ERC FUNDED PROJECTS

Asymmetric cell division is a key mechanism for the generation of cell diversity in eukaryotes. During this process, a polarized mother cell divides into non-equivalent daughters. These may differentially inherit fate determinants, irreparable damages or age determinants. Our aim is to decipher the mechanisms governing the individualization of daughters from each other. In the past ten years, our studies identified several lateral diffusion barriers located in the plasma membrane and the endoplasmic reticulum of budding yeast. These barriers all restrict molecular exchanges between the mother cell and its bud, and thereby compartmentalize the cell already long before its division. They play key roles in the asymmetric segregation of various factors. On one side, they help maintain polarized factors into the bud. Thereby, they reinforce cell polarity and sequester daughter-specific fate determinants into the bud. On the other side they prevent aging factors of the mother from entering the bud. Hence, they play key roles in the rejuvenation of the bud, in the aging of the mother, and in the differentiation of mother and daughter from each other. Recently, we accumulated evidence that some of these barriers are subject to regulation, such as to help modulate the longevity of the mother cell in response to environmental signals. Our data also suggest that barriers help the mother cell keep traces of its life history, thereby contributing to its individuation and adaption to the environment. In this project, we will address the following questions: 1 How are these barriers assembled, functioning, and regulated? 2 What type of differentiation processes are they involved in? 3 Are they conserved in other eukaryotes, and what are their functions outside of budding yeast? These studies will shed light into the principles underlying and linking aging, rejuvenation and differentiation.

2 200 000 €

Start date: 2010-05-01, End date: 2015-04-30

Cells use circuits of interacting proteins to respond to their environment. In the past decades, molecular biology has provided detailed knowledge on the proteins in these circuits and their interactions. To fully understand circuit function requires, in addition to molecular knowledge, new concepts that explain how multiple components work together to perform systems level functions. Our lab has been a leader in defining such concepts, based on combined experimental and theoretical study of well characterized circuits in bacteria and human cells. In this proposal we aim to find novel principles on how circuits resist fluctuations and errors, and how they can be controlled by drugs: (1) Why do key regulatory systems use bifunctional enzymes that catalyze antagonistic reactions (e.g. both kinase and phosphatase)? We will test the role of bifunctional enzymes in making circuits robust to variations in protein levels. (2) Why are some genes regulated by a repressor and others by an activator? We will test this in the context of reduction of errors in transcription control. (3) Are there principles that describe how drugs combine to affect protein dynamics in human cells? We will use a novel dynamic proteomics approach developed in our lab to explore how protein dynamics can be controlled by drug combinations. This research will define principles that unite our understanding of seemingly distinct biological systems, and explain their particular design in terms of systems-level functions. This understanding will help form the basis for a future medicine that rationally controls the state of the cell based on a detailed blueprint of their circuit design, and quantitative principles for the effects of drugs on this circuitry.

2 261 440 €

Start date: 2010-03-01, End date: 2015-02-28

The ribosome is a large cellular organelle that plays a central role in the process of protein synthesis in all organisms. Currently, structural information at atomic resolution exists only for bacterial ribosomes and some of their functional complexes. Eukaryotic ribosomes are larger and significantly more complex than their bacterial counterparts. They consist of two unequal subunits with a combined molecular weight of approximately 4 million Daltons and contain 70-80 different protein molecules and four different RNAs. Currently the only structural information on eukaryotic ribosomes is available from cryo electron microscopic reconstructions in the nanometer resolution range, which is insufficient to derive information about the function of the eukaryotic ribosome at the atomic level. The aim of this proposal is to use X-ray crystallography to obtain structural and functional information on the eukaryotic ribosome and its functional complexes at high resolution. The key targets of the structural work will be: i) the structure of the small ribosomal subunit, ii) the structure of the large ribosomal subunit, and iii) structures of complexes involved in the initiation of protein synthesis. Besides the obvious fundamental importance of this research for understanding protein synthesis in eukaryotes the proposed studies will also be the prerequisite for understanding the structural basis of the regulation of protein synthesis in normal cells and how it is perturbed in various diseases. Finally, comparing the structures of bacterial and eukaryotic ribosomes is important for understanding the specificity of various clinically used antibiotics for the bacterial ribosome.

2 446 725 €

Start date: 2010-07-01, End date: 2015-06-30

Immunological memory confers long term protection against pathogens and is the basis of successful vaccination. Following antigenic stimulation long lived plasma cells and memory B cells are maintained for a lifetime, conferring immediate protection and enhanced responsiveness to the eliciting antigen. However, in the case of variable pathogens such as influenza virus, B cell memory is only partially effective, depending on the extent of similarity between the preceding and the new viruses. The B cell response is dominated by serotype-specific antibodies and heterosubtypic antibodies capable of neutralizing several serotypes appear to be extremely rare. Understanding the basis of broadly neutralizing antibody responses is a critical aspect for the development of more effective vaccines. In this project we will explore the specificity and dynamics of human antibody responses to influenza virus by using newly developed technological platforms to culture human B cells and plasma cells and to analyze the repertoire of human naïve and memory T cells. High throughput functional screenings, structural analysis and testing in animal models will provide a thorough characterization of the human immune response. The B cell and T cell analysis aims at understanding fundamental aspects of the immune response such as: the selection and diversification of memory B cells; the individual variability of the antibody response, the mechanisms of T-B cooperation and the consequences of the original antigenic sin and of aging on the immune response. This analysis will be complemented by a translational approach whereby broadly neutralizing human monoclonal antibodies will be developed and used: i) for passive vaccination against highly variable viruses; ii) for vaccine design through the identification and production of recombinant antigens to be used as effective vaccines; and iii) for active vaccination in order to facilitate T cell priming and jump start the immune responses.

1 979 200 €

Start date: 2010-09-01, End date: 2015-08-31

Small RNA-mediated regulation of gene expression is a recent addition to fundamental gene regulatory mechanisms that directly or indirectly affect possibly every gene of a eukaryotic genome. The predominant sources of small RNA in somatic tissues are microRNA genes that encode short dsRNA hairpins of evolutionary conserved sequence. Disorders of metabolism, such as obesity and type 2 diabetes are poorly understood at a molecular level. In this application we propose to explore if miRNA regulatory networks play a role in these diseases. We will employ state of the art methods for identification of small RNAs and their regulated targets and use biochemical, cell and animal model systems to study the detailed molecular mechanisms of metabolic gene regulation by miRNAs. In addition, we will investigate the underlying principles of how RNAs are taken up by cells and develop methods that will improve delivery of miRNA mimetics or inhibitors through cell-specific uptake. The specific aims of this study are: Aim 1: To define the small regulatory miRNA content of liver, muscle and adipose tissue that are associated with abnormal glucose and lipid homeostasis and to dissect the underlying molecular pathways that govern their expression. Aim 2: To characterize the functions of miRNAs in insulin resistance, glucose uptake and production, fatty acid oxidation and lipogenesis. Aim 3: To identify factors and dissect the pathways that regulate RNA uptake by cells and to develop novel pharmacological treatment strategies to manipulate miRNA-expression. Together, this proposal will shed light on the function that miRNA regulatory networks play in metabolism and in the pathophysiology of obesity/type 2 diabetes. In addition, these studies will contribute to the development of new RNA delivery technologies that are urgently needed as experimental tools as well as for novel therapeutic strategies.

2 021 235 €

Start date: 2010-07-01, End date: 2015-06-30

There is an immediate need to increase our understanding of the genetic basis for fitness differences in natural populations (Ellegren and Sheldon Nature 452:169-175, 2008). Fortunately, technological developments within genome research, notably the recent ability to retrieve massive amounts of DNA sequence data based on next generation sequencing , will make possible completely novel investigations of the link between genotypes and phenotypes in non-model organisms. With our background as major players in molecular ecology and evolutionary genomics of non-models for the last 15-20 years, we are excellently placed to take on a leading role in this process, developing a Next Generation Molecular Ecology . This research program will combine studies of candidate genes with large-scale gene expression analysis, several mapping approaches and comparative genomics to study the genetic basis of trait evolution in wild bird populations. First, we will search for and analyse loci involved with reproductive isolation and adaptive population divergence in a well-known system for speciation research the pied flycatcher and the collared flycatcher. A milestone of this program will be genome sequencing of the two flycatcher species. Second, we will track the genetic basis of behaviour using a unique breeding population of zebra finches and benefitting from the recently obtained genome sequence of this species. Third, we will identify the targets for adaptive evolution during avian evolution using comparative genomics. Overall, the program will be able to reveal the molecular genetic architecture behind phenotypic variation. The potential for scientific break-through in this interdisciplinary program should be significant.

2 500 000 €

Start date: 2010-04-01, End date: 2015-09-30

The hallmark of social insect colonies is reproductive division of labour which is often associated with dramatic morphological and behavioural differences between queens, workers and males. The aim of this proposal is three-fold. First, we will use our recently developed fiducial identification system to investigate the general principles of social organisation and division of labour. The video tracking of workers labelled with markers derived from the augmented reality library ARTag allows us for the first time to distinguish up to 2000 individuals and precisely locate them every 500ms, hence allowing large-scale experiments addressing the question of how the behaviour of individual workers is influenced by the joint effects of environmental factors and social interactions. The second related aim is to investigate how the level of altruism within colonies and the reliability of communication systems are shaped by colony kin structure. Because it is not possible to conduct artificial evolution with social insects we will use a new experimental system consisting of colonies of small mobile robots with simple vision and communication abilities. This system permits to conduct hundreds of generations of experimental evolution in colonies with variable group composition to identify the factors affecting the evolution of altruism and communication. Finally, we will complement these studies with a genetic perspective using a remarkable genetic social polymorphism that we recently discovered in the fire ant Solenopsis invicta. The advent of new ultra high-throughput sequencing techniques will allow us to document the steps involved in the evolution of this genetic social polymorphism and test the suggestion that the chromosome involved in the social polymorphism has the properties of a sex chromosome. This project will be highly interdisciplinary, involving skills in evolutionary biology, the study of animal behaviour, bioinformatics, engineering and molecular biology

2 497 500 €

Start date: 2010-05-01, End date: 2016-04-30

One of the central mysteries of immunology is self-tolerance. How does the human body select ~10e12 T lymphocytes, that are reactive to foreign pathogens but tolerant to normal cellular constituents of the host? Over the last few years, my laboratory identified 2 fundamental mechanisms used by thymocytes to establish T cell tolerance. We demonstrated that the affinity threshold for negative selection is a constant for all thymocytes expressing MHC I restricted TCRs. This binding affinity threshold (KD H 6 ¼M; estimated T1/2 H 2 sec) is the fundamental biophysical parameter used by TCRs to delete autoimmune T cells. We also established how the TCR generates distinct signals for positive and negative selection. At the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signaling intermediates. The ability to compartmentalize signaling molecules differentially within the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the molecular basis for central tolerance. In the present application, we plan to fully understand 1-how the biophysical events during antigen binding to the TCR initiate an intracellular signal; 2-how these signals program an unambiguous cell fate and 3-how the system fails, when an autoimmune T cell is generated and activated. We will use a combination of transgenic and knockout mice, biochemistry and molecular imaging to fully define how the TCR functions as a molecular switch.

1 930 000 €

Start date: 2010-04-01, End date: 2015-03-31