10 aa of CHMP6 show only mild inhibition of abscission and no cell death (Figure 4, B, C, and E, and Supplemental Videos S7 and S8), and the only construct that is able to recapitulate the inhibition observed for CHMP6-N–GFP is CHMP6-N-1-42–GFP (Figure 4, D and E, and Supplemental Video S9). This indicates that although binding to VPS25 is sufficient for CHMP6-N localization to the intercellular bridge, it is not sufficient for CHMP6-N–induced inhibition of abscission. The first 10 aa of CHMP6 include a myristoylation moiety and a basic patch of amino acids that presumably assigns CHMP6 affinity to the plasma membrane (Saksena et al., 2007). Perhaps membrane binding of CHMP6-N is essential for its ability to inhibit abscission.


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)

Deletions in CHMP6-N suggest a role for the first 10 aa in CHMP6-N–induced inhibition of ESCRT-driven abscission. (A) Schematic view of the CHMP6-N deletion constructs used in the study. VPS25-binding domain is depicted in purple. All constructs were fused to GFP/mCherry fluorescent tags on their C-terminal. (B–D) MDCK cells expressing CHMP6-N-11-52–GFP (B), CHMP6-N-11-42–GFP (C), or CHMP6-N-1-42–GFP (D) together with mCherry-tubulin were imaged during cytokinesis using a spinning-disk confocal microscope at 7-min intervals. Each panel shows a maximum-intensity projection of different time points taken from a representative movie sequence (Supplemental Videos S7–S9 respectively). Top, tubulin signal alone (bars, 10 μm); middle, overlay of the CHMP6-N construct (green) and tubulin (red); bottom, enlargement of the intercellular bridge from the overlay images (bars, 5 μm). All constructs localize to the center of the intercellular bridge during abscission (arrows). Of note, whereas exogenous expression of either CHMP6-N-11-52–GFP or CHMP6-N-11-42–GFP has only a mild effect on abscission and has no effect on cell viability, expression of CHMP6-N-1-42–GFP delays abscission and increases cell death. Asterisks indicate dying cells exhibiting acute cell contraction and membrane blebbing. (E) Percentage of cells that completed abscission successfully and percentage of daughter cell death observed within the time course of the experiments (3–4 h) upon expression of the different constructs. Cell death was determined based on cell morphology (see asterisks in D). CHMP6-N-11-52–GFP (n = 43); CHMP6-N-11-42–GFP (n = 49); and CHMP6-N-1-42–GFP (n = 40).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Deletions in CHMP6-N suggest a role for the first 10 aa in CHMP6-N–induced inhibition of ESCRT-driven abscission. (A) Schematic view of the CHMP6-N deletion constructs used in the study. VPS25-binding domain is depicted in purple. All constructs were fused to GFP/mCherry fluorescent tags on their C-terminal. (B–D) MDCK cells expressing CHMP6-N-11-52–GFP (B), CHMP6-N-11-42–GFP (C), or CHMP6-N-1-42–GFP (D) together with mCherry-tubulin were imaged during cytokinesis using a spinning-disk confocal microscope at 7-min intervals. Each panel shows a maximum-intensity projection of different time points taken from a representative movie sequence (Supplemental Videos S7–S9 respectively). Top, tubulin signal alone (bars, 10 μm); middle, overlay of the CHMP6-N construct (green) and tubulin (red); bottom, enlargement of the intercellular bridge from the overlay images (bars, 5 μm). All constructs localize to the center of the intercellular bridge during abscission (arrows). Of note, whereas exogenous expression of either CHMP6-N-11-52–GFP or CHMP6-N-11-42–GFP has only a mild effect on abscission and has no effect on cell viability, expression of CHMP6-N-1-42–GFP delays abscission and increases cell death. Asterisks indicate dying cells exhibiting acute cell contraction and membrane blebbing. (E) Percentage of cells that completed abscission successfully and percentage of daughter cell death observed within the time course of the experiments (3–4 h) upon expression of the different constructs. Cell death was determined based on cell morphology (see asterisks in D). CHMP6-N-11-52–GFP (n = 43); CHMP6-N-11-42–GFP (n = 49); and CHMP6-N-1-42–GFP (n = 40).
Mentions: To test whether binding of CHMP6-N to VPS25 is sufficient for inhibiting abscission, we generated a series of CHMP6-N truncations without perturbing the VPS25-CHMP6 interaction domain (aa 11–42; Im et al., 2009). Three additional CHMP6-N truncations were conjugated to GFP: CHMP6-N-11-52, CHMP6-N-1-42, and CHMP6-N-11-42 (Figure 4A). All three constructs efficiently localize to the intercellular bridge of dividing MDCK cells (Figure 4, B–D, bottom, arrows, and Supplemental Videos S7–S9), confirming that CHMP6-N localization to the intercellular bridge is mediated through binding to VPS25. Of interest, constructs lacking the first 10 aa of CHMP6 show only mild inhibition of abscission and no cell death (Figure 4, B, C, and E, and Supplemental Videos S7 and S8), and the only construct that is able to recapitulate the inhibition observed for CHMP6-N–GFP is CHMP6-N-1-42–GFP (Figure 4, D and E, and Supplemental Video S9). This indicates that although binding to VPS25 is sufficient for CHMP6-N localization to the intercellular bridge, it is not sufficient for CHMP6-N–induced inhibition of abscission. The first 10 aa of CHMP6 include a myristoylation moiety and a basic patch of amino acids that presumably assigns CHMP6 affinity to the plasma membrane (Saksena et al., 2007). Perhaps membrane binding of CHMP6-N is essential for its ability to inhibit abscission.

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