Identification of abscission checkpoint bodies as structures that regulate ESCRT factors to control abscission timing
- Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, United States
- Department of Biochemistry, University of Utah School of Medicine, United States
Figures
Figure 1 with 1 supplement
System for abscission checkpoint enrichment.
(A) Schematic of the CHMP4C protein. (B) Timeline for synchronizing cells with an active abscission checkpoint using siNup153/siNup50 (siNups), followed by thymidine treatment. (C) Western blot of …
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Figure 1—source data 1
Source Data for Figure 1.
- https://cdn.elifesciences.org/articles/63743/elife-63743-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Nup depletion and cell synchronization delay abscission without increasing chromatin bridges.
(A) Quantification of % midbody-stage cells with chromatin bridges marked by Lap2ß under asynchronous conditions (48 hr after transfection with siNups or siControl). N = 300 midbodies, n = 3 …
Figure 2 with 3 supplements
Abscission checkpoint activity delays ALIX recruitment to the midbody.
Immunofluorescence and time course quantifications of CEP55 (A, B) and ALIX (C, D) recruitment to early-stage midbodies in control and checkpoint-active cells. N = 300 midbodies/timepoint from n = 3 …
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Figure 2—source data 1
Source Data for Figure 2.
- https://cdn.elifesciences.org/articles/63743/elife-63743-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
The abscission checkpoint delays ALIX recruitment in the total midbody population.
(A, B) Timecourse quantification of CEP55 (A) or ALIX (B) recruitment to midbodies in control (siControl) and checkpoint-active (siNup153/50) cells. N = 300 midbodies scored for each timepoint, n = 3…
Figure 2—figure supplement 2
The abscission checkpoint does not delay TSG101/ESCRT-I recruitment to the midbody.
(A, B) Immunofluorescence and timecourse quantification of TSG101 recruitment to midbodies in control and checkpoint-active cells. N = 400 midbodies scored/timepoint from n = 4 biological …
Figure 2—figure supplement 3
The abscission checkpoint delays IST1 recruitment to the midbody.
(A, B) Immunofluorescence and timecourse quantification of IST1 recruitment to midbodies in control and checkpoint-active midbodies. N = 300 midbodies scored/timepoint from n = 3 biological …
Figure 3 with 2 supplements
pppCHMP4C localizes to Abscission Checkpoint Bodies when the abscission checkpoint is active.
(A, B) Immunofluorescence and time course quantification of pppCHMP4C recruitment to midbodies in control and checkpoint-active cells, and (C) time course quantification of midbody-stage cells with …
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Figure 3—source data 1
Source Data for Figure 3.
- https://cdn.elifesciences.org/articles/63743/elife-63743-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
The abscission checkpoint does not delay CHMP4C recruitment to the midbody.
(A–C) Quantification of % midbody-stage cells (A), immunofluorescence (B), and timecourse quantification of HA-CHMP4C recruitment to midbodies (C) in control and checkpoint-active cells, using a …
Figure 3—figure supplement 2
CHMP4C is detected in ACBs, which are maintained in stable numbers when the abscission checkpoint is active.
(A) Confocal z-projections of pre-permeabilized midbody-stage cells under asynchronous (48 hr) siControl and siNups conditions co-stained for CHMP4C and an ACB marker (detected by mAb SC35, see Figur…
Figure 4 with 4 supplements
ACBs are related to MIGs and contribute to abscission delay.
(A) Confocal z-projections of pre-permeabilized (Pre-Perm) midbody-stage cells showing that SC35 antibody and antibody against pAurB co-stain ACBs (asynchronous cultures, 48 hr after transfection …
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Figure 4—source data 1
Source Data for Figure 4.
- https://cdn.elifesciences.org/articles/63743/elife-63743-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
ACBs do not colocalize with a variety of subcellular organelles and structures.
(A, B) Immunofluorescence of asynchronous (48 hr) control and checkpoint-active (siNups) midbody-stage cells stained to detect ACBs (marked by (A) α-pAurB or (B) α-CHMP4B or (D) mAb-SC35), and …
Figure 4—figure supplement 2
Colocalization of ACB components.
(A, B) Confocal z-projections of pre-permeabilized midbody-stage cells under asynchronous (48 hr) control and checkpoint-active conditions, co-stained for ACB components as indicated.
Figure 4—figure supplement 3
Interfering with timely resolution of MIGs delays cytokinetic abscission.
(A) Time schematic of fixed-imaging experiments after treatment with DMSO, 1 µM DYRK3i, or 1 µM CLK1i. Inhibitors were added 13 hr post-thymidine release, after most cells had completed metaphase, …
Figure 4—figure supplement 4
CLK1 expression partially dissolves ACBs to mitigate abscission arrest.
(A) Quantification of percent midbody-stage cells with ACBs present after treatment as in Figure 4H, with a representative western blot show below. N = 1200 midbodies from n = 6 biological …
Figure 5 with 5 supplements
ACBs are conserved and dependent upon abscission checkpoint factor CHMP4C.
(A, B) Confocal z-projections of pre-permeabilized control and checkpoint-active RPE1 cells following 14 hr thymidine release, stained for ACB markers as indicated. (C) Quantification of control and …
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Figure 5—source data 1
Source Data for Figure 5.
- https://cdn.elifesciences.org/articles/63743/elife-63743-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
ACB formation and ALIX recruitment delay occur following replication stress.
(A, B) Timeline schematic (A) and quantification (B) of synchronized % midbody-stage cells after treatment with 0.4 µM aphidicolin or DMSO following thymidine release. N = 900 cells scored/timepoint …
Figure 5—figure supplement 2
ACB formation and ALIX recruitment delay occur when intercellular tension is heightened.
(A) Quantification of % midbody-stage cells plated at high density (low tension) or low density (high tension). N = 1200 cells scored/condition from n = 4 biological replicates. (B, C) …
Figure 5—figure supplement 3
Chromatin bridges do not significantly promote ACB formation or ALIX recruitment delay.
(A) Immunofluorescence of representative cells with and without chromatin bridges. (B) Quantification of dividing cells with ACBs both with/without siNups treatment (for comparison) and with/without …
Figure 5—figure supplement 4
RPE1 cells display hallmarks of the abscission checkpoint, including ALIX recruitment delay and ACB formation.
(A, B) Quantification of % midbody-stage cells and western blot of lysates from RPE1 cells after siCon or siNups treatment 14 hr following thymidine release. N = 800 cells scored/condition from n = 3…
Figure 5—figure supplement 5
Confirmation of efficiency and specificity of protein depletion when CHMP4C and Nup153/50 are simultaneously targeted.
Western blots of the experiment shown in Figure 5D.
Figure 6 with 2 supplements
ACBs contain ALIX and cells with ACBs have delayed ALIX midbody recruitment.
(A) Confocal z-projections of pre-permeabilized midbody-stage HeLa cells under asynchronous conditions (48 hr after transfection with siNups or siControl), stained as indicated. (B) Confocal …
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Figure 6—source data 1
Source Data for Figure 6.
- https://cdn.elifesciences.org/articles/63743/elife-63743-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
ACB size but not abscission checkpoint arrest is dependent on ALIX.
(A, B) Representative immunofluorescence and quantification of cells treated with indicated siRNAs for 72 hr. White arrowhead: midbody. Yellow arrowhead: multinucleate cell. Blue arrowhead: failed …
Figure 6—figure supplement 2
ALIX localizes specifically to ACBs.
(A–B) Confocal z-projections of pre-permeabilized cells (A) and quantification (B) of ALIX intensity per area in ACBs under asynchronous conditions (72 hr after transfection with siCon/siNups or …
Author response image 1
AurB inhibition decreases but does not completely dissolve ACBs.
(A) Quantification of midbody-stage cells (Intact) and recently-abscised midbody pairs (Cut) with ACBs marked by α-pAurB after treatment with siCon or siNups and thymidine synchronization. With and …
Author response image 2
Specific CLK1/2 inhibition delays abscission timing.
(A) Quantification of midbody-stage cells after 5 h asynchronous treatment with DMSO, 0.5 – 10 µM Pan-CLK inhibitor (Pan-CLKi, TG003, Sigma), or 1 µM specific CLK1/2 inhibitor (CLK1i, 534350, …
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, ID confirmed by STR profiling | |
| Cell line (Homo sapiens) | RPE1 | Bruce Edgar Lab | RRID:CVCL_4388 | Non-transformed, ID confirmed by STR profiling |
| Transfected construct (Homo sapiens) | pLVX-CLK1 | This paper | Addgene Cat#:174088 | Lentiviral construct to inducibly express CLK1 |
| Antibody | anti-ALIX (Rabbit polyclonal) | Covance (This Lab) | RRID:AB_2892637 | IF (1:500), WB (1:500) |
| Antibody | anti-pAurB (Rabbit polyclonal) | Rockland | Cat#: 600-401-677 RRID:AB_206164 | IF (1:500) |
| Antibody | anti-pppCHMP4C (Rabbit polyclonal) | Pier Paolo D’Avino | N/A | IF (1:500) |
| Antibody | anti-SC35 (mouse monoclonal) | Abcam | Cat#: ab18826 RRID:AB_298608 | IF (1:5000) |
| Antibody | anti-SRRM2 (rabbit polyclonal) | Thermofisher | Cat#: PA5-66827 RRID:AB_2665182 | IF (1:1000) |
| Sequence-based reagent | siNup153 | Mackay et al., 2010 | siRNA | GGACUUGUUAGAUCUAGUU |
| Sequence-based reagent | siNup50 | Mackay et al., 2010 | siRNA | GGAGGACGCUUUUCUGGAU |
| Chemical compound, drug | CLK1/2 Inhibitor | Millipore | 534350 | 1 µM |
| Chemical compound, drug | DYRK3 Inhibitor | Tocris | GSK 626616 | 1 µM |
| Chemical compound, drug | AurB Inhibitor | Bio-Techne | ZM 447439 | 2 µM |
| Chemical compound, drug | Thymidine | CalBiochem | CAS 50-89-5 | 2 mM |
| Software, algorithm | Fiji | NIH | RRID:SCR_002285 |
Additional files
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Supplementary file 1
Antibodies used for western blot and immunofluorescence in this study.
- https://cdn.elifesciences.org/articles/63743/elife-63743-supp1-v2.xlsx
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Supplementary file 2
Plasmids and siRNAs used in this study.
- https://cdn.elifesciences.org/articles/63743/elife-63743-supp2-v2.xlsx
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Supplementary file 3
Adjusted p-values calculated from this study.
- https://cdn.elifesciences.org/articles/63743/elife-63743-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/63743/elife-63743-transrepform-v2.docx
