To understand how brains integrate sensory information and generate behavioural responses is a central goal of systems neuroscience. Larval Zebrafish have emerged as a very promising vertebrate model system to address this challenge because their small size and transparency enable optical access to the majority of neurons within the brain at cellular resolution. Important insights on circuit function have already been gained by two-photon imaging of neuronal populations in restrained larvae behaving in virtual environments – however, critical limitations of virtual environments as a replacement for real environments are widely acknowledged and remain a barrier to progress. An ideal solution to this problem would be to image larvae during natural, unrestrained behaviour, and this is the goal we aim to achieve in this project. It has so far not been possible to track naturally moving zebrafish, nor any other vertebrates, while imaging at cellular resolution. Here we propose to overcome this limitation by forming a novel collaboration across disciplines and three different laboratories. We will pool our collective expertise in optical systems design, wavefront-shaping, electrical engineering, and zebrafish neuroscience to perform an experiment that has until now been impossible: imaging comprehensive neural activity in a freely moving, untethered vertebrate. We will collectively design and validate a real-time optical tracking and imaging system to measure brain activity with single neuron resolution in a freely moving vertebrate. This will allow us to monitor, with unprecedented detail, the population activity of the reticulospinal system in larval zebrafish and understand how this population of ~ 300 supraspinal neurons combinatorially codes for the full range of locomotor and postural behaviors.

Lymph nodes (LNs) are situated at junctures of the blood vascular and the lymphatic system where antigens drain from peripheral tissues via afferent lymphatics. During the development of malignant breast cancer, new LNs emerge within the glandular tissues that are normally devoid of LNs. However, we do not understand the mechanism of development of these LNs and their role in antitumor immunity. We hypothesize that de novo LNs form in the vicinity of mammary carcinomas as an accessory “base camp” for the initiation and maintenance of antitumor immunity. Here, we combine and leverage our expertise in immunology, vascular biology, and bioengineering to address the hypothesis in three specific aims: (1) To molecularly dissect the pathways of tumor-induced development of accessory LNs with a particular emphasis on lymphovasculokines. (2) To assess to which extent accessory LNs support antitumor immunity. (3) To stimulate formation of accessory LNs in mammary tissues to foster antitumor immunity. The potential impact of this project is high as a deeper understanding of the interplay between accessory LNs and tumors may lead to a novel cancer treatment approach.

The objectives of the planned research are to characterize the dynamic structure of a G-protein coupled receptor in micelle and phospholipid nanodisc environments in solution. The research will rely on the directed evolution technologies developed in the laboratory of Dr. Andreas Plückthun at the University of Zurich for expressing functional GPCRs at high yield in E.coli. This approach has enabled bacterial expression of isotope labeled neurotensin receptor NTR1 suitable for NMR studies in solution. The Plückthun group also contributes numerous functional assays and important insights into GPCR biology. The Wagner laboratory will pursue a characterization of the dynamic structure of NTR1 using NMR. We will use new 15N-detected and other advanced experiments to assign backbone and methyl-bearing side chains of the receptor. We will use relaxation dispersion and other methods to characterize internal motions of the receptor in the presence of agonists and antagonists in order to elucidate allosteric mechanisms by which the GPCR transmits signals. We have furthermore developed methods to place receptors in phospholipid nanodiscs where the surrounding membrane scaffolding protein is covalently circularized to produce circular nanodiscs (cNDs) of exact size with tunable diameters from 11 to 80 nm. Thus, the diameter of the cND nanodisc can be tuned to include a single copy of the receptor. The cND-enbedded NTR1 can be complexed with heterotrimeric G protein and/or arrestin, expressed in insect cells, which has also already been worked out by the team. Dynamic changes of the GPCR upon such interactions and upon agonist antagonist interactions will be followed by observing changes of methyl group chemical shifts and dynamics. Our preliminary results provide for the first time a complete map of NMR backbone and methyl signals of a GPCR suitable to characterize dynamics of signal transduction. The technology of monitoring dynamic events upon ligand binding in the near-native membrane environment of cNDs offers unique tools for elucidating molecular mechanisms of dynamic GPCR signaling. This project will significantly complement and extend our knowledge about GPCR mechanisms beyond recent advances in GPCR x-ray crystallography, as it is not constrained by the necessity to grow crystals of particular states, and adds information on dynamics previously inaccessible.

We propose to apply a unique combination of molecular biology, physical chemistry and nuclear magnetic resonance to addressing a major unsolved problem in the study of biological systems: how to observe the dynamics of cellular function deep inside living organisms with high sensitivity and spatial resolution. Existing optical technologies such as fluorescent proteins are difficult to detect in opaque tissues, while high-resolution non-invasive techniques such as magnetic resonance imaging (MRI) lack sensitive genetically encoded reporters. To bridge this gap, we will develop genetically encoded imaging agents for hyperpolarized MRI – an advanced physical technique capable of increasing the sensitivity of MRI by up to five orders of magnitude. These imaging agents are based on a special class of proteins that interact with xenon, a biocompatible noble gas that can be hyperpolarized and introduced into the body by inhalation. Xenon dissolves in blood and is distributed throughout the body, enabling xenon-based MRI (Xe-MRI) of tissues such as the brain. We recently discovered that gas vesicles (GVs), a unique class of genetically encoded protein nanostructures, can bind hyperpolarized xenon and produce Xe-MRI contrast at picomolar concentrations (an improvement of ~10,000-fold over the state of the art). Based on this discovery, we propose to develop GVs as ultra-sensitive genetic reporters of mammalian cell migration, viability and function in vivo. To achieve this goal, we will characterize the physico-chemical properties of GVs and optimize them via their DNA sequence for optimal Xe-MRI detection. We will then transplant the genes encoding GVs into mammalian cells of interest, and develop methods to use Xe-MRI to image GV-labeled cells in vivo. To demonstrate utility, we will use this technology to image the trafficking of immune monocytes to sites of neuroinflammation. This research is enabled by the unique and complementary expertise of the co-investigators. Schröder introduced the NMR methodology enabling sensitive imaging of Xe-MRI reporters and is an expert in physical chemistry and NMR. Shapiro discovered the use of GVs as genetically encoded imaging agents and is an expert in biomolecular and cellular engineering and in vivo MRI. Successful implementation of this proposal will transform multiple areas of pre-clinical biomedical research.

To beat wings 50-60 times per second to hover a ruby-throated hummingbird (Archilochus colubris) must burn calories faster than any other vertebrate. To provide these calories, hummingbirds ingest little more than simple sugars (glucose and fructose) found in floral nectar. Thus, their incredible flight can be powered by the simplest fuels at rates 55× those of mice, rats, or even aerobic dogs. When a tiny hummingbird stops feeding at night, it must keep itself warm by burning energy dense stored fats accumulated during evening hours, as opposed to sugars that are too heavy to carry. Similarly, when a hummingbird heads south in the fall on a long migratory journey to Central America, its journey is fueled by fat stores accounting for ~30% of its mass, built from the sugar in ingested nectar over just a few days prior to departure. Just what adaptations of hummingbird metabolism permit these extreme rates of sugar breakdown and fat deposition are not well understood. Our understanding of the function and regulation of enzyme pathways that catabolize sugars and produce fatty acids comes largely from studies of mice, rats, and humans, energetic sloths compared to hummingbirds. What we know from studies of these and other model organisms fails to explain how hummingbirds do what they do. For example, insulin plays a central role in control over blood sugar and the mixture of fuels our muscles burn, but birds are unresponsive to this hormone. Further, the metabolic pathways at work in hummingbirds are not simply faster versions of those in mice. Instead, the same metabolic machinery must operate with increased efficiency at the molecular level. Our team will use a variety of approaches, from studies of fuel use in freely-behaving hummingbirds (Welch), to studies of their enzymes (Wong), the genes that encode them (Timp), and the atomic structure of this molecular machinery for an improved understanding of its function (Valle) to build a fundamental understanding of the unique aspects of hummingbird physiology and behavior that enable their superior performance. In doing so, we will establish the hummingbird as a new model organism in which to understand the limits to physiological performance and function.

Like all living cells, bacteria need an adequate supply of energy for vital processes. When under stress, for example when poisoned or exposed to light, bacteria face a difficult juggling act – use energy to respond to the stress or keep vital functions going. In fact, they do both but we do not understand how bacteria manage this difficult balance. We propose to investigate the strategies bacteria adopt to coordinate their free energy by directly measuring the flow of energy under stress.
To do this we will develop techniques that can measure in real time and in a single cell the distribution of the two main sources of free energy: the proton motive force (PMF) and the internal ATP levels. We will then monitor how this changes when the bacteria is stressed. With this information we can start to build a mathematical model that will allow us to understand just how bacteria manage their free energy supplies when stressed. Put simply, we hope to uncover the very fundamental principles of how bacteria manage energy flow and offer a very scientific definition of ‘dead’ and ‘alive.’

Microorganisms are the most abundant living organisms on earth and they thrive in virtually every possible habitat, including in our bodies and those of other organisms. Understanding the causes and consequences of their diversity is therefore essential for controlling microbes in health, agriculture and biotechnology. Studies of microbial diversity are also increasingly important for advancing general ecological and evolutionary theory, because microbes are powerful experimental model systems. We seek to advance our understanding of the mechanisms underlying microbial diversity by focusing on the role of extra-cellular products (EPs). EPs play an interesting but largely unknown role in the evolution of microbial diversity. Given possible effects of EPs on the fitness of cells other than the producer, a growing perception is that EP production is a form of cooperative behavior, suggesting that it has evolved due to benefits conferred on the population of EP producers rather than on individual producers. However, this view rests on models that make simplifying assumptions that need to be rigorously tested. Our international collaborative project combines the expertise of: (1) two biologists, who will use existing and newly developed millifluidic droplet technology to test for collective benefit and its evolutionary consequences in experiments with two bacterial EP systems with different fitness effects and availability to others, (2) a colloid chemist, who will develop novel millifluidic tools that provide a unique opportunity to quantitatively assess the evolutionary consequences of EP production, and (3) a theoretical biophysicist, who will develop computational models that incorporate biological details of both experimental EP systems, and will both generate specific predictions that will be tested experimentally and facilitate the results from these experiments. By generating unique quantitative data on two very different bacterial EP systems and comparing results against predictions from realistic models, we expect that our project will provide conclusive answers and set new standards for the study of microbial interactions.

Successful defense against pathogens is critical for survival. Microbes have developed many ways to invade larger organisms which in turn have evolved numerous immunity mechanisms. Lipid droplets (LDs) are dynamic and complex organelles that provide all eukaryotic cells with lipidic substrates for energy metabolism, membrane synthesis, and production of lipid-derived molecules. Probably for this reason, LDs are part of the infection cycle of viral, fungal, and bacterial pathogens. Mechanistic details of LD/pathogen interactions are largely unknown, but many pathogens stimulate LD biogenesis. The current view is that LDs are “hijacked” to provide lipids and energy to pathogens for effective and rapid growth.
Our recent work has challenged this dogma and demonstrated that LDs are organelles that actively participate in the Drosophila organismal antibacterial response. This new type of innate immunity could well be at work in every infected tissue of our body since all cells accumulate LDs. Preliminary data, specially generated for this project, supports the idea that this innate immune system has been conserved during evolution.
Thus, based on solid preliminary data, we here propose a multidisciplinary research project to characterize cells, sites, and mechanisms of action of this innate immunity. Whole animal studies will determine cells of interaction, high-resolution ultra-structural analysis of infected tissues and cellular models will determine sub-cellular locations and details, single-organelle biophysical studies and in vitro LD-bacteria killing assays combined with ultra-structural studies will develop a mechanistic understanding of the process, and comparative proteomics will identify protein complexes conferring chemotaxis and antibiotic activity to LDs.
In conclusion, here we present a project designed to identify and characterize a new innate immune system that will be paradigm-shifting in Immunology, Physiology, and Cell Biology. Further, the combination of the detailed single-organelle killing assays with the super-resolution studies specially developed for these studies will allow unprecedented mechanistic details establishing an entirely new dimension in the ongoing host pathogen conflict.

There is now a large body of experimental evidence indicating that the ability of many newly synthesized proteins to reach full functionality in a cell depends strongly on the rate at which individual codons are translated by the ribosome during protein synthesis. These codon translation rates can determine whether a nascent protein will fold and function, misfold and malfunction, aggregate or efficiently translocate to a different cellular compartment. That is, changing the translation rate of a codon can change the fate of a protein in living cells. This indicates that the sequence of nucleotides in a transcript not only encodes genomic information but also kinetic information that is used by cells to coordinate co-translational processes and influence nascent protein behavior. Therefore, quantifying and predicting the influence codon translation rates on nascent protein behavior is an immediate and pressing need that if met will provide fundamental insights into the coordination of co-translational processes and nascent protein maturation. This proposal addresses this challenge by proposing a novel collaboration between the theoretical biophysics lab of Ed O’Brien and the experimental molecular biology lab of Bernd Bukau. The goals of this proposal are (1) to experimentally quantify the influence that codon translation rates have on E. coli’s nascent protein behavior at the proteome level, (2) to create an experimentally verified model that accurately predicts the consequences of a transcripts’ translation-rate profile for the behavior of the newly synthesized protein, and (3) to address fundamental biology questions that include (i) how robust are nascent protein behaviors to changes in codon translation rates? (ii) What codon positions in an open reading frame cause major changes in a nascent protein’s behavior upon a change in their translation rate? (iii) How does changing codon translation rates affect downstream cellular processes involving the nascent protein? By bringing together the complementary skill sets of the O’Brien and Bukau Labs, this research will significantly advance our quantitative understanding of the temporal coordination of translation and co-translational processes affecting nascent protein behavior.

We aim to establish a blueprint for how organisms tell time over the course of the year. This is not so simple as merely responding to the changing seasons, requiring preparation well in advance for changes in the environment. A key adaptation involves an internal annual timing mechanism (sometimes known as a “circannual clock”), which free-runs in constant conditions but is normally synchronized by environmental signals (entrainment). Circannual clocks are a fundamental feature of life, necessary for such diverse phenomena as the timed emergence of ground squirrels or bats from their hibernacula, the autumn migration of birds, and transitions between life-cycle stages in unicells. We aim to dissect the mechanisms driving the circannual clock and propose that these will turn out to be strongly conserved (i.e. similar) in different groups of organisms. We propose that circannual clocks are driven by specialist timer cells, with the clock based on cycling between resting and active phases (reversible cellular quiescence, RCQ).
We have two objectives. First (Objective 1, Functional biology), we will test the hypothesis that the RCQ is driven by a mechanism involving chemical modification of the cell’s DNA (epigenetics). Focusing on those tissues in which we think the circannual clock resides, we will explore circannual effects on the epigenetic make up of cells. We will test the generality of these mechanisms in other vertebrate groups and in a distantly related marine micro-organism. Additionally, we will test for the action of a conserved circadian input mechanism in driving seasonal timers over wide evolutionary divides.
Second (Objective 2, Evolutionary Biology), we predict that key genes in these ancestral long-term timing pathways have been subject to selection in response to evolutionary pressures on the function of these timers. Here, we will study the evolution of breeding seasons in at different latitudes in birds and fish, and test the hypothesis that natural selection tunes the circannual clockwork and photoperiodic input pathways to match local seasonal environments. We will also explore how seasonal timing circuits have been subject to selection in domestication in birds and mammals.
Integration of results from these two Objectives will give new insights into seasonal time-keeping mechanisms.

Transmission is a key step in the infection cycle of all pathogens. Pathogens have developed sophisticated strategies to survive through this crucial phase. Use of vectors that shuttle pathogens to a new host is a common strategy employed by many infectious agents. Shuttling may require internalization of pathogens in vectors, or shuttling occurs with the pathogens attached only to external vector parts. Both cases involve intimate interactions between vectors and pathogens. The latter mode of transmission is used by many arthropod-vectored animal (including human) and plant viruses and is referred to as mechanical and non-circulative transmission, respectively.
We will interrogate the non-circulative transmission of representative plant viruses that are spread by insects in the order Hemiptera. Insects in this order are sucking and piercing feeders that imbibe nutrients from the plant host’s phloem. We will focus on molecular and biophysical aspects of three related steps in the transmission process: 1) virus uptake, 2) virus inoculation, and 3) the step in-between: virus retention in the vector; specifically targeting mechanisms that mediate virus uptake from, and inoculation to, the phloem and, importantly, the biophysical changes occurring in virion-bound vectors. Re: 1) Uptake. Evidence for an exemplary plant virus, Cauliflower mosaic virus (CaMV), indicates that non-circulative transmission is mediated by specific interactions (among the virus, vector and host plant) that boost transmission efficiency. Indeed, CaMV responds to its aphid vector’s presence on the host by instantly forming transmission forms, a phenomenon named transmission activation (TA). However, neither ubiquity nor the mechanisms that drive TA are known. Here, we will test generality of TA of tissue-acquired viruses like CaMV, and for a first time TA of phloem-restricted viruses. Re: 2) Inoculation. We will determine dietary conditions and identify components required for virus release from vectors and compare different viruses and vectors to reveal common and diverging parameters in this never before investigated step of the transmission process. Re: 3) Retention. How viruses attach to, are retained in, and are released from vectors, is a black box. Using advanced biophysical methods, we will study virus attachment and release in dissected mouthparts and eventually life insects.

Living organisms, particularly mammals, constantly shift between different behavioral states according to their external environment. These shifts are largely regulated by specific molecules, termed "neuromodulators" that convey information to a wide range of brain systems. The mechanisms by which these neuromodulators regulate distinct behavioral states remain enigmatic. One important brain neuromodulator is the neuropeptide oxytocin, which recently became well known for its positive, “pro-social” effects on human social behavior. Oxytocin is synthesized and released by a small number of hypothalamic neurons that send their projections to various forebrain regions, to regulate a plethora of social behaviors ranging from aggression to empathy.
Relying on anatomical and physiological distinction between subsets of oxytocin neurons, we hypothesize that these neurons are segregated into functional modules associated with distinct types of social behavior. To test this hypothesis we will focus on one hypothalamic region – the paraventricular nucleus, which is the main source for oxytocin projections throughout the entire brain. We propose a comprehensive multidisciplinary study aiming to identify, analyze and mathematically model functional oxytocin neuronal modules activated during various forms of social behavior.
To pursue this hypothesis we will employ a novel genetic technique named vGAIT (virus-mediated genetic activity-induced tagging), which enables tagging and manipulating oxytocin neurons that were active during a given social behavior, ranging from simple social interaction to a complex “human-like” cooperative behavior. Using this technique in freely moving rats, we will explore the activity and connectivity, as well as the intrinsic electrophysiological and molecular properties of activated oxytocin modules at the single-cell level. Building on this information we will develop a computational model of the oxytocin system and investigate the mechanistic principles underlying its function. The unique combination of cutting-edge techniques and approaches will allow an unprecedented insight into the mode of operation of a central brain neuromodulatory system that controls mammalian social behavior. Furthermore, the expected findings will pave the way for the development of interventions directed towards specific pathologies of social behavior in humans.

We inherit our traits from our parents through genes embedded in the DNA of the sperm and the oocyte. It was already known that besides DNA, other components of the egg are also inherited that might contribute to the offspring. For example, a mother might pass on effects of the environment to its baby either during pregnancy, or in case of externally fertilizing animals such as fish, by deposited materials in the egg. In recent years it is becoming clear that environmental effects on the father (from various forms of stress to diet changes) can also be inherited by the offspring and even passed on to the next generations and result in changes in traits. How this inheritance occurs puzzles scientists. Increasingly, scientists are considering the possibility of the inheritance of environmental effects through 'epigenetic' marks and they call this process epigenetic inheritance. Epigenetic marks include chemical tags, which influence the way DNA is packaged (termed chromatin). Chromatin in the sperm and the egg may then influence how genes get switched on and off in the offspring and can have lasting impact by changing the traits of the offspring. In this consortium, an evolutionary biologist (Immler) a biochemist (Cairns) and a developmental geneticist (Mueller) join together to address the mechanisms of epigenetic inheritance by proposing a new model to study epigenetic inheritance: the zebrafish. Zebrafish, like us is a vertebrate, which develops outside of the mother as a transparent embryo, which makes it popular for developmental biologists and geneticists. Immler has recently discovered that zebrafish embryos develop faster than their siblings if the father was competing among males for breeding with female. This observation serves as an exciting model to study epigenetic effects - and the biochemist Cairns together with the developmental geneticist Mueller will use the male competition model alongside feeding experiments and genetic manipulation tools to elucidate how and what chemical tags are responsible for the observed changes in development. Together with Immler they will study whether the observed changes in development may also have a lasting impact on adult fitness of the animal and may be inherited over several generations. The findings will contribute to answering an important question about how vertebrates (including ourselves) pass on experience through the father to their offspring.

RNA viruses represent an existing and emerging global threat to human and animal health. Vaccination is the only known approach to prevent viral infection but suffers from several limitations, one of which is restoration of the virulent phenotype as a result of recombination between the vaccine strain and a circulating, wild strain. In general, recombination is one of the most, if not the most, important molecular process driving the expansion of the host range of viruses. Recombination is also a major contributor to viral evolution, leading to the emergence of vaccine-resistant strains. Unfortunately, we lack sufficient knowledge of the molecular mechanism of RNA virus recombination to prevent these issues from thwarting vaccine development and long-term efficacy. Therefore, an understanding of recombination is of potentially broad, practical value. For example, detailed knowledge of the mechanism of recombination may establish principles that can be exploited for development of strategies to suppress recombination and/or design of recombination-deficient vaccine strains. The overarching goal of the proposed research is to create fundamental knowledge on the mechanism of RNA virus recombination. Our model system will be enterovirus 71 (EV71), a member of the Picornavirus family of viruses. EV71 outbreaks are on the rise and are driven by the emergence of strains created by recombination.
Milestones for this project are as follows:
• We will provide a complete biochemical and biophysical description of the molecular events that ensue when a viral RNA polymerase encounters a putative RNA trigger for recombination.
• We will elucidate properties of the viral RNA polymerase that contribute to recombination frequency.
• We will determine whether recombination contributes to viral fitness in vitro and in vivo and perhaps even the extent to which new recombinant viral strains have outbreak potential by exhibiting increased virulence.
To achieve these goals, we have assembled a multi-disciplinary, multi-national team of scientists that brings a broad base of intellectual and experimental resources to the problem of RNA virus recombination. These resources include single-molecule biophysics, single-cell virology, a small-animal model to assess pathogenesis and to access currently circulating viral strains.

In variable environments (either natural or altered by humans) organisms can adjust to new conditions for better success in producing offspring. In many cases, organisms do this through cognitive abilities: their ability to gather information from their surroundings, process that information, and make decisions to improve their lot. We now realize that there is extensive variation in cognitive capacity both between species and within species suggesting that cognitive abilities evolve, yet we still know very little about how cognition evolves. Most organisms have a number of different cognitive abilities (memory, attention, learning, etc…) and comparative research suggests that certain environments select for improvement in some, but not all, of these abilities, but this has not been demonstrated directly. Furthermore, while our understanding of the neural basis of cognition has improved in model species, we still know very little about the ecological importance of most cognitive capacities and their underlying neural structure. Our project will investigate the evolution of cognitive abilities and neural structure in a wild great tits, and thus provide a big step forward in our understanding of cognitive evolution. To do this, we will study 6 populations in the French Pyrenees that differ in ecology (altitude and urbanization), and thus likely favor different cognitive traits. Our team, specialists in cognition, brain imaging, computer science, and evolution will develop new tools (touchscreens) and analytical approaches (automated video analysis, MRI of non-model species) to examine cognitive traits that could present a clear advantage under some, but not all, ecological contexts. We will measure cognitive abilities of birds in these contrasted populations and quantify their fitness to understand what cognitive traits and neural structures are favored in different ecological settings. In doing so, we can measure natural selection on cognitive abilities and neural structure, understand what factors in the environment drive this selection, and ultimately gain a much better understanding of how evolution shapes minds in the wild. This interdisciplinary project provides advances to a number of different fields of research while together contributing to an important stride forward in our understanding of the neural basis of cognition and how cognitive evolves.

Successful acclimation of photosynthetic organisms to variable environments requires a tight management of source (light harvesting) and sink (CO2 assimilation) relationships. Photosynthetic organisms optimize photosynthesis in low light, where excitation energy is limiting CO2 fixation and minimize photo-oxidative damage in high light, dissipating excess photons. Despite extensive studies of these phenomena, the mechanism governing light utilization in plants is still poorly understood. Our results pinpointing a K+ channel (TPK3) as an essential component of light-biomass conversion have pioneered a new research field (photosynthesis optimization by ion flux). By counterbalancing proton pumping with potassium flux, TPK3 modulates specific functions of the proton motive force in the light (non-photochemical dissipation of excess light, electron flow efficiency), ultimately improving plant fitness. Very recently, other channels/exchangers have been suggested to play similar roles. Here, we propose to establish the rules governing ion modulation of photosynthesis using a comprehensive and integrative approach indifferent model organisms. Complementary expertise in our team (electrophysiology, spectroscopy, bioinorganic chemistry and molecular genetics) will allow to identify chloroplast ion channels, generate down-regulated mutants, characterize their function in vitro (electrophysiology) and in vivo (using Stark spectroscopy) and collate their phenotype to ion dynamics in vivo (developing new fluorescent sensors for ion flux imaging in living cells). This will allow the complex regulation of light conversion into chemical energy to be dissected. Focusing on multiple phylogenetically relevant organisms (green algae, diatoms, mosses and vascular plants) is feasible due to expertise in their physiology and molecular biology in this consortium, and will provide essential insights into the evolution of photosynthesis regulation. Gathering these four investigators in a structured network will enable a cutting-edge, interdisciplinary and integrative approach to answer fundamental biological questions, which no individual lab could solve on its own.

Fundamental to sexual reproduction in eukaryotes is the generation of haploid gametes from diploid germ cells during meiosis, errors in which lead to chromosome gain or loss (aneuploidy), a major cause of miscarriage and developmental disorders such as Down Syndrome. Orderly reduction of chromosome numbers requires that homologous chromosomes (homologs) pair and become physically linked through DNA recombination events called crossovers, prior to the first meiotic division. Crossover formation entails the introduction of DNA double strand breaks, which are repaired by a specialized homologous recombination pathway. To protect genome integrity and avoid aneuploidy, meiotic recombination is tightly regulated and meiotic progression is prevented by checkpoints until all homologs have paired and all DNA breaks have been repaired. Recently, a conserved meiosis-specific structure called the chromosome axis has been identified as a vital regulator of recombination: prior to homolog pairing, the axis is believed to promote DNA break formation and block meiotic progression. Upon successful homolog pairing, the axis is incorporated into the conserved synaptonemal complex, which is proposed to suppress DNA breaks and alleviate the block to meiotic progression.
We aim to uncover the molecular mechanisms of axis- and synaptonemal complex-mediated control of meiotic recombination, using a combination of biochemistry, structural biology, and genetics. We will use chemical cross-linking and mass spectrometry to first define the components and overall architecture of chromosome axes and synaptonemal complexes purified from mouse spermatocytes. Based on these data, we will reconstitute defined sub-complexes, biochemically dissect important interactions, and determine subunit and sub-complex structures using x-ray crystallography. These combined data will enable us to build detailed mechanical models of the chromosome axis and synaptonemal complex. We will validate these models using targeted mutations in in vitro-reconstituted complexes, and test functional predictions in vivo using genetic manipulations in mice. This full-circle approach will achieve a comprehensive mechanistic understanding of meiotic regulation by the chromosome axis and synaptonemal complex, and will aid the understanding and prevention of human aneuploidies and their associated developmental disorders.

Since Weismann formulated the distinction between innate and acquired characteristics in the 19th century, the debate about the inheritance of acquired traits has raised many controversies in the scientific community. Following convincing arguments against, e.g. by William Bateson, this debate was then largely set aside. However, a number of phenomena and epigenetic mechanisms involving RNA, histone modification or DNA methylation have renewed interest in this area. Transgenerational epigenetic effects likely have wide-ranging implications for human health, biological adaptation and evolution, however their mechanisms remain poorly understood. Already, a number of human diseases including metabolic syndromes, cancer and heart disease have been shown to be mediated by transgenerational effects.
We have identified the recent discovery of RNA-mediated multigenerational stable inheritance in the nematode C. elegans as a unique opportunity to dissect the systems biology and mechanism as well as the organismal and species impact of transgenerational epigenetic inheritance (TEI). C. elegans is an ideally suited animal for this undertaking due to its short generation time of three days (organismal studies), small genome size (genomics and functional genomics), transparency (imaging) and genetic tool kit (genomic engineering).
To advance this area of biomedical research an integrated multi-disciplinary approach will be essential. Our team collectively has the skill set to have a transformative impact. We will integrate genetic, genomic, dynamic imaging, biochemistry and proteomic technologies into a quantitative description of RNAi-mediated TEI. This work will provide a mechanistic understanding of the expression of TEI in an animal. Furthermore, this project will test if and how TEI provides an environmental memory (“Lamarckism”) and examine its implications for the adaption of individuals and the evolution of a population. We anticipate that this work will help guide the analysis of TEI in other species including humans.

The germinal center (GC) is a specialized tissue microenvironment of the immune system in which T, B and stromal cells cooperate in the evolution and production of high affinity antibodies and immunological memory. While GCs are a prominent feature of lymphoid tissues, there is a limited understanding of information transfer between the component cells and recent evidence for communication between different GCs further enlarges the spectrum of information processing and evolution of optimal functionality.
The main aim of this project is to develop an experimentally validated, predictive, and scale-bridging theory of the GC reaction, i.e. a theory that connects experiments on different spatial scales. Bringing together experts for quantitative GC experimentation at the molecular, cellular, and organismal level with multi-scale mathematical modelling, we expect to uncover the molecular mechanisms important for the development of optimized antibodies in the fight against invading pathogens. Research will unravel principles of the three-dimensional organization of the immunological synapse at cellular interfaces, of the evolution of high affinity clones at the cellular level of individual GCs, of the exchange of cells between different GCs within the same lymphoid organ, and of the coordination of system-wide humoral immune responses. With the resulting scale-bridging GC modeling platform we will explore a new degree of complexity in multi-scale modeling, which will rely on the exchange of sophisticated and innovative experimental approaches between the partner labs. All partners will have to go beyond their established research themes in order to tackle this challenge because the research will be focused on the interface of the different spatial scales.
Human GCs have not been investigated so far for technical reasons. With a highly innovative approach combining the expertise of all partners, the project will explore the possibilities and limits of extending GC research from the murine to the human organism. This will only be possible based on a team effort and transforms the research program of all partners.
The results of GC research in mouse and human will provide a fundamental understanding of information processing in a complex biological system, in which evolution of new functionality can be assessed on a timescale of weeks.

In order to understand the brain, tools need to be developed to allow the investigation of interactions between many individual neurons. Noninvasive tools (MRI, EEG) provide structural and functional information, but at low spatial resolution. Invasive recording methods (patch-clamp, multi-electrode array) provide high temporal resolution data from single neurons, but are bulky and lack spatial resolution and throughput. For this reason, considerable efforts have been invested in developing optical detection methods including the utilization of voltage-sensitive dyes (VSDs) and genetically-encoded fluorescent voltage-sensor proteins (VSPs). Despite these advances, VSDs and VSPs are not yet preforming at the needed level of detection.
We therefore propose to develop solid-state nanoscale voltage sensors nanoparticles (vsNPs) that report on action potentials (APs) on the nanoscale. These devices could dramatically change the way we image and study nervous system activities, and how we interface these systems to provide novel therapeutic approaches and prosthetic strategies. The proposed vsNPs are based on the quantum confined Stark effect. They will be designed to be injectable, targetable, and to be self-inserted into the membrane. They will optically record, non-invasively, APs at the single particle level at the nanoscale, at multiple sites and across a large field-of-view. They will display high voltage sensitivity, high brightness, single-particle detection sensitivity, large spectral shift, fast temporal response, minimal photobleaching, large Stokes shifts, large two-photon cross sections, excellent NIR performance, and compatibility with lifetime imaging.
We will pursue the following aims: (i) synthesis of heterostructure vsNPs (Weiss & Oron) and characterization of their performance (Enderlein, Weiss & Oron); (ii) development of functionalization methods based on ligand exchange with transmembrane (amphiphilic) peptides by self-assembly (Weiss) and optimization of vsNPs insertion into the membrane (Weiss and Enderlein); (iii) development of optical vsNPS recording methodologies and benchmarking of vsNPS performance (Enderlein, Oron, & Weiss); (iv) demonstration of vsNPs utility in model neuronal systems (Triller), exploration of their use in studying synapses and neuronal integration of excitatory and inhibitory inputs (Triller).