The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of 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 with 4 supplements
CAPN7 binds IST1 through tandem microtubule-interacting and trafficking (MIT) domains.
(A) Domain organization of CAPN7 and IST1, depicting the binding interaction between tandem MIT domains of CAPN7 and MIT-interacting motif (MIM) elements of IST1 (red double-headed arrows). Domain …
Figure 1—figure supplement 1
NMR titration of 15N-labeled IST1314-343 with unlabeled CAPN71-75.
(A) Overlaid 2D [1H,15N]-HSQC NMR spectra showing 15N-labeled IST1314-343 resonances across a CAPN71-75 titration. Resonances are color coded from blue (no CAPN71-75) to red (five molar equivalents …
Figure 1—figure supplement 2
NMR spectra of free and CAPN7(MIT)2-bound 15N-labeled IST1303-366.
Overlaid 2D [15N,1H]-HSQC spectra from uniformly 15N-labeled IST1303-366 in the absence (black contours) and presence (orange contours) of a 1.3-fold molar excess of CAPN7(MIT)2. Resonance color …
Figure 1—figure supplement 3
CAPN7(MIT)2 binds equally well to IST1316-366 and the minimal IST1322-366 construct.
Fluorescence polarization anisotropy binding isotherms showing CAPN7(MIT)2 binding to fluorescently labeled peptides spanning IST1316-366 or the minimal construct defined by NMR chemical shift …
Figure 1—figure supplement 4
Raw fluorescence polarization anisotropy binding isotherms, best-fit models, and associated statistics corresponding to the normalized binding isotherms presented in Figure 1B.
Figure 2 with 3 supplements
Crystal structure of the CAPN7(MIT)2-IST1322-366 complex.
(A) Ribbon representation of CAPN7(MIT)2 (blue) in complex with IST1322-366 (green, with buried core interface sidechains shown) (PDB 8UC6). Locations of residues that were mutated in CAPN7(MIT)2 …
Figure 2—figure supplement 1
Unbiased electron density omit map for IST1322-366.
(A) IST1324-332 Fo-Fc omit map (magenta, contoured at 2.0 σ) overlaid with the CAPN74-68-IST1324-332 complex. The CAPN7 Val18 residue is highlighted in magenta. (B) IST1349-364 Fo-Fc omit map …
Figure 2—figure supplement 2
Alignment of IST1325-336 and CHMP6168-179 in the CAPN74-68 binding groove.
(A) Structural alignment of the CAPN74-68–IST1324-332 complex (gray and green) with CHMP6168-179 (light blue) from the VPS4A3-75-CHMP6168-179 complex (PDB 2K3W). CAPN74-68 is shown as a surface …
Figure 2—figure supplement 3
Binding isotherms for individual CAPN7 microtubule-interacting and trafficking (MIT) domains binding to individual IST1 MIT-interacting motif (MIM) elements.
Fluorescence polarization anisotropy binding isotherms show individual CAPN7 MIT domains binding to individual fluorescently labeled IST1 peptides spanning the N-terminal (IST1316-343) or C-terminal …
Figure 3 with 3 supplements
Mutational analyses of the CAPN7-IST1 complex.
(A) Fluorescence polarization anisotropy binding isotherms showing CAPN7(MIT)2 constructs binding to IST1316-366. Isotherm data points and dissociation constants are means from three independent …
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Figure 3—source data 1
Annotated and uncropped Coomassie-stained SDS-PAGE for Figure 3B.
- https://cdn.elifesciences.org/articles/84515/elife-84515-fig3-data1-v2.zip
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Figure 3—source data 2
Annotated and uncropped western blots and raw images for Figure 3C.
- https://cdn.elifesciences.org/articles/84515/elife-84515-fig3-data2-v2.zip
Figure 3—figure supplement 1
Circular dichroism spectra of recombinant CAPN7(MIT)2 proteins and co-immunoprecipitation of full-length CAPN7 and full-length IST1 from cells.
(A) Circular dichroism spectra of purified, recombinant wt and mutant CAPN7(MIT)2 proteins. Spectra are displayed as the mean of triplicate measurements. (B) Co-immunoprecipitation of full-length …
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Figure 3—figure supplement 1—source data 1
Annotated and uncropped western blots and raw images for Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/84515/elife-84515-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
Size-exclusion chromatographic analyses of CAPN7 and IST1 complex formation.
(A) Calibration and individual protein chromatograms showing the elution volume of each peak and its corresponding molecular weight estimation from the calibration on a S200 Superdex column. Note …
Figure 3—figure supplement 3
CAPN7(MIT)2 binding to IST1316-366 is diminished by mutations in either IST1 MIT-interacting motif (MIM) element.
Fluorescence polarization anisotropy binding isotherms showing CAPN7(MIT)2 binding to a fluorescently labeled wt IST1 peptide spanning both MIM elements (IST1316-366), or to IST316-366 peptides with …
Figure 4 with 1 supplement
IST1 binding is required for CAPN7 midbody localization.
(A) Representative immunofluorescence images showing the extent of midbody colocalization of mCherry-CAPN7 constructs (or the mCherry control), with endogenous IST1 in synchronous, NoCut …
Figure 4—figure supplement 1
Western blot confirmation of rescue construct expression and siRNA knockdown efficiency.
Western blot analyses showing CAPN7-mCherry protein expression levels and knockdown efficiencies of endogenous CAPN7 and Nup153 in the experiments shown in Figure 4A and B. siNT is a non-targeting …
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Figure 4—figure supplement 1—source data 1
Annotated and uncropped western blots and raw images for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/84515/elife-84515-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
IST1-binding and catalytic activity are required for CAPN7 abscission and NoCut functions.
(A, C) Quantification of midbody-stage and multinucleate HeLa cells from unperturbed asynchronous cultures (A), or cells in which NoCut checkpoint activity was sustained by Nup153 depletion (C). (B, …
Figure 5—figure supplement 1
Western blot confirmation of rescue construct expression and siRNA knockdown efficiency.
(A, B) Western blot analyses showing mCherry-CAPN7 protein expression levels and knockdown efficiencies of endogenous CAPN7 and Nup153 in the experiments shown in Figure 5A and C, respectively.
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Figure 5—figure supplement 1—source data 1
Annotated and uncropped western blots and raw images for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/84515/elife-84515-fig5-figsupp1-data1-v2.zip
Figure 6
CAPN7 and SPAST are required to maintain the NoCut checkpoint in response to DNA bridges and replication stress.
Quantification of midbody-stage and multinucleate HeLa cells from asynchronous cultures in which NoCut checkpoint activity was sustained by inducing DNA bridges with ICRF-193 treatment (A) or …
Author response image 1
Mock figure showing the structure model presented in the manuscript, overlaid with another possible model with alternative connectivity between the two copies of the complex within the crystallographic asymmetric unit.
The two structures were aligned to the first MIT domain to highlight the divergence between possible interdomain linkers and C-terminal domain positions.
Tables
Table 1
CAPN7(MIT)2–IST1322-366 complex (PDB: 8UC6) crystallographic data and refinement statistics.
| Data collection, integration, and scaling | |
|---|---|
| Programs | XDS, AIMLESS |
| Source/wavelength (Å) | SSRL 14–1/1.19499 |
| Space group (unit cell dimensions) | P6522 (87.84, 87.84, 183.89, 90.0, 90.0, 120.0) |
| Resolution (high-resolution shell) (Å) | 40.0–2.70 (2.83–2.70) |
| # reflections measured | 1,398,023 |
| # unique reflections | 12,228 |
| Redundancy (high-resolution shell) | 114 (104) |
| Completeness (high-resolution shell) (%) | 100.0 (99.9) |
| <I/σI> (high-resolution shell) | 11.0 (1.5) |
| <CC1/2> | 0.998 (0.650) |
| Rpim (high-resolution shell) | 0.080 (0.666) |
| Mosaicity (°) | 0.12 |
| Refinement | |
| Program | Phenix.refine |
| Resolution (Å) | 40.0–2.70 |
| Resolution (Å) – (high-resolution shell) | (2.81–2.70) |
| # reflections | 12,169 |
| # reflections in Rfree set excluded from refinement | 1221 |
| Rcryst | 0.211 (0.284) |
| Rfree | 0.285 (0.371) |
| RMSD: bonds (Å)/angles (°) | 0.008/0.976 |
| B-factor refinement | Group B |
| <B> (Å2): all atoms/# atoms | 49/2,779 |
| <B> (Å2): water molecules/#water | 46/42 |
| Φ/ψ most favored (%)/additionally allowed (%) | 97/1.8 (0.9 outlier) |
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CC1/2, correlation coefficient; Rpim, precision-indicating merging R-factor; RMSD, root-mean-square deviation.
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-FLAG (M2, mouse monoclonal) | Sigma | F1804 | WB (1:5000) |
| Antibody | Anti-MYC (4A6, mouse monoclonal) | Millipore | 05-724 | WB (1:2500) |
| Antibody | Anti-RFP (rat monoclonal) | ChromoTek | 5F8 | IF (1:500) |
| Antibody | Anti-RFP (mouse monoclonal) | ChromoTek | 6G6 | WB (1:1000) |
| Antibody | Anti-alpha-tubulin (DM1A, mouse monoclonal) | Cell Signaling Technologies | DM1A | IF (1:2000) |
| Antibody | Anti-alpha-Tubulin (chicken polyclonal) | Synaptic Systems | 302 206 | IF (1:1000) |
| Antibody | Anti-CAPN7 (rabbit polyclonal) | Proteintech | Cat# 26985-1-AP | IF (1:500) WB (1:4000) |
| Antibody | Anti-IST1 (rabbit polyclonal) | Sundquist Lab/Covance | UT560 | IF (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) | |
| Antibody | Anti-GAPDH (mouse monoclonal) | Millipore | ||
| Sequence-based reagent | siNT | Mackay et al., 2010 | siRNA | GCAAAUCUCCGAUCGUAGA |
| Sequence-based reagent | siCAPN7 | Wenzel et al., 2022 | siRNA | GCACCCAUACCUUUACAUU |
| Sequence-based reagent | siNUP153 | Mackay et al., 2010 | siRNA | GGACUUGUUAGAUCUAGUU |
| Chemical compound, drug | Doxycycline Hyclate | Sigma | 324385 | 1–2 µg/mL |
| Chemical compound, drug | Thymidine | CalBiochem | CAS 50-89-5 | 2 mM |
| Chemical compound, drug | Oregon Green 488 maleimide | Life Technologies/Molecular Probes | O6034 | Fluorescent label for peptides |
| Software, algorithm | Fiji | NIH | RRID:SCR_002285 | |
| Software, algorithm | Prism 9 | GraphPad |
Additional files
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Supplementary file 1
ESI-MS mass confirmation of purified proteins.
- https://cdn.elifesciences.org/articles/84515/elife-84515-supp1-v2.docx
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Supplementary file 2
Plasmids.
- https://cdn.elifesciences.org/articles/84515/elife-84515-supp2-v2.docx
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Supplementary file 3
siRNA sequences.
- https://cdn.elifesciences.org/articles/84515/elife-84515-supp3-v2.docx
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Supplementary file 4
Antibodies.
- https://cdn.elifesciences.org/articles/84515/elife-84515-supp4-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84515/elife-84515-mdarchecklist1-v2.pdf
