ERC FUNDED PROJECTS

A cell’s decision to die is governed by multiple input signals received from a complex network of programmed cell death (PCD) pathways, including apoptosis and programmed necrosis. Additionally, under some conditions, autophagy, whose function is mainly pro-survival, may act as a back-up death pathway. We propose to apply new approaches to study the molecular basis of two important questions that await resolution in the field: a) how the cell switches from a pro-survival autophagic response to an apoptotic response and b) whether and how pro-survival autophagy is converted to a death mechanism when apoptosis is blocked. To address the first issue, we will screen for direct physical interactions between autophagic and apoptotic proteins, using the protein fragment complementation assay. Validated pairs will be studied in depth to identify built-in molecular switches that activate apoptosis when autophagy fails to restore homeostasis. As a pilot case to address the concept of molecular ‘sensors’ and ‘switches’, we will focus on the previously identified Atg12/Bcl-2 interaction. In the second line of research we will categorize autophagy-dependent cell death triggers into those that directly result from autophagy-dependent degradation, either by excessive self-digestion or by selective protein degradation, and those that utilize the autophagy machinery to activate programmed necrosis. We will identify the genes regulating these scenarios by whole genome RNAi screens for increased cell survival. In parallel, we will use a cell library of annotated fluorescent-tagged proteins for measuring selective protein degradation. These will be the starting point for identification of the molecular pathways that convert survival autophagy to a death program. Finally, we will explore the physiological relevance of back-up death mechanisms and the newly identified molecular mechanisms to developmental PCD during the cavitation process in early stages of embryogenesis.

2 500 000 €

Start date: 2013-03-01, End date: 2018-02-28

The endoplasmic reticulum (ER) is the cellular organelle that serves as the entry site into the secretory pathway. Although the ER has a single continuous membrane, it is functionally divided into subdomains (SDs). These specialized regions allow the ER to carry out a multitude of functions such as folding, maturation, quality control and export, of all secreted and most membrane bound proteins; lipid biosynthesis; ion homeostasis; and communication with all other organelles. The ER is therefore not only the largest single copy organelle in most eukaryotic cells, but, thanks to the presence of SDs, also one of the more functionally diverse and structurally complex. Changes in ER functions have been shown to contribute to the progression of many diseases such as heart disease, neurodegeneration and diabetes. Moreover, a robustly functioning ER is required for development of dedicated secretory cells such as antibody producing plasma cells and insulin secreting pancreatic cells. The past years have brought about a revolution in our understanding of basic ER functions and the homeostatic responses coordinating them. However, despite their obvious importance for robust activity of the ER, we still know very little about SD biogenesis and function. Therefore, the time is now ripe to extend our understanding by facing the next challenges in the field. Specifically, it is now of major importance to understand how cells ensure accurate SD biogenesis and function. This proposal tackles this question by three independent but complementary screens each aimed at revealing one aspect of SDs: their structure/function, biogenesis or dynamics. The merging of all three aspects of information will give us a holistic picture of this process – one that could not have been attained by the pixilated view of any single piece of data. We propose to explore these facets in both yeast and mammals utilizing systematic tools such as high content microscopic screens followed up by the creation of genetic interaction maps and follow-up hypothesis based biochemical and genetic experiments. By combining several approaches and different organisms we hope to enable a more efficient reconstruction of this complex process. When completed this proposal will have shed light on a little explored but central question in cellular biology. More broadly, the mechanisms that arise as guiding SD biogenesis may help us in understanding how membrane domains form in general. Due to the novelty of our approach and the cutting-edge tools used to tackle this fundamental problem in cell biology, this work will provide a paradigm for addressing complex biological questions in eukaryotic cells. It may very well be that it is this aspect of the proposal that may ultimately most broadly impact the biological community.

1 499 999 €

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

Signaling, genetic regulatory circuits, and tissue morphology are inherently coupled to each other during embryonic development. Although changes in cellular and tissue morphology are commonly treated as a downstream consequence of cell fate decision processes, there are multiple examples where morphological changes occur concurrently with the differentiation processes. This suggests that a feedback between cell morphology and regulatory processes can play an important role in coordinating tissue development. Currently, however, we lack the experimental, theoretical, and conceptual tools to understand this interplay between cell morphology, signaling, and regulatory circuits. In particular, we need to understand (1) how intercellular signaling depends on the cellular morphology and on the properties of the boundary between cells, and (2) how intercellular signaling, genetic circuits, and cell morphology integrate to generate robust differentiation patterns. Here, I propose to combine quantitative in-vitro and in-vivo experiments with mathematical modeling to address these questions in the context of Notch signaling and Notch mediated patterning, typically used for coordinating differentiation between neighboring cells during development. We will utilize novel reporters and micropatterning technology to analyze Notch signaling between pairs of cells. We will elucidate how the geometry and the molecular composition of the boundary between cells affect signaling. At the tissue level, we will study how the interplay between cell morphology and Notch signaling gives rise to robust patterning in the mammalian inner ear. We will use cochlear inner ear explant imaging to track the transition from disordered undifferentiated state to ordered pattern of hair and supporting cells in the cochlea. Together with a novel hybrid modeling approach, we will provide the foundation for a systems level understanding of development that interconnects morphology and regulatory circuits.

2 000 000 €

Start date: 2016-06-01, End date: 2021-05-31

Cellular growth and migration depend on intracellular communication webs mediated by polypeptide growth factors. One example comprises EGF-like growth factors and their ErbB receptor tyrosine kinases. EGFR and HER2 are frequently involved in cancer progression, and they serve as targets for cancer therapeutics. The existence of a kinase-dead receptor, as well as the emergence of resistance in patients treated with EGFR and HER2 blockers, instigated a paradigm shift from a linear EGF-to-ErbB cascade to a robust network characterized by multiple feedback loops. We assume that deregulation of feedback loops plays essential roles in human cancer. Because of the abundance of feedback regulation, we predict subtle, multi-component impact on disease. Aiming at the natural richness of feedback regulation in breast cancer, we will develop in vitro models of normal mammary cells, and introduce genetic, disease-mimicry manipulations. Two time domains of feedback regulation will be addressed: (i) the early domain of post-translational modifications, which we will explore using proteomic approaches. And (ii) the late domain comprising alterations in transcription, micro-RNAs and alternative splicing, processes we will investigate using deep sequencing and array technologies. Once verified and characterized in normal cells, we will survey the operational status of the unravelled feedback loops in genetically manipulated cell systems and in tumour specimens, using immunological and bio-informatical approaches. Detailed knowledge of feedback regulation of multi-layered signalling networks, such as ErbB, is expected to shed light on the currently elusive basis of signal integration and elimination of noise, as well as identify markers of prognosis.

2 228 180 €

Start date: 2011-08-01, End date: 2016-07-31