Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna Biocenter, Austria
- Marine Biological Laboratory, United States
- Institute of Science and Technology Austria, Austria
Figures
Figure 1 with 2 supplements
Cleavage furrow ingression initiates by contraction of a randomly oriented actin filament network, which subsequently gradually aligns at the cell equator.
(A) Schematics of actin filament (grey) and fluorescent dipole orientation (green) relative to the optical section of the microscope. Upper panel shows actin filament parallel to the focal plane of …
Figure 1—figure supplement 1
LC-PolScope fluorescence polarization microscopy and analysis pipeline.
(A) Confocal LC-PolScope employs liquid crystal-based universal compensator for modifying the linear polarization state of the excitation laser. This setup allows exciting the fluorophores in the …
Figure 1—figure supplement 2
Regression model to determine cytokinesis timing in fixed cells.
(A) Cytokinesis staging by measurement of the distance between center points of segregating chromosome masses and cleavage furrow diameter. Lines indicate measured distances. Live hTERT-RPE-1 cells …
Figure 2 with 3 supplements
Quantification of actin reorganization by live-cell confocal polarization microscopy.
(A) Fluorescence polarization microscopy of stress fibers in live interphase hTERT-RPE-1 cells labeled with SiR-actin using the LC-PolScope. Color saturation indicates degree of fluorophore …
Figure 2—figure supplement 1
Polarization microscopy with linear polarizers in the emission beam path.
(A) Confocal fluorescence polarization microscope setup used for live-cell imaging. A linearly polarized excitation laser that was aligned with the X-axis of the optical table, and two …
Figure 2—figure supplement 2
Analysis pipeline of live-cell polarization microscopy.
(A) Analysis pipeline for a representative live hTERT-RPE-1 cell stained with SiR-actin recorded with the confocal fluorescence polarization microscope setup. Cells were recorded with horizontal and …
Figure 2—figure supplement 3
Cortex organization in para-nitroblebbistatin-treated anaphase cells and lateral distribution of actin and myosin in untreated cells.
(A) Diameter at equatorial position measured in hTERT-RPE-1 cells expressing H2B-mRFP
and stained with SiR-actin, in presence of 50 µM para-nitroblebbistatin, an inhibitor of myosin II. Line indicates mean, shaded area indicates s.d. of 10 cells. Anaphase onset = 0 s. (B, C) Quantification of SiR-actin fluorescence at cell poles and equator as illustrated for untreated and para-nitroblebbistatin treated cell in Figure 2B. Anaphase onset = 0 s (D) Confocal image of live hTERT-RPE-1 cell expressing non-muscle myosin IIc-EGFP (MLC-12B-EGFP), stained with SiR-actin. Dashed line indicates measurement region for line profiles, arrowhead indicates cell equator position. (E) Quantification of lateral distribution of MLC-12B-EGFP and actin filaments at late furrow ingression stage as in (D); lines represent mean fluorescence in cortical line profiles centred to the cell equator (position = 0 μm), shaded areas represent s.d.; n = 37 cells. Scale bars = 10 µm.
Figure 3 with 1 supplement
Equatorial actin filaments align on planar surfaces of confined cells.
(A) Schematics of cell confinement chamber used to enforce flat geometry of top- and bottom cell cortex during cytokinesis. (B, C) Confocal 3-D images of live hTERT-RPE-1 cells stably expressing …
Figure 3—figure supplement 1
3D-imaging of cleavage furrow ingression in unconfined cells.
(A, B) Confocal 3-D images of live hTERT-RPE-1 cells stained with SiR-actin at early (A) and late (B) furrow ingression stages. The x/z and y/z sections are at positions as indicated by blue or …
Figure 4 with 1 supplement
Laser microsurgery reveals cortical tension orientation during cytokinesis.
(A) Total internal reflection fluorescence (TIRF) microscopy of hTERT-RPE-1 under confinement stably expressing LifeAct-mCherry. Representative images of n = 11 cells. Time = 0 s at anaphase onset, …
Figure 4—figure supplement 1
Analysis of laser cutting experiments.
(A) Laser cutting of an hTERT-RPE-1 cell stably expressing LifeAct-mCherry (grey) stained with Hoechst (magenta), cultured in a confinement chamber. Dashed line indicates site of cutting with a …
Figure 5
Model for actomyosin network reorganization during cytokinesis of vertebrate cells.
For details see main text.
Author response image 1
Videos
Video 1
TIRF microscopy of hTERT-RPE-1 expressing LifeAct-mCherry under confinement stably.
Time = 0 s at anaphase onset, as determined in phase contrast images of the same cell. Video shows same cell as Figure 4A,B.
Additional files
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Source code 1
- https://doi.org/10.7554/eLife.30867.015
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Transparent reporting form
- https://doi.org/10.7554/eLife.30867.016
