Mapping of dynamic connectomes during fear processing holds promise for transforming our understanding of fear regulation mechanisms and for providing novel insight into the cause of anxiety disorders, in which fear deregulation is the core feature. To achieve this goal, we need to be able to manipulate and record, the activity of single neurons throughout the brain fear circuits in behaving animals. Optogenetics combined with holographic phase shaping enables in vitro and in vivo “all-optical” readout and manipulation of activity in neural circuits with single-spike and single-neuron precision. However, light scattering limits optical imaging and focusing in mammalian brain to shallow depths (<300 µm). Here, we aim at overcoming these limitations by combining the development of new ultratargeted opsins with innovative endoscopy techniques based on emerging concepts of wavefront shaping and wave propagation in complex scattering media. This novel toolbox will be developed through a unique synergetic approach will permit optogenetics and minimally-invasive imaging with single cell precision at unprecedented depths, and will be used to decipher the neural response dynamics underlying the learning and expression of divergent fear responses in the central amygdala circuits in behaving animals.

From the stunning aerial displays of large bird flocks to the collective coordination of human culture, how do the interactions among individuals give rise to such interesting and emergent group behavior, and how are these interactions constructed and controlled within the brain and the body? While collective animal behavior has long fascinated and engaged scientists across disciplines, quantitative studies in three-dimensional environments in which animals naturally live are rare and phenomenological descriptions of collective interactions, on which many models are based, have not previously been connected with their foundation in biological circuits. Here, we propose a novel, cross-disciplinary effort to measure and model the three-dimensional collective dynamics of shoaling in the zebrafish (Danio rerio) and to use genetic, pharmacological and neural perturbations to connect these dynamics to underlying biological mechanisms. We will combine expertise in zebrafish genetics and biology with precision 3D motion tracking and theoretical ideas drawn from statistical physics to quantify and elucidate the interactions that drive such collective motion. We will design a custom apparatus consisting of multiple, fast cameras and image-processing software to capture, with high-resolution in space and time, the simultaneous dynamics of interacting fish in a fully three-dimensional arena. Using this apparatus and leveraging the power of the zebrafish model system, we will quantify the collective dynamics of wild-type shoals and compare them to shoals from zebrafish deficient in their visual and lateral line sensory systems. We will also analyze shoals with disruptions to social neural circuits in the brain, focusing in particular on oxytocin and dopaminergic signaling, using optogenetic imaging. Exploiting this rich abundance of shoaling trajectory data, we will construct maximum entropy and agent-based models of group dynamics, and we will use the parameterization of these models to quantitatively characterize the collective shoaling state and changes of this state between conditions. While shoaling is the focused target of our initial efforts, zebrafish exhibit a variety of social behaviors that can also be studied with our proposed capabilities, both computational and experimental.

The paths we follow when we speak.

The analysis of language processes is traditionally based on discrete, categorical variables, such as noun phrase, suffix or phoneme, quite different in nature from the continuously varying neuronal variables (firing rates, or even spike emission times) that at a microscopic level necessarily underlie them. Forms of analog-to-digital conversion have then to be assumed to link linguistic phenomena, in particular in relation to memory, to cortical network operations. Other memory-related phenomena, however, such as navigation in rodents, are beginning to be understood in detail and to reveal computations that remain analog even at the cognitive level – e.g., the choice of a trajectory in space. Are there analog computations that are relevant to understanding language, in humans?

We address this question at two different scales of complexity. First, in the choice of successive phonemes while uttering a word, which we take to be produced by a well-localized network, perhaps in the left inferior frontal gyrus. It may be envisaged as a continuous trajectory on a ‘phoneme manifold’ which expresses, in the space of all possible vocalizations, the phonological memory of one’s own language(s). We ask how the structure of such a manifold would reflect the statistical learning process with which it is gradually acquired during development, and how it would itself be reflected in the patterns of errors observed when reading aloud. To this end, we shall use network models comprised of individual neuronal units and psycholinguistic tests.

Second, to contrast analog with digital computations within the same paradigm, we consider the memory devices that have culturally evolved to remember extended verbal material, and have crystallized in poetry. Some of them, such as meter, can be thought of as expressing a quasi-continuous trajectory, while others, such as rhyme, are more punctuate and essentially digital. We intend to assess the effectiveness of devices of different nature by manipulating them in network models and in psycholinguistic and EEG experiments involving poetry recall. Since meter and rhyme are embedded in complex constructs including meaning, syntax and other components, which are beyond our scope, we shall represent the whole cortex as a network of Potts units, effectively a model of interacting cortical patches.

Understanding the regulation of somatic stem cell (SSC) self-renewal and their regenerative capacity is essential to understand the decline in SSC function during aging. SSCs are thought to produce adenosine triphosphate (ATP) by glycolysis because of the hypoxic environment, but my hypothesis is that glycolytic energy production is actively switched on to minimize the generation of reactive oxygen species (ROS). ROS are important signaling molecules and I hypothesize they have a relevant role in the regulation of HSC self-renewal and differentiation. Accumulation of mitochondrial DNA point mutations during aging could lead to changed ROS levels as well as translational stress within mitochondria, inducing a mitochondrial unfolded protein response (UPRmt). My aim is to elucidate how mitochondrial function and ROS status change in HSCs and other hematopoietic cells during aging.
A growing amount of data suggest that metabolic regulation is important for SSC homeostasis. Mitochondria are central players in the cellular metabolic pathways, by producing metabolic intermediates and reducing equivalents, and recent data suggest that mitochondrial DNA integrity is essential for normal SSC function. Deciphering the function of mitochondria in SSCs, which has been studied only to a limited extent, particularly in the context of aging, will shed light on the mechanisms of SSC dysfunction underlying aging. I aim to understand how mitochondrial function changes with age in HSCs and how this affects the HSC properties over time and the capacity to regenerate the hematopoietic system.

The cross-talk between integrin-based adhesions to the extracellular matrix and dynamic microtubules plays a crucial role in cell polarity and migration. Integrin-based adhesions can promote cortical microtubule stabilization in their vicinity. In turn, microtubules can strongly affect the formation and turnover of the adhesion sites. However, the molecular basis of these connections remains enigmatic.
Here, we propose to decipher the molecular chain of events induced by mechanotransduction at integrin adhesions that leads to cortical microtubule stabilization and analyze the morphogenetic impact of this process. To achieve this goal, we will bring together four distinct sets of expertise: cell biology of the cytoskeleton (Akhmanova); structural biology, biochemistry and biophysics of protein-protein interactions (Goult); mechanobiology and single molecule analysis of force dependence of protein-protein interactions (Yan) and genetic analysis of morphogenesis in flies (Tanentzapf).
We will use advanced cell manipulation assays to directly test whether mechanotransduction at adhesions affects microtubule capture and stabilization at the adjacent cortical sites. To uncover the molecular basis of this process, we will focus on a recently discovered connection between a cortical adaptor protein, which constitutes a part of the microtubule stabilization complex, and an adhesion component. At the same time, we will perform experiments, which will allow us to identify and explore additional links between focal adhesions and the microtubule-stabilizing complex, and thus generate a comprehensive functional map of these connections. Given that the investigated proteins are highly conserved, we will combine genetics, imaging and modeling to address their impact on morphogenesis and tissue maintenance in flies. Our work will shed light on how cellular mechanics and cytoskeletal dynamics are integrated at the molecular level.

Our body consists of trillions of cells, which derive from a single fertilized egg. The divisions that generate these cells constitute a genealogical tree, with a single root (the fertilized egg) and trillions of terminal branches (each of our cells). Knowing the shape and branching order of this tree is important because it provides vital information about how we developed; cells that are on the same branch shared the same cell precursors and a common history of developmental decisions. The cell lineage tree of an individual is also important for understanding cancer; its origins and history of colonizing the body.
Discovering the cell lineage of an organism is a huge challenge that has been solved only in the simplest cases. In the nematode worm Caenorhabditis, whose body consists of approximately 1000 cells, the complete cell lineage was painstakingly determined by direct observation of each cell division under the microscope. In larger organisms we can only infer partial cell lineages by direct observation, or through a method called clonal analysis in which genetic marks are used to label the progeny of individual cells. Each of these methods has its limitations. Direct observation is only applicable to transparent, tiny organisms and to events that occur within hours or days. Clonal analysis can inform us about the rough structure of the lineage tree, but its precise branching patterns are unresolved.
Here we propose a new strategy. It is based on the idea that random mutations, which accumulate in cells during the lifetime of an organism, can reveal the structure of its lineage tree: cells belonging to a major branch will uniquely share mutations that occurred in their common ancestors, those that lie together on a finer branch will share additional mutations, and so on. In principle, if a sufficient number of mutations were detected, we could infer the complete lineage tree of the individual. In practice, this approach is limited by our ability to find those rare somatic mutations within the genome of individual cells. Our strategy resolves this problem by relying on two new technologies: a method that allows us to generate mutations at specific sites in the genome, combined with a method for detecting these mutations in the organism, with single-cell precision.

Dengue virus (DENV), a mosquito-borne RNA virus, is a leading cause of illness and death affecting up to 100 million people worldwide annually. Similar to all other viruses, DENV relies on cellular ribosomes to produce viral proteins and inhibit the innate immune response, and has therefore developed various strategies to seize control of the translation machinery. In response, host cells have evolved various mechanisms to subvert these attempts and prevent viral replication. The proposed project will combine different approaches to study how DENV interacts with the translation machinery and triggers changes in protein synthesis that facilitate or hinder production, folding and assembly of viral proteins. Specifically, I will use ribosome profiling and mass-spectrometry (MS) to monitor translation in infected cells and better characterize the dynamics of viral RNA translation and host response. Then, I will use CirSeq, which allows high-resolution sequencing of viral populations, to search for mutations in the viral genome that affect association with ribosomes and thus alter translation rates. Finally, I will identify host factors involved in the synthesis of DENV proteins by using quantitative MS to analyze the composition of translating ribosomes from infected cells. This will be followed by biochemical and functional characterization of selected candidates to investigate their possible role in viral replication. This work on DENV will expand our understanding of virus-host interactions and may inform approaches to prevention and treatment.

Although potentially of fundamental importance for many phototrophs on Earth, the physiological mechanisms and molecular underpinnings that allow survival over long periods of dark remain a mystery. Diatoms, the dominant oceanic eukaryotic photosynthetic organisms, specifically Fragilariopsis cylindrus (polar pennate diatom with a sequenced genome) and Thalassiosira gravida (representative Arctic centric diatom), will serve as models to characterize the physiological, cellular, genomic, epigenomic, and metabolic state of cells during prolonged darkness and the return of light. Complementary expertise in our team (culturing of polar species, optics, photochemistry, genomics) will allow us to identify key adaptive mechanisms used by diatoms.
Realistic light transitions from fall to early spring will then be simulated to grow F. cylindrus and T. gravida in specially designed bioreactors under stable nutrient and cold temperature conditions. Cellular energy flow and allocation will be monitored before, during and after a simulated 6-month polar night: light absorption coefficients and pigment composition, absorption cross-sections and concentrations of PS1 and PS2, electron production by PS2, redox states of the plastoquinone pool, linear and alternative electron flows, cellular content of electron acceptors, concentrations and activity of RUBISCO, rates of C-fixation, respiration, heterotrophy, and reduction level of storage compounds. F. cylindrus cells will also be examined for their transcriptional and translational activity, and will be subject to transcriptome and metabolome analyses. Mass spectrometry and immunoblotting performed on chromatin will identify major changes in DNA methylation and histone tail modifications. Features of interest will be subject to chromatin immunoprecipitation and DNA sequencing. DNA methylation and histone marks will be aligned to the reference genomes. Chromatin and gene expression states arising in response to prolonged darkness and the return of light will be defined, informing metabolic maps and physiologies. In parallel, organellar structures will be assessed by electron microscopy. Candidate genes encoding key processes for dark survival and recovery will be studied by producing transgenic strains of F. cylindrus, in which these specific genes will be knocked-out.

Animals have to constantly make decisions about what to do next. Especially, when navigating in the natural world, they have to cope with an abundance of potentially conflicting cues. Hence, in order to interact with the environment in a meaningful way, animals have to extract the currently relevant sensory features and select an appropriate, experience- or internal state-based, behavioral action. How this is achieved by the nervous system is poorly understood in any sensory modality and organism.

Landmark studies in primates have already revealed some principles of decision making but, due to technical limitations, could only focus on confined brain regions. However, understanding the complex process of how an animal selects the right behavior requires complete explorations of the hierarchical and parallel processing steps within neuronal assemblies in different parts of the entire brain.

The larval zebrafish is becoming the model of choice for studying brain-wide sensory processing as it offers a unique combination of tools: Larval zebrafish possess a variety of innate and robust behaviors in response to sensory stimulation, while giving access to state-of-the-art rapid whole-brain imaging techniques with cellular resolution.

Adapting successful concepts used in classical primate literature, I will use larval zebrafish as a modern model to investigate general principles of sensory integration, stimulus competition, decision making, and action selection. This will permit unprecedented detailed explorations of how neuronal circuits in an entire vertebrate brain extract features from a complex sensory world in order to decide what to do next.

In living cells, molecules organize into complex assemblies on the micrometer scale. Among these structures is the bacterial cell wall - a rigid exoskeleton composed of peptidoglycan chains cross-linked by short peptides. This two-dimensional polymer surrounds the cell and defines its shape during growth and division. Although its synthesis is the target of many antibiotics, how bacteria build the cell wall is largely unknown, even for a relatively simple cell like E. coli. To shed light on the mechanism of cell wall assembly I will develop an experimental assay to reconstitute peptidoglycan synthesis in vitro. Starting from the enzyme PBP1b and its substrate lipid II I will build a PG layer on a biomimetic membrane and quantitatively characterize the interactions, which can lead to a continuous, two-dimensional polymer. Using this novel reconstituted system, I will not only access the molecular mechanism of cell wall assembly, but also identify how local interactions contribute to the large-scale structure of cell wall, which ultimately determines the shape of the bacterial cell.

Filopodia are thin, actin-rich plasma membrane protrusions, which function as sensory organelles of cells. Despite a wealth of information on the physiological functions, the molecular mechanisms underlying their assembly and dynamics are incompletely understood. Filopodia are characterized by high negative membrane curvature and high density of phosphoinositides. Their formation and dynamics are controlled by an array of actin-binding and signaling proteins, many of which interact also with phospholipids. However, the mechanisms and biological roles of these lipid-interactions are largely unknown. We will collaboratively examine the molecular principles of filopodia assembly, with a specific focus on the roles of membrane curvature sensing and phosphoinositide clustering in this process. We will use advanced cell biology methods (genome editing combined with live-cell imaging approaches) to identify the mechanisms by which central filopodial proteins are recruited to these membrane protrusions in cells. Phosphoinositide and curvature sensing properties as well as potential synergetic effects of these proteins will be quantitatively measured by in vitro experiments (optical tweezers, confocal microscopy, FCS) using model membrane systems (membrane nanotubes pulled from Giant Unilamellar Vesicles) with controlled curvature, and also in silico (multiscale simulations) and in vivo with relevant mutant proteins. These computationally efficient simulations are based on systematic coarse-graining methods, in which the molecular resolution is reduced but the effects of key molecular features retained, thus adding insight into the cooperative processes underlying filopodia formation. Such insight will also help to connect the results of the in vivo and in vitro experiments. Finally, once a minimal set of proteins responsible for this synergy will have been identified, we will include actin and reconstruct a synthetic filopodium. Additional positive feedback is eventually expected to take place due to actin polymerizing against the membrane. Thus, by virtue of reconstituted systems, we will aim to identify the essential physical mechanisms underlying the very first steps of filopodia formation.

The cohesin-complex mediates sister chromatid cohesion from S-phase until mitosis and is involved in the formation of higher-order chromatin structure. To fulfill these vital functions, cohesin is loaded and positioned in the genome by mechanisms that are only poorly understood. In vitro, loading of cohesin on DNA only requires ATP and a loading-complex formed by Scc2-Scc4, while loading in vivo on chromatin is regulated by additional factors. For example, in Xenopus laevis oocytes, cohesin loading strictly depends on pre-replication complexes (pre-RCs), which are formed in telophase/G1.
Mechanistic studies are required to understand how cohesin-loading occurs at the molecular level. I will first determine the mechanism by which Scc2-Scc4 loads cohesin on DNA. Using single-molecule FRET and optical tweezers, I will monitor the effect of Scc2-Scc4 on conformational changes of cohesin as it is loaded on a DNA template. After characterizing this minimal loading reaction, I will reconstitute cohesin-loading during telophase/G1 using a purified system. With these experiments I will address why and how loading of cohesin is regulated by the formation of pre-RCs.

The capacity of cells to move and migrate is fundamental to their viability, whether they originate from multicellular or single-celled organisms. This is exemplified in the process of infection, such as that by the the protozoan parasite Plasmodium, the causative agent of malaria disease in humans. During an infection, cell migration for both the human immune cell or the malaria parasite both rely on force generation from structures within each that link them to, and propel them across, the extracellular environment. For the immune cell, its amoeboid-like movement is the product of polymerising actin filaments combined with force generation from a myosin motor, which together drive changes in cell shape propelling the cell at speeds of several micron/min. In contrast, while relying on very same actin-myosin proteins, the malaria parasite does not change its shape, yet can move at speeds of >1 micron/sec, an order of magnitude above our fastest cells. Whilst a great deal is understood about amoeboid migration, we know little about how malaria parasites achieve directional motility or such great speed.
Underpinning Plasmodium cell migration across its lifecycle, whether in the liver, the blood circulatory system or mosquito, is an unconventional myosin (XIV), lacking many of the canonical features associated with myosin motors. Together with dynamic parasite actin filaments these somehow generate a force that drives the parasite forwards, however, the mechanics of how this actually works is far from understood. Here, building on insights we gained into actin regulation and organisation in the malaria parasite from our first HFSP program, we turn our attention firmly on myosin to understanding how it interacts with actin inside the cell to produce directional cell migration. Combining the state-of-the-art in biochemical methods, molecular and cellular parasitology, biophysics and structural biology (including cryoelectron microscopy), we aim to dissect at every level - from single molecule to whole cell - how motor organisation inside the malaria parasite leads to directional, fast cell movement. This will uncover profound insights into the workings of an ancient, supremely fast cell migration machine, and may potentially reveal weaknesses that could be targeted to cure one of mankind’s greatest diseases

Macrophages play beneficial roles in protective immunity. However, they also favor the progression of several pathologies when they massively infiltrate diseased tissues. A present challenge in cancer, for instance, is to control macrophage tissue infiltration, which involves the mesenchymal motility. This motility is characterized by the ability of the cell to form protrusive structures called podosomes. A podosome constitutes a submicron core of actin filaments surrounded by a ring of integrin-based adhesion complexes. Our working model postulates a mechanical connection that counterbalances the actin-rich protrusive core by traction at the adhesive ring, likely embodied by acto-myosin contractile cables. Therefore core and cables would form a unique two-module protrusive force generator that balances forces at the level of a single podosome to ultimately contribute to the mechanics of macrophage migration.
Our objective is to build a sound experimental corpus to substantiate this two-module mechanism of force generation and determine its implications for macrophage 3D motility. To this end, we will characterize podosome molecular architecture and formulate its relationship to force generation. Furthermore, we will identify the mechanical role of key podosome components in cell migration.
We assembled a multidisciplinary team that combines cutting-edge expertise in: i) macrophage 3D migration, ii) 3D nanoscale imaging, iii) live super-resolution imaging, iv) cryo-electron tomography technologies allowing resolution of actin filaments within cellular networks, v) measurement of podosome protrusion forces and vi) mechanics of 3D cell migration in custom microfabricated environments.
Our ambitious research plan will deliver the nanoscale localization of podosome molecular components (applicants A1, A3), determine how the architecture evolves along with force dynamics (A2), investigate the mechanics of 3D migrating cells (A4) and reveal the role of podosome components in these uncharted features (A1-4). Thanks to this groundbreaking study, we will articulate mechanical and architectural insights into an integrated portrait of podosomes from the molecular scale up to the biological context of 3D cell migration and thus identify molecular means for the modulation of pathological macrophage tissue infiltration.

Neural oscillations, the byproduct of periodic fluctuations in synchronized brain activity, are a defining feature of neural circuits. While oscillatory activity is closely correlated with both an animal’s behavioral and cognitive state, it is unclear whether oscillations are causally involved in behavior and cognition. In this research proposal, we will focus on thalamo-cortical spindle oscillations, examining their possible causal role in memory consolidation. Our research proposal consists of two main research aims: 1) quantifying the causal relationship between thalamo-cortical spindles and memory-related oscillatory dynamics known as hippocampal sharp-wave ripples and 2) behaviorally measuring the causal relationship between spindle generation and memory consolidation. This will be accomplished by a synergistic merger of molecular-biology (Halassa lab) and a computational toolbox (Bendor lab), while performing large-scale neural recordings (Bendor and Halassa lab). Our combined, state of the art toolbox provides a more temporally and spatially precise method of recording, stimulating and blocking spindle activity, while directly addressing causal interactions using sophisticated Bayesian-based computational methods. Using these methods we will directly test two hypotheses, that thalamo-cortical spindles and hippocampal ripples have causal, bi-directionally interactions (aim 1), and that the amount of thalamo-cortical spindle activity is related to the strength of the encoded memory, with this effect being both modality-specific and sensitive to the timing of hippocampal ripples (aim 2).

Inorganic polyphosphate (polyP) is a biopolymer that serves multiple critical functions in biology. PolyP has been mainly studied in bacteria and shown to be involved in such diverse processes as the bacterial stress response, biofilm formation, plasmid uptake and antibiotic resistance. In comparison, its functions in mammals are significantly less well understood. Even so, polyP has been linked to multiple important physiological phenomena, such as blood clotting, chaperone function, posttranslational protein modification and neuronal signaling.
While in bacteria the enzymes that generate polyP are known (polyphosphate kinases Ppks), the mammalian enzymes remain elusive to date. In fact, we know that polyP is present in different cell types and that it is enriched in different organelles, but we do not know how and where it is made and how it is shuttled. Moving polyP research forward will critically require identification of the elusive enzymes that make and traffic polyP. Currently, a dearth of chemical tools has precluded such deeper insight into mammalian polyP physiology.
Our proposed research will culminate in new technologies required to make, modify, analyze, transport and deliver polyP both ex and in vivo. We will develop and combine multiple novel synthetic approaches to generate a range of modified polyP analogs that are not yet available. Furthermore, we will develop technologies to deliver and release those synthetic analogs in a controlled way outside and inside of cells. Together, these technologies will provide valuable novel insight into how cells make, regulate and distribute polyP. We will use these tools to identify the mammalian enzymes involved in polyP turnover, investigate how polyP levels are affected by other signaling molecules such as inositol pyrophosphates, and try to understand how polyP affects different biological processes. For example, an in-depth evaluation of the role of polyP in blood clotting will be achieved using our novel approaches. These studies will lay the foundation for further research into polyP physiology. By virtue of the combination of synthetic organic chemistry, drug delivery technology, and cell and animal biology, we will be able to develop and apply powerful novel tools dedicated to the elucidation of polyP physiology in mammals.

Genome architecture is emerging as a key epigenetic property providing either positional or topological cues that regulate gene expression. By following certain folding principles and controlling gene positioning within the nucleus or chromosomal interactions, the three-dimensional organization of chromatin seems to exert a pivotal role in coordinating the emergence of unique cellular phenotypes. During early mouse embryo development, the nuclear volume decreases ten times, while encompassing the same amount of genetic material. At the same time, transcriptional networks undergo drastic changes, switching from maternally-controlled to zygote-specific programs. While these expression patterns must unfold in a robust manner from one stage of development to the next, they also need to incorporate some degree of flexibility and cell-to-cell variability, eventually making every cell unique throughout differentiation. Deciphering how the interplay between genome organization and expression operates during this highly dynamic period in a multicellular organism’s lifespan is a formidable challenge. Here, I propose to establish a pioneering framework for assessing various aspects of genome organization while monitoring gene expression profiles in exactly the same single blastomeres, tracing their astounding dynamics while the embryo is developing into a blastocyst. By combining multi-assay measurements in the same single blastomeres with high-dimensional correlative data analyses, I aim at identifying general design principles governing DNA folding, as well as the crosstalk between genome architecture and gene expression.

Tuberculosis is an ancient disease caused by Mycobacterium tuberculosis that still exerts a massive burden on public health in many parts of the world. The success of M. tuberculosis is primarily due to its ability to survive within macrophages for months and even years in an asymptomatic latent state. It is still largely unknown what makes this pathogenic invader successful, or what determines if the immune cells eliminate the assault or succumb to it. Thus far studies of host-pathogen encounters were performed at the population level, averaging molecular signatures, while masking any cell-to-cell variation. The heterogeneous, stochastic, and dynamic nature of both host and pathogen suggest that descriptions of average behavior may fail to accurately characterize these interactions. Here, I propose to take a systematic quantitative approach to study the process of infection by M. tuberculosis, establishing a fluorescent method to specifically differentiate the outcomes of infection, and combining it with single-cell RNA-seq. Using this method I will examine whether variation in transcriptional profile of individual M. tuberculosis-macrophage encounters translate to distinct infection outcomes. I will apply this method to determine the degree to which genetic diversity among clinical strains is reflected in cell-to-cell variability of the transcriptional profile and translates to distinct of infection outcomes among these strains. Collectively my studies will expose mechanisms by which transcriptional heterogeneity affects the outcome of infection, and provide a holistic view on the process of M. tuberculosis infection.

Birds are paedomorphic dinosaurs: they exhibit in their adult skeleton some traits that were present in the juveniles of their dinosaur ancestors. The mechanisms underlying this paedomorphosis are largely unknown. We hypothesize that it is caused by differences in the rates of skeletal growth and differentiation, which are known to be higher in birds than in dinosaurs. The aim of this research proposal is to investigate the mechanisms that control how fast skeletal cells differentiate, and how their rates of differentiation influence the morphology of the skeleton. Most of the vertebrate skeleton develops by replacement of cartilage extracellular matrix by bone, a process called endochondral ossification. We propose to undertake this investigation by comparing endochondral ossification in embryos of two species of birds with different rates of development – quails and zebra finches – to embryos of alligators, which will be used as out-group. We will use two novel experimental approaches: (1) the adaptation of recently developed whole-mount techniques and cutting edge imaging technologies (micro-computed tomography and confocal light microscopy) to the study of skeletal development, and (2) the compared analysis of in vitro culture of cartilage cells obtained from the three species. Those experiments will allow us to characterize, in each species, the temporal and spatial expression of molecular regulators of endochondral ossification, its relation to changes in the shape and growth of the skeleton, the relative duration of cell cycle progression, and the rates of chondrocyte differentiation.

Upon antigen recognition, T cells switch from a fast migratory to a stationary state. T-cell receptor (TCR) recognises specific peptide bound to major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells (APCs). TCR interaction with pMHC of high affinity leads to a migration stop and robust TCR signalling. However, how a molecular recognition event can coordinate global cellular changes remains unclear. Here, I propose that membrane tension is the cellular coordinator that translates antigen recognition into a stop signal and ultimately enables T cell activation. I hypothesize that TCR engagement in motile cells creates mechanical stress that increases membrane tension, leading to extracellular calcium entry through mechanosensitive ion channels, which results in arrest of locomotion, cytoskeletal rearrangement and TCR signalling. Using a FRET-based force sensor and micro-fabricated migration channels, I will quantify the mechanical forces that act on the TCR when motile T cells encounter immobilised pMHC molecules. I will then measure membrane tension with optical tweezers and membrane topography with 3D super-resolution microscopy as a function of pMHC affinity. Next, I will investigate the role of mechanosensitive ion channels and image cytoskeletal remodelling and trafficking of signalling vesicles in T cell during the transition phase. Taken together, the proposed project will provide a better understanding of how mechanical forces derived from cell motility mediate antigen-induced T cell arrest and activation.