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