ERC FUNDED PROJECTS

The mechanism of cell division is conserved in many eukaryotes, from yeast to man. A contractile ring of filamentous actin and myosin II motors generates the force to bisect a mother cell into two daughters. The actomyosin ring is among the most complex cellular machines, comprising over 150 proteins. Understanding how these proteins organize themselves into a functional ring with appropriate contractile properties remains one of the great challenges in cell biology. Efforts to generate a comprehensive understanding of the mechanism of actomyosin ring assembly have been hampered by the lack of structural information on the arrangement of actin, myosin II, and actin modulators in the ring in its native state. Fundamental questions such as how actin filaments are assembled and organized into a ring remain actively debated. This project will investigate key issues pertaining to cytokinesis in the fission yeast Schizosaccharomyces pombe, which divides employing an actomyosin based contractile ring, using the methods of genetics, biochemistry, cellular imaging, DNA origami, genetic code expansion, and click chemistry. Specifically, we will (1) attempt to visualize actin filament assembly in live cells expressing fluorescent actin generated through synthetic biological approaches, including genetic code expansion and click chemistry (2) decipher actin filament polarity in the actomyosin ring using total internal reflection fluorescence microscopy of labelled dimeric and multimeric myosins V and VI generated through DNA origami approaches (3) address when, where, and how actin filaments for cytokinesis are assembled and organized into a ring and (4) reconstitute actin filament and functional actomyosin ring assembly in permeabilized spheroplasts and in supported bilayers. Success in the project will provide major insight into the mechanism of actomyosin ring assembly and illuminate principles behind cytoskeletal self-organization.

2 863 705 €

Start date: 2015-11-01, End date: 2020-10-31

AFRIGOS investigates the process of 'respacing' Africa, a political drive towards regional and continental integration, on the one hand, and the re-casting of Africa's engagement with the global economy, on the other. This is reflected in unprecedented levels of investment in physical and communications infrastructure, and the outsourcing of key functions of Customs, Immigration and security agencies. AFRIGOS poses the question of how far respacing is genuinely forging institutions that are facilitating or obstructing the movement of people and goods; that are enabling or preventing urban and border spaces from being more effectively and responsively governed; and that take into account the needs of African populations whose livelihoods are rooted in mobility and informality. The principal research questions are approached through a comparative study of port cities, border towns and other strategic nodes situated along the busiest transport corridors in East, Central, West and Southern Africa. These represent sites of remarkable dynamism and cosmopolitanism, which reflects their role in connecting African urban centres to each other and to other global cities. AFRIGOS considers how governance 'assemblages' are forged at different scales and is explicitly comparative. It works through 5 connected Streams that address specific questions: 1. AGENDA-SETTING is concerned with policy (re-)formulation. 2. PERIPHERAL URBANISM examines governance in border towns and port cities. 3. BORDER WORKERS addresses everyday governance emerging through the interaction of officials and others who make their livelihoods from the border. 4. CONNECTIVE INFRASTRUCTURE looks as the transformative effects of new technologies. 5. PEOPLE & GOODS IN MOTION traces the passage of people and goods and the regimes of regulation to which they are subjected. AFRIGOS contributes to interdisciplinary research on borderland studies, multi-level governance and the everyday state.

2 491 364 €

Start date: 2016-01-01, End date: 2020-12-31

Imagine being able to design into living cells and organisms de novo vesicle transport mechanisms that do not naturally exist? At one level this is a wild-eyed notion of synthetic biology. But we contend that this vision can be approached even today, focusing first on the process of exocytosis, a fundamental process that impacts almost every area of physiology. Enough has now been learned about the natural core machinery (as recognized by the award of the 2013 Nobel Prize in Physiology or Medicine to the PI and others) to take highly innovative physics/engineering- and DNA-based approaches to design synthetic versions of the secretory apparatus that could someday open new avenues in genetic medicine. The central idea is to introduce DNA-based functional equivalents of the core protein machinery that naturally form (coats), target (tethers), and fuse (SNAREs) vesicles. We have already taken first steps by using DNA origami-based templates to produce synthetic phospholipid vesicles and complementary DNA-based tethers to specifically capture these DNA-templated vesicles on targeted bilayers. Others have linked DNA oligonucleotides to trigger vesicle fusion. The next and much more challenging step is to introduce such processes into living cells. We hope to break this barrier, and in the process start a new field of research into “synthetic exocytosis”, by introducing Peptide-Nucleic Acids (PNAs) of tethers and SNAREs to re-direct naturally-produced secretory vesicles to artificially-programmed targets and provide artificially-programmed regulation. PNAs are chosen mainly because they lack the negatively charged phosphate backbones of DNA, and therefore are more readily delivered into the cell across the plasma membrane. Future steps, would include producing the transport vesicles synthetically within the cell by externally supplied origami-based PNA or similar cages, and - much more speculatively - ultimately using encoded DNA and RNAs to provide these functions.

3 000 000 €

Start date: 2015-09-01, End date: 2021-08-31

Amine containing compounds are ubiquitous in everyday life and find applications ranging from polymers to pharmaceuticals. The vast majority of amines are synthetic and manufactured on large scale which creates waste as well as requiring high temperatures and pressures. The increasing availability of biocatalysts, together with an understanding of how they can be used in organic synthesis (biocatalytic retrosynthesis), has stimulated chemists to consider new ways of making target molecules. In this context, the iterative construction of C-N bonds via biocatalytic hydrogen borrowing represents a powerful and unexplored way to synthesise a wide range of target amine molecules in an efficient manner. Hydrogen borrowing involves telescoping redox neutral reactions together using only catalytic amounts of hydrogen. In this project we will engineer the three key target biocatalysts (reductive aminase, amine dehydrogenase, alcohol dehydrogenase) required for biocatalytic hydrogen borrowing such that they possess the required regio-, chemo- and stereo-selectivity for practical application. Recently discovered reductive aminases (RedAms) and amine dehydrogenases (AmDHs) will be engineered for enantioselective coupling of alcohols (1o, 2o) with ammonia/amines (1o, 2o, 3o) under redox neutral conditions. Alcohol dehydrogenases will be engineered for low enantioselectivity. Hydrogen borrowing requires mutually compatible cofactors shared by two enzymes and in some cases will require redesign of cofactor specificity. Thereafter we shall develop conditions for the combined use of these biocatalysts under hydrogen borrowing conditions (catalytic NADH, NADPH), to enable the conversion of simple and sustainable feedstocks (alcohols) into amines using ammonia as the nitrogen source. The main deliverables of BIO-H-BORROW will be a set of novel engineered biocatalysts together with redox neutral cascades for the synthesis of amine products from inexpensive and renewable precursors.

2 337 548 €

Start date: 2017-06-01, End date: 2022-05-31