Wenzel, D. M. | Search bioRxiv

bioRxiv2022

Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that recruits essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 pure recombinant human MIT domains. We measured 228 pairwise interactions, quantified 58 positive interactions, and discovered 16 previously unreported interactions. We also report the crystal structure of the SPASTIN MIT domain in complex with the IST1 C-terminal tail. Three MIT enzymes were studied in detail and shown to: 1) localize to cytokinetic midbody membrane bridges through interactions with their specific ESCRT-III binding partners (SPASTIN-IST1, KATNA1-CHMP3, and CAPN7-IST1), 2) function in abscission (SPASTIN, KATNA1, and CAPN7), and 3) function in the "NoCut" abscission checkpoint (SPASTIN and CAPN7). Our studies define the human MIT-ESCRT-III interactome, identify new factors and activities required for cytokinetic abscission and its regulation, and provide a platform for analyzing ESCRT-III and MIT cofactor interactions in all ESCRT-mediated processes.

biochemistry↗

bioRxiv2018

A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery and is tightly regulated by Charged Multivesicular body Protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4CT232 (rs35094336), increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: the CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome mis-segregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint, and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.\n\nSignificance StatementThe final step of cell division, abscission, is temporally regulated by the Aurora B kinase and CHMP4C in a conserved pathway called the abscission checkpoint which arrests abscission in the presence of lingering mitotic problems. Despite extensive study, the physiological importance of this pathway to human health has remained elusive. We now demonstrate that a cancer predisposing polymorphism in CHMP4C disrupts the abscission checkpoint and results in DNA damage accumulation. Moreover, deficits in this checkpoint synergize with p53 loss and generate aneuploidy under stress conditions that increase the frequency of chromosome missegregation. Therefore, cells expressing the cancer-associated polymorphism in CHMP4C are genetically unstable, thus suggesting a novel oncogenic mechanism that may involve the dysregulation of abscission.

cell biology↗