ERC FUNDED PROJECTS

Neural circuits, composed of interconnected neurons, represent the basic unit of the nervous system. One way to understand the highly complex arrangement of cross-talking, serial and parallel circuits is to resolve its developmental and evolutionary emergence. The rationale of the research proposal presented here is to elucidate the complex circuitry of the vertebrate and insect forebrain by comparison to the much simpler and evolutionary ancient “connectome” of the marine annelid Platynereis dumerilii. We will build a unique resource, the Platynereis Neuron Type Atlas, combining, for the first time, neuronal morphologies, axonal projections, cellular expression profiling and developmental lineage for an entire bilaterian brain. We will focus on five days old larvae when most adult neuron types are already present in small number and large part of the axonal scaffold in place. Building on the Neuron Type Atlas, the second part of the proposal envisages the functional dissection of the Platynereis chemosensory-motor forebrain circuits. A newly developed microfluidics behavioural assay system, together with a cell-based GPCR screening will identify partaking neurons. Zinc finger nuclease-mediated knockout of circuit-specific transcription factors as identified from the Atlas will reveal circuit-specific gene regulatory networks, downstream effector genes and functional characteristics. Laser ablation of GFP-labeled single neurons and axonal connections will yield further insight into the function of circuit components and subcircuits. Given the ancient nature of the Platynereis brain, this research is expected to reveal a simple, developmental and evolutionary “blueprint” for the olfactory circuits in mice and flies and to shed new light on the evolution of information processing in glomeruli and higher-level integration in sensory-associative brain centres.

2 489 048 €

Start date: 2012-03-01, End date: 2017-02-28

The vast majority of genetic diseases are complex traits, conditioned by multiple genetic and environmental factors. Yet our understanding of the genetics underlying such traits in humans remains extremely limited, due largely to the statistical complexity of inferring the effects of allelic variants in a genetically diverse population. Novel tools for the dissection of the genetic architecture of complex traits, therefore, can be most effectively developed in model organisms, where the contribution of individual alleles can be quantitatively determined in controlled genetic backgrounds. We have previously established the yeast Saccharomyces cerevisiae as a model for complex traits by unravelling complex genetic architectures that govern quantitative phenotypes in this organism. We achieved this by pioneering approaches that have revealed crucial information about the complexity of the underlying genetics. Here we propose to advance to the next level of complex trait dissection by developing systematic, genome-wide technologies that aim to identify all of the variants underlying a complex trait in a single step. In particular, we will investigate traits involved in mitochondrial function, which are both clinically relevant and highly conserved in yeast. Our combination of genomic technologies will allow us to: 1) systematically detect, with maximal sensitivity, the majority of genetic variants (coding and non-coding) that condition these traits; 2) quantify the contributions of these variants and their interactions; and 3) evaluate the strengths and limitations of current methods for dissecting complex traits. Taken together, our research will yield fundamental insights into the genetic complexity of multifactorial traits, providing valuable lessons and establishing novel genomic tools that will facilitate the investigation of complex diseases.

2 499 821 €

Start date: 2012-11-01, End date: 2017-10-31

"Translation of the genetically encoded information into polypeptides, protein biosynthesis, is a central function executed by ribosomes in all cells. In the case of membrane protein synthesis, integration into the membrane usually occurs co-translationally and requires a ribosome-associated translocon (SecYEG/Sec61). This highly coordinated process is poorly understood, since high-resolution structural information is lacking. Although single particle cryo-electron microscopy (cryo-EM) has given invaluable structural insights for such dynamic ribosomal complexes, the resolution is so far limited to 5-10 Å for asymmetrical particles. Thus, the mechanistic depth and reliability of interpretation has accordingly been limited. Here, I propose to use single particle cryo-EM at improved, molecular resolution of 3-4 Å to study two fundamental ribosome-associated processes: (i) co-translational integration of polytopic membrane proteins and (ii) recycling of the eukaryotic ribosome. First, we will visualize nascent polytopic membrane proteins inserting into the lipid bilayer via the bacterial ribosome-bound SecYEG translocon. Notably, the translocon will be embedded in a lipid environment provided by so-called nanodiscs. Second, we will visualize in a similar approach membrane protein insertion via the YidC insertase, the main alternative translocon. Third, as a novel research direction, we will determine the structure and function of eukaryotic ribosome recycling complexes involving the ABC-ATPase RLI. The results will allow, together with functional biochemical data, an in-depth molecular structure-function analysis of these fundamental ribosome-associated processes. Moreover, reaching molecular resolution for asymmetrical particles by single particle cryo-EM will lift this technology to a level of analytical power approaching X-ray and NMR methods. ERC funding would allow for this highly challenging research to be conducted in an internationally competitive way in Europe."

2 995 640 €

Start date: 2012-05-01, End date: 2017-04-30

"The degradation of mature mRNAs has emerged as a key step in the regulation of eukaryotic gene expression. Modulation of the half-life of mRNAs via their degradation is a powerful and versatile mechanism to swiftly alter the expression of proteins in response to changes in physiological conditions. The decay of mRNAs is performed by a set of macromolecular complexes that act in a sequential and coordinated manner, progressively eroding the ends of the transcript until its degradation is complete. These macromolecular assemblies contain only a few catalytically active subunits and a large number of regulatory components. How and why the activities are regulated within the architecture of the complexes is largely unknown. Also unclear are the mechanisms with which the complexes communicate with each other and/or with the changing composition of the nucleic acid. In this project, we will reconstitute the key protein complexes in mRNA decay from recombinant proteins in vitro. Specifically, we will focus on the evolutionary conserved deadenylation, decapping and exosome-Ski complexes. The reconstituted complexes will be used for structural studies to derive atomic models of the holoenzymes using a combination of X-ray crystallography and cryoelectron microscopy. In parallel to obtaining static views of the individual steps in the pathway, we will establish the assays to study how information from one processing step is passed on to the next in a dynamic manner. We will address the basis for the timing and interrelationship of the conserved enzymatic machineries and the influence of the mRNP composition on their activity. Our final goal is to recapitulate the complex behavior of the mRNA decay pathway in vitro. Our lab has extensive experience in biochemical reconstitution of protein complexes, in vitro biochemical assays and X-ray crystallography. In the next five years, we plan to implement cryoelectron microscopy within the scope of this proposal."

2 499 344 €

Start date: 2012-04-01, End date: 2017-03-31