Figure 1. 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 the microscope, lower panel shows an actin filament that is perpendicularly oriented to the focal plane of the microscope. On perpendicularly oriented actin filaments, fluorescent dipoles are oriented point symmetrically in every direction and the ensemble of molecules therefore does not yield a fluorescence anisotropy signal. (B) Fluorescence polarization microscopy of stress fibers in fixed interphase hTERT-RPE-1 cells labeled with Alexa Fluor 488-phalloidin using the LC-PolScope. Color saturation indicates degree of fluorophore alignment (anisotropy), hue indicates mean orientation of fluorescence dipoles as shown in upper left corner. (C) Quantification of polarization normalized to calibration samples: stress fibers as in (B) and fluorescent plastic with random fluorescence dipole orientation. Dots indicate individual measurements, bars indicate median. (D) Geometry and normalized polarization predicted by canonical purse-string model of cytokinesis at the equator (red) or cell poles (blue). (E) Images of Alexa Fluor 488-phalloidin-stained hTERT-RPE-1 cells at representative stages during cytokinesis. Blue and red arrows indicate polar and equatorial positions of quantification regions, respectively (upper panel). Lower panel shows the orientation map of the fluorescent dipole as calculated by the different orientations. Color saturation indicates degree of fluorophore alignment (anisotropy), hue indicates mean orientation of fluorescence dipoles as shown in upper left corner. Yellow arrowhead indicates edge of cleavage furrow. (F) Lateral distribution of polarization factor measured along the cell cortex in central sections of late furrow ingression-stage cells, aligned for the cleavage furrow edge as in (E). Line indicates median, shaded area indicates s.d. of 22 cells. (G) Quantification of normalized polarization as indicated in (E). Dots indicate individual cells (n = 136, mean + s.e.m. of both measurements at opposing cortical cell regions; ****p<0.0001 by Kolmogorov-Smirnov test). Scale bars = 10 µm.

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 sample with polarized laser light in four different angles. Schematic of the beam path of LSM780, the position of the liquid crystal is indicated. (B) Acquisition and analysis pipeline of LC-PolScope data. Four images were sequentially recorded whereby the linearly polarized excitation laser light was reoriented into four different angles as indicated. These images enable to calculate an ‘average image’ of the fluorophore localization and an ‘anisotropy image’ that contains information about the net fluorophore orientation in each pixel. These information can be visualized in a merged image, in which the brightness indicates overall fluorescence intensity, color saturation indicates the degree of fluorophore alignment (anisotropy), and hue indicates mean orientation of fluorescence dipoles as shown in upper left corner. The cell cortex was then segmented based on the ‘average image’ and the furrow midpoints and pole midpoints were determined. The cortex segmentation was then transferred to the ‘anisotropy image’ and analysis regions along the cortex were determined based on the furrow midpoints and the pole midpoints, respectively. Only these indicated sub-region of the entire segmentation mask (yellow regions overlaying ‘anisotropy image’) were used for quantification. (C) Distribution of average fluorescence intensity and normalized polarization factor along the contour of the segmented cell cortex of an early anaphase cell (same cell as shown for 0–100 s in Figure 1E). Upper panels indicate original images and segmented cortex regions, lower panels indicate the straightened cortex contours from the same images. Scale bar = 10 µm.

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 stably expressing H2B-mRFP and stained with SiR-actin were recorded as time-lapse movies, (B) quantified and binned into four bins. Line indicates mean and shaded area indicates s.d. of n = 14 cells. Scale bar = 10 µm.