Biology subjects

Pecreaux, J.

Publications and source records attributed to Pecreaux, J..

10 recordsLinked to original sources

bioRxiv2026

Semi supervised GAN for smart microscopy, fast and data efficient cell cycle classification

Modern optical microscopes are fully motorised; however, transforming them into truly smart systems requires real-time adjustment of acquisition settings in response to detected objects and dynamic biological events. At the core are classification algorithms that commonly depend on customised softwares and are generally designed for narrowly-defined biological applications. In addition, they often require substantial annotated datasets for effective training. We introduce a semi-supervised generative adversarial network (SGAN) for robust cell-cycle stage classification under low-resource conditions, adaptable to diverse cellular structures. The framework combines unlabelled microscopy images with synthetically generated samples to mitigate limited annotation, while preserving stable performance even when the unlabelled subset is class-imbalanced. Tested on the Mitocheck dataset, which features five mitosis classes, the model achieved 93{+/-}2% accuracy using only 80 labelled per class and 600 unlabelled images. The proposed algorithm is generic and can be readily adapted to new labeling schemes, classification targets, cell lines, or microscopy modalities through transfer learning. SGAN is well suited for integration into automated microscopes, enabling efficient and adaptable image analysis across diverse biological and microscopy applications.

bioinformatics↗

bioRxiv2024

Microtubule stiffening by doublecortin-domain protein ZYG-8 contributes to spindle orientation during C. elegans zygote division

In the Caenorhabditis elegans zygote, mutations in zyg-8DCLK1, the sole Doublecortin-family member, disrupt mitotic spindle positioning, as seen by immunofluorescence. Doublecortin proteins bind microtubules and are thought to stabilise or rigidify them. In the zygote, ZYG-8 only modestly affects microtubule growth and nucleation. We thus investigated whether these moderate dynamic perturbations alone could explain the spindle mispositioning observed in zyg-8 mutants. Using three complementary genetic perturbations--RNAi-mediated depletion of ZYG-8, its overexpression, and the thermosensitive zyg-8(or484ts) mutant (that disrupts microtubule binding)--we observed altered spindle pole oscillations and changes in microtubule cortical-contact behaviour, indicative of impaired cortical forces. Importantly, these phenotypes could not be fully explained by previously reported alterations in microtubule dynamics, suggesting an additional mechanism. Our findings indicate that ZYG-8 increases microtubule rigidity: ZYG-8 depletion or mutation led to more frequent microtubule bending and higher curvature and tortuosity. Simulations confirmed that reduced rigidity prolongs cortical contact lifetimes, an effect we experimentally observed in zyg-8(RNAi) embryos. Using custom biophysical assays, we showed that microtubule softening in zyg-8(RNAi) embryos and zyg-8 mutants reduced the efficiency of centring forces, leading to exaggerated spindle-pole oscillations. In mutants, the largest oscillations caused spindle poles to move closer to the cell periphery, preventing re-centring and resulting in spindle mispositioning and misorientation during late anaphase. Importantly, reducing cortical pulling forces rescued orientation defects, highlighting the importance of balanced pulling-pushing forces for proper spindle positioning. We propose that sufficient microtubule rigidity is essential for generating effective cortical pushing forces, potentially in synergy with other microtubule properties, which contribute to centring mechanisms that ensure accurate spindle orientation in late mitosis. Given that DCLK1 is frequently deregulated in human cancers and that accurate spindle positioning is essential for maintaining cell proliferation-differentiation balance, these findings may have implications for understanding how disruptions in microtubule mechanics contribute to carcinogenesis. Author summaryDuring cell division, the mitotic spindle--a structure made of microtubules that separates chromosomes in daughter cells--must position itself precisely to ensure healthy cell growth and differentiation. Microtubules are tiny, dynamic, semi-flexible fibers that notably generate forces at the cell periphery, cortical pulling and pushing. These two opposite forces contribute to position the spindle. In the worm C. elegans, the protein ZYG-8 (similar to human DCLK1) stabilizes the microtubules. Our study reveals that ZYG-8 also makes microtubules stiffer, a property critical for generating strong enough pushing forces. When zyg-8 was impaired (mutation or RNAi), microtubules bent excessively, weakening the pushing forces that keep the spindle correctly centered and oriented. As a result, softer microtubules in zyg-8 mutants caused the spindle to drift toward the cells periphery, resulting in mispositioning and misorientation when mitosis ends. By reducing the opposing cortical pulling forces in zyg-8 mutants, we restored normal spindle orientation, highlighting that microtubule rigidity helps balance the forces that position the spindle correctly. Our findings suggest that microtubule stiffness is essential for accurate cell division--a process vital for both normal development and disease prevention, including cancers where homologous proteins are often disrupted.

cell biology↗

bioRxiv2024

The Roboscope: Smart and Fast Microscopy for Generic Event-Driven Acquisition

Automation of fluorescence microscopy is a challenge for capturing rare or transient events in biology and medicine. It relies on smart devices that integrate and interpret the observed data, and react to the targeted biological event. We report on the Roboscope, a novel autonomous microscope combining sequence interruption and deep learning integration, allowing generic event-driven acquisitions. This system distinguishes itself by its adaptability to various experiments, quick capture of dynamic events, and minimal data greediness - training with less than 100 images per class. The Roboscopes capability is demonstrated in non-synchronized cells by capturing the metaphase, a 20-minute event happening once per day or less. Conversely, double thymidine-block synchronisation, despite occurring during DNA replication, may perturb mitotic-spindle mechanics. The Roboscopes versatility and efficiency offer significant advancements to tackle the current challenges of cell biology, spreading out advanced microscopy methods to fundamental research as well as high content screening and precision medicine.

bioengineering↗

bioRxiv2023

Lack of chromokinesin Klp-19 creates a more rigid midzone and affects force transmission during anaphase in C. elegans

Recent studies have highlighted the significance of the spindle midzone, the region between the segregating chromosomes, in ensuring proper chromosome segregation. By combining 3D electron tomography, cutting-edge light microscopy and a novel single cell in vitro essay allowing single molecule tracking, we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans by the chromokinesin KLP-19, and its relevance for proper spindle function. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and KLP-19 in C. elegans, the homologs of PRC1 and KIF4a, suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length and thus microtubule-based interactions in the spindle midzone, which affects spindle dynamics and force transmission. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone. SummaryKLP-19 controls microtubule length in the spindle midzone of C. elegans, affecting spindle dynamics and force transmission during mitosis.

cell biology↗

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↗

bioRxiv2020

Neural network fast-classifies biological images using features selected after their random-forests-importance to power smart microscopy.

Artificial intelligence is nowadays used for cell detection and classification in optical microscopy, during post-acquisition analysis. The microscopes are now fully automated and next expected to be smart, to make acquisition decisions based on the images. It calls for analysing them on the fly. Biology further imposes training on a reduced dataset due to cost and time to prepare the samples and have the datasets annotated by experts. We propose here a real-time image processing, compliant with these specifications by balancing accurate detection and execution performance. We characterised the images using a generic, high-dimensional feature extractor. We then classified the images using machine learning for the sake of understanding the contribution of each feature in decision and execution time. We found that the non-linear-classifier random forests outperformed Fishers linear discriminant. More importantly, the most discriminant and time-consuming features could be excluded without any significant loss in accuracy, offering a substantial gain in execution time. It suggests a feature-group redundancy likely related to the biology of the observed cells. We offer a method to select fast and discriminant features. In our assay, a 79.6 {+/-} 2.4 % accurate classification of a cell took 68.7 {+/-} 3.5 ms (mean {+/-} SD, 5-fold cross-validation nested in 10 bootstrap repeats), corresponding to 14 cells per second, dispatched into 8 phases of the cell cycle using 12 feature-groups and operating a consumer market ARM-based embedded system. Interestingly, a simple neural network offered similar performances paving the way to faster training and classification, using parallel execution on a general-purpose graphic processing unit. Finally, this strategy is also usable for deep neural networks paving the way to optimising these algorithms for smart microscopy.

bioinformatics↗

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↗