Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission
- Department of Biochemistry, University of Utah School of Medicine, United States
- Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, United States
Figures
Figure 1
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 …
Figure 2 with 6 supplements
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 …
Figure 2—figure supplement 1
Binding isotherms for MIT domains that bind promiscuously to ESCRT-III protein tails.
Representative binding curves for pairwise interactions between MIT domains and ESCRT-III C-terminal tails. Binding isotherms with KD <200 μM are fit with colored curves. Data points for weak and …
Figure 2—figure supplement 2
Binding isotherms for MIT domains that bind specifically to ≤3 different ESCRT-III tails.
Representative binding curves for pairwise interactions between MIT domains and ESCRT-III C-terminal tails. Binding isotherms with KD <200 μM are fit with colored curves. Data points for weak and …
Figure 2—figure supplement 3
USP54 MIT binds weakly to all ESCRT-III C-terminal tails.
Representative binding curves for pairwise interactions between USP54 MIT and ESCRT-III C-terminal tails. Binding isotherms with KD <200 μM are fit with colored curves, whereas non and weak binders …
Figure 2—figure supplement 4
Raw binding data for MIT domains that do not bind any ESCRT-III tails.
Representative raw FP values for pairwise interactions between ESCRT-III C-terminal tails and MIT domains are shown for KATNAL2 (A), NRBF2 (B), and VPS9D1 (C).
Figure 2—figure supplement 5
Competitive binding analyses of CHMP4C tails binding to MIT domains.
(A) Comparison of the C-terminal peptides from different CHMP4 paralogs. Serines are colored red to highlight the unique CHMP4C insert residing between the MIM2 sequence and the ALIX binding site, …
Figure 2—figure supplement 6
ESCRT-III binding is not conserved across ULK family members.
(A) HEK 293T cells were co-transfected with constructs encoding One-Strep-Flag (OSF) ULK3(MIT)2 or ULK1(MIT)2 and the indicated Myc-tagged proteins. OSF-tagged proteins were bound to Strep-Tactin …
-
Figure 2—figure supplement 6—source data 1
Annotated uncropped western blots and raw images for Figure 2—figure supplement 6A and B.
- https://cdn.elifesciences.org/articles/77779/elife-77779-fig2-figsupp6-data1-v1.zip
Figure 3
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 …
Figure 4 with 2 supplements
Identification of MIT-MIM binding mutants.
(A and B) Two orientations of the SPASTIN MIT-IST1 Type 3 structure (PDB 7S7J) displaying the locations of binding mutants (stick representation). See Figure 4—figure supplement 1A and B, for a …
Figure 4—figure supplement 1
Analysis of SPASTIN-IST1 complex and comparisons with SPASTIN-CHMP1B and SPARTIN-IST1.
(A) Fo-Fc IST1 omit map contoured at 3σ overlaid with the SPASTIN-IST1 structure (left) and expanded view (right) highlighting the omit map quality. SPASTIN and IST1 are shown in blue and green …
Figure 4—figure supplement 2
Binding isotherms for WT and mutant SPASTIN MIT domains with the CHMP3 MIM element.
Cartoon inset depicts the position of the groove mutations. Both H1-H3 groove mutations disrupt CHMP3 binding.
Figure 5 with 1 supplement
ESCRT-III-interacting proteins SPASTIN, KATNA1, and CAPN7 are recruited to the midbody and are differentially required for abscission and maintenance of the abscission checkpoint.
Immunofluorescence images of SPASTIN (A, D), KATNA1 (B, E), and CAPN7 (C, F) after treatment with the indicated siRNAs. Checkpoint Active samples (D–F) were generated by additional treatment with …
Figure 5—figure supplement 1
Confirmation of the efficiency and specificity of protein depletion by siRNA treatments.
(A) Western blots from experiments in Figure 5. (B) Western blots using a second, independent siRNA for SPASTIN (SPAS-b), KATNA1 (KATNA1-b), and CAPN7 (CAPN7-b). (C, D) Quantification of abscission …
-
Figure 5—figure supplement 1—source data 1
Annotated uncropped western blots and raw images for Figure 5—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/77779/elife-77779-fig5-figsupp1-data1-v1.zip
Figure 6 with 2 supplements
ESCRT-III binding to SPASTIN, KATNA1, and CAPN7 is required for midbody localization when the abscission checkpoint is sustained.
(A) Immunofluorescence of DOX-inducible cell lines expressing siRNA-resistant mCherry-SPASTIN-WT, mCherry-SPASTIN-F124D, and mCherry-SPASTIN-L177D constructs under sustained abscission checkpoint …
Figure 6—figure supplement 1
Confirmation of the efficiency and specificity of protein depletion by siRNA and DOX-inducible protein expression.
(A–C) Western blots from experiments in Figure 6.
-
Figure 6—figure supplement 1—source data 1
Annotated uncropped western blots and raw images for Figure 6—figure supplement 1 (A–C).
- https://cdn.elifesciences.org/articles/77779/elife-77779-fig6-figsupp1-data1-v1.zip
Figure 6—figure supplement 2
The KATNA1 V55D mutation does not disrupt KATNB1 binding.
(A) Structure of KATNA1 MIT domain showing locations of V55 (cyan, stick) and R14 (red, stick). Left image shows the MIT domain alone and right image shows the MIT domain in complex with KATNB1 (PDB …
-
Figure 6—figure supplement 2—source data 1
Annotated and uncropped western blots and raw images for Figure 6—figure supplement 2B.
- https://cdn.elifesciences.org/articles/77779/elife-77779-fig6-figsupp2-data1-v1.zip
Author response image 1
Cells were treated as in Manuscript Figure 5D-H (active checkpoint), fixed in either PFT or Methanol and stained with KATNA1-specific antibodies from Proteintech or Abcam.
Two examples from each treatment are shown.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Hela-N | Maureen Powers Lab | HeLa cells selected for transfectability | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 | |
| Antibody | Anti-CAPN7 (Rabbit polyclonal) | Proteintech | Cat#26985–1-AP | IF (1:1000) WB (1:5000) |
| Antibody | Anti-CEP55 (Sheep polyclonal) | Bastos and Barr, 2010 | IF (1:3500) | |
| Antibody | Anti-IST1 (Rabbit polyclonal) | Sundquist Lab/Covance | UT560 | WB (1:1000) |
| Antibody | Anti-CHMP4C (Rabbit polyclonal) | Sadler et al., 2018 | WB (1:500) | |
| Antibody | Anti-KATNA1 (Rabbit polyclonal) | Proteintech | 17560–1-AP | IF (1:1000) |
| Antibody | Anti-KATNA1 (Rabbit polyclonal) | Abcam | ab111881 | IF (1:500) WB (1:1000) |
| Antibody | Anti-SPASTIN (Mouse monoclonal) | Sigma | S7074 | IF (1:1000) WB (1:1000) |
| Antibody | Anti-NUP153 (SA1) (Mouse monoclonal) | Brian Burke | WB (1:50) | |
| Antibody | Anti-NUP50 (Rabbit polyclonal) | Mackay et al., 2010 | WB (1:2500) | |
| Sequence-based reagent | siNT | Mackay et al., 2010 | siRNA | GCAAAUCUCCGAUCGUAGA |
| Sequence-based reagent | siCHMP4C | Strohacker et al., 2021 | siRNA | CACUCAGAUUGAUGGCACA |
| Sequence-based reagent | sIST1 | Bajorek et al., 2009a | siRNA | AGAUACCUGAUUGAAAUUG |
| Sequence-based reagent | siNUP153 | Mackay et al., 2010 | siRNA | GGACUUGUUAGAUCUAGUU |
| Sequence-based reagent | siNUP50 | Mackay et al., 2010 | siRNA | GGAGGACGCUUUUCUGGAU |
| Sequence-based reagent | siCAPN7 | This Paper | siRNA | GCACCCAUACCUUUACAUU |
| Sequence-based reagent | siCAPN7-b | This Paper | siRNA | GGCCGUUACUGAUUGAGCU |
| Sequence-based reagent | siKATNA1 | This Paper | siRNA | GGACAGCACUCCCUUGAAA |
| Sequence-based reagent | siKATNA1-b | Horizon Discovery | CAT# L-005157 | ON-TARGET-PLUS siRNA- SMARTPOOL |
| Sequence-based reagent | siSPAS | This Paper | siRNA | GAACAGUGUGAAAGAGCUA |
| Sequence-based reagent | siSPAS-b | This Paper | siRNA | CGUUAUUGAUACUUGGAUA |
| Chemical compound, drug | Thymidine | CalBiochem | CAS 50-89-5 | 2 mM |
| Chemical compound | Oregon Green 488 maleimide | Life Technologies/Molecular Probes | O6034 | Fluorescent label for peptides |
| Software, algorithm | Fiji | NIH | RRID:SCR_002285 | |
| Software, algorithm | KaleidaGraph | Synergy Software |
Author response table 1
| MIT Domain | ESCRT-III Tail | KD (direct binding) (µM) | KI competition experiment (µM) |
|---|---|---|---|
| MITD1 | CHMP4B | 15 ± 1 (n≥3) | 21.7 ± 0.4 (n≥3) |
| USP8 | CHMP4B | 11.7 ± 0.9 (n≥3) | 38.9 ± 0.3 (n=2; error is the range of the measurement) |
| USP8 | IST1-MIM1/3 | 0.6 ± 0.01 (n≥3) | 1.1 ±- 0.2 (n=2; error is the range of the measurement) |
Author response table 2
| MIT Domain | ESCRT-III Tail | KD (N-terminal Fluor) (µM) | KD (C-terminal Fluor) (µM) |
|---|---|---|---|
| CAPN7 | IST1 | 0.07 ± 0.02 (n≥3) | 0.09 ± 0.01 (n=3) |
Author response table 3
| MIT Domain | ESCRT-III Tail | KD (FP) (µM) | KD (SPR) (µM) |
|---|---|---|---|
| SPASTIN | IST1 | 0.5 ± 0.1 (n≥ 3) | 0.57 ± 0.08 (n=2) |
Additional files
-
Supplementary file 1
MIT domain proteins and ESCRT-III peptides used for the fluorescence polarization screen and X-ray crystallography.
(A) MIT domain protein constructs used in our screen. (B) Fluorescently labeled ESCRT-III C-terminal tails used in the fluorescent polarization screen. (C) Unlabeled ESCRT-III C-terminal peptide tails used for competition experiments and structural biology.
- https://cdn.elifesciences.org/articles/77779/elife-77779-supp1-v1.docx
-
Supplementary file 2
Plasmids, siRNA and antibodies used in this study.
(A) Plasmids, (B), siRNA sequences, and (C) antibodies used for this study.
- https://cdn.elifesciences.org/articles/77779/elife-77779-supp2-v1.docx
-
Supplementary file 3
SPASTIN MIT-IST1 complex data collection and refinement statistics.
- https://cdn.elifesciences.org/articles/77779/elife-77779-supp3-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/77779/elife-77779-transrepform1-v1.pdf
