ERC FUNDED PROJECTS

Biological cells possess a chemical “sense of smell” and a physical “sense of touch”. Structure, dynamics, development, differentiation and even apoptosis of cells are guided by physical stimuli feeding into a regulatory network integrating biochemical and mechanical signals. Cells are equipped with both, force-generating structures, and stress sensors including force-sensitive structural proteins or mechanosensitive ion channels. Pathways from force sensing to structural and transcriptional controls are not yet understood. The goal of the proposed interdisciplinary project is to quantitatively establish such pathways, connecting the statistical physics and the mechanics to the biochemistry. We will measure and model the complex non-equilibrium mechanical structures in cells, and we will study how external and cell-generated forces activate sensory processes that (i) act (back) on the morphology of the cell structures, and (ii) lead to cell-fate decisions, such as differentiation. The most prominent stress-bearing and -generating structures in cells are actin/myosin based, and the most prominent mechanoactive and -sensitive cell types are fibroblasts in connective tissue and myocytes in muscle. We will first focus on actin/myosin bundles in fibroblasts and in sarcomeres in developing heart muscle cells. We will observe cells under the influence of exactly controlled external stresses. Forces on suspended single cells or cell clusters will be exerted by laser trapping and sensitively detected by laser interferometry. We furthermore will monitor mechanically triggered transcriptional regulation by detecting mRNA in the nucleus of mouse stem cells differentiating to cardiomyocytes. We will develop fluorescent mRNA sensors that can be imaged in cells, based on near-IR fluorescent single-walled carbon nanotubes. Understanding mechanical cell regulation has far-ranging relevance for fundamental cell biophysics, developmental biology and for human health.

2 425 200 €

Start date: 2014-06-01, End date: 2019-05-31

Kainate receptors (KARs) are often regarded as the last frontier of glutamate receptor research, since much less is known about their physiological roles compared with that of the other glutamate receptor subtypes. This field of research is very important not just because of the unique role that KARs play in neuronal function, including specific forms of synaptic plasticity, but because of the increasing evidence that KARs are involved in a plethora of brain diseases and that KAR antagonists are promising novel therapeutic targets. I propose to lead a highly multidisciplinary approach, in collaboration with colleagues at Bristol and strategic collaborators worldwide, to develop novel pharmacological and genetic tools, which will be rapidly disseminated to the neuroscience community. These tools will be used here to test hypotheses regarding functions of KARs in granule cells (GCs) in the dentate gyrus of the hippocampal formation, with a focus on mossy fibre long-term potentiation (LTP). We propose four interrelated objectives: (i) to develop potent and selective antagonists for the GluK2 subunit of KARs, (ii) to generate GC specific knockouts of the five KAR subunits, by floxing GluK1-5 and crossing with a GC-specific Cre recombinase mouse line, (iii) to use these and existing tools in a combined pharmacological and genetic approach, to understand the functions of KARs at mossy fibre synapses in acute and organotypic hippocampal slices. A new development will be to record simultaneously from synaptically coupled GC-CA3 neuronal pairs and to image Ca2+ from participating mossy fibre boutons, (iv) to extend these investigations to the study of mossy fibre function, in particular LTP, in anaesthetised animals and to establish the function of mossy fibre LTP in hippocampus-dependent learning and memory. Although highly ambitious, the proposal is based on a long track record of KAR research by the PI and his collaborators plus a substantial amount of preliminary data.

2 500 000 €

Start date: 2014-02-01, End date: 2019-01-31

Optogenetics are used to activate or silence specific sets of neurons, intracellular signaling pathways, or to examine brain structure at an unprecedented detail. The only optogenetic approach lagging behind all others is the genetically encoded optical monitoring of multi-neuronal activity at a time resolution of single action potentials. Such ultrafast (<1 ms) resolution is important to understand network function in healthy and diseased nervous systems because timing of neuronal firing and synchrony are the foremost determinants of brain function. Techniques exist to measure membrane voltage and/or cellular activity using optical probes, but all have drawbacks either in their genetic targeting, optical sensitivity and/or temporal resolution. Recent developments using genetically encoded hybrid voltage sensor (GEVOS) methodology showed that this approach has an excellent potential to become an ultrafast voltage sensing system. The GEVOS technique can easily be adapted to work with multiple colors simultaneously, thus recording the activities of genetically distinct cell types in the same preparation. The overall objective of this proposal is to advance the GEVOS method so that it can be used with multiple colors simultaneously in at least two different genetically targetable cell types. Two major advances are sought after in this proposal: a technical/ methodological innovation (improve upon the GEVOS technique and extend it to two fluorescent proteins) and a scientific vision. The latter relates to gaining insights into the parallel functioning of local microcircuits and the optical recording of the concurrent behavior of pre- and postsynaptic elements during GABAergic inhibition. These studies will advance high temporal resolution optical voltage sensing (the other side of optogenetics) and will provide an unprecedented look at the functioning of cortical microcircuits with their specific components monitored at the same time..

3 437 138 €

Start date: 2014-02-01, End date: 2019-01-31

Neurons exhibit the most marked size differences and diversity in intrinsic growth rates of any class of cells. How then can a neuron coordinate between biosynthesis rates in the soma and the growth needs of different lengths of axons? The central hypothesis of this proposal is that neurons sense the lengths of the axonal microtubule cytoskeleton on an ongoing basis by bidirectional motor-dependent axon-nucleus communication, and that the oscillating retrograde signal generated by this mechanism provides input for the coordinated regulation of neuronal biosynthesis and axonal growth. We will test this hypothesis in a multidisciplinary work program that will characterize and quantify the link between biosynthesis levels and axon outgrowth rates and identify and validate the roles and functions of key molecules underlying this mechanism. This research program will elucidate how neuronal biosynthesis and axon growth are co-regulated. New mechanistic insights on this fundamental aspect of neuronal cell biology will have far-reaching implications. From the basic science perspective, this work will establish a new modality for encoding spatial information in biological signals, providing a one-dimensional solution to the three-dimensional problem of sensing cell size. Moreover, the proposed mechanism can explain intrinsic limits on regenerative neuronal growth and raises the intriguing possibility of opening new avenues to bypass such limits towards acceleration of axonal growth for effective neural repair.

2 498 040 €

Start date: 2013-10-01, End date: 2018-09-30