Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission
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Evaluation Summary:
The authors quantitatively characterize binding interactions between all known MIM motifs of ESCRT-III proteins with the MIT motifs of several AAA+ ATPases. In addition to the analysis of these interactions, which will be an important resource to the ESCRT community, the authors also identify new roles of the ATPases SPASTIN, KATNA1 and CAPN7 in cytokinesis. Therefore, the work will be of broad interest to biologists interested in membrane-associated complexes and in cell cycle and cytokinesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
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 is used to recruit essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 recombinant human MIT domains. We measured 228 pairwise interactions, quantified 60 positive interactions, and discovered 18 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.
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Evaluation Summary:
The authors quantitatively characterize binding interactions between all known MIM motifs of ESCRT-III proteins with the MIT motifs of several AAA+ ATPases. In addition to the analysis of these interactions, which will be an important resource to the ESCRT community, the authors also identify new roles of the ATPases SPASTIN, KATNA1 and CAPN7 in cytokinesis. Therefore, the work will be of broad interest to biologists interested in membrane-associated complexes and in cell cycle and cytokinesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The work by Wenzel and colleagues exhaustively studies the network of interactions between MIT-containing proteins with the MIM containing proteins of the ESCRT-III complex. They discover 16 new interactions and at least one unknown factor interacting with ESCRT-III, CAPN7. Several findings are very interesting and provides essential stepping stones on which the community will continue progressing. In particular, the finding that IST1 clusters a very important network of MIT-containing proteins is reinforcing the view that IST1 is an essential factor of abscission, as proposed by the authors in previous papers. Also, that specific interactions between one ESCRT-III-MIM containing subunit and one MIT.containing partner can be disrupted through single point mutations, not affecting other MIT-MIM interactions …
Reviewer #1 (Public Review):
The work by Wenzel and colleagues exhaustively studies the network of interactions between MIT-containing proteins with the MIM containing proteins of the ESCRT-III complex. They discover 16 new interactions and at least one unknown factor interacting with ESCRT-III, CAPN7. Several findings are very interesting and provides essential stepping stones on which the community will continue progressing. In particular, the finding that IST1 clusters a very important network of MIT-containing proteins is reinforcing the view that IST1 is an essential factor of abscission, as proposed by the authors in previous papers. Also, that specific interactions between one ESCRT-III-MIM containing subunit and one MIT.containing partner can be disrupted through single point mutations, not affecting other MIT-MIM interactions is very striking and will provide excellent tools for the community to study the physiological role of these interactions in the future. Finally, the authors continue studying the role in cytokinetic abscission of several of these new interactions, in particular between IST1 and KATNA1, SPASTIN and CAPN7. They convincingly show that these co-factors are required for the completion of cytokinetic abscission, and that they are recruited by ESCRT-III subunits at the midbody. Overall, the study is very well conducted, interesting, providing a lot of essential information for the community working on ESCRT-III.
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Reviewer #2 (Public Review):
Wenzel et al., present a focussed manuscript describing the many possible combinations of MIT-domain/MIM interactions between ESCRT-III subunits and their MIT-domain containing counterparts. Using elegant in-vitro studies, they describe and quantify binding affinities between many of these pairs (some of which have not been previously described), provide new highly detailed structural data describing how some of these interactions (SPASTIN MIT/IST1 MIM) occur. Building on the discovery of these new interactions, they describe MIT/MIM-dependent midbody localisation for new MIT-domain containing factors and their function in abscission, both in unperturbed cells and in cells in which abscission checkpoint signalling had been engaged. The strengths of this manuscript are the quality of the in-vitro data and the …
Reviewer #2 (Public Review):
Wenzel et al., present a focussed manuscript describing the many possible combinations of MIT-domain/MIM interactions between ESCRT-III subunits and their MIT-domain containing counterparts. Using elegant in-vitro studies, they describe and quantify binding affinities between many of these pairs (some of which have not been previously described), provide new highly detailed structural data describing how some of these interactions (SPASTIN MIT/IST1 MIM) occur. Building on the discovery of these new interactions, they describe MIT/MIM-dependent midbody localisation for new MIT-domain containing factors and their function in abscission, both in unperturbed cells and in cells in which abscission checkpoint signalling had been engaged. The strengths of this manuscript are the quality of the in-vitro data and the comprehensive nature of the interactome analysis. The collection of affinities for the various MIT-MIM interactions are valuable, believable and strong. The crystal structures describe well how the IST1 MIM binds SPASTIN's MIT domain and exposes differences between this and how SPASTIN's MIT binds the CHMP1B-MIM. The weaknesses are that besides documenting new players in abscission and/or abscission checkpoint operation, I'm not sure that we are learning much new about how the biological processes studied operate. I think that there is some interesting biology in the manuscript, but the cell biological side currently feels a little underdeveloped.
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Reviewer #3 (Public Review):
In this manuscript, Wenzel et al. have sought to quantitatively understand the mechanism of AAA+ ATPase recruitment at the midbody during cytokinesis through ESCRT-III interactions. All known ESCRT-III proteins (12 in humans) possess MIM motifs, which bind to the MIT motifs of AAA+ ATPases such as VPS4. Additional MIT-domain containing proteins also exist, >20 in humans. An important feature of ESCRT-III proteins is their ability to form polymers - therefore at the midbody or other ESCRT-related locations, multiple MIM motifs (in multiple ESCRT-III) exist which can recruit MIT containing proteins.
While we can hypothesize that multiple MIM-motifs of ESCRT-III proteins may enable higher-avidity interactions to recruit the ATPases, the reason behind the existence of the MIM motifs in all ESCRT-III proteins is …
Reviewer #3 (Public Review):
In this manuscript, Wenzel et al. have sought to quantitatively understand the mechanism of AAA+ ATPase recruitment at the midbody during cytokinesis through ESCRT-III interactions. All known ESCRT-III proteins (12 in humans) possess MIM motifs, which bind to the MIT motifs of AAA+ ATPases such as VPS4. Additional MIT-domain containing proteins also exist, >20 in humans. An important feature of ESCRT-III proteins is their ability to form polymers - therefore at the midbody or other ESCRT-related locations, multiple MIM motifs (in multiple ESCRT-III) exist which can recruit MIT containing proteins.
While we can hypothesize that multiple MIM-motifs of ESCRT-III proteins may enable higher-avidity interactions to recruit the ATPases, the reason behind the existence of the MIM motifs in all ESCRT-III proteins is less intuitive. In this regard, the authors provide a useful resource to the community by quantitating all known MIM-MIT interactions in humans. As ESCRT-related events occur throughout biology, this work should be of broad interest beyond the cytokinesis field.
In addition to the analysis of the binding interactions, the authors also find previously unknown binding interactions between ESCRT-III and three ATPases SPASTIN, KATNA1 and CAPN7. The authors solve the structure of SPASTIN-MIT with IST1-MIM, and compare it to the previously solved structure of SPASTIN-MIT with CHMP1B-MIM. This analysis allowed the authors to create a specific mutation that disrupts SPASTIN's binding to CHMP1B but not to IST1. This mutant still localizes to the midbody compared to a mutant that disrupts binding to both CHMP1B and IST1, suggesting that CHMP1B binding of SPASTIN is dispensable for midbody localization in the presence of IST1 binding.
Furthermore, the authors find that a specific set of ESCRT-III proteins: IST1, CHMP3 and CHMP1B bind promiscuously to many MIT motifs, and provide a structural explanation behind it, owing to multiple binding modes between the MIM of CHMP3 with MIT of KATNA1.
The strength of the work lies in the comprehensive quantitative analysis of the different sets of interactions between MIM and MIT motifs, further structural and cellular correlation of these binding interactions, and the finding of new roles for previously undescribed interactions. The limitations of the work are a lack of acknowledgement/analysis of possible effects of labeled MIM peptides in the quantitation of binding affinities and an unsatisfying description of how the different number of interactions play a role in cytokinetic abscission, and generally in ESCRT-related events.
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