Contractile acto-myosin network on nuclear envelope remnants positions human chromosomes for mitosis
- University of Dundee, United Kingdom
- University of Sussex, United Kingdom
- University of Minnesota, United States
Figures
Figure 1 with 5 supplements
Chromosome scattering volume is reduced in early prometaphase, independently of spindle MTs.
(A) Diagrams show how the chromosome scattering volume (CSV) was defined. Left-hand diagram shows how the convex hull was generated in two dimensions (2D) to represent chromosome distribution. A …
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Figure 1—source data 1
Data at individual time points in individual cells.
- https://doi.org/10.7554/eLife.46902.008
Figure 1—figure supplement 1
Method for estimation of the NEBD timing.
Images of the same cell shown in Figure 1B. Top-row images show SiR-DNA-stained chromosomes. Bottom-row images show GFP-LacI-NLS (nuclear localization signal). The timing of NEBD was determined …
Figure 1—figure supplement 2
CSV decreases and actin accumulates on the NE around NEBD in asynchronous wild-type U2OS cells (without cdk1-as).
Images show single z-sections of a U2OS cell with SiR-DNA-stained chromosomes (blue), expressing mCherry-Lifeact (red) and undergoing NEBD. Yellow arrowheads indicate location of the actin network. …
Figure 1—figure supplement 3
Confirmation that MTs are absent after nocodazole treatment.
Immunostaining of fixed cells to test the presence or absence of microtubules (MTs) within cells. Cells were analyzed by live-cell imaging for Figure 1C, and then fixed and immunostained for MTs …
Figure 1—figure supplement 4
The chromosome distribution in three-dimensional space with and without nocodazole treatment.
The 3D convex hull of the cell in Figure 1C (top) is shown over the time course. The 3D convex hull (green) is viewed on two planes, that is x-y and z-x/y planes. ‘a’ and ‘b’ show the corresponding …
Figure 1—figure supplement 5
Chromosome mass volume did not considerably change after NEBD.
Diagram (left) shows how CSV (chromosome scattering volume) and CMV (chromosome mass volume) were determined. Graph (right) shows the mean of CMV (n = 3) and CSV (n = 8). CSV data are from Figure 3A.…
Figure 2 with 2 supplements
Actin accumulates outside of the NE in prophase, and its network shrinks after NEBD.
(A) Actin accumulates on the NE around NEBD. Images (z-projections) show a representative cell expressing GFP-tubulin and mCherry-Lifeact (that fluorescently marks F-actin). Time is relative to …
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Figure 2—source data 1
Data at individual time points in individual cells.
- https://doi.org/10.7554/eLife.46902.013
Figure 2—figure supplement 1
The volume inside of the actin network is rapidly reduced after NEBD.
Graph shows the mean of normalized volume inside the actin network (normalized to the volume at NEBD in each cell) after NEBD (n = 7). Error bars, s.e.m. p-value was determined by one-way ANOVA for …
Figure 2—figure supplement 2
During contraction of the actin network, outermost chromosomes are located right beneath the network.
Images show projections of five z-sections (A, top row) and two z-sections (A, other rows) of a representative cell, expressing histone 2B (H2B)-cerulean, GFP-Tubulin and mCherry-Lifeact, in early …
Figure 3 with 6 supplements
Myosin II activity is required for the CSV reduction after NEBD.
(A) Inhibition of myosin II activity alleviates CSV reduction after NEBD. Images (z-projections) show representative cells with SiR-DNA-stained chromosomes, which entered mitosis in the presence of …
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Figure 3—source data 1
Data at individual time points in individual cells.
- https://doi.org/10.7554/eLife.46902.024
Figure 3—figure supplement 1
Actin depolymerization inhibitor does not change CSV reduction after NEBD.
Images (z-projections) show SiR-DNA-stained chromosomes in representative cells incubated in the presence of 1 µM jasplakinolide (Jasplak., bottom) or DMSO (control, top). Time is relative to NEBD. …
Figure 3—figure supplement 2
Myosin II co-localizes with the actin network on the NE in prophase.
Images on left (single Z-sections) show a representative cell expressing mCherry-Lifeact and GFP-NMHCII-A (GFP-tagged Myosin II-A heavy chain). MHC denotes myosin II heavy chain. Scale bars, 10 µm. …
Figure 3—figure supplement 3
Myosin II inhibition alleviates contraction of the actin network on the NE remnant after NEBD.
(A) Images (projections of two consecutive z-sections where spindle poles were in focus) show representative cells expressing GFP-tubulin and mCherry-Lifeact in the presence of 50 µM …
Figure 3—figure supplement 4
Myosin II inhibition does not affect chromosome mass volume (CMV) after NEBD.
Graphs show means of CSV and CMV in the presence (right, pnBB) and absence (left, control) of 50 µM pnBB (CSV n = 10, CMV n = 3; error bars. s.e.m.). CSV data are from Figure 3A. Graph at left-hand …
Figure 3—figure supplement 5
Myosin II inhibition often leaves the actin network extending beyond the spindle poles soon after NEBD.
Images (projections of two consecutive z-sections where spindle poles were in focus) show representative cells expressing GFP-tubulin, mCherry-Lifeact and H2B-cerulean in the presence of 50 µM …
Figure 3—figure supplement 6
Atypical spindle morphology is found in LINC-DN-expressing and control cells, soon after NEBD.
We observed that LINC-DN-expressing and pnBB-treated cells sometimes showed atypical spindle morphology, which includes (A) spindle MTs extending from a pole in wide-ranging directions (A) in the …
Figure 4
Local inhibition of myosin II activity results in local alleviation of CSV reduction.
(A) Diagram shows that azidoblebbistatin (azBB) is covalently linked to the myosin II heavy chain and inhibits its activity in the half of the nucleus exposed to infrared light. (B) Images …
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Figure 4—source data 1
Data at individual time points in individual cells.
- https://doi.org/10.7554/eLife.46902.028
Figure 5 with 4 supplements
Evidence that removal of actin network delays chromosome congression and anaphase onset, and increases frequency of abnormal chromosome segregation.
(A) Images (z-projections) show representative cells expressing histone 2B (H2B)-cerulean and GFP-tubulin. They progress from NEBD to anaphase, expressing either LINC-DN or a control construct. …
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Figure 5—source data 1
Data in individual cells.
- https://doi.org/10.7554/eLife.46902.034
Figure 5—figure supplement 1
LINC-DN expression did not change the distance between centrosomes and did not cause deviation of the spindle from the center of the nucleus.
(A) Images acquired for Figure 5A were analyzed further. Graph shows the mean centrosome distance after NEBD in cells expressing a control construct (n = 33) or LINC-DN (n = 35). Error bars, s.e.m. …
Figure 5—figure supplement 2
The presence of back-of-spindle chromosomes soon after NEBD leads to a delay in chromosome congression.
Graph compares time (minutes) from NEBD to chromosome congression in cells expressing LINC-DN with (right, n = 16) versus without (left, n = 14) back-of-spindle chromosomes (magenta arrows) at 20 …
Figure 5—figure supplement 3
pnBB treatment delays chromosome congression and anaphase onset, and increases frequency of abnormal chromosome segregation.
(A) Images (z-projections) show representative pnBB-treated and control cells with GFP-tubulin and SiR-DNA-stained chromosomes. They progress from NEBD to anaphase. Orange arrows indicate time …
Figure 5—figure supplement 4
RPE cells show accumulation of actin on the NE in early mitosis, but HeLa cells do not.
(A) RPE cells show accumulation of actin on the NE in early mitosis. RPE cells expressing mCherry-Lifeact and H2B-cerulean were arrested with RO-3306 treatment and released to fresh media. 10 min …
Figure 6
Summary diagram.
Prior to NEBD, the LINC complex promotes the accumulation of acto-myosin to the cytoplasmic side of the nuclear envelope (i, ii). Upon NEBD, myosin II promotes contraction of the acto-myosin network …
Videos
Video 1
CSV decreases after NEBD even in the absence of MTs.
Video of representative cells (Control and Nocodazole-treated) shown in Figure 1C (SiR-DNA staining). Frame intervals are 2 min. Video is shown at 600x speed.
Video 2
Actin accumulates on the NE around NEBD.
Video of representative cells shown in Figure 2A (expressing GFP-tubulin and mCherry-Lifeact). Frame intervals are 1 min. Video is shown at 600x speed.
Video 3
During contraction of the actin network, outermost chromosomes are located right beneath the network.
Video of representative cells shown in Figure 2—figure supplement 2 (expressing mCherry-Lifeact, GFP-tubulin and H2B-cerulean). GFP-tubulin is not shown in the Video. Frame intervals are 1 min. …
Video 4
Removal of the actin network results in slower CSV reduction after NEBD.
Video of representative cells (Control and LINC-DN expressing) shown in Figure 2D (SiR-DNA staining). Frame intervals are 2 min. Video is shown at 600x speed.
Video 5
Inhibition of myosin II activity alleviates CSV reduction after NEBD.
Video of representative cells (Control and pnBB treated) shown in Figure 3A (SiR-DNA staining). Frame intervals are 2 min. Video is shown at 600x speed.
Video 6
CSV reduction is almost completely alleviated when myosin II activity is inhibited in the absence of MTs.
Video of representative cells (Nocodazole and Nocodazole +pnBB treated) shown in Figure 3B (SiR-DNA staining). Frame intervals are 2 min. Video is shown at 600x speed.
Video 7
Evidence that removal of actin network delays chromosome congression and anaphase onset.
Video of representative cells (Control and LINC-DN expressing) shown in Figure 5A (expressing GFP-tubulin and H2B-cerulean). Frame intervals are 2 min. Video is shown at 600x speed.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identi-fiers | Additional information |
|---|---|---|---|---|
| Cell line (Human) | U2OS cdk1-as | Rata et al., 2018 | ||
| Cell line (Human) | wild-type U2OS | ATCC | HTB-96 | |
| Cell line (Human) | HeLa | ATCC | CCL-2 | |
| Cell line (Human) | hTERT RPE | ATCC | CRL-4000 | |
| Transfected construct (Human) | p3'SSclonedimer pF9-poly-EGFP-lacI-NLS (GFP-LacI-NLS) | Hori et al., 2013 | ||
| Transfected construct (Human) | mCherry-Lifeact-7 (mCherry-Lifeact) | Addgene | #54491 | A gift from Michael Davidson |
| Transfected construct (Human) | pGFP-alphaTub (GFP-Tubulin) | Other | A gift from Jason Swedlow lab | |
| Transfected construct (Human) | mRFP1-SR-KASH (LINC-DN) | Luxton et al., 2010 | ||
| Transfected construct (Human) | mRFP1-KASH-ΔL (Control) | Luxton et al., 2010 | ||
| Transfected construct (Human) | pCS-H2B-cerulean (H2B-Cerulean) | Other | A gift from Sean Megason Lab | |
| Transfected construct (Human) | CMV-GFP-NMHC II-A (MHC-GFP) | AddGene | #11347 | A gift from Robert Adelstein |
| Antibody | anti-Tubulin (Rat monoclonal) | Cell Signalling Technology | YL1/2 | Used at 1:1000 dilution |
| Antibody | anti-LaminB1 (Rabbit polyclonal) | Abcam | ab16048 | Used at 1:1000 dilution |
| Antibody | anti-Actin (Rabbit polyclonal) | Thermo-Fisher | PA1-183 | Used at 1:1000 dilution |
| Antibody | A488 goat anti-Rabbit | Thermo-Fisher | A-11034 | Used at 1:1000 dilution |
| Antibody | A488 donkey anti-Rat | Thermo-Fisher | A-21208 | Used at 1:1000 dilution |
| Antibody | A594 goat anti-Rabbit | Thermo-Fisher | A-11037 | Used at 1:1000 dilution |
| Chemical compound, drug | Phalloidin DyLight 650 | Invitrogen | 13454309 | Used at 1:1000 dilution |
| Chemical compound, drug | Hoechst 33342 | Sigma-Aldrich | 14533 | Used at 1:2000 dilution |
| Chemical compound, drug | SiR-DNA | tebu-bio | SC007 | Used at 100 nm |
| Chemical compound, drug | 1NM-PP1 | Merck Millipore | 529581 | Used at 1 μM |
| Chemical compound, drug | RO-3306 | Merck Millipore | 217699 | Used at 10 μM |
| Chemical compound, drug | nocodazole | Sigma | M1404 | Used at 3.3 μM |
| Chemical compound, drug | paranitroblebbistatin (pnBB) | motorpharmacology | Used at 50 μM | |
| Chemical compound, drug | azidoblebbistatin (azBB) | motorpharmacology | Used at 5 μM | |
| Chemical compound, drug | jasplakinolide | Merck | J4580 | Used at 1 μM |
| Chemical compound, drug | MK-1775 | Selleck Chemicals | S1525 | Used at 500 nM |
| Software, algorithm | Imaris, 3D surface tool | Bitplane | ||
| Software, algorithm | Imaris, Convex Hull generation tool | Bitplane | Convex Hull tool from filament tool adapted for 3D surface tool | |
| Software, algorithm | Fiji | https://fiji.sc/ |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46902.037
