ERC FUNDED PROJECTS

Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy, yet despite this pivotal importance it has taken over twenty years for its first downstream target to be discovered. However, whilst our identification of the autophagy related membrane protein Atg9 as the first Atg1 substrate is an important advance, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase will enable us to provide a definitive mechanistic understanding of autophagy. We have devised a novel permeabilized cell assay that reconstitutes the pathway in vitro, allowing us to recapitulate key steps in the autophagic process and thereby determine how the individual steps that lead up to autophagy are controlled. We will use this system to dissect the functional role of Atg1 kinase in autophagosome-vacuole fusion (Objective 1), and to determine the origin of the autophagic membrane and the role of Atg1 in expanding these (Objective 2). To reveal how Atg1/ULK1 kinase is activated in mammalian cells, we will apply the unique and carefully tailored synthetic in vivo approaches that we have recently developed (Objective 3). By focusing on the activation of the Atg1 kinase and the molecular events that it executes, we will be able to explain its central role in regulating the autophagic process and define the mechanistic steps in the pathway.

1 955 666 €

Start date: 2018-06-01, End date: 2023-05-31

Eukaryotic cells constantly convert signals between biochemical energy and mechanical work to timely accomplish many key functions such as migration, division or development. Filopodia are essential finger-like structures that emerge at the cell front to orient the cell in response to its chemical and mechanical environment. Yet, the molecular interactions that make the filopodia mechanosensitive are not known. To tackle this challenge we propose unique biophysical in vitro and in vivo experiments of increasing complexity. Here we will focus on how the underlying actin filament bundle regulates filopodium growth and retraction cycles at the micrometer and seconds scales. These parallel actin filaments are mainly elongated at their barbed-end by formins and cross-linked by bundling proteins such as fascins. We aim to: 1) Elucidate how formin and fascin functions are regulated by mechanics at the single filament level. We will investigate how formin partners and competitors present in filopodia affect formin processivity; how fascin affinity for the side of filaments is modified by filament tension and formin presence at the barbed-end. 2) Reconstitute filopodium-like actin bundles in vitro to understand how actin bundle size and fate are regulated down to the molecular scale. Using a unique experimental setup that combines microfluidics and optical tweezers, we will uncover for the first time actin bundles mechanosensitive capabilities, both in tension and compression. 3) Decipher in vivo the mechanics of actin bundles in filopodia, using fascins and formins with integrated fluorescent tension sensors. This framework spanning from in vitro single filament to in vivo meso-scale actin networks will bring unprecedented insights into the role of actin bundles in filopodia mechanosensitivity.

1 499 190 €

Start date: 2016-03-01, End date: 2021-02-28

Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear. My lab has a long-standing track record in elucidating molecular mechanisms underlying key complement reactions. We have revealed, for example, how the interplay between assembly and proteolysis of these large multi-domain protein complexes achieves elementary regulatory functions, such as localization, amplification and inhibition, in the central (so-called alternative) pathway of complement. Results from my lab underpin research programs for the development of novel therapeutic approaches in academia and industry. Here the goal is to understand how the molecular mechanisms of complement attack and defense on cell membranes determine clearance of a cell. Enabled by new mechanistic insights and preliminary data we can now address both long-standing and novel questions. In particular, we will address the role of membrane organization and dynamics in complement attack and defense. Facilitated by recent technological developments, we will combine crystallography, cryo-EM, cryo-ET and high-resolution microscopy to resolve complement complex formations and reactions on membranes. Thus, this project aims to provide an integrative understanding of the molecular complement mechanisms that determine cell fate. Results will likely be of immediate importance for novel therapeutic approaches for a range of complement-related diseases. Furthermore, it will provide clarity into the general, and possibly fundamental, role of complement in tissue maintenance in mammals.

2 332 500 €

Start date: 2018-07-01, End date: 2023-06-30

The interest on autophagy, an evolutionarily conserved process in eukaryotes, has enormously increased in the last years, since autophagy is involved in many diseases such as cancer and neurodegenerative disorders. Autophagosome formation is the key process in autophagy. Despite extensive work, the model of autophagosome formation is not yet well established. Some important questions on autophagosome biogenesis remain to be elusive, such as where the bona fide marker protein of autophagosome, LC3, is lipidated, how lipidated LC3 functions in autophagosome formation, and how the proteins for LC3 lipidation and delipidation are involved in autophagosome formation. Although genetic approaches have been useful to identify genes involved in autophagy, they are chronic and thereby the dynamics of phenotypic change cannot be followed, making them not suited for study highly dynamic process such as autophagosome formation. Herein, I propose to develop and use novel chemical probes to address these issues. First, I plan to prepare semi-synthetic caged LC3 proteins and apply them to monitor dynamics of autophagosome formation in the cell in order to address those questions on autophagosome formation. The semi-synthetic LC3 proteins are expected to confer a temporal control and to realize manipulation of protein structure, which renders such studies possible. Second, I intend to develop a versatile approach targeting specific endogenous proteins using a reversible chemically induced dimerization (CID) system, termed as “knock on and off” strategy. I plan to use this approach to elucidate the function of two distinct PI3K complexes in autophagosome formation. On one hand, the establishment of novel approaches will open up a new avenue for studying biological processes. On the other hand, the use of the tool will reveal the mechanism of autophagy.

1 500 000 €

Start date: 2016-09-01, End date: 2021-08-31