Astral microtubule dynamics regulate anaphase oscillation onset and set a robust final position of the C. elegans zygote spindle

Background: The correct positioning of the mitotic spindle during the asymmetric division of the nematode C. elegans zygote relies on the combination of centering and cortical–pulling forces. These forces, revealed by centrosome anaphase oscillations, are regulated through the dynamics of force generators, related to mitosis progression. Recently, we reported the control of oscillation onset by the posterior spindle pole position in related species C. briggsae, necessitating a re-evaluation of the role of astral microtubules dynamics. Results: After exhibiting such a positional switch in C. elegans, we mapped the microtubule ends at the cortex and observed a correlation between the proximity of the centrosomes and the density of microtubule contacts. To explore the functional consequences, we extended the “tug–of–war” model and successfully accounted for the positional switch. We predicted and experimentally validated that the control of oscillation onset was robust to changes in cell geometry or maximum number of attached force generators. We also predicted that the final position of the posterior centrosome and thus the spindle has a reduced dependence upon the force generator dynamics or number. Conclusion: The outburst of forces responsible of spindle anaphase oscillations and positioning is regulated by the spindle position through the spatial modulation of microtubule contacts at the cortex. This regulation superimposes that of force generator processivity putatively linked to the cell cycle. This novel control provides robustness to variations in zygote geometry or detailed properties of cortical force generators

Microtubule Feedback and LET-99-Dependent Control of Pulling Forces Ensure Robust Spindle Position

International audienceDuring asymmetric division of the Caenorhabditis elegans zygote, to properly distribute cell fate determinants, the mitotic spindle is asymmetrically localized by a combination of centering and cortical-pulling microtubule-mediated forces, the dynamics of the latter being regulated by mitotic progression. Here, we show a, to our knowledge, novel and additional regulation of these forces by spindle position itself. For that, we observed the onset of transverse spindle oscillations, which reflects the burst of anaphase pulling forces. After delaying anaphase onset, we found that the position at which the spindle starts to oscillate was unchanged compared to control embryos and uncorrelated to anaphase onset. In mapping the cortical microtubule dynamics, we measured a steep increase in microtubule contact density after the posterior centrosome reached the critical position of 70% of embryo length, strongly suggesting the presence of a positional switch for spindle oscillations. Expanding a previous model based on a force-generator temporal control, we implemented this positional switch and observed that the large increase in microtubule density accounted for the pulling force burst. Thus, we propose that the spindle position influences the cortical availability of microtubules on which the active force generators, controlled by cell cycle progression, can pull. Importantly, we found that this positional control relies on the polarity-dependent LET-99 cortical band, the boundary of which could be probed by microtubules. This dual positional and temporal control well accounted for our observation that the oscillation onset position resists changes in cellular geometry and moderate variations in the active force generator number. Finally, our model suggests that spindle position at mitosis end is more sensitive to the polarity factor LET-99, which restricts the region of active force generators to a posterior-most region, than to microtubule number or force generator number/activity. Overall, we show that robustness in spindle positioning originates in cell mechanics rather than biochemical networks

Two dynamical behaviours of the microtubules at cell cortex reveal pulling and pushing forces that position the spindle in C. elegans embryo

In the Caenorhabditis elegans zygote, astral microtubules generate forces, pushing against and pulling from the cell periphery. They are essential to position the mitotic spindle and in turn the cytokinesis furrow, ensuring the proper distribution of fate determinants to the daughter cells. By measuring the dynamics of astral microtubules, we revealed the presence of two populations, residing at the cortex during 0.4 s and 1.8 s, proposed to reflect the pulling and pushing events, respectively. This is a unique opportunity to unravel the time and space variations of these both spindle-positioning forces, to study their regulation under physiological conditions. By an investigation at the microscopic level, we first confirmed that the asymmetry in pulling forces was encoded by an anteroposterior imbalance in dynein-engaging rate, and that this asymmetry exists from early metaphase on and accounts for the final spindle position. More importantly, we obtained direct proof of the temporal control of pulling forces through the forcegenerator processivity increase during anaphase. Lastly, we discovered an anti-correlation between the long-lived population density and the stability of the spindle position during metaphase, which strongly suggests that the pushing forces contribute to maintaining thespindle at the cell centre