The correct position of the spindle during asymmetric cell division, that of stem cells, ensures the proper fate of the daughter cells. Astral microtubules, highly dynamic semi-flexible filaments, help generate the forces involved. In this article published in the journal EMBO reports, the scientists measured the individual behaviors of these filaments, deciphering the multiple regulations at work. To do this, they combined in vivo experiments on the nematode model organism with computer vision.
How to control the choreography of the mitotic spindle during stem cell divisions.
During asymmetric divisions, it is essential to position the mitotic spindle correctly, offset from the cell center. The spindle plays the primary role during cell division. It allows the separation of chromosomes thanks to the microtubules connecting the centrosomes located at the spindle poles and the chromosomes located in the middle. Moreover, its final position dictates the position of the groove initiating cytokinesis, i.e. the division of the cell contents and its membrane. In the case of stem cells, which are polarized, the two daughter cells do not inherit the same contents and therefore have different fates. One keeps a stem character and the other differentiates into tissue. This process is very common in the animal kingdom, including humans. However, a defect in the positioning of the spindle distorts the cellular destiny and eventually leads to abnormal proliferation until the development of a cancer.
© Hélène Bouvrais & Jacques Pécréaux
Scientists use a classical model organism to study the regulation of this positioning: the single-cell polarized embryo of the nematode Caenorhabditis elegans, a small, non-pathogenic worm that occurs naturally in the soil of our regions. In this cell, the pronuclei as well as the centrosomes, and then the spindle, perform a complex ballet. They move from the posterior end of the cell to the center and then back to the posterior side. This very stereotyped choreography must be respected so that the posterior daughter cell keeps a stem character (it will give in particular the germ line) whereas the anterior cell by dividing will lead to the formation of part of the worm tissues.
Astral microtubules (which emanate from the centrosomes to the cell periphery) are very dynamic, alternating elongation and shortening. They generate forces that position the spindle, either by pushing against the cell periphery or by transmitting traction forces generated by the dynein molecular motor located transiently at the cell periphery. The actors of these forces are well known, but their coordination remains poorly understood.
This work proposes a threefold control of tensile forces, by polarity, spindle position itself, and temporal progression during division, with these controls acting independently of each other. Anteroposterior asymmetry in the rate of dynein attachment to the microtubule alone is sufficient to cause posterior displacement of the spindle, reflecting polarity. Positional control reinforces this imbalance only at the end of sister chromatid separation (anaphase). Temporal control, on the other hand, is a function of the dynein pull time, which increases during division. Finally, these measurements suggest that the pushing forces, constant and symmetrical during mitosis, are dominant when the chromosomes are "at the equator" of the cell (metaphase) and contribute to the maintenance of the spindle at the center of the cell. On the contrary, during anaphase, the traction forces dominate and allow the posterior displacement of the spindle.
This work combines innovative approaches of computer image analysis and the statistical study of microtubule contacts at the cell periphery, thanks to their fluorescent labeling. Two populations of contacts have been measured in physiological conditions, revealing for one the distribution of tensile forces, for the other the distribution of push forces. This was made possible by a resolutely interdisciplinary approach, from an innovation patented by the laboratory in the control of microscopes to the design of software for the analysis of cell mechanics based on images.
These results show a robustness of mitotic spindle positioning that emerges from networks of dynamic actors (microtubules, motors), able to adapt to the forces generated and to probe the spindle position. They also suggest that symmetric and asymmetric divisions may share the same spindle positioning mechanisms, more or less dominant depending on the cellular context. Finally, this work opens the way to understand how such adaptation mechanisms could help cancer cells to cope with chromosomal defects and resist antimitotic drugs used in chemotherapy.
© Hélène Bouvrais & Jacques Pécréaux
(A) (Left) Push forces dominate in metaphase and keep the spindle in the center of the cell. (Right) Pulling forces dominate in anaphase and lead to posterior displacement of the spindle.
(B) The scientists demonstrated a triple control of the tensile forces that position the spindle, through three independent mechanisms: (left) spindle position dictates the number of contacts in the posterior region, creating a feedback loop; (middle) polarity regulates pulling forces by modulating the rate of dynein attachment to microtubules, with the latter being higher on the posterior side; (right) mitotic progression regulates forces through the rate of dynein detachment, i.e., the duration of pull on the microtubule.
(C) (From left to right) The scientists used the model organism nematode (Caenorhabditis elegans) from which they extracted the eggs. They then imaged the single-cell embryo by fluorescence microscopy after labeling the microtubules by modifying a corresponding gene to make it fluoresce. The images were then denoised and the microtubule contacts detected by an adapted image analysis. A statistical study of these measured contacts allows to map, along the axis of the embryo and over time, short contacts revealing traction forces (illustration map) and long contacts revealing push forces.
For more information :
The coordination of spindle-positioning forces during the asymmetric division of the C. elegans zygote.
Hélène Bouvrais, Laurent Chesneau, Yann Le Cunff, Danielle Fairbrass, Nina Soler, Sylvain Pastezeur, Thierry Pécot, Charles Kervrann, Jacques Pécréaux. EMBO reports