Sallé J et al. (2019), Asymmetric division through a reduction of micr...

ECB-ART-46834

J Cell Biol 2019 Mar 04;2183:771-782. doi: 10.1083/jcb.201807102.

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Asymmetric division through a reduction of microtubule centering forces.

Sallé J , Xie J , Ershov D , Lacassin M , Dmitrieff S , Minc N .

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Asymmetric divisions are essential for the generation of cell fate and size diversity. They implicate cortical domains where minus end-directed motors, such as dynein, are activated to pull on microtubules to decenter asters attached to centrosomes, nuclei, or spindles. In asymmetrically dividing cells, aster decentration typically follows a centering phase, suggesting a time-dependent regulation in the competition between microtubule centering and decentering forces. Using symmetrically dividing sea urchin zygotes, we generated cortical domains of magnetic particles that spontaneously cluster endogenous dynein activity. These domains efficiently attract asters and nuclei, yielding marked asymmetric divisions. Remarkably, aster decentration only occurred after asters had first reached the cell center. Using intracellular force measurement and models, we demonstrate that this time-regulated imbalance results from a global reduction of centering forces rather than a local maturation of dynein activity at the domain. Those findings define a novel paradigm for the regulation of division asymmetry.

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Genes referenced: dnah3 LOC100887844 LOC100893907 LOC115919910

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	Figure 1. Magnetic cortical pulling domains drive marked asymmetric divisions. (A) Magnetic beads injection, cap formation, and induced asymmetric division. (B–E) Time-lapse images of the cleavage of sea urchin zygotes. (B) The cap is formed with beads with strong MT minus end activity and maintained; (C) a noninjected control embryo; (D) the pulling cap is not maintained; and (E) a control using a cap formed with beads that do not interact with MTs. Yellow stars: female/zygote nucleus; red stars: blastomere nuclei after division. (F) Divided blastomere area ratio in conditions B to E (n = 10, n = 8, n = 11, and n = 10 cells). (G) Quantification of the angle (Δα) between the division axis and the axis from the cell center to the cap (n = 12, n = 17, and n = 10 cells). (H) Blastomere area ratio plotted as a function of the angle (β) formed by the edges of the pulling cap and the cell center. The dashed line is a visual guide. Results were compared by using a two-tailed Mann–Whitney test. ns, P > 0.05; ****, P < 0.0001. Error bars are SD. Bars, 20 µm.
	Figure 2. Consecutive aster centration and decentration. (A) Time-lapse of the consecutive centering motion of the male nucleus followed by a decentration of the zygote nucleus toward the cap. Arrowheads: nuclei at the aster center; red stars: blastomere nuclei. Contrast is adjusted to compensate for an increase in DNA labeling (Hoechst), and bright blobs correspond to excess sperm on the fertilization envelope. (B) Aster trajectories from fertilization to metaphase onset, aligned with the pulling cap (n = 32 cells). (C) Aster position with and without cortical caps (n = 18 and n = 9, respectively). (D) Aster velocity during centration and decentration (n = 18, n = 17, and n = 17 cells). (E) 1D force competition model with a pulling cap force (in red) and length-dependent pulling forces (in blue). (F) Simulated aster position under no, weak, or strong cortical forces. (G) Simulated aster position, with a decreasing centering constant, a (purple) or an increasing cap force, Fcap, after centration (red). Results were compared by using a two-tailed Mann–Whitney test. ns, P > 0.05; ****, P < 0.0001. Error bars are SD. Bars, 20 µm.
	Figure 3. Cortical pulling forces remain constant during and after aster centration. (A) The method used to measure cortical pulling forces. (B) Time-lapse of aster centration, and concomitant reduction of the magnetic force holding the cap. Top: Low-magnification view of the embryo moving away from the probe (left). Bottom: Details of cap detachment dynamics. Yellow stars: the aster center; arrowheads: the cap. (C) Typical example of the evolution of the distance between the cell cortex and the cap (gray), and of the magnetic force (red), as a function of the distance between the egg and magnet. Arrowhead: the cap “take-off.” (D) Average distance between cap and cortex plotted as a function of the magnetic force, during centration (in green; n = 16) and after centration (in red; n = 18). Dashed lines are SD. Take-off forces (arrows) correspond to cortical forces exerted by domains onto asters and were determined using the fits (continuous lines; see Materials and methods). (E) Cortical forces plotted as a function of cap volumes. Red dashed line: linear regression. Pearson r and P value are displayed on the graph. Bars, 20 µm.
	Figure 4. Aster centering stiffness decreases after centration. (A) Time-lapse of a centering aster subjected to two consecutive external magnetic forces. Top: Low-magnification views of the embryo and magnet tip. Bottom: Position of aster centers (nuclei) and beads aggregate. Orange line: the centration path (fertilization from the top); orange arrows: the direction of the magnetic forces. Bars, 20 µm. (B) Time-lapse projection of aster deviation from the centration path upon the first force application. (C) Kymograph of aster deviation from the cell center from the second force application. Black and white arrowheads: the initial centered position and the maximum deviation (plateau) during force application, respectively. Bars, 5 µm. (D) Quantification of paired aster centering spring constants (connected by gray dashed lines [n = 10]). The “single-pull” category corresponds to force measurement performed only once after aster centration (n = 15). (E) Quantification of paired aster drags. (F) Average displacement/force plotted as a function of time. Error bars are SEM. Dashed lines: Voigt model fit (see Materials and methods). Results were compared using a two-tailed Mann–Whitney test. ns, P > 0.05; **, P < 0.01.
	Figure 5. Reduction in aster centering stiffness drives aster decentration and asymmetric division. (A) Time-lapse of a 3D simulation with constant pulling cap and aster centering stiffness reduction. The red hemisphere is the cap, MTs are in green and the aster trajectory in blue. (B) Examples of comparisons between simulated (top) and experimental (bottom) aster trajectories. (C) Aster position relative to the cell center in 3D simulations using positions of caps and sperm entry from experiments (in blue; n = 18). Blue dashed lines: SD. The red dashed line: the experimental curve (shown in Fig. 2 B). (D and E) Comparison of aster distances from the cell center and aster speeds in simulations (blue) and experiments (red). Error bars are SEM. (F) Schematized consecutive aster centration and decentration and model for asymmetric division driven by a reduction in aster MT centering stiffness under constant cortical forces.

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