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2019 -
Grant Awardees - Program Grants

Molecular approaches to study individually identified mechanosensory neurons of the leech

WEISBLAT David (USA)

Dept. of Molecular and Cell Biology - University of California, Berkeley - Berkeley - USA

FERNANDEZ DE MIGUEL Francisco (MEXICO)

Instituto de Fisiologia Celular-Neurociencias - Universidad Nacional Autónoma de México - Ciudad de Mexico - MEXICO

SZCZUPAK Lidia (ARGENTINA)

Institute FBMC - University of Buenos Aires - Caba - ARGENTINA

KUO Dianhan (CHINA, REPUBLIC OF (TAIWAN))

Dept. of Life Science - National Taiwan University - Taipei - CHINA, REPUBLIC OF (TAIWAN)

To study how nervous systems arise and function, scientists use animal models in which it is possible to integrate research on fundamental processes across different levels of organization from genes to behavior, and from the zygote to the adult. The medicinal leech (genus Hirudo) provides one useful model, because its nervous system is much simpler and easier to work with than vertebrate or mammalian nervous sytems, even though it functions in a similar manner--Hirudo has been used to study phenomena of general importance such as: how glial cells function; how neurotransmitter are released; how synapses form and regenerate; and how neural circuits function to control behavior. Another leech (genus Helobdella) is used to study development and how development changes during evolution, giving rise to kinds of animals over hundreds of millions of years. These two leech species exhibit marked similarities of course, but also some differences. Technical considerations (small embryos for Hirudo; small adults for Helobdella) have made it difficult to integrate these two models, e.g. by applying molecular approaches in Hirudo or to study the adult nervous system in Helobdella. The goals of our project are: 1) to enhance the power of the Hirudo model by introducing newly-developed molecular approaches; 2) to implement approaches in Helobdella that will enable us to unite molecular and cellular approaches to developmental and behavioral neurobiology; 3) to develop new optical techniques for stimulating and recording neuronal activity without exogenous dyes or genetic manipulations.
The intellectual significance of the proposed work is twofold. First, it will enhance our abilities to answer fundamental questions regarding how nervous systems function and development by introducing cutting edge technical approaches to the cellularly simple, physiologically accessible leech models. Of equal importance, it will provide a new evolutionary perspective into neurobiology, by allowing us to examine similarities and differences between leech and other models, including arthropods, nematodes, and vertebrates which have all been evolving separately for more than half a billion years.

2019 -
Grant Awardees - Program Grants

3D atomic-scale movies of molecular machines in action

WEISS Shimon (USA)

Dept. of Chemistry and Biochemistry, Dept. of Physiology - University of California, Los Angeles - Los Angeles - USA

SCHULER Benjamin (AUSTRIA)

Dept. of Biochemistry - University of Zurich - Zurich - SWITZERLAND

BAKER David (USA)

Dept. of Biochemistry - University of Washington - Seattle - USA

GULINATTI Angelo (ITALY)

Dipartimento di Elettronica, Informazione e Bioingegneria - Politecnico di Milano - Milan - ITALY

Capturing the dynamic 3D atomic-scale structure of a macromolecular machine while it performs its biological function remains an outstanding goal of biology. Conventional structural tools (e.g. X-ray crystallography, NMR & cryoEM) only provide ‘snapshots’ of stable states along a reaction pathway. Reaction intermediates, and in particular short-lived intermediates, are hard to capture and characterize with such conventional techniques. Here we propose to combine (prior) information from multiple existing static structures of stable states with dynamic datasets of inter-atomic distances obtained by high-throughput non-equilibrium single-molecule FRET (smFRET) measurements in a microfluidic mixer using novel time-resolved multi-pixel single-photon avalanche diode detector. These measurements will be performed on libraries of molecular constructs, sampling multiple inter-atomic distances as function of reaction time. These measured distance distributions will then serve as multiple intra- and inter-domain distance constraints which, together with prior information (available structures), will enable the Rosetta software to achieve large-scale energy optimization-based refinement of time-resolved ‘snap shots’ of complex structures with improved accuracy. These time-resolved Rosetta structures together with intermediary molecular dynamics simulations will allow solving the 3D atomic-level structure of the macromolecule for each sampled reaction time point, eventually producing a 3D structural dynamic movie of the macromolecule in action. To demonstrate the utility of the proposed method, we will solve the dynamic structure of RNA polymerase during transcription initiation (promoter binding, bubble opening, abortive initiation, promoter clearance) and a pair of intrinsically disordered proteins (IDPs) involved in transcription regulation (ACTR and NCBD) that adopt a fully folded structure during a coupled folding and binding reaction. In addition to elucidating outstanding questions in transcription by combining detector developments, high-throughput and time-resolved out-of-equilibrium single-molecule FRET measurements with new experimentally-constrained molecular structure computational approaches, this multidisciplinary project will result in a new generic toolkit applicable to a large array of enzymes, proteins and molecular machines.

2019 -
Long-Term Fellowships - LTF

The coevolution of transposable elements and zinc finger proteins across the vertebrate phylogeny

WELLS Jonathan (UK)

Department of Molecular Biology and Genetics - Cornell University - Ithaca - USA

FESCHOTTE Cédric (Host supervisor)

Transposable elements (TEs) comprise a substantial proportion of all vertebrate genomes, including more than half of our own. They are particularly active in the germ line and during development, and impose an significant evolutionary burden on organisms. As such, numerous mechanisms have evolved to control their spread, including the KRAB zinc finger (KZF) family - the largest and most diverse group of transcription factors in mammals. Strong evidence points to an arms race between TEs and KZFs, and their genome frequencies are highly correlated across diverse vertebrate species. However, KZFs are involved in numerous biological processes beyond TE repression, and we do not yet understand how coevolution with TEs has produced this functional diversity in vertebrates. In addition, whilst the KZFs are mostly limited to tetrapods, other large ZF subfamilies exist outside of this lineage, and the roles of many of these are completely unknown.

Using a comparative and functional genomics approach, I will investigate the dynamics of TE-ZF coevolution, in order to better understand how this phenomenon has shaped the architecture and regulation of vertebrate genomes. Specifically, I will test the hypothesis that TE insertion near ZFs leads to co-regulation of their activity, and furthermore, that this process drives the genomic clustering of ZFs. In parallel, I will use functional techniques and long read re-sequencing to undertake a comprehensive study of of a large, un-described and mysterious family of ZFs in zebrafish. In doing so, I hope to open new lines of research in this essential model organism of embryogenesis and development.

2019 -
Grant Awardees - Program Grants

Spatiotemporal neurochemical dynamics of behavioral flexibility in the striatum

WICKENS Jeffery (NEW ZEALAND)

Neurobiology Research Unit - Okinawa Institute of Science and Technology - Onna-Son, Kunigami - JAPAN

TIAN Lin (USA)

Dept. of Biochemistry and Molecular Medicine/ Tian Lab - Universiy of California, Davis/School of Medicine - Davis - USA

GOLDBERG Joshua A. (ISRAEL)

Dept. of Medical Neurobiology - IMRIC - The Faculty of Medicine - Jerusalem - ISRAEL

The overarching goal of this proposal is to investigate the spatiotemporal coding of acetylcholine (ACh) and dopamine (DA) with high-resolution and precision in the striatum using state-of-the-art genetically encoded biosensors combined with modern optics in awake animal imaging. The striatum is crucial for movement, learning and flexible behavior, with striatal DA and ACh both playing key roles in these functions. While the role of DA is relatively well established, the role of ACh in natural behavior still remains enigmatic. Cholinergic interneurons (CINs), the major source of striatal ACh, are involved in processing contextual information that guides flexible behavior. Locally, CINs also exert control over striatal DA release, hijacking DA axons and making them release ACh by activating nicotinic receptors near their terminals. We propose to image the spatiotemporal dynamics of striatal DA and ACh using two-photon microscopy and endoscopy in awake mice engaged in tasks requiring behavioral flexibility. To image DA and ACh simultaneously during behavior we will extend the color-spectrum of DA and ACh biosensors. We will also further optimize the performance of these biosensors to make them suitable for robust in vivo application. Our combined interdisciplinary but complementary expertise – in biosensor engineering, imaging, modelling and behavior – is essential for our aims. We will ensure a coherent, interactive approach by sharing procedures, behavioral tasks, and biosensor technology, with regular planning sessions and feedback of results. A successful outcome of this program will reveal, for the first time, the spatiotemporal coding of neuromodulatory signaling by DA and ACh and how it shapes the function of striatal circuits during flexible behavior. We will also obtain a mathematical understanding of the genesis of the spatiotemporal dynamics. The newly engineered sensors developed in the program will have further broad applications in various biological systems of interest, which will ultimately pave the way toward a more complete understanding of brain function at synaptic, microcircuit, and behavioral levels.

2019 -
Long-Term Fellowships - LTF

Manipulation of linear plasmids in vivo for enhanced homologous recombination

WILLIS Julian (UK)

Broad Institute of MIT and Harvard - Harvard University - Cambridge - USA

LIU David (Host supervisor)

The genome editing field is currently limited in its control over how double-strand breaks can be selectively repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. By providing a donor DNA template, HDR offers potential for corrective therapies targeting a much larger number of genetic diseases which cannot be cured by either single nucleotide substitutions or gene inactivation alone. Yet the efficiency of HDR remains considerably lower than that of NHEJ, hampering its usefulness for therapeutic applications. The use of linear plasmids for enhanced homologous recombination in this proposal directly aims to tackle this challenge and should drive efficient genome editing towards clinical translation.

Unlike circular plasmids which are a ubiquitous tool used throughout modern molecular biology, linear plasmids are an underappreciated biological phenomenon despite being widespread in nature. What constitutes their biological function and what genes they encode is largely unknown. I propose a new method for the in vivo generation of a linear plasmid. The ability to manipulate linear plasmids in vivo will provide a powerful tool to study and understand their biological significance for the first time. Linear plasmids are double-stranded DNA genetic elements characterised by terminal proteins covalently attached to the 5'-end of each linear DNA strand. In particular, the feature of a terminal protein covalently linked to DNA is a strong prospect for biotechnology applications: I will exploit such linear plasmid systems in mammalian cells for enhanced homologous recombination.

2019 -
Cross Disciplinary Fellowships - CDF

Applying surface chemistry techniques to study electron-transfer in conductive proteins and biofilms

WOOD Mary (UK)

Laboratory of Nanobiotechnology - EPFL - Lausanne - SWITZERLAND

BOGHOSSIAN Ardemis (Host supervisor)

The overall aim of the proposed work is to develop new physical chemistry/bioelectrochemistry techniques in order to better understand the electron-transfer mechanisms of conductive biofilms at the electrode surface. This will have immediate benefits in the development of the promising renewable biophotovoltaic (BPV) technology as well as long-term significance for improvements in understanding the role of key biological electron-transfer species across a broad range of systems.

BPVs use photosynthetic biofilms to harvest solar energy and then convert this into electrical current. They are, however, limited in power output due to unexpectedly slow electron-transfer at the biofilm/electrode interface, and it remains unclear why this is. There is an urgent need to improve fundamental understanding of these electron-transfer mechanisms in order to direct future development of BPVs and remove this bottleneck for their maximum efficiency.

Here, we propose the combination of a number of sophisticated and highly-specialised surface-study techniques in combination with the bioelectrochemistry, synthetic biology and electrode design already in place in the host group, to study the key species (such as the individual redox proteins and complexes) both individually and in situ within model lipid bilayers and finally within the biofilms themselves. Importantly, this will allow us to build up a mechanistic picture of each aspect of these complex buried biointerfaces and hence distinguish between different electron-transfer mechanisms (e.g. via pili aromatic amino acids, outer-membrane redox proteins or diffusional shuttles) that are currently proposed.

2019 -
Long-Term Fellowships - LTF

Elucidating the functional asymmetry of echolocation and communication systems in odontocete brains

WRIGHT Alexandra (USA)

School of Biology - University of St. Andrews - St. Andrews - UK

TYACK Peter Lloyd (Host supervisor)

Most odontocete cetaceans (toothed whales, dolphins, and porpoises) produce echolocation and communication sounds using bilateral pairs of sound-generating phonic lips. The production of high-frequency echolocation clicks and analysis of the returning echoes allow foraging odontocetes to detect, localize, and capture prey. Communication between odontocete conspecifics is mediated by family-specific sounds, with dolphins producing low-frequency whistles and porpoises emitting high-frequency social clicks. Previous research on the lateralization of sound production in dolphins suggested a left hemisphere bias for echolocation and a right hemisphere bias for conspecific communication; however, similar to other behavioral studies of cetacean lateralization, hemispheric specialization was inferred, rather than measured. Here, I aim to perform the first assessment of hemispheric specialization for the production and perception of echolocation and communication sounds in the bottlenose dolphin (Tursiops truncatus) and harbor porpoise (Phocoena phocoena). Using bioacoustics approaches, novel species-customized electroencephalography-functional near-infrared spectroscopy (EEG-fNIRS) arrays, and functional magnetic resonance imaging (fMRI), I will directly and indirectly measure neural activity underlying motor control and auditory sensation in these odontocetes. In addition to providing unprecedented insights into cetacean brain function, this project has the potential to redefine the limits of neuroscience and animal behavior research through the introduction of portable, non-invasive, and customizable EEG-fNIRS equipment that can be adapted for taxonomically diverse species.

2019 -
Long-Term Fellowships - LTF

Designing a new class of protein ligands to control cell surface receptor signaling

YANG Aerin (KOREA, REPUBLIC OF (SOUTH KOREA))

Department of Molecular and Cellular Physiology - Stanford University - Stanford - USA

GARCIA K. Christopher (Host supervisor)

Cytokines act as essential regulators of immunity by eliciting signaling outputs from cell surface receptors. There have been extensive efforts to use cytokines for a wide range of immune disease, including cancer, but their functional pleiotropy has limited their clinical utility. Precedent studies in the Garcia lab, demonstrated that surrogate cytokine ligands can alter signaling output by modifying receptor dimer geometry. For more precise topological control of receptor-ligand interactions and signaling, we hope to create a new class of modular binding scaffold proteins which are applicable to diverse receptor targets. The new scaffolds will be computationally designed and engineered to have three major characteristics: (i) high binding specificity to a target receptor, (ii) self-assembly to form dimers or trimers, (iii) drug-like properties including minimal immunogenicity. These scaffolds will be used for manipulating the geometry of cell surface receptors to fine-tune of signaling outputs. These novel scaffolds can also be utilized to drive the formation of non-natural receptor pairings to elicit completely new functional outputs. Proof-of-concept experiments from Garcia lab have shown that non-natural receptor dimers of receptor tyrosine kinases (RTK) can induce different signaling outputs from each of their parental receptors. We believe that the new scaffolds can efficiently combine any cytokine receptor and RTK to form diverse combinations of dimeric or trimeric receptor pairs. In this way, it will help us to broaden our knowledge on kinase-linked receptor signaling and also have a chance of finding new agonistic and therapeutically meaningful protein ligands.

2019 -
Long-Term Fellowships - LTF

Single-cell DNA methylation dynamics of stem cell differentiation across intestinal crypts

YEUNG Jake (CANADA)

Laboratory of Quantitative Biology - Hubrecht Institute - Utrecht - NETHERLANDS

VAN OUDENAARDEN Alexander (Host supervisor)

Intestinal stem cells, which reside in crypts, fuel self-renewal, maintenance, and repair of the epithelial lining of the gut. The regulatory mechanisms that govern maintenance and differentiation of intestinal stem cells, as well as structural and functional differences along regions of the intestine are poorly understood. DNA methylation (DNAme) holds potential in deciphering these mechanisms by revealing gene regulation and clonal structure of cell populations.

The goal is to investigate the dynamics, diversity, and function of DNAme patterns regulating intestinal epithelial homeostasis at single-cell resolution. This proposal will survey single-cell DNAme of intestinal crypts at three anatomical scales: dynamics within individual crypts, diversity between neighboring crypts, and region-specific diversity from proximal to distal sections of the small intestine. Our hypothesis, which builds on preliminary data, is that DNAme patterns of intestinal cells are largely determined by the clonal history of these cells and therefore hold promise as an endogenous lineage tracing tool. To test this hypothesis, I will use a range of single-cell sequencing techniques and develop computational methods to pursue three specific aims:

Aim 1: Determine the single-cell DNAme dynamics within intestinal crypts.

Aim 2: Analyze the diversity of DNAme patterns between crypts along different regions of the intestine.

Aim 3: Disentangle the contribution of cell-type and clonal history in DNAme patterns.

This proposal combines computational with experimental approaches, and leverages existing collaborations between the van Oudenaarden and Clevers labs at the Hubrecht Institute.

2019 -
Long-Term Fellowships - LTF

Unwrapping myelin plasticity in health and disease

YEUNG WONG Maggie Sui Yan (SWEDEN)

Department of Neurology - UC San Francisco - San Francisco - USA

CHAN Jonah R. (Host supervisor)

The brain has a remarkable capacity for combining long-term memory with exquisite plasticity allowing for adaptation to the environment. A growing aspect of neural plasticity is modulation of myelination. How can myelination be altered? Myelination can be altered by the generation of new myelinating oligodendrocytes or alternatively by mature myelinating oligodendrocytes rendering their existing myelin. The current dogma is that continuous generation of oligodendrocytes underlies myelin remodeling—even though the contribution of mature oligodendrocytes remains poorly understood. Here I propose a multidisciplinary approach, combining my expertise in human cell biology with Dr. Chan’s extensive proficiency in oligodendrocyte biology to study the cellular and molecular mechanisms underlying myelin alterations upon induced neuronal activity and after injury. I plan to explore the myelinogenic potential of mature oligodendrocytes and their contribution to myelin remodeling. This strategy combines genetic fate-mapping and manipulation of oligodendrocyte lineage cells, in vivo pharmacogenetic neuronal stimulation (DREADDs) with single cell, temporal-resolved gene expression analysis and behavioral assessment. More specifically, genetically altered progenitors unable to generate new oligodendrocytes and mature oligodendrocytes will be labeled and profiled using single cell RNA-seq. This will allow for the longitudinal assessment of genes involved in activity-dependent myelination and the consequence of this manipulation. Knowledge of myelin plasticity is essential for the development of new treatments for demyelinating disease such as multiple sclerosis.

2019 -
Career Development Awards

A molecular approach to brain sexual dimorphism: cell types and circuits in the medial amygdala

ZEISEL Amit (ISRAEL)

Faculty of Biotechnology and Food Engineering - Technion - Israel Institute of Technology - Haifa - ISRAEL

Neurons are the most diverse class of nervous system cell types, based on thorough characterization of single cells into molecular taxonomies. Individually neurons are fascinating and complicated, but at the core of computation are their interactions. The unique arrangement of circuits between specialized neuronal types results in a wide range of sophisticated functions and complex behaviors. While female and male brains largely perform the same sophisticated functions, the neurobiological basis for gender differences in some behaviors has fascinated researchers over decades. Sexual dimorphism in the amygdala (specifically the posterodorsal medial amygdala, MePD) shapes gender differences in reproductive behavior, aggression or social recognition, with well-established contribution of gonadal steroid hormones. At the core of this dimorphism are neuroanatomical differences of the MePD, such as regional and cell volume, cell number and cell circuitry. While the phenomenon is well established using relatively simple methods, a more global, systematic characterization of differences in circuits in the context of cell types and gene expression has not been technically possible. To this end, I propose to develop and apply an integrated approach for simultaneous connectomics and transcriptomics measurements of the medial amygdala at single cell resolution. This study will reveal the contribution of circuits, cell types and gene expression to gender-based amygdala dimorphism. Further, the proposed framework to understanding circuit implementations in the context of cell types will advance other studies of nervous system function and behavioral neuroscience.

2019 -
Long-Term Fellowships - LTF

Investigating heterochromatin dynamics underlying early development and reprogramming

ZHANG Jingchao (CHINA, PEOPLE'S REPUBLIC OF)

Department of Cell and Developmental Biology - University of Pennsylvania - Philadelphia - USA

ZARET Kenneth S. (Host supervisor)

The goal of this proposal is to understand how heterochromatin functions impose major roadblocks to cell fate changes in development and iPS cell reprogramming. Reprogramming somatic cells into pluripotency by the Yamanaka factors has enormous potential in the regenerative field, although the efficiency remains low. Heterochromatin functions as a major epigenetic barrier to cell fate changes and the Zaret lab showed that its erasure on pluripotent genes at the late stage of reprogramming is a rate-limiting step. However, so far little is known about how heterochromatin domains are established, maintained and reset during development and reprogramming. To fill in this gap, we propose to map the dynamics of heterochromatin formation during mouse peri-implantation, when heterochromatin domains are first established, and to map heterochromatin dynamic resetting in reprogramming intermediates. Taking both natural and reprogramming contexts together, we will construct lineage relationships of heterochromatin dynamics at pluripotency genes as they related to transcription. Finally, to gain temporal and mechanistic insights into heterochromatin regulations of pluripotent genes, we will use a live imaging system to assess directly the consequences of systematic knockdown heterochromatin-associated proteins on the transitions between chromatin topologies and transcriptional activation of pluripotent genes and reprogramming efficiency. Our novel approach of combining datasets and analysis of early development and late reprogramming will contribute to designing robust reprogramming strategies with higher efficiencies and realizing full the potential of iPS cells.

2018 -
Cross Disciplinary Fellowships - CDF

Self-organization of bacterial biofilms as active matter

ALERT Ricard (SPAIN)

Lewis-Sigler Institute for Integrative Genomics - Princeton University - Princeton - USA

WINGREEN Ned S. (Host supervisor)

Biofilms are surface-adhered communities of bacterial cells embedded in a polymeric matrix, and they constitute a major form of bacterial life. Biofilms originate in one of two ways: by clonal proliferation from a single founder cell, or by aggregation of many migrating cells. What are the respective benefits and challenges of these two modes of biofilm growth? To answer this question, we need to understand the physical principles that underlie both modes of biofilm formation. Specifically, for clonal proliferation, it is unclear how cell-level processes, such as directional growth and division, cell adhesion, and heterogeneous matrix secretion, give rise to the evolving macroscopic organization and properties of the mature biofilm. For aggregation, the mechanics of how collective cell migration leads to a biofilm remains controversial. Here, under Prof. N. Wingreen’s supervision, I propose an interdisciplinary approach that will address biofilm formation in two such contrasting systems. Collaborating with Prof. B. Bassler, I will develop an active liquid crystal model to understand the growth and internal ordering of Vibrio cholerae biofilms. Working with Prof. J. Shaevitz, I will investigate whether the aggregation of Myxococcus xanthus constitutes a phase separation or a dewetting transition. Their research programs, respectively, provide single-cell resolution of developing biofilms and traction-force maps of collective cell migration. These data call for active matter models — first to elucidate general principles of self-organization that apply broadly across species, and second to understand the evolutionary selection of species-specific modes of biofilm growth.

2018 -
Grant Awardees - Program Grants

Controlling cellular biochemistry with electronic signals – a step towards bioelectronic hybrids

ALEXANDROV Kirill (AUSTRALIA)

Molecular Engineering Group - Centre for Tropical Crops and Biocommodities - Brisbane - AUSTRALIA

KATZ Evgeny (USA)

Dept. of Chemistry & Biomolecular Science - Clarkson University - Potsdam, NY - USA

O' SULLIVAN Ciara (IRELAND)

Dept. Chemical Engineering - Universitat Rovira i Virgili - Tarragona - SPAIN

The idea of combining man-made and natural systems has fascinated humans for centuries. However, despite the explosion of life science advances, the integration of electronic and biological systems remains woefully underdeveloped. To address this, we will apply principles of Synthetic Biology to develop biological cells controlled by electronic signals.

We propose to create synthetic cellular receptors selectively activated by electrochemically activated peptide. In order to endow the system with the ability to activate a selected ligand:receptor pair in the presence of other polypeptides, we propose to construct peptide-selective bio-electrodes. Electro activation of the peptides decreases their affinity for the electrode and leads to its relocation to the two component cellular receptor that utilizes intramolecular proteolysis to activate transcription of the reporter genes. Using the team’s expertise in protein, cellular and electronic engineering we will design an integrated system where the activation of the reporter expression can be both controlled and monitored using electrode array in a few or even in a single mammalian cell.

This proposed approach enables the construction of a potentially unlimited number of orthogonal electrode/ peptide/receptor systems that allow multichannel information transfer between computing devices and biological cells. While the first embodiment relies on the relatively slow gene expression readout, the same approach can be used to control practically any biochemical process in real time. Such synthetic signaling pathways capable of performing logical operations will exert complex and precise control on cellular biology, leading to a first generation of bioelectronic hybrids.

2018 -
Long-Term Fellowships - LTF

Development of a microtubule nucleation biosensor

ALI Aamir (INDIA)

Department of Cell and Developmental Biology - Institute for Research in Biomedicine - Barcelona - SPAIN

LUDERS Jens (Host supervisor)

Organizing microtubules (MTs) into arrays is fundamental for many cellular functions and critically depends on MT nucleation by the gamma-Tubulin Ring Complex (gammaTuRC). MT Nucleation occurs from the centrosome as well as from dispersed non-centrosomal sites and is temporally regulated. Although the role of gammaTuRC in MT nucleation is widely accepted, the tools for measuring this activity are severely limited. Current methods rely on indirect readouts using MT plus end tracking proteins, based on the rationale that nucleation generates growing MT plus ends. However, polymerization of MT ends generated by severing enzymes or MT rescue can also result in growing plus ends and may be mis-interpreted as nucleation. To address this, I propose to develop a biosensor for the specific detection of gammaTuRC-dependent nucleation within the cells. I will explore several alternative approaches designed to detect interaction between gamma-tubulin molecules in the gammaTuRC and alpha-beta-tubulin oligomers, as it occurs during nucleation. I will use tagged gamma-tubulin in combination with chemical or protein-based probes to generate a fluorescence signal upon formation of the gammaTuRC/MT interface. The signal will be based on FRET, SPLIT-GFP fluorescence complementation, or aggregation-induced emission by a “turn on” chemical probe. I will then use the biosensor to characterize MT nucleation in cycling cells and in post-mitotic neurons differentiating in vitro. This work will provide unprecedented insights into gammaTuRC-dependent nucleation and its regulation in space and time, and will significantly advance our understanding of how cells assemble and remodel MT arrays.

2018 -
Long-Term Fellowships - LTF

Dissecting the role of dopamine to supervise limbic neural ensembles during emotional learning

AMADEI Elizabeth (USA)

Institute of Neuroinformatics - ETH Zurich - Zurich - SWITZERLAND

GREWE Benjamin F. (Host supervisor)

The ability to associate sensory stimuli with appropriate emotional meaning while computing an optimal behavioral response is essential for survival. One key regulator of this emotional learning is the neurochemical dopamine (DA) acting in multiple brain areas such as the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). DA’s modulation of neural plasticity during emotional learning has traditionally been studied using electrophysiology in individual neurons, but how it occurs on the level of large neuronal networks (ensembles) remains unknown. To address this gap, I will investigate DA’s influence on dynamic ensemble activity within the mPFC and BLA during pavlovian fear conditioning, a classic model of emotional learning. Using in vivo calcium imaging and optogenetic control of DA release in mice, I will test DA’s role in establishing ensemble representations of conditioned stimuli. The long-term goals of this project are to (1) reveal key principles by which DA modulates neuronal ensemble coding and (2) lay the groundwork for future studies investigating how abnormal changes in ensemble coding relate to DA-sensitive anxiety disorders such as post-traumatic stress disorder.

2018 -
Long-Term Fellowships - LTF

The origin of thoughts: neural mechanisms of spontaneous thought generation in wakefulness and sleep

ANDRILLON Thomas (FRANCE)

Monash Institute of Cognitive and Clinical Neuroscience - Monash University - Clayton - AUSTRALIA

PEARSON Joel (Host supervisor)
TSUCHIYA Naotsugu (Host supervisor)

The brain is a ceaseless worker, endlessly integrating the information it receives into a continuous stream of conscious thoughts. Interestingly, even falling asleep does not completely suppress conscious thoughts, as exemplified by dreams. This is all the more surprising since wakefulness and sleep differ so greatly in terms of neural activity and phenomenology. What is then the core neural substrate enabling the generation of conscious thoughts?
I will focus here on spontaneous thought generation across wakefulness and sleep. Spontaneous thoughts will be here defined as conscious representations that are not triggered by a specific external event (e.g. an image, a word). Mind-wandering is a classical example of spontaneous thoughts during wakefulness, and dreams during sleep.
My goal will be to identify the neural correlates of spontaneous thought generation, i.e. the minimal set of brain regions that are jointly sufficient to spontaneously produce conscious representations even in the absence of sensory stimulation. I will use both correlational and interventional approaches. By directly stimulating the human brain, I will seek to understand how brain activations constrain the content of thoughts.
Comparing the neuronal correlates of spontaneous thoughts in wakefulness and sleep will be quite powerful because spontaneous thoughts arise without external stimuli, removing confounds due to sensory stimulations. Furthermore, the resulting neural correlates of spontaneous thoughts will, by construct, generalize across different conscious states, allowing us to isolate purer neural correlates of consciousness than previously proposed.

2018 -
Long-Term Fellowships - LTF

Functional metagenomic discovery of host-microbiome effectors using massively parallel scRNA-seq

ANTONOVSKY Niv (ISRAEL)

Laboratory of Genetically Encoded Small Molecules - Rockefeller University - New York - USA

BRADY Sean (Host supervisor)

Over a 100 years ago, Élie Metchnikoff’s visionary work laid the foundations to our understanding regarding the importance of the gut microbiota for the physiology of the host. Recently, technological breakthroughs sparked a renaissance in the research of the human microbiome. While many observational studies identify clinically relevant correlations between the structure of the microbiome and states of disease, our true aim is to uncover the molecular mechanisms and functional roles of host-microbe interactions. Here I describe an innovative sequencing-based functional metagenomic approach, termed FunMet-Seq, which utilizes transcriptional profiling of single human cells towards the discovery and characterization of microbially produced effector molecules. To this end, a library of microbiome-derived DNA, extracted from stool samples, is heterologously expressed in E. coli. Next, culture broth from the library is sampled, sterilized, and used to treat genetically barcoded HEK293-TN cells. Finally, massively parallel transcriptional profiling of single cells enables to identify culture broth samples which contain an effector molecule, and to trace its origin to a specific clone in the array of metagenomic DNA. While the realization of the FunMet-Seq pipeline is an ambitious goal, it will catapult forward our ability to understand the molecular mechanisms underlying the he complex microbial ecology in the human body. Moreover, as the role of the microbiome in human physiology becomes evident, deciphering the interkingdom crosstalk within the human body is critical to reveal the etiology of microbiota-related diseases, and is expected to produce therapeutic applications.

2018 -
Long-Term Fellowships - LTF

Characterizing circadian rhythms in red blood cells; a multi-level approach

ARMONY Gad (ISRAEL)

Department of Pharmaceutical Sciences - Utrecht University - Utrecht - NETHERLANDS

HECK Albert J. R. (Host supervisor)

Circadian rhythms are present in almost all light-sensitive organisms from cyanobacteria through plants, flies, and mammals. Human hormone levels and metabolism are tied to circadian rhythms and affect sleep and wakefulness timings. Disruption of the synchronous action of circadian clocks leads to several human diseases, mainly sleep and metabolic disorders such as non-24-hour sleep wake disorder and diabetes. Most known circadian clocks stem from a transcription-translation feedback loop. Protein and mRNA levels oscillate with a 24 hour period, matching the day-night cycle. An intriguing exception is circadian oscillations in cultured red blood cells (RBCs); since RBCs are anuclear, the circadian rhythm must be non-transcriptionally controlled. RBCs are a major blood component, so their circadian rhythm might affect some of the many metabolic signals carried by the blood stream. Furthermore, the RBC circadian rhythms in individuals with hematological disorders may be out of sync or even cause the disorders’ phenotypes. I aim to use methods that complement each other (e.g., proteomics, metabolomics, and interactomics) to investigate in depth the molecular mechanism of the RBC circadian rhythms, which will advance our understanding of the rhythm’s role in metabolism signaling and hematological disorders. This strategy is suitable for resolving this question since the circadian rhythm is non-transcriptionally controlled and not much is known about it. Revealing the mechanism of the RBC clock will be the first time a eukaryotic non-transcription-translation based circadian rhythm is characterized.

2018 -
Career Development Awards

Investigating the circuit basis of adolescence impulsivity

ARRUDA-CARVALHO Maithe (BRAZIL)

Department of Psychology - University of Toronto - Scarborough - Toronto - CANADA

Adolescence is a unique transitional period marked by deficits in impulse control and behavioral inhibition, often leading to high-risk behaviors. It has long been believed that adolescent impulsivity is a product of a delay in the maturation of the brain circuit supporting decision-making, but due to technical limitations, this link remains elusive. While research has correlated adolescent changes in isolated brain regions with changes in decision-making, no study to date has probed how these brain regions act as functional circuits to modulate adolescence impulsivity. Here we will take advantage of recent technological advances to (1) investigate the pattern of brain circuit activity during live decision-making in adolescent and adult animals, and (2) determine the microcircuit dynamics underlying this behavior to identify the circuit basis of adolescent impulsivity. This will pave the way for future experiments in which we will manipulate circuit function in vivo to modulate adolescent impulsivity in behaving animals. Approaching behavior from the standpoint brain circuit maturation will consolidate a major conceptual and technical shift in systems neuroscience. This shift will shed light on the biological mechanisms underlying behavioral changes during early life and adolescence, but will also fuel a novel approach to the study of the etiology of mental and neurodevelopmental disorders with early onset, thereby informing novel, more effective circuit-specific therapeutic strategies.