Biology subjects

Chesneau, L.

Publications and source records attributed to Chesneau, L..

5 recordsLinked to original sources

bioRxiv2023

Only three principal components account for inter-embryo variability of the spindle length over time.

How to quantify inter-individual variability? When measuring many features per experiment/individual, this question becomes non-trivial. One challenge lies in choosing features to recapitulate high-dimensional data. This paper focuses on spindle elongation phenotypes to highlight how a data-driven approach can help tackle this challenge. We showed that only three typical elongation patterns describe spindle elongation in C. elegans one-cell embryo. We called them archetypes. These archetypes were automatically extracted from the experimental data using principal component analysis (PCA) rather than defined a priori. They accounted for more than 95% of inter-individual variability in a dataset of more than 1600 experiments across more than 100 different experimental conditions (RNAi, mutants, changes in temperature, etc.). The two first archetypes were consistent with standard measures in the field, namely the average spindle length and the spindle elongation rate in late metaphase and anaphase. However, our archetypes were not strictly corresponding to these classic, manually-set, features. The third archetype, accounting for 6% of the variability, was novel and corresponded to a transient spindle shortening in late metaphase. We revealed that it is part of spindle elongation dynamics in all conditions. It is reminiscent of the spindle elongation pattern observed upon kinetochore function defects. Interestingly, because these archetypes were all three present from metaphase on, it implied that spindle elongation around the anaphase onset is sufficient to predict its late anaphase length. We validated this idea using a machine-learning approach. The inter-individual differences between embryos depleted from cell division-related proteins have the same underlying nature as inter-individual differences naturally arising between wild-type embryos. The same conclusion holds also when analysing embryos dividing at various temperatures. We thus propose that beyond the apparent complexity of the spindle and variability in the phenotypes of various gene depletions, only three independent mechanisms account for spindle elongation, weighted differently in the various conditions; meanwhile, no mechanism is specific to any condition. As such, given amounts of these three archetypes could represent a quantitative phenotype.

bioinformatics↗

bioRxiv2022

Kinetochore microtubules flux poleward along fixed centrosome-anchored microtubules during the metaphase of C. elegans one-cell embryo.

The mitotic spindle, a key structure to partition chromosomes during cell division, connects its poles to the chromosomes through microtubules. Their plus-ends, oriented towards the chromosomes, exhibit dynamic instability crucial for kinetochore correct attachments. Involved in this process, the poleward flux implicates the displacement of microtubules towards the spindle poles, coordinated with polymerisation at the plus ends. The mechanisms behind this are diverse. It includes treadmilling powered by microtubule depolymerisation at the spindle poles, sliding of spindle microtubules by molecular motors like Kinesin-5, and pushing microtubules away from the chromosomes by chromokinesins. Interestingly, no such flux was reported in the Caenorhabditis elegans zygote, although all proteins contributing to flux in mammals have homologs in the nematode. To explore this, we fluorescently labelled microtubules and conducted photobleaching. We found no global poleward flux; the bleached zones edges moved inward. The centrosome-side front motion was caused by dynamic instability, while the chromosome-side front exhibited faster recovery, suggesting an additional mechanism. This larger slope was detected only near the chromosomes, indicating that only kinetochore microtubules undergo flux. Consistently, this flux depended on proteins ensuring the chromosome attachment and growth of the kinetochore microtubules, notably NDC-80, CLS-2CLASP, and ZYG-9XMAP215. Furthermore, this flux decreased as metaphase progressed and attachments transitioned from side-to end-on; it was reduced by SKA-1 recruitment. Treadmilling was unlikely to account for these observations, as most kinetochore microtubules do not reach spindle poles in the zygote spindle. Conversely, the depletion of kinesin-12 KLP-18KIF15, which cross-links and focuses microtubules at meiosis, reduced the front rate. Ultimately, we propose that the sole kinetochore microtubules slide along spindle microtubules, likely powered by KLP-18, contrasting with solid displacement in other systems. It aligns with observations in human cells of decreasing flux with increasing chromosome distance.

cell biology↗

bioRxiv2019

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. By measuring the dynamics of astral microtubules at the cortex, we revealed the presence of two populations, residing there for 0.4 s and 1.8 s, which correspond to the pulling and pushing events, respectively. Such an experiment offers a unique opportunity to monitor both forces that position the spindle under physiological conditions and study their variations along the anteroposterior axis (space) and the mitotic progression (time). By investigating pulling-force-generating events at the microscopic level, we showed that an anteroposterior asymmetry in dynein on-rate - encoding pulling-force imbalance - is sufficient to cause posterior spindle displacement. The regulation by spindle position - reflecting the number of microtubule contacts in the posterior-most region - reinforces this imbalance only in late-anaphase. Furthermore, we exhibited the first direct proof that the force-generator increasing persistence to pull (processivity) accounts for the temporal control of pulling force throughout mitosis. We thus propose a three-fold control of pulling force, by the polarity, spindle position and mitotic progression. Focusing on pushing force, we discovered a correlation between its density and the stability of the spindle position during metaphase, which strongly suggests that the pushing force contributes to maintaining the spindle at the cell centre. This force remains constant and symmetric along the anteroposterior axis during the division. The pulling one increases in intensity and becomes dominant at anaphase. In conclusion, the two-population study enabled us to decipher the complex regulation of the spindle positioning during cell division.

biophysics↗

bioRxiv2017

Dynamics Of Dynein At Microtubule Plus-Ends And The Cortex During The Division Of The C. elegans Zygote

During asymmetric cell division, dynein generates forces, which position the spindle to reflect polarity and ensure correct daughter cell fates. The transient cortical localization of dynein raises the question of its targeting. We found that it accumulates at the microtubule plus-ends like in budding yeast, indirectly hitch-hiking on EBP-2EB1 likely via dynactin. Importantly, this mechanism, which modestly accounts for cortical forces, does not transport dynein, which displays the same binding/unbinding dynamics as EBP-2EB1. At the cortex, dynein tracks can be classified as having either directed or diffusive-like motion. Diffusive-like tracks reveal force-generating dyneins. Their densities are higher on the posterior tip of the embryos, where GPR-1/2LGN concentrate, but their durations are symmetric. Since dynein flows to the cortex are non-polarized, we suggest that this posterior enrichment increases dynein binding, thus accounts for the force imbalance reflecting polarity, and supplements the regulation of mitotic progression via the non-polarized detachment rate.

biophysics↗

bioRxiv2017

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

During the asymmetric division of the Caenorhabditis elegans nematode zygote, the polarity cues distribution and daughter cell fates depend on the correct positioning of the mitotic spindle, which results from both centering and cortical pulling forces. Revealed by anaphase spindle rocking, these pulling forces are regulated by the force generator dynamics, which are in turn consequent of mitotic progression. We found a novel, additional, regulation of these forces by the spindle position. It controls astral microtubule availability at the cortex, on which the active force generators can pull. Importantly, this positional control relies on the polarity dependent LET-99 cortical band, which restricts or concentrates generators to a posterior crescent. After delaying anaphase onset, we detected this positional pulling force regulation in C. elegans as a precocious spindle rocking with respect to anaphase onset. We ascribed this control to the microtubule dynamics at the cortex. Indeed, in mapping the cortical contacts, we found a correlation between the centrosome-cortex distance and the microtubule contact density. In turn, it modulates pulling force generator activity. We modelled this control, predicting and experimentally validating that the posterior crescent extent controlled where the anaphase oscillations started, in addition to mitotic progression. We found in particular that the oscillation onset position resists changes in cellular geometry and moderate variations of active force generator count. Finally, we propose that spatially restricting force generator to a posterior crescent sets the spindles final position, reflecting polarity through the LET-99 dependent restriction of force generators to a posterior crescent. This regulation superimposes that of force generator processivity. This novel control confers a low dependence on microtubule and active force generator exact numbers or dynamics, provided that they exceed the threshold needed for posterior displacement. Interestingly, this robustness originates in cell mechanics rather than biochemical networks.

biophysics↗