In most instances, the growth of solid tumors occurs in a constrained environment and requires a competition for space. The competition for space results in a bidirectional mechanical coupling between the tumor and its close environment, the stroma. The expanding neoplastic tissue compresses the stroma, thus builds up, and stores an internal stress; in parallel, the stroma is contractile and exerts a mechanical stress on the tumor. To evaluate the effect of such a stress, we grow multicellular aggregates made of cancer cells under a controlled mechanical pressure. We observe that a gentle compression (500 Pa) drastically reduces the growth rate of spheroids made of cancer cells, whereas it has no impact on the same cells, when grown individually. A major difference between individual cells and multicellular aggregates is the presence of Extracellular Matrix (ECM) in the multicellular context. Contrarily to cells, which are almost incompressible, the ECM is permeable to water and highly compliant. Thus, a gentle compression generates a much larger strain in the ECM than in the cells. Thus, we model and characterize the compressibility and the permeability of composite aggregate, made of cells and ECM. We find that ECM properties mainly determine the mechanical response of a multicellular aggregate to external stimuli. In addition, we observe that the ECM compression has a large impact on cell proliferation, migration and morphology. We conclude that the ECM play the role of a pressure sensor in a multicellular context.

“Immunotherapy has recently transformed the outcome for patients with some lethal cancers such as melanoma and lung cancer. Immunotherapy now shows promise in treating breast and other cancers. However, less than 50% of patients respond to current immunotherapies and many responders become resistant. What are the intrinsic and induced mechanisms of resistance to immunotherapies? This important topic will be addressed by Dr Roger Lo, a pioneer in dissecting the complex biological mechanisms that determine sensitivity and resistance to ground-breaking therapies”    

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L'ADN, molécule dépositaire du message génétique et support de l’hérédité, est soumis quotidiennement à l'agression par des agents exogènes ou endogènes. Ces altérations génétiques, directement liées au processus de mutagenèse, sont également associées au vieillissement et impliquées dans les phénomènes de cancérogenèse et de létalité cellulaire. Le problème qui se pose aux biologistes est de pouvoir évaluer et de comprendre le rôle précis de chaque défaut. Ainsi, l’utilisation de fragments d’ADN synthétiques (oligonucléotides), préparés par des approches chimiques ou biochimiques, de séquences parfaitement définies et contenant une lésion déterminée, constitue un outil précieux pour étudier précisément l'implication biologique d'une lésion particulière au niveau moléculaire (mécanisme de formation, stabilité, réparation, réplication, pouvoir mutagène, modification structurale…). Nous exposerons dans la première partie du séminaire, les principales approches synthétiques développées au laboratoire pour la préparation des sondes nucléiques substrats ainsi que les études biochimiques, physico-chimiques et structurales menées, permettant une meilleure compréhension du mode d'action de divers agents génotoxiques issus de processus radiatifs, chimiques ou physiologiques.

Dans un second temps, basées sur les propriétés d’auto-assemblage à l’échelle nanométrique, les capacités de la biochimie et de la chimie moderne à synthétiser et fonctionnaliser à façon l’ADN, nous verrons comment ce biopolymère peut être utilisé pour le nano-manufacturing. Ainsi, après avoir définies les propriétés structurales et d’auto-assemblages intrinsèques uniques de l’ADN, nous aborderons au travers de plusieurs exemples récents, ses potentialités en fabrication bio-inspirée de nano-édifices et nano-objets utilisables dans les technologies du futur.

The follicular epithelium in the Drosophila ovary is an ideal model for the study of epithelial biology. It possesses many classical epithelial features, such as a columnar cell shape, apical/basal polarity, and canonical cell adhesion complexes, and yet is a relatively simple tissue and is highly tractable for molecular and cell biological analysis.

See: http://anatomy.ucsf.edu/nystul/

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The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost 'generic' marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium.
Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called 'mini-guts' in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of human stomach, liver, pancreas, prostate and kidney. Using CRISPR/Cas9 technology, genes can be efficiently modified in organoids of various origins.

It is an open question how development can result in highly reproducible structures despite strong intrinsic variability on the molecular and cellular level. The anchor cell organizes the development of the reproductive organs of the nematode C. elegans. In this talk, I will discuss two events in the life of the anchor cell that are strongly impacted by intrinsic variability. First, the anchor cell is born and specified in a stochastic manner that we found to rely strongly on variability in cell signaling and the cell cycle. Second, the anchor cell induces an invariant cell fate pattern in the vulva precursor cells in a distance-dependent manner. However, we found that the position of the anchor cell relative to the vulva precursor cells shows strong animal-to-animal variability and we uncovered a mechanism, involving cell signaling and cell migration, that corrects for anchor cell misplacement  to generate the correct vulva cell fate pattern in all animals.

The human brain is the most fascinating and most complex organ of all. Despite this enormous complexity, the billions of neurons in our brain arise from a limited number of progenitors following a specific set of lineage decisions. We combine Drosophila genetics with 3D organoid cultures derived from pluripotent human stem cells to unravel the basic principles of brain development.
In the fruitfly Drosophila, neural stem cells called neuroblasts generate all neurons in a series of asymmetric cell divisions. Defects in asymmetric cell division result in the formation of lethal brain tumors that can be transplanted indefinitely. We have recently discovered an unexpected and unconventional mechanism involving a long non-coding RNA that explains why the neural stem cells in those tumors are immortalized and no longer obey the proliferation control mechanisms that act in normal, healthy Drosophila neural stem cells.
To investigate whether our results hold true in humans as well, we have developed cerebral organoids. Cerebral organoids are pluripotent stem cell derived 3D cultures that are remarkably similar to a human embryonic brain. With intriguing precision, they recapitulate key brain morphogenetic events like the formation of distinct progenitor and differentiation layers, the generation of neuronal subclasses and the establishment of coordinated neuronal activity. We have used patient derived iPS cells to model microcephaly, a human neurodevelopmental disorder difficult to recapitulate in mice. To recapitulate more complex neuropsychiatric disorders, we have reconstituted the dorso-ventral brain axis. By fusing differentially patterned organoids, we can monitor long distance interneuron migration between distinct areas of the human brain. Our data describe an in vitro approach that recapitulates development of even the most complex organ and can be used to gain insights into disease mechanisms.

We are using the C. elegans zygote as a model to understand basic principles that govern self-organizing cortical polarity.  Zygotic polarity is specified by the asymmetric enrichment of highly conserved polarity determinants, known as PAR proteins, into complementary anterior and posterior cortical domains. PAR asymmetries are established in response to a transient sperm-derived cue and then dynamically maintained, after the cue is gone, through local PAR protein interactions and local coupling to the cortical actomyosin cytoskeleton. Our goal is to understand how robust, self-stabilizing, cortical polarity emerges through these local interactions.  In this talk, I will summarize our current understanding of how this works, focusing on three main organizing principles: (1) Mutual antagonism, in which anterior and posterior PAR proteins compete with one another to inhibit cortical binding and/or activity; (2) local clustering of polarity proteins, which leads to ultrasensitive dependence of cortical binding on the densities of proteins and their local antagonists, and which couples polarity proteins to the actomyosin cortex; and (3) mechanochemical feedback coupling in which PAR proteins modulate actomyosin contractility and cortical flows to promote (or inhibit) their own redistribution.
 

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Organised by Danny Reinberg, Howard Hughes Medical Institute, NYU Langone School of Medicine at Smilow Research Center, New York, USA (Sabbatical 2017 - Blaise Pascal Research Chair)

With Edith Heard, UMR3215/U934 and Geneviève Almouzni, Dir. Of the Research Center, UMR3664, Institut Curie

Registration is free: http://sondage.curie.fr/index.php?sid=24693&lang=en

Deadline September 18th

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G Protein Coupled receptors (GPCRs) are integral membrane protein and constitute the largest family of membrane proteins. GPCRs are localised at the plasma membrane of eukaryotic cells. They mediate signal transduction from the extracellular to the intracellular compartment and play an important role in cell communication for physiological and pathological processes. Once activated by their respective ligands, GPCRs undergo conformational changes required for activating intracellular G protein, or arrestin and Kinase recruitment. GPCRs activation can be define by an ensemble of conformational states that are in dynamic equilibrium in the cell. Understanding the molecular basis of ligand binding mode and signal transduction require solving the structure of these different receptor conformation bound to ligand and to intracellular signalling partners. Over the last decade, recent advance in protein engineering and micro crystallography, allowed solving the structure of a large number of GPCRs, representing different conformational states. I will review and illustrate the recent advances in the field by using the human adenosine receptor A2A as a case study. In the second part of my talk, I will show how recent methodology can be apply to other receptors with an example that is currently under investigation in our lab: the human metabotropic glutamate receptor dimer. High-resolution structures of GPCRs provide unprecedented source of information for designing better drugs.

SAVE THE DATE 

La demi-journée Scientifique du Centre de Recherche se tiendra le vendredi 20 octobre 2017 à partir de 8h30 en Amphi BURG. 

Chairs 2017 : Aura Carreira, UMR3348 et Yohanns Bellaïche, UMR3215/U934 

More information on: https://www.rockefeller.edu/our-scientists/heads-of-laboratories/899-eric-d-siggia/

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Human embryonic stem cells when confined within two dimensional micropatterns with diameters in the 0.5-1mm range and subject to uniform Wnt or BMP stimulation, will spontaneously form radially symmetric spatial patterns that recapitulate the proximal-distal axis of the mammalian embryo at the time of gastrulation. This assay, when combined with inducible cell lines that express the canonical morphogens or inhibitors, becomes a very powerful technique to understand how an epithelium with ~2000 cells can self organize. To eliminate the boundaries that are inherent in 2D culture we have extended the assay to cells in defined hydrogels. They form closed apical-basal polarized shells that form a localized primitive streak in response to suitable morphogens. This quantitative assay shows in a context very different from the embryo how the canonical signaling pathways, generate spatial patterns over large scales, but it also reveals cell biological aspects of signaling that are difficult to study in mammalian embryos.

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. We recently investigated whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2 or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. This work therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.
 

Degradation of essential amino acids is an immunosuppressive tumour escape strategy exploited by many tumours to hamper tumour-specific immune responses. However, the mechanisms by which cancer cells compensate for the shortage of essential amino acids in the local microenvironment remain unclear. We performed RNA-seq analysis of tumour cells expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which revealed extensive remodelling of tumour cell metabolism, amino acid biosynthesis and amino acid transport gene expression. In particular, our findings demonstrated that expression of IDO or tryptophan 2,3-dioxygenase (TDO) induces up-regulation of several amino acid transporter genes, which allows improved glutamine and tryptophan import into tumour cells. We extended these results by demonstrating that re-programming of the amino transporter profile is caused by shortage of tryptophan, rather than accumulation of the tryptophan degradation by-product kynurenine, and is dependent on ATF4 expression, as ATF4 knockdown cells failed to up-regulate appropriate amino acid transporters under low tryptophan conditions. Our studies shed light on the compensatory mechanisms employed by tumours to allow survival under conditions of nutritional stress and have relevance for the identification of novel targets for pharmacological inhibition of tumours expressing amino acid-degrading enzymes IDO and TDO.

Gisou van der Goot studied engineering at the Ecole Centrale de Paris, then did a PhD in Molecular Biophysics at the Nuclear Energy Research Center, Saclay, France, followed by a postdoc at the European Molecular Biology Laboratory (EMBL) in Heidelberg. She started her own group in 1994 in the department of Biochemistry, University of Geneva, became Associate professor at the Faculty of Medicine (Univ. Geneva) in 2001 and finally Full Professor at the EPFL in 2006, where she co-founded the Institute of Global Health. In 2014, she was appointed Dean of EPFL's School of Life Sciences.

The integration of distinct endocytic routes is critical to regulate receptor signaling. A non-clathrin endocytic pathway (NCE) of the epidermal growth factor receptor (EGFR) is activated at high ligand concentrations, in parallel to clathrin-mediated endocytosis (CME). EGFR-NCE requires receptor ubiquitination, which also occurs at high EGF doses, and targets EGFRs to degradation, attenuating signaling. Despite its critical role in restricting EGFR signaling, the molecular mechanism of EGFR-NCE was poorly defined. Through an unbiased proteomic approach coupled to siRNA screening, we identified NCE-specific regulators, including the endoplasmic reticulum (ER)-resident protein reticulon-3 (RTN3), and a specific cargo, CD147. RTN3 was critical for EGFR/CD147 internalization via NCE, promoting the creation of plasma membrane (PM)–ER contact sites that were required for the formation/elongation of NCE tubular invaginations. Local Ca2+ release at these sites, triggered by IP3-dependent activation of ER Ca2+ channels, was needed for the completion of EGFR internalization and vesicle fission, in concert with the action of the GTPase dynamin. Thus, we identified a novel mechanism of EGFR endocytosis that relies on ER-PM contact sites and local Ca2+ signaling. This modality of internalization integrates numerous cellular functions towards its execution - as NCE regulators are proteins belonging to different cellular compartments, including ER, mitochondria and nucleus - and it is critical to mediate EGF-dependent cellular responses.

Cell membranes have developed a tremendous complexity of lipids and proteins geared to perform the functions cells require. To facilitate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane sub-compartmentalization. Key to understanding the principles underlying liquid-liquid de-mixing in cell membranes is the mutual weak interactions between sterols, sphingolipids
and raft proteins. The potential for sphingolipid-cholesterol self-assembly combines with protein specificity to dynamically regulate protein segregation within the membrane plane. The regulation of the two dimensional separation of lipids and proteins in membranes into dynamic liquid membrane rafts, separating from the surrounding bilayer, is dependent on the propensity for liquid phase separation. Most importantly, cellular plasma membranes seem to be poised close to a phase transition, facilitating dynamic sub-compartmentalization with little energetic cost.
The capability of liquid-liquid phase transitions depends on a tight control of cellular lipid composition. Amazingly, this stringent regulation translates into a new measure for metabolic status. We have developed a shotgun mass spectrometry platform that can analyse hundreds of lipids in only a few minutes with absolute quantification. This effective routine of shotgun lipidomics became possible by introducing a unique workflow that combined improved extraction protocols with cutting edge mass spectrometry and novel software. We have used this technology to analyse blood lipidomes from thousands of human subjects. The data show that the blood lipidomics can be used to measure health and disease.