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.