Author response: Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation The importance of the information provided to the community, their quality and the new questions they open will certainly make this paper an essential step forward in understanding the regulation of ESCRT-III. https://doi.org/10.7554/eLife.77779.sa0 Decision letter Reviews on Sciety eLife's review process Introduction To complete cell division, a cell must equally and fully partition a faithfully duplicated genome into two nascent progeny cells through mitosis, and these cells must physically separate through cytokinesis. In animal cells, cytokinesis initiates when an actomyosin contractile ring ingresses to create a cleavage furrow between the two spindle poles following chromosome segregation (Addi et al., 2018). The plasma membrane furrow ultimately narrows into an intercellular membrane bridge containing a dense central array of microtubules (the midbody), which then undergoes abscission to create two new cells. Premature abscission in the presence of aberrantly segregated DNA can lead to tension-induced double strand breaks in chromatin bridges that traverse the cleavage furrow (Janssen et al., 2011), chromosome instability (Umbreit et al., 2020), and formation of micronuclei and extensive DNA damage through chromothripsis (Zhang et al., 2015; Crasta et al., 2012). Hence, abscission is required for cell proliferation and its misregulation can contribute to DNA damage and even cancer development (Pharoah et al., 2013; Sadler et al., 2018). The ESCRT (Endosomal Sorting Complexes Required for Transport) pathway mediates abscission in cultured mammalian cells (Carlton and Martin-Serrano, 2007; Morita et al., 2007), and in neural progenitor cells in vivo (Tedeschi et al., 2020; Little et al., 2021). Approximately 30 human ESCRT factors comprise five subcomplexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and VPS4, which assemble sequentially to constrict and sever membranes (McCullough et al., 2018). During cytokinesis, ESCRT complexes are recruited to the central Flemming body within the midbody by the CEP55 adaptor protein. CEP55 binds the early-acting ESCRT protein, ALIX (Lee et al., 2008; Christ et al., 2016; Morita et al., 2007; Carlton and Martin-Serrano, 2007), which in turn recruits ESCRT-III subunits of the CHMP4 family (Christ et al., 2016; McCullough et al., 2008; Katoh et al., 2003; Katoh et al., 2004; Kim et al., 2005). In a parallel pathway, CEP55 binds the TSG101 subunit of ESCRT-I/II complexes (Morita et al., 2007; Carlton and Martin-Serrano, 2007; Christ et al., 2016; Lee et al., 2008), again leading to ESCRT-III recruitment. ESCRT-III recruitment is also promoted by the midbody protein SEPT9 (Karasmanis et al., 2019), and other CEP55-independent pathways (Merigliano et al., 2021; Addi et al., 2020). Humans express 12 homologous ESCRT-III proteins that fall into eight different families, termed CHMP1-7 and IST1. The different ESCRT-III subunits co-polymerize to form spiraling filaments within the midbody (Guizetti et al., 2011; Mierzwa et al., 2017; Pfitzner et al., 2021; Nguyen et al., 2020; Elia et al., 2011). These filaments recruit VPS4 AAA+ ATPases that in turn promote dynamic ESCRT-III subunit exchange, midbody constriction, membrane fission, and daughter cell separation (Pfitzner et al., 2021; Mierzwa et al., 2017; Elia et al., 2012; Pfitzner et al., 2020). Abscission timing and progression are regulated by the evolutionarily conserved abscission/NoCut cell cycle checkpoint (Norden et al., 2006; Steigemann et al., 2009). This checkpoint pauses abscission progression so that upstream mitotic events can be completed correctly before the cell irreversibly separates. Several different conditions are known to sustain abscission checkpoint activation, including incompletely segregated DNA within the midbody (Norden et al., 2006; Mendoza and Barral, 2008; Amaral et al., 2016), nuclear pore subunit depletion (Mackay et al., 2010), DNA damage resulting from replication stress (Mackay and Ullman, 2015), and midbody tension (Lafaurie-Janvore et al., 2013; Strohacker et al., 2021; Caballe et al., 2015). These conditions all converge on phosphorylated Aurora B (pAurB) kinase, which maintains checkpoint signaling and inhibits ESCRT activity. AurB is targeted to the intercellular bridge as a component of the multi-protein Chromosomal Passenger Complex (CPC), where it phosphorylates multiple substrates, including the regulatory ESCRT-III protein CHMP4C (pCHMP4C) (Carlton et al., 2012; Capalbo et al., 2012; Steigemann et al., 2009). pCHMP4C associates with ANCHR, and this complex sequesters VPS4 AAA+ ATPases from abscission sites, thereby inhibiting abscission progression (Thoresen et al., 2014). pAurB and pCHMP4C also localize to cytoplasmic Abscission Checkpoint Bodies (ACBs), where pro-abscission machinery, including ALIX and ESCRT-III proteins, are sequestered from the midbody (Strohacker et al., 2021). The importance of the abscission checkpoint and its regulation by CHMP4C is underscored by the discovery that a missense mutation in the penultimate CHMP4C residue (A232T) disrupts ALIX binding, inactivates the checkpoint, and predisposes carriers to several types of cancers (Sadler et al., 2018; Pharoah et al., 2013). In addition to constricting the midbody, ESCRT-III filaments also recruit cofactors that contribute to abscission and checkpoint regulation. Human ESCRT-III proteins have conserved helical core domains that mediate filament formation (McCullough et al., 2018; Pfitzner et al., 2021; Bajorek et al., 2009b), and variable C-terminal tails that contain short peptide elements called MIT-Interacting Motifs (MIMs) (Figure 1A). MIMs bind cofactors that contain MIT (Microtubule-Interacting and Trafficking) domains (Hurley and Yang, 2008; Figure 1B). MIT domains are simple three helix bundles, but they can bind MIM elements in at least seven distinct ways (Skalicky et al., 2012; Obita et al., 2007; Scott et al., 2005; Stuchell-Brereton et al., 2007; Kieffer et al., 2008, Yang et al., 2008; Solomons et al., 2011; Fujioka et al., 2014), which we have termed Type 1–7 binding modes (Figure 1C). Figure 1 Download asset Open asset Domain organization of ESCRT-III and MIT domain-containing proteins. (A) Generalized ESCRT-III schematic, depicting the conserved helical core domain and the variable C-terminal tail that contains MIT-interacting Motif (MIM) elements. (B) Human proteins with MIT domains, grouped by enzymatic categories. Three dimensional structures (PDB IDs in parenthesis) are available for the MIT domains from human VPS4A (1YXR, 2JQ9, 2K3W), VPS4B (2JQH, 2JQK, 4U7Y, 1WRO, 2CPT), SPASTIN (3EAB), USP8 (2A9U), AMSH (2XZE), NRBF2 (4ZEY), LIP5 (2LXL, 2LXM, 4TXP, 4TXQ, 4TXR, 4U7E), SPARTIN (4U7I), MITD1 (4A5X), and ULK3 MIT2 (4WZX). Abbreviations: MIT, Microtubule Interacting and Trafficking; MTB, Microtubule Binding Domain; S/T Kinase, Serine/Threonine Kinase domain; PSK, Pseudokinase domain; PX, Phosphoinositide binding domain; CysPc, Calpain protease domain; USP, Ubiquitin Specific Protease Domain; PLD, Phospholipase D-like domain; VSL, Vta1-SBP1-LIP5 domain; JAMM, JAB1/MPN/Mov34 metalloenzyme domain; CBSW, calpain-type beta-sandwich domain. (C) Gallery showing the different types of binding interactions formed between MIT domains (blue) and MIM elements (green). For clarity, the three helix bundles of the different MIT domains are shown in approximately equivalent orientations. Type 1 interaction: a helical MIM binds in the groove between MIT helices 2 and 3 (H2/H3 groove), and is oriented parallel to MIT helix 3. Type 2 interaction: the MIM forms an extended strand that binds in the MIT H1/H3 groove, parallel to MIT helix 3. Type 3 interaction: a helical MIM binds in the MIT H1/3 groove, parallel to MIT helix 3. Type 4 interaction: Similar to Type 1, except that the longer MIM helix binds lower in the H2/H3 groove and interacts with the H2/H3 loop. Type 5 interaction: Two MIM helices and adjacent linkers wrap nearly completely around the MIT domain. Type 6 interaction: a mixed helix/strand MIM element binds in the MIT H1/3 groove, antiparallel to MIT helix 3. Type 7 interaction: a helical MIM element binds in the MIT 2/3 groove, antiparallel to helix 3. The specific complexes shown in the figure are labeled below, together with their PDB accession codes. Humans express more than 20 MIT domain-containing proteins, which also contain a variety of associated activities that could function in cytokinesis (Figure 1B). Well-characterized examples of MIT domain-containing proteins that bind ESCRT-III proteins and perform important midbody functions include: (1) VPS4. The archetypal MIT domains of the related VPS4A and VPS4B ATPases bind promiscuously to different ESCRT-III filaments. These interactions promote assembly of VPS4 hexamers and activate ATPase activity (Han et al., 2015; Azmi et al., 2008; Norgan et al., 2013). VPS4 MIT domains can alternatively bind a MIM element within ANCHR, which sequesters the ATPases from the abscission zone (Thoresen et al., 2014). (2) ULK3 kinase. The second ULK3 MIT domain binds IST1, which localizes this kinase to the midbody where it phosphorylates ESCRT-III proteins and inhibits membrane fission (Caballe et al., 2015). (3) SPASTIN. The MIT domain of the microtubule severing AAA+ ATPase SPASTIN binds CHMP1B (Yang et al., 2008), which reportedly localizes the enzyme to the midbody where it clears spindle microtubules to allow abscission (Connell et al., 2009). (4) MITD1. The MIT domain of MITD1 binds preferentially to CHMP1A, CHMP1B, CHMP2A and IST1 and localizes this phospholipase-D family member to midbodies where it stabilizes the bridge, preventing abscission failure (Hadders et al., 2012; Lee et al., 2012). (5) SPARTIN. The MIT domain of the hereditary spastic paraplegia protein SPARTIN preferentially binds IST1, which localizes the protein to the midbody to support abscission (Renvoisé et al., 2010). The 12 ESCRT-III and >20 MIT human proteins have the potential to form a complex combinatorial network of ESCRT-III-MIT interactions that help mediate and regulate abscission. However, the ESCRT binding and cytokinetic functions of many human MIT proteins have not yet been tested, and prediction of MIT-MIM interactions can be difficult owing to their remarkable variety of different possible binding modes (Figure 1C). To address these limitations, we systematically quantified the binding of all 12 human ESCRT-III MIM elements to nearly all known human MIT domains. Three MIT enzymes with interesting activities and ESCRT-III binding patterns were further tested for midbody localization, and roles in cytokinetic abscission and the abscission checkpoint. These studies revealed that SPASTIN, KATANIN-P60 (KATNA1), and CALPAIN-7 (CAPN7) all localize to midbodies through specific MIT-ESCRT-III interactions and are required for efficient abscission, and that SPASTIN and CAPN7 are also required for abscission checkpoint maintenance. These studies define the human MIT-ESCRT-III interactome and identify new factors and activities required for cytokinetic abscission and its regulation. Results MIT-MIM interaction screen We defined the ESCRT-III-MIT interactome by using fluorescence polarization anisotropy (FP) to quantify the binding interactions between recombinant human MIT domains and ESCRT-III MIM peptides. These experiments employed fluorescently labeled C-terminal peptides (52–88 residues) that encompass the known MIM elements of the 12 human ESCRT-III proteins (Figure 1A, Supplementary file 1B). The ESCRT-III tails were fluorescently labeled at their N-termini. Literature analyses were used to identify 21 candidate human MIT domains (Ciccarelli et al., 2003; Rigden et al., 2009; Skalicky et al., 2012; Xiao et al., 2008; Fujioka et al., 2014; Figure 1B; see Materials and methods), which were expressed as recombinant proteins and purified to homogeneity. Constructs encoding the tandem MIT domains of LIP5, CAPN7, and ULK3 spanned both domains, thereby allowing intramolecular interactions to occur and native structures to form. (Skalicky et al., 2012; Vild et al., 2015; Guo and Xu, 2015). 19/21 of the human MIT domain constructs could be purified in sufficient quantities for FP binding titrations (Figure 2). The two exceptions were the C-terminal tandem MIT domains of ULK1 and ULK2, which function in autophagy (Fujioka et al., 2014) and apparently do not bind ESCRT-III proteins (see below). Figure 2 with 6 supplements see all Download asset Open asset ESCRT-III-MIT domain interaction network. Equilibrium dissociation constants (μM) for pairwise binding interactions are displayed for each ESCRT-III-MIT pair and are reported as averages of at least three independent measurements. Values in parenthesis denote ± standard deviation. Interactions are color-coded based on strength of measured binding affinity (see key bar). Asterisks denote KI values that were determined from competition experiments (see Materials and methods and Figure 2—figure supplement 5B-E). Binding constants for ULK3(MIT)2 and IST1 are from Caballe et al., 2015, but were measured in the same fashion as the other interactions and are reproduced here for comparison. Pairwise FP binding isotherms were fit to 1:1 equilibrium binding models (Figure 2, Figure 2—figure supplements 1–4, Figure 3). Dissociation constants (KD) for the 228 interactions tested are summarized in Figure 2 and color-coded based upon interaction strengths. Our screen generally recapitulated binding interactions reported previously using orthogonal techniques (See Supplemental Discussion), but we cannot rule out the possibility that in some cases the position of the label could have interfered with or artificially enhanced binding interactions for some ESCRT-III-MIT pairs. Pairwise binding isotherms for weak binding pairs often did not reach saturation (Figure 2—figure supplements 1–3), and the KDs for those interactions should therefore be considered approximations. In cases where isotherms did not reach half saturation at the highest MIT concentration tested (usually 100 µM), we did not attempt to estimate the KD. Figure 3 Download asset Open asset MIT-MIM Binding Interactions of SPASTIN, KATNA1 and CAPN7 and IST1. (A) Binding isotherms from human ESCRT-III C-terminal tails and the MIT domains from SPASTIN (left), KATNA1 (middle), and CAPN7 (right). Representative binding data are shown for each ESCRT-III-MIT pairwise binding interaction. Binding isotherms with KD <200 µM are fit with colored curves. Weak and non-binders (KD >200 µM) are plotted as black and white points. (B) Domain schematic of the ESCRT-III protein IST1 showing the position of the Type 2, MIM316-343 and Type 1/3, MIM344-366 elements within the full-length protein. Data for the ‘IST1’ peptide encompasses residues 316–366. (C) Dissociation constants for MIM344-366 and MIM316-343 peptides with IST1-binding MIT domains. Dissociation constants shown are averages calculated from at least three independent experiments with standard deviation reported in parentheses. Interactions are color-coded based on strength of measured binding affinity (see key bar). Asterisk denotes KI values determined from competition experiments (see Materials and methods and Figure 2—figure supplement 5E). Binding data for ULK3(MIT)2 and IST1, IST1-MIM344-366 and IST1-MIM316-366 are from Caballe et al., 2015, but are reproduced here for comparison. Our binding survey confirmed a series of previously reported ESCRT-III-MIT domain interactions (See Supplemental discussion), and also revealed 18 previously unreported interactions (Figure 2), including establishing new direct links between ESCRT-III proteins and the MIT domains from KATNA1, KATNAL1, RPS6KC1, RPS6KL1, SNX15, and AMSHLP. These new interactions should facilitate future investigations of ESCRT-associated activities. ESCRT-III binding behaviors MIT domains displayed a range of different ESCRT-III binding behaviors that we classified as promiscuous (>3 ESCRT-III binding partners) (Figure 2—figure supplement 1), specific (≤3 ESCRT-III binding partners) (Figure 3A, Figure 2—figure supplement 2), non-specific (Figure 2—figure supplement 3), and non-binding (Figure 2—figure supplement 4). The seven promiscuous binders bound ESCRT-III tails with high (KD <2 µM) or moderate (KD <100 µM) affinities. The MIT domain of the deubiquitinase AMSH exhibited the greatest promiscuity, binding all but two of the 12 different ESCRT-III subunits. The eight specific binders interacted with CHMP3 (KATNA1, KATNAL1, RPS6KL1, RPS6KC1, and AMSHLP), IST1 (RPS6KC1, CAPN7, SNX15, and SPARTIN), or CHMP1B, CHMP3, and IST1 (SPASTIN). The predicted MIT domain of the catalytically inactive deubiquitinase, USP54 (Rigden et al., 2009), bound weakly (KD >100 µM) to nearly all ESCRT-III proteins, indicating that these interactions were probably non-specific (Figure 2—figure supplement 3). Finally, two MIT domains (NRBF2 and VPS9D1) did not bind any ESCRT-III proteins (Figure 2—figure supplement 4). The MIT domain of NRBF2 has been characterized structurally (PDB 4ZEY, 2CRB) and shown to interact with the VPS15 kinase to promote autophagosome biogenesis (Young et al., 2019). The putative VPS9D1 MIT domain was predicted through bioinformatics, has not been characterized structurally, and has no known ESCRT connections (Rigden et al., 2009). The tightest MIM-MIT binding pair was LIP5-CHMP5 (KD = 2.5 nM), which reflects the fact that CHMP5 tail helices 5 and 6 and adjacent linkers form an amphipathic ‘leucine collar’ that wraps almost completely around the second LIP5 MIT domain (Figure 1C; Skalicky et al., 2012). The ESCRT-III proteins IST1, CHMP3, and CHMP1B partnered with the largest subset of MIT domains (Figure 2). IST1 bound 11/19 MIT domains with high affinity, and this promiscuity likely reflects the presence of two MIMs in the IST1 tail; a helical Type 1 or Type 3 (Type 1/3) element and an extended Type 2 element et al., Guo and Xu, 2015; Figure To the of each IST1 MIM element to we tested their binding to each of the MIT domains, using peptides MIM (Figure and These experiments revealed that the IST1 Type element bound MIT domains, and the Type 2 element bound MIT domains. Three binding patterns for the IST1 MIMs with MIT domains (1) the Type element (SPASTIN, and SPARTIN), (2) the Type 2 element or (3) both MIMs CAPN7, SNX15, LIP5, and (Figure we did not binding between the IST1 Type 2 element and the LIP5 and CAPN7 MIT domains, the IST1 Type 2 element to contribute to LIP5 and CAPN7 binding its binding affinity the IST1 both IST1 MIM elements can bind MIT domains, thereby to both MIM elements can MIT proteins in some thereby binding affinity et al., Bajorek et al., the other of the a fluorescently labeled CHMP4C peptide did not bind to any MIT domain This was as and AMSH all bound the other two CHMP4 and (Figure 2). CHMP4C is in containing a that is phosphorylated by AurB (Carlton et al., 2012; Capalbo et al., 2012; Figure 2—figure supplement We that this the fluorescence anisotropy in bound thereby To this we the CHMP4C peptide could MITD1 and USP8 MIT binding to labeled (Figure 2—figure supplement These experiments revealed that CHMP4C bound AMSH MIT µM), but to bind MITD1 or CHMP4C a in abscission checkpoint regulation (Carlton et al., 2012; Capalbo et al., and this function have the to some MIT binding interactions. we were to express and the ULK1 or tandem MIT domains in sufficient quantities for direct binding we it was important to the MIT domains from these related could bind ESCRT-III proteins (1) the MIT domains of and human bind within autophagosome complexes et al., 2009; Fujioka et al., 2014), and (2) the MIT domain of the related ULK3 kinase binds to IST1, and weakly to CHMP1A, CHMP1B, and CHMP3 (Caballe et al., and (Figure 2). We therefore from human cell to could bind ESCRT-III proteins (Figure 2—figure supplement and recapitulated the known interactions, as as However, did not bind to any of the ESCRT-III binding partners of that these ESCRT-III interactions are not conserved in the family of protein In MIT proteins can ESCRT-III proteins and their interactions a range of and indicating that they to ESCRT pathway functions in a variety of different SPASTIN, KATNA1, and CAPN7 as for MIT-ESCRT-III interactions Three MIT proteins with specific ESCRT-III interactions and interesting associated enzymatic domains were for further (1) SPASTIN CHMP1B, and CHMP3 (2) KATNA1 binding and (3) CAPN7 binding (Figure ESCRT-III interactions with SPASTIN et al., 2005; Yang et al., 2008; et al., and CAPN7 et al., 2008; et al., have been SPASTIN and KATNA1 binding to CHMP3 have The MIT domain of SPASTIN bound to the IST1 MIM344-366 (Type and CHMP1B (Type The CAPN7 tandem MIT domain binds both MIM elements et al., 2010), and in this multiple different binding modes are SPASTIN and KATNA1 are microtubule severing AAA+ ATPases and 2012). have been in midbody microtubules to abscission (Yang et al., 2008; et al., 2009; et al., 2012; et al., but has been to abscission checkpoint activity. CAPN7 is an protease that has not previously been to any cytokinetic and of the SPASTIN complex The SPASTIN MIT domain is in binding to three different ESCRT-III CHMP1B, CHMP3, and IST1. The SPASTIN complex has been characterized structurally (Yang et al., 2008; Figure the SPASTIN and interactions have We determined a high crystal structure of the SPASTIN complex using a SPASTIN (Figure and Figure supplement and and Supplementary file PDB The structure revealed that the IST1 MIM344-366 an amphipathic helix that its in the SPASTIN MIT H1/H3 groove (Type 3 binding, Figure Figure supplement The IST1 and CHMP1B MIM elements binding modes (Figure but with two (Figure supplement (1) The longer CHMP1B helix three the IST1 and (2) the MIT H1/H3 groove to the longer CHMP1B with a of 2.5 at the of helix 3. The structure of is nearly to which also a Type 3 interaction and Xu, 2015; Figure supplement IST1 binding is than CHMP1B binding, likely owing to enhanced interactions with the SPASTIN H1/H3 groove (Figure supplement 1C). IST1 two and two the CHMP1B binding element residues and three Figure 4 with 2 supplements see all Download asset Open asset of MIT-MIM binding and Two of the SPASTIN Type 3 structure (PDB the of binding Figure supplement and for a of the and Supplementary file 3 for data and structure (C) depicting the of mutation sites in the SPASTIN MIT H1/H3 structure (PDB showing the of binding Figure supplement for an of both SPASTIN Binding isotherms for and MIT domains with the MIM elements. KATNA1-CHMP3, and For and binding was measured using IST1 peptides of both MIM elements MIT groove binding within the of in the MIT domain. also Figure supplement 2 for binding isotherms for and SPASTIN MIT domains with the CHMP3 MIM on we SPASTIN MIT to the binding of both CHMP1B and IST1 in Figure and to CHMP1B binding IST1 binding in Figure shown in Figure and these as thereby a of that we could to the of SPASTIN binding to both CHMP1B and IST1 binding to CHMP1B CHMP3 binding was also by both SPASTIN and CHMP3 also likely binds as an extended Type 3 helix in the SPASTIN MIT H1/H3 groove (Figure supplement 2), and IST1 binding to the SPASTIN owing to its Type 3 KATNA1 and CAPN7 binding to ESCRT-III proteins We also for MIT that could ESCRT-III binding to KATNA1 and CAPN7 by a series of in the MIT H1/H3 and H2/H3 and these binding in fluorescence polarization anisotropy binding In the a mutation in the MIT H1/H3 groove CHMP3 binding (Figure Our data therefore again a Type 3 interaction for the helical CHMP3 MIM CAPN7 has tandem MIT domains, and we that IST1 binding was by in the H1/H3 groove of the or second CAPN7 MIT domains (Figure with MIM experiments (Figure these data that both CAPN7 MIT domains both IST1 MIM Type 2 and Type 3 binding SPASTIN, KATNA1, and CAPN7 localize to midbodies an screen for cytokinetic we SPASTIN, KATNA1, CAPN7 localize to the midbodies of cells (Figure were for microtubules and which is recruited to of the Flemming body where it initiates ESCRT complex assembly et al., 2005; Carlton and Martin-Serrano, 2007; Morita et al., was confirmed in parallel experiments in which each MIT protein was using (Figure Figure 5 with 1 supplement see all Download asset Open asset
Source: eLife
Institutions: University of Utah, Huntsman Cancer Institute
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
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