There is a common perception that larger brains mediate higher ‘intelligence’ or cognitive capacity. Many insects, however, demonstrate that sophisticated social structures and cognition are possible with very small brains. Foraging honeybees, for example, memorize flower signals and display higher-order learning such as categorization, concept learning and ‘counting’. How do bees perform these feats with small nervous systems? Despite the extensive knowledge on the cognitive abilities of bees, to date, no study has attempted to elucidate the neural mechanisms underpinning their higher-order learning. Here we will pool our expertise in cognitive, computational and neuroscience methods to unravel the neural circuits and architecture underpinning such cognitive performances in the honeybee. We will combine behavioral recordings of bees walking stationarily on a locomotion compensator in a virtual environment with access to its brain via multielectrode recordings. Data will be fed into computational models providing testable hypotheses about minimal neural architectures for cognition, and ultimately working towards whole-brain modeling (connectomics).
This project will expand the currently available information on the neurobiology of insect learning, and will provide the first complete computational model for the neuronal mechanisms that underlie cognition in a miniature nervous system. Our expected results have far-reaching implications for psychophysics, neuroscience and behavioral biology as well as the potential to revolutionize the field of visual processing in animals and robotics.

We propose to use DNA-based scaffolds to build lectins of pre-specified stoichiometry and precisely defined three-dimensional geometry to test mechanochemical requirements of plasma membrane invagination mediated by their membrane-resident glycosphingolipid (GSL) binding partners.
It is striking that numerous pathogenic GSL-binding lectins including the B-subunits of Shiga and cholera toxins and the VP1 protein of SV40 (i) consist of a pentameric fold (ii) bind GSLs with similar geometries (i.e., angles and distances between carbohydrate recognitions sites (CRDs)); and (iii) finally adopt a curvature-active state that enables their clathrin-independent uptake into cells despite the fact that they do not exhibit any sequence similarity. This suggests the exciting possibility that their clustering, membrane bending and invagination capacities are the result of parallel, convergent evolution.
Probing lectin functionality experimentally is infeasible using conventional approaches such as targeted mutagenesis because it is impossible to alter lectin oligomerization state and CRD geometry in a controlled manner. Therefore, we will engineer synthetic scaffolds composed of DNA to display GSL-binding CRDs in specific natural and non-natural geometries and stoichiometries. Such designer DNA-based lectins indeed offer the ability to address structural questions with unprecedented molecular precision. Innovative computational and chemical biological tools will be developed that should be of general interest to the fields of DNA devices and synthetic biology. Novel DNA-based GSL-binding scaffolds will be characterized as to their capacities to cluster on membranes, to adopt a curvature-active state (i.e. to induce or sense curvature), and to traffic into cells via defined pathways. Based on structural input, experiments will be modeled at length scales from 0.1 nm to 10 microns to provide a molecular understanding of underlying mechanistic aspects, and guide the design of new scaffolds. We expect to achieve an unprecedented understanding of GSL-based membrane mechanical processes, including the discovery of novel physical principles. Beyond the world of pathogens, our study bears relevance to the GSL-dependent functions of signaling receptors and the design of novel delivery tools for therapeutic intervention against GSL-expressing tumors.

Ribosomes stand amongst the most complicated structures in biology. Their assembly is a question of fundamental interest, but is still poorly understood in spite of efforts of excellent groups. We propose to address this challenging question using a quantitative approach, inspired by recent results in cell-free translation and in single-molecule biophysics. By applying a force to specially designed DNA/RNA duplexes, we can repetitively release and re-anneal RNA with full control over velocity and orientation of the strands. We can thus mimic the gradual appearance and folding of rRNA in co-transcriptional assembly of bacterial ribosomes, including varying transcription rate and pauses. Combined force and fluorescence experiments will allow us to follow in real time the binding of ribosomal (r-)proteins to the gradually released rRNA. We will focus with our single molecule measurements on the initial phase of the assembly of the large ribosomal subunit of E. coli. This involves the 23S rRNA, the five r-proteins that have been identified as essential and sufficient for the first assembly stage (L4, L13, L20, L22 and L24), and a selection of assembly helper proteins. The single-molecule studies will be complemented by coupled transcription-assembly experiments, in which the assembly will be followed in fluorescence experiments using Förster Resonance Energy Transfer between fluorescent labels on r-proteins adjacent in the ribosome and by testing the ribosomal capacity of protein synthesis. Using this combination of approaches, we will provide a dynamic view of the choreography of ribosome assembly, identify essential assembly factors among the about 15 known assembly helpers and identify their roles. Our interdisciplinary team combines two biophysics groups, well known for single-molecule studies on nucleic acids and associated proteins, and two biochemistry groups, well known in the fields of ribosome assembly and cell-free translation. Close collaboration between these four groups will bring together the expertise required to successfully perform the proposed experiments and will bring our understanding of ribosome assembly to a new level.

Ribosomes stand amongst the most complicated structures in biology. Their assembly is a question of fundamental interest, but is still poorly understood in spite of efforts of excellent groups. We propose to address this challenging question using a quantitative approach, inspired by recent results in cell-free translation and in single-molecule biophysics. By applying a force to specially designed DNA/RNA duplexes, we can repetitively release and re-anneal RNA with full control over velocity and orientation of the strands. We can thus mimic the gradual appearance and folding of rRNA in co-transcriptional assembly of bacterial ribosomes, including varying transcription rate and pauses. Combined force and fluorescence experiments will allow us to follow in real time the binding of ribosomal (r-)proteins to the gradually released rRNA. We will focus with our single molecule measurements on the initial phase of the assembly of the large ribosomal subunit of E. coli. This involves the 23S rRNA, the five r-proteins that have been identified as essential and sufficient for the first assembly stage (L4, L13, L20, L22 and L24), and a selection of assembly helper proteins. The single-molecule studies will be complemented by coupled transcription-assembly experiments, in which the assembly will be followed in fluorescence experiments using Förster Resonance Energy Transfer between fluorescent labels on r-proteins adjacent in the ribosome and by testing the ribosomal capacity of protein synthesis. Using this combination of approaches, we will provide a dynamic view of the choreography of ribosome assembly, identify essential assembly factors among the about 15 known assembly helpers and identify their roles. Our interdisciplinary team combines two biophysics groups, well known for single-molecule studies on nucleic acids and associated proteins, and two biochemistry groups, well known in the fields of ribosome assembly and cell-free translation. Close collaboration between these four groups will bring together the expertise required to successfully perform the proposed experiments and will bring our understanding of ribosome assembly to a new level.