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Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently-developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new standalone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled setup for long-term experimental evolution.

SummaryCyclin-dependent kinase (CDK) plays an essential role in determining the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK, it remains elusive how they coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. This could be due to the lack of tools to monitor CDK activity in living cells. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by using a newly developed CDK activity biosensor, Eevee-spCDK, based on Förster Resonance Energy Transfer (FRET). Taking advantage of this system, we unravel the profile of CDK activity in vegetatively growingS. pombecells. Thus, we detect a transient increase in S phase followed by a gradual increment during G2 phase. CDK activity then reaches its maximum in early M phase and rapidly decreases at mitotic exit. During G2 phase, CDK activity exhibits a biphasic pattern,i.e., an early slow increase and a late fast rise prior to the G2/M phase transition, as predicted from mathematical studies. Remarkably, although CDK activity does not necessarily correlate with cyclin levels, we find that it converges to the same level around mitotic onset in several mutant backgrounds, includingpom1Δcells andwee1orcdc25overexpressing cells. These data provide the first direct evidence that cells enter M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.

Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, due in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from models such as budding and fission yeast, but even for these morphologically stereotypical cells, assessment of cell volume has relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle difference in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work reveals how coupling FXm with yeast genetics will bring new insights into the complex biology of cell growth.SUMMARY STATEMENTFluorescence exclusion provides a unique method to accurately measure the volume of yeast cells at both the population and single-cell levels.

Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building artificial genetic circuits and studying their behaviours has rapidly demonstrated its potential in a broad range of fields in the life sciences. From the assembly of synthetic genomes to the generation of novel biological functions, yeast cells have imposed themselves as the most powerful eukaryotic model for this approach. However, we are only beginning to explore the possibilities of synthetic biology, and the perspectives it offers in a genetically amenable system such as yeasts are endless. Copyright © 2016 John Wiley & Sons, Ltd.

Microfluidic technologies have become a standard tool in cell biological studies, offering unprecedented control of the chemical and physical environment of cells grown in microdevices, the possibility of multiplexing assays, as well as the capacity to monitor the behavior of single cells in real time while dynamically manipulating their growth medium. However, the properties of the materials employed for the fabrication of microchips that are compatible with live-cell imaging has limited the use of these techniques for a broad range of experiments. In particular, the strong absorption of a large panel of small molecules by these materials prevents the accurate delivery of compounds of interest. Here we describe a novel microsystem dedicated to live-cell imaging that (1) uses alternative materials devoid of absorptive properties, and (2) allows for dynamic in-chip control of sample temperature. Based on a proof-of-concept design that we have routinely used with non-adherent fission yeast cells, this chapter details all the steps for the fabrication and utilization of these microdevices.

Background Real-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with precision and high time resolution, and the simultaneous alteration of multiple parameters is a major challenge. Recently, microfluidics has provided powerful solutions for such analyses, bringing an unprecedented level of control over the conditions and the medium in which cells under microscopic observation are grown. However, such technologies have remained under-exploited, largely as a result of the complexity associated with microfabrication procedures. Results In this study, we have developed simple but powerful microfluidic devices dedicated to live-cell imaging. These microsystems take advantage of a robust elastomer that is readily available to researchers and that presents excellent bonding properties, in particular to microscopy-grade glass coverslips. Importantly, the chips are easy-to-build without sophisticated equipment, and they are compatible with the integration of complex, customized fluidic networks as well as with the multiplexing of independent assays on a single device. We show that the chips are re-usable, a significant advantage for the popularization of microfluidics in cell biology. Moreover, we demonstrate that they allow for the dynamic, accurate and simultaneous control of multiple parameters of the cellular environment. Conclusions While they do not possess all the features of the microdevices that are built using complex and costly procedures, the simplicity and versatility of the chips that we have developed make them an attractive alternative for a range of applications. The emergence of such devices, which can be fabricated and used by any laboratory, will provide the possibility for a larger number of research teams to take full advantage of these new methods for investigating cell biology. Electronic supplementary material The online version of this article (10.1186/s12860-018-0158-z) contains supplementary material, which is available to authorized users.

International audience; One of the most important areas of research on microfluidic technologies focuses on the identification and characterisation of novel materials with enhanced properties and versatility. Here we present a fast, easy and inexpensive microstructuration method for the fabrication of novel, flexible, transparent and biocompatible microfluidic devices. Using a simple hot press, we demonstrate the rapid (30 s) production of various microfluidic prototypes embossed in a commercially available soft thermoplastic elastomer (sTPE). This styrenic block copolymer (BCP) material is as flexible as PDMS and as thermoformable as classical thermoplastics. It exhibits high fidelity of replication using SU-8 and epoxy master molds in a highly convenient low-isobar (0.4 bar) and iso-thermal process. Microfluidic devices can then be easily sealed using either a simple hot plate or even a room-temperature assembly, allowing them to sustain liquid pressures of 2 and 0.6 bar, respectively. The excellent sorption and biocompatibility properties of the microchips were validated via a standard rhodamine dye assay as well as a sensitive yeast cell-based assay. The morphology and composition of the surface area after plasma treatment for hydrophilization purposes are stable and show constant and homogenous distribution of block nanodomains (similar to 22 degrees after 4 days). These domains, which are evenly distributed on the nanoscale, therefore account for the uniform and convenient surface of a "microfluidic scale device". To our knowledge, this is the first thermoplastic elastomer material that can be used for fast and reliable fabrication and assembly of microdevices while maintaining a high and stable hydrophilicity.

International audience; Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

In this review, we look at the processes that occur `behind the scenes' in Wnt signalling, within the Wnt-producing cells. The Wnt community has long been focused upon events that occur downstream of Wnt binding to its receptors, but the recent discovery that the maturation of the Wnt protein may

In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model’s predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities., Author Summary The eukaryotic cell division cycle is driven by fluctuating activities of cyclin-dependent kinases (Cdk), which are activated and inactivated by several mechanisms, including cyclin synthesis and degradation. Although the cell cycle is driven by many different Cdk-cyclin complexes in present-day eukaryotes, experiments with fission yeast demonstrate that a single Cdk-cyclin complex is sufficient to order the events of the cell cycle. Surprisingly, a Cdk-inhibitory mechanism working through tyrosine phosphorylation of the kinase subunit, which is essential for modern fission yeast, becomes dispensable in the Minimal Cdk Network (MCN). By developing both deterministic and stochastic models of the MCN, we show that a different inhibitory mechanism based on a stoichiometric Cdk inhibitor (called Rum1) can compensate for the lack of inhibitory Cdk phosphorylation in the MCN. We also demonstrate that this compensation mechanism is suppressed in wild-type fission yeast cells by the other Cdk-cyclin complexes, which down-regulate the level of Rum1. These predictions of computational modelling are supported by our experimental data. Our work provides new insights into the interplay between the structure of the control network and the physiology of the cell cycle.

Wnt proteins function as morphogens that can form long-range concentration gradients to pattern developing tissues. Here, we show that the retromer, a multiprotein complex involved in intracellular protein trafficking, is required for long-range signaling of the Caenorhabditis elegans Wnt ortholog EGL-20. The retromer functions in EGL-20–producing cells to allow the formation of an EGL-20 gradient along the anteroposterior axis. This function is evolutionarily conserved, because Wnt target gene expression is also impaired in the absence of the retromer complex in vertebrates. These results demonstrate that the ability of Wnt to regulate long-range patterning events is dependent on a critical and conserved function of the retromer complex within Wnt-producing cells.

Abstract The secreted glycoproteins of the Wnt family activate a highly conserved signalling pathway that controls several developmental processes. Wnt ligands function as morphogens that act at a distance from their source by establishing a concentration gradient. The various fates of the Wnt responding cells along the morphogenetic field contribute to the precise patterning of the embryo and depend on the concentration of morphogen to which they are exposed. However, the different mechanisms that allow the controlled formation of the Wnt gradient are unclear. Here we give an overview of current understanding of the mechanisms that potentially control the spreading of the Drosophila Wnt ortholog Wingless.

Axin, APC, and the kinase GSK3β are part of a destruction complex that regulates the stability of the Wnt pathway effector β-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3β, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/β-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway,pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibitsmab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/β-catenin signaling in C. elegans.

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-beta chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vbeta13 and Vbeta2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vbeta, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vbeta13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vbeta families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vbeta13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

Summary Fifteen years ago, it was proposed that the cell cycle in fission yeast can be driven by quantitative changes in the activity of a single protein kinase complex comprising a cyclin – namely cyclin B – and cyclin dependent kinase 1 (Cdk1). When its activity is low, Cdk1 triggers the onset of S phase; when its activity level exceeds a specific threshold, it promotes entry into mitosis. This model has redefined our understanding of the essential functional inputs that organize cell cycle progression, and its main principles now appear to be applicable to all eukaryotic cells. But how does a change in the activity of one kinase generate ordered progression through the cell cycle in order to separate DNA replication from mitosis? To answer this question, we must consider the biochemical processes that underlie the phosphorylation of Cdk1 substrates. In this Commentary, we discuss recent findings that have shed light on how the threshold levels of Cdk1 activity that are required for progression through each phase are determined, how an increase in Cdk activity generates directionality in the cell cycle, and why cell cycle transitions are abrupt rather than gradual. These considerations lead to a general quantitative model of cell cycle control, in which opposing kinase and phosphatase activities have an essential role in ensuring dynamic transitions.

Background: The switch from cellular proliferation to differentiation occurs to a large extent through specific programs of gene expression. In fission yeast, the master regulator of sexual differentiation, ste11, is induced by environmental conditions leading to mating and meiosis. Results: We show that phosphorylation of serine 2 (S2P) in the C-terminal domain of the largest subunit of the RNA polymerase II (Po111) enzyme by the Lsk1 cyclin-dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. The recruitment of the Lsk1 kinase initiates in the vicinity of the transcription start site of ste11, resulting in a marked increase of S2P on the ste11 unit, including an extended 5 ' untranslated region (5 ' UTR). This pattern contrasts with the classical gradient of S2P toward the 3 ' region. In the absence of S2P, both PoIII occupancy at the ste11 locus and ste11 expression are impaired. This results in sterility, which is rescued by expression of the ste11 coding sequence from the adh1 promoter. Conclusion: Thus, the S2P polymerase plays a specific, regulatory role in cell differentiation through the induction of ste11.

International audience; Control of eukaryotic cell proliferation involves an extended regulatory network, the complexity of which has made it difficult to understand the basic principles of the cell cycle. To investigate the core engine of the mitotic cycle we have generated a minimal control network in fission yeast that efficiently sustains cellular reproduction. Here we demonstrate that orderly progression through the major events of the cell cycle can be driven by oscillation of an engineered monomolecular cyclin-dependent protein kinase (CDK) module lacking much of the canonical regulation. We show further that the CDK oscillator acts as the primary organizer of the cell cycle, imposing timing and directionality to a system of two CDK activity thresholds that define independent cell cycle phases. We propose that this simple core architecture forms the basic control of the eukaryotic cell cycle.

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila. To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.

SummaryWnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.

Disabled proteins are a conserved family of monomeric adaptor proteins that in mammals are implicated in the endocytosis of lipoprotein receptors. Previous studies have shown that the sole Caenorhabditis elegans Disabled homologue, DAB-1, is involved in the lipoprotein receptor-mediated secretion of a fibroblast growth factor. We show here that DAB-1 is essential for the uptake of yolk protein by developing oocytes, and for the localisation of the yolk receptor RME-2. The localisation of DAB-1 in oocytes is itself dependent upon clathrin and AP2, consistent with DAB-1 acting as a clathrin-associated sorting protein during yolk protein endocytosis. DAB-1 is also required for the endocytosis of molecules from the pseudocoelomic fluid by the macrophage-like coelomocytes, and is broadly expressed in epithelial tissues, consistent with a general role in receptor-mediated endocytosis. We also show that dab-1 mutations are synthetic lethal in combination with loss-of-function mutations affecting the AP-1 and AP-3 complexes, suggesting that the reduced fluid and membrane uptake exhibited by dab-1 mutants sensitises them to defects in other trafficking pathways.

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.

The transactivation of TCF target genes induced by Writ pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). We show that disruption of beta-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21(CIP1/WAF1) promoter. Following disruption of beta-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21(CIP1/WAF1), transcription, which in turn mediates G1 arrest and differentiation. Thus, the beta-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells

Cell cycle progression is an essential process whose regulation is highly conserved among eukaryotes. Cyclin-dependent protein kinases (Cdks) are key components of the cell cycle control network and interact with various cyclins to drive robust cell proliferation. A regulatory mechanism of Cdk activity, known as the Wee1/Cdc25 feedback loop, is highly conserved in eukaryotic cells and is essential in wild type cells. We previously demonstrated that MCN cells lacking the conserved Wee1/Cdc25 feedback loops (referred to as MCN-AF) are surprisingly viable. However, MCN-AF cells show a significantly reduced growth rate. After performing experimental evolution with MCN-AF cells, we identified a number of mutations linked to an improvement of growth in the absence of the Wee1/Cdc25 feedback loop. Spo12 is a small nuclear protein of unknown function, which we have shown, when disrupted, will improve the growth of MCN-AF cells and shorten G1 phase length. Indeed, I demonstrate that Spo12 has a role in a novel feedback loop on Cdk activity, which when disrupted will reduce overall Cdk activity during the cell cycle. Furthermore, I have also successfully adapted an automated image analysis pipeline to provide high-throughput data acquisition from complex time-lapses of cell cycle markers, which will be a useful tool for the fission yeast community. Lastly, I have also contributed to the development of a time-lapse volume measurement methodology, as part of a larger project to accurately measure volume in fission yeast.; La progression du cycle cellulaire est un processus essentiel dont la régulation est hautement conservée chez les eucaryotes. Les protéines kinases dépendantes des cyclines (Cdks) sont des composants clés du réseau de contrôle du cycle cellulaire et interagissent avec diverses cyclines pour conduire une prolifération cellulaire robuste. Un mécanisme de régulation de l'activité des Cdk, connu sous le nom de boucle de rétroaction Wee1/Cdc25, est hautement conservé dans les cellules eucaryotes et est essentiel dans les cellules de type sauvage. Nous avons précédemment démontré que les cellules MCN dépourvues des boucles de rétroaction Wee1/Cdc25 conservées (appelées MCN-AF) sont étonnamment viables. Cependant, les cellules MCN-AF présentent un taux de croissance significativement réduit. Après avoir réalisé une évolution expérimentale avec les cellules MCN-AF, nous avons identifié un certain nombre de mutations liées à une amélioration de la croissance en l'absence de la boucle de rétroaction Wee1/Cdc25. Spo12 est une petite protéine nucléaire de fonction inconnue, dont nous avons montré que, lorsqu'elle est perturbée, elle améliore la croissance des cellules MCN-AF et raccourcit la durée de la phase G1. En effet, je démontre que Spo12 joue un rôle dans une nouvelle boucle de rétroaction sur l'activité de la Cdk, qui, lorsqu'elle est perturbée, réduit l'activité globale de la Cdk au cours du cycle cellulaire. En outre, j'ai également adapté avec succès un pipeline d'analyse d'images automatisé pour permettre l'acquisition de données à haut débit à partir de laps de temps complexes de marqueurs du cycle cellulaire, ce qui constituera un outil utile pour la communauté des levures de fission. Enfin, j'ai également contribué au développement d'une méthodologie de mesure du volume en time-lapse, dans le cadre d'un projet plus large visant à mesurer avec précision le volume chez la levure de fission.