Insights from synthetic yeasts Coudreuse D Yeast 33(9):483-92



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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.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging Chen T, Gomez-Escoda B, Munoz-Garcia J, Babic J, Griscom L, Wu PY, Coudreuse D Open Biology 6(8)



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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.

Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription Banyai G, Baïdi F, Coudreuse D*, Szilagyi Z* (*senior authors) Nature Communications 7:11161



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Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression.

Cell cycle control by a minimal cdk network Gérard C, Tyson JJ, Coudreuse D*, Novák B1* (*senior authors) PLoS Comput Biol. 2015 Feb 6;11(2):e1004056 2015-02-06



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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.

Phosphorylation network dynamics in the control of cell cycle transitions Fisher D, Krasinska L, Coudreuse D, Novák B J Cell Sci 125(Pt 20):4703-11;



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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.

Driving the cell cycle with a minimal CDK control network Coudreuse D, Nurse P. Nature 468(7327):1074-9;



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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.

A gene-specific requirement of RNA polymerase II CTD phosphorylation for sexual differentiation in S. pombe. Coudreuse D, van Bakel H, Dewez M, Soutourina J, Parnell T, Vandenhaute J, Cairns B, Werner M, Hermand D. Curr Biol 20(12):1053-64;



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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 (PolII) 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 PolII 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.

Mammalian Wnt3a is released on lipoprotein particles Neumann S, Coudreuse DY, van der Westhuyzen DR, Eckhardt ER, Korswagen HC, Schmitz G, Sprong H. Traffic 10(3):334-43;



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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.

Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells Yang PT, Lorenowicz MJ, Silhankova M, Coudreuse DY, Betist MC, Korswagen HC. Dev Cell 14(1):140-7;



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Wnt 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.

Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans Oosterveen T, Coudreuse DY, Yang PT, Fraser E, Bergsma J, Dale TC, Korswagen HC. Dev Biol 308(2):438-48;



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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.

C. elegans Disabled is required for cell-type specific endocytosis and is essential in animals lacking the AP-3 adaptor complex. Holmes A, Flett A, Coudreuse D, Korswagen HC, Pettitt J. J Cell Sci 120(Pt 15):2741-51;



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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.

The making of Wnt: new insights into Wnt maturation, sorting and secretion Coudreuse D and Korswagen HC Development 134(1):3-12;



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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 have a profound effect on its signalling properties has excited great interest. In the last 2 years, several key regulators of Wnt production have been discovered, but our global understanding of this process remains relatively poor. Several models that reconcile former and recent observations of Wnt modification, sorting and secretion, and which highlight the potential of this emerging field, are presented here.

Wnt gradient formation requires retromer function in Wnt-producing cells Coudreuse D, Roël G, Betist MC, Destrée O, Korswagen HC. Science 312(5775):921-4;



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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.

The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis AP, Tjon-Pon-Fong M, Moerer P, van den Born M, Soete G, Pals S, Eilers M, Medema R, Clevers H. Cell 111(2):241-50;



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The transactivation of TCF target genes induced by Wnt 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.

The Axin-like protein PRY-1 is a negative regulator of a canonical Wnt pathway in C. elegans Korswagen HC, Coudreuse DY, Betist MC, van de Water S, Zivkovic D, Clevers HC. Genes Dev 16(10):1291-302



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Axin, APC, and the kinase GSK3 beta are part of a destruction complex that regulates the stability of the Wnt pathway effector beta-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/GSK3 beta, 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/beta-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 inhibits mab-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/beta-catenin signaling in C. elegans.