The muscular systems of humans and chimpanzees differ in a variety of traits, with human muscles built for dexterity and endurance, whereas chimpanzee muscles are built for strength. However, the genetic basis of this variation is little understood. Here, I propose to use a novel system of hybrid human-chimpanzee tetraploid induced pluripotent cells to study how the regulation of genes affecting muscle physiology has evolved along our lineage. This hybrid cell line offers a unique and unprecedented opportunity to tease out genetic from environmental factors, as well as cis- from trans-acting factors. I will first differentiate the parental and hybrid cells into skeletal myocytes, the main cells determining fitness capabilities. Then, I will identify allele-specific expression by comparing the gene expression levels of the hybrids to their parental cells. This will allow me to establish the first catalogue of cis- and trans-regulatory divergence in muscle cells. Finally, I offer to pick ten genes, where it is most probable to identify the variant the drives their diverged regulation, and to genetically edit them to investigate the phenotypic effect of each sequence change. This novel system will allow me to circumnavigate a key ethical barrier in great ape studies – obtaining great ape cells. Moreover, this system and pipeline could be used to study a variety of cells, thus establishing a novel approach to identify the regulatory variants that drove species-specific traits.

While hematopoietic stem cell (HSC) transplantation has become the standard of care for treating many hematologic diseases, wider use is restrained by immunologic incompatibility and inefficient reconstitution of hematopoiesis from allogeneic sources. Pluripotent stem cells (PSCs) promise an attractive alternative source of HSCs, but current PSC-derived HSCs display fetal phenotypes that lead to stem cell exhaustion and immature progeny function. We hypothesize that human PSC-derived definitive hemogenic endothelium (HE) can be efficiently converted to HSCs with adult-like functionality. To achieve this, we first propose to develop a robust reporter system that differentially marks fetal versus adult HSCs. By combining this with recently-developed technology to transgenically convert HE into fetal HSC-like cells, we can execute in vivo targeted and high-throughput genetic screens to identify TFs and pathways that regulate HSC maturity. We will subsequently find small molecules or morphogens that modulate activity of these regulatory factors to establish transgene-free conditions for the specification of adult-like HSCs. To ensure physiological relevance of the resultant cells, they will be assessed for long-term engraftment capability, functionality of their terminally-differentiated cells and appropriate response to stress conditions in vivo. This proposal will provide proof-of-concept that adult functionality can be conferred to PSC-derived cells, provide a sustainable platform for modeling adult hematologic diseases, and most importantly, bring us one step closer towards clinical grade PSC-derived HSCs for transplantation.

Tissue stem cells (SCs) depend upon their niche microenvironment, suggesting that spatially localized signals play key roles in determining SC identity and plasticity. Evidence from the hematopoietic system suggests that vasculature is a critical niche component. Vasculature also governs tumor-initiating SCs of squamous cell carcinomas. The spatial and temporal organization of capillary networks in healthy dermis suggests that vasculature also impacts normal hair follicle SC (HFSC) behavior, a notion further fueled by hair growth sensitivity to systemic changes. Despite several recent advances, the molecular basis by which a SC can sustain regeneration is still poorly understood. Here, I aim to perform the first spatiotemporal screening to dissect the molecular crosstalk between HFSCs and vasculature remodeling, and elucidate how crosstalk dictates ‘stemness’ identity to meet regenerative demands during normal hair cycling and wound-repair. Using mouse as model, I aim to tackle these objectives by: (1) Mapping and defining the spatial and temporal localization of the skin vasculature network relative to HFSCs during development and hair cycling. (2) Elucidate the molecular crosstalk between vasculature and HFSCs, and how it changes during capillary network remodeling. (3) Dissect mechanistically how vasculature-mediated signaling influences SC physiology during homeostasis and wound-repair by using in utero lentiviral delivery to skin epithelium of HFSC-specific activation of Cas9/guide gene silencing. By understanding how the vasculature dynamically redistributes and reshapes the niche, I hope to uncover new avenues for therapeutics in skin regeneration and wound-repair.

Cell survival requires efficient adaptation to external changes. Bacteria use complex signaling networks to sense and respond to these changes, particularly nutrient availability. The current knowledge of bacterial responses to carbon starvation is based mainly on E. coli and involves small molecule second messengers like cAMP. However, intracellular responses to starvation and cAMP regulation may differ in other species. The alpha-proteobacterium Caulobacter crescentus is a powerful model for understanding how intracellular signals are transduced and impact cell-cycle progression in bacteria. Carbon starvation somehow prevents cell differentiation and a G1-S transition during the Caulobacter cell cycle. This differentiation step is normally triggered by the synthesis of a regulator called SpmX. I propose that the absence of a carbon source prevents SpmX synthesis through the inhibition of its transcriptional regulator, TacA. I will use ChIP-qPCR and in vivo phosphorylation assays to test this model and dissect the mechanisms that temporally regulate SpmX. Further, I hypothesize that, like E. coli, the main intracellular signal following carbon starvation in Caulobacter is cAMP, and that cAMP directly regulates TacA activation. I will measure cAMP intracellular levels using a FRET-based method and assess the impact of cAMP on TacA activity and SpmX synthesis. I will also use capture-compound mass spectrometry to identify the cAMP interactome in Caulobacter. I anticipate that this study will provide new insights into cAMP-dependent cellular responses and nutrient-sensing mechanisms in Caulobacter, which may differ significantly from what is known in E. coli.

During development, embryonic tissues develop into organs of stereotyped size and shape. Morphogens are secreted from discrete regions in developing organs and form spatial concentration gradients that guide gene activation, pattern formation and tissue growth. Morphogen gradients scale with tissue size, ensuring that morphological patterns remain proportionate in organs of different size. The mechanisms of morphogen dispersal and scaling are not yet clear. Two models for morphogen spreading attracted much attention in the field: i) fast extracellular diffusion versus ii) cycles of internalization and recycling (transcytosis). I will develop a generalized theory of transport that captures extracellular diffusion and transcytosis simultaneously: I will consider extracellular diffusion, morphogen/receptor binding/unbinding and various endocytic trafficking steps. This novel theoretical method will uncover the parameter space where either extracellular diffusion or transcytosis dominates the dynamics of dispersal. I will also explore whether both phenomena could contribute significantly to the length scale of the steady-state gradient. My model system will be the Decapentaplegic (Dpp) gradient in the fly wing. Using novel experimental endocytic assays from the host lab, I will parameterize Dpp trafficking dynamics to establish the prevalence of extracellular diffusion versus transcytosis. Unpublished work from the host lab shows that trafficking is spatially heterogeneous in the wing. I will upgrade my theory to a heterogeneous scenario with the potential to yield, as an emerging property, a novel mechanism of gradient scaling.

When we navigate and make decisions in familiar environments we rely on an internal, mental map of the features and general shape of the environment. Remarkably, work over the last four decades has revealed how different locations in space and different navigational plans are represented as patterns of activity in thousands of neurons in the brains of rodents. It stands to reason that this 'neural representation' of an environment should remain stable as long as an animal is using it, to allow the animal the reliably navigate. However, we found that the neural activity that makes up this map changes over time so that different neurons represent specific features of a familiar environment after several days. We do not have a theory to explain how the brain can build and use such a continually changing mental map, nor do we know why the map changes, or what purpose this continual change might serve. In this project we will build and experimentally test new theories and models of how the brain represents environments, potentially changing our understanding of how the brain works.

The random selection of an allele to be exclusively expressed affects gene expression and any possible phenotypes related to the allele. This may be one of the key mechanisms through which phenotypic variation is established. Estimates of the prevalence of autosomal random monoallelic expression (aRME) vary widely between different experiments and cell types and importantly, the frequencies of fixed and dynamic aRME have not been addressed in the same experiments. I aim to address the balance between fixed and dynamic aRME in an in vivo as well as in vitro model. By combining single cell, allele sensitive transcriptomics approaches, which have been developed in the Sandberg lab, combined with an inducible cell-lineage reporter system, I will be uniquely able to characterize the extent of both fixed and dynamic aRME. We hypothesize that the balance between fixed and dynamic aRME depends on the stabilization of expression on a given allele. According to that model, fixed as well as dynamic aRME is caused by stochastic expression that is then stabilized to different extents. By studying this in vivo we will be able to characterize the stability of aRME in different tissues where aRME stabilization may differ (eg. neurons vs. fibroblasts). The in vitro model on the other hand would give me the possibility to perturb the system and study the mechanisms that establish and stabilize aRME. Additionally, the data resulting from the experiments I propose will also provide a novel and unique insight into the relationship between gene expression and cell clonality in a wide range of mouse tissues and cell types.

Chemical probes are small molecules that can perturb the activity of a target macromolecule, and thus can be used to understand the biological roles of receptors. Currently, identifying a chemical probe for one specific target requires screening many chemicals to find ones that bind that target. If one screen could be used to find chemical probes for many targets, one could dramatically increase the rate of probe discovery.

Our first goal is to identify novel chemical probes for approximately 40 receptors in S. cerevisiae. This will be done using a novel high throughput metabolomics approach. Recent advances in the Sauer lab allow for the speedy generation of a metabolomic fingerprint of the effect of a compound treatment on the cell. Therefore it should be possible to adopt an innovative approach to chemical screening where compounds are identified that are able to mimic the metabolomic profile of yeast where receptors are genetically perturbed. This will allow us to find chemical probes for those targets. Thus, we will be able to identify many chemical probes for many receptors from a single chemical screen.

Our second goal will be to investigate the roles of these receptors in vivo using our chemical probes. We will investigate what specific metabolomic and transcriptomic changes individual S. cerevisiae nutrient sensors elicit when they activated by a non-metabolizable ligand. This is of interest because it can isolate what the cellular response to receptor activation is, and potentially show why it is adaptive for the cell to respond in that way. Additionally, we will use them to investigate the biological roles of poorly characterized receptors in S. cerevisiae.

The neural circuits underlying vision share a lot of structural and functional similarities between Drosophila and mammals, with motion detection being the best-studied visual modality. The highly stereotyped columnar and layered organization of Drosophila optic lobe neuropils (the visual processing centres) allows a precise study of circuit wiring. Two parallel motion channels have been described in Drosophila, one for detecting moving light edges (ON pathway) and one for detecting moving dark edges (OFF pathway). The main components of these channels have been identified, comprising neurons in the lamina, medulla, lobula and lobula plate neuropils. However, little is known about the specific mechanisms leading to the orderly and stereotypic assembly of these cell types to form functional circuits. In this proposal we offer to study by live imaging the spatiotemporal dynamics of motion detection circuit assembly and the determinants of such formation. It has been proposed that circuits are assembled in the order of neuron generation. To test this hypothesis, we will analyse the assembly order of ON and OFF motion circuit components and study how the developmental specification of cell types is linked to circuit formation. Furthermore, we will investigate whether there are neurons with pioneer roles in circuit assembly, the influence of neuronal soma positioning on axonal and dendritic layer targeting, and the role of spontaneous activity in circuit wiring. Altogether, this project will advance our understanding of the mechanisms governing functional circuitry assembly, which is likely to have broader implications for other neuronal systems.

Autophagy is a ubiquitous process of eukaryotic cell biology, which occurs by the formation and growth of the isolation membrane (IM). The IM engulfs bulk cytosol or targets such as protein aggregates, damaged organelles, and invading pathogens. The IM closes to become the autophagosome, fuses with the lysosome, and its contents are degraded. More than 40 Atg proteins are dedicated to autophagy, thus the “parts list” is known. How the autophagosome initiates, grows, and closes, using these parts is a world-class mystery. We propose to use biochemical reconstitution, cell imaging, and computational biophysics to dissect the uptake of the intracellular pathogen Salmonella (“xenophagy”) and so reveal the physical basis of autophagosome formation. In one major model, derived from yeast data, the IM nucleates from several small vesicles that then fuse into a sheet. The sheet grows by fusion of more small vesicles. In another model, derived from mammalian cell data, the IM is extruded out of a domain of the endoplasmic reticulum (ER). We propose to resolve which of these mechanisms is operative, by reconstituting autophagosome formation, and by directly imaging the process both in vitro and in cells using real-time super-resolution imaging. We will then move beyond distinguishing between these qualitative models, to a quantitative, biophysical paradigm. The arrival, departure, copy numbers, and structures of the relevant protein complexes on and off membranes will be determined in vitro and in vivo. Changes in membrane shape will be correlated with these parameters. These will be input to a computational model accounting for membrane physical properties and protein structures. The complexity of this system, with 10s-100s of copies of more than 40 different proteins, operating on a time scale of minutes and a length scale of two microns, makes this one of the most complex systems to be tackled with such biochemical and biophysical detail.

This project will investigate locomotion control in undulatory animals and transitions between different environmental media. It will test the idea that a single control principle could explain different modes of locomotion in vertebrate and invertebrate animals for different morphologies in different environmental media (water and ground). The control principle is based on “sensory synchronization” of local neural oscillators. It merges ideas from central pattern generators and from sensory-driven locomotor models. We postulate that flexible motor patterns for body-limb coordination during swimming, crawling and walking can be obtained in a distributed network of neural oscillators that rely to a large extent on (multimodal) sensory feedback signals for synchronization. In other words, we postulate that the synchronization between oscillations in body and limbs, and hence the generation of gaits, is due to mechanical interactions of the body with the environment and the resulting sensory signals more than to direct neuronal (or central) coupling between the involved neuronal oscillators. We also postulate that many observed features of various gaits are due to changes in the environment rather than to changes in the descending commands.
This idea and related predictions will be tested by investigating Polypterus (a walking fish), salamander, and centipede locomotion. These three species represent a diversity of life that has overcome the problems associated with neuro-control in amphibious environments. Our research tools combine animal locomotion studies, neuromechanical simulations, and robotic experiments. The neuromechanical simulations and the robots will play an essential role in decoding the complex interplay between central pattern generators, sensory feedback, the musculoskeletal system, and the different environments. The interdisciplinary approach will allow us to investigate whether the same control principle could not only facilitate adaptations to growth, lesions, perturbations, and different environments during the lifetime of the animal, but also to evolutionary adaptations (e.g. changes of ecological niches and of morphologies) in particular the transition from aquatic to terrestrial environments in vertebrates and invertebrates, a key step in evolution.

In photosynthesis, light-harvesting complexes (LHCs) capture solar energy and feed it to the downstream molecular machinery. However, when light absorption exceeds the capacity for utilization, the excess energy can cause damage. Thus, LHCs have evolved a feedback loop that triggers photoprotective energy dissipation. The critical importance of photoprotection for plant fitness has been demonstrated, as well as its impact on crop yields. However, the mechanisms of photoprotection ? from fast chemical reactions of molecules to slow conformational changes of proteins ? have not yet been resolved. Despite extensive studies of the native photosynthetic apparatus, previous experiments have not been able to control architecture in order to distinguish between hypotheses of the mechanisms of photoprotection. To overcome this barrier, we take a novel synthetic structural biology approach. We build minimal regulatory units using model membranes that we measure and model to gain a multi-timescale understanding of photoprotection, which is core to natural light harvesting. Taking this synthetic structural biology approach is a significant experimental challenge in three distinct fields; biochemical and molecular biology development is required to construct a well-defined and well-characterized sample, new spectroscopic analyses and collection strategies are required to adapt optical experiments to probe these samples, and theoretical advances are required to model these length scales.
Uniquely, photoprotection regulates a photophysical process, providing an intrinsic spectroscopic handle to uncover how information flows within the multi-timescale phenomena of biological regulation. LHCs and other biological systems perform meso- and macroscopic functions efficiently and robustly even when exposed to the changing and fluctuating environment that is a hallmark of natural systems. Furthermore, biological systems adapt and even reprogram their function in response to the environment. Unveiling the design and working principles of such responsive and highly autonomous behaviors exhibited by molecular systems is one of today’s grand challenge areas from the standpoint of both theoretical and experimental research; we incisively explore such behaviors in the context of photosynthetic light harvesting through the work proposed here.

Mitochondria are organelles that perform fundamental functions in eukaryotic cells. They are responsible of cellular respiration, energy generation and an array of biosynthetic processes. Additionally, they play a key regulatory role in programmed cell death. In medicine they are widely studied, as mitochondrial dysfunction/disease have enormous health consequences, due to their involvement in aging and in a number of pathologies including, cancer, metabolic disorders and heart disease.
Despite their importance, and the effort of numerous researches, multiple aspects of mitochondrial biology are still unclear. In this project we propose to exploit a unique system to develop novel tools to study and to interact with mitochondria, with potential groundbreaking applications. Midichloria mitochondrii is an intracellular bacterium that presents a characteristic that makes it unique: it is able to enter the mitochondria of its host cells. This unique interaction has been discovered recently, and has still widely uncharacterized. Our multidisciplinary team envisioned an integrated experimental approach, using state of the art technologies in the fields of bioinformatics, -omics, imaging, biochemistry, and cellular biology to investigate this novel biological system.
This research project will allow us to understand the evolution and the functional details of the relationship between M. mitochondrii and its host at the organismal, cellular and molecular level. We will use this novel information to develop an in vitro model system that will allow us to use this unique bacterium to interact with mitochondria in novel ways. This approach will ultimately lead to the development of new experimental tools to characterize mitochondrial mechanisms, including mitochondrial fusion/fission, inner and outer membrane repair, metabolism and apoptotic signaling.

Demography (i.e. a population’s history of size changes, gene flow, and divergence events) affects patterns of genetic diversity, and has implications for studying evolution, adaptations, and disease. However, demographic inference is complicated by “ghost populations,” unobserved populations in the past that admixed with the ancestors of sampled populations. Inferring this hidden structure is challenging both because the number of ghost populations is unknown, and also because signals of cryptic structure may be distorted by other evolutionary forces. I will address this problem by developing a flexible method capable of estimating unknown population structure jointly with other sources of genetic diversity. Our method will combine models from Coalescent theory with machine learning techniques for inferring graph structure, and will estimate the number of ghost populations, their demographic history, and the distribution of fitness effects for mutations arising in these ghost populations. Furthermore, our approach will allow us to estimate allele frequencies in ancestral populations, which will be useful for identifying adaptive introgressed regions from ghost populations, and for addressing questions such as the adaptive consequences of gene flow from ghost populations. We will apply our new method to diverse human whole-genomes to shed light on open questions in recent human evolutionary history, such as the relationship between modern African populations and the out-of-Africa population that gave rise to modern Europeans and Asians, or the extent to which variants selected in particular populations have spread more broadly by admixture and introgression.

Propagation of a-synuclein in the brain is the hallmark feature in the pathogenesis of Parkinson’s disease (PD). Patients with PD exhibit marked differences in the rate of a-synuclein propagation, but the reasons for this variability are unknown. I hypothesize that the rate of a-synuclein propagation varies because it is affected by various genetic modifiers. In addition, I hypothesize that individuals with PD are mosaics for a-synuclein mutations in the brain, a type of genetic variation that is missed by standard screening methods, which further impacts on a-synuclein propagation. In order to investigate these hypotheses, I am going to generate human iPSc-derived neuronal cultures expressing a construct in which mutant a-synuclein that is fused with mCherry on its C-terminus is also fused to a GFP tag on its N-terminus through a 2A self-cleaving peptide. Neuronal cultures expressing wild type (WT) a-synuclein fused with BFP on its C-terminus will also be generated. I am then going to mix these neuronal cultures to generate cultures of various mosaic percentages for the a-synuclein mutation. This system will enable the identification of single neurons that have uptaken WT or mutant a-synuclein through propagation based on their positivity to GFP, mCherry and/or BFP. I am then going to use a CRISPR-Cas9 high throughput screening system combined with high content imaging techniques developed at the host institution to assess the effect of thousands of genetic factors on the rate of propagation in the mosaic neuronal cultures, followed by RNA sequencing to determine molecular signatures that underlie the propagation process and that are specific for each genetic modifier.

Monitoring membrane potential is a necessary step in understanding neuronal activity. Invasive methods cannot provide the spatial resolution and coverage required for such information. On the other hand, optical methods that provide high spatio-temporal information demand optical sensors with large signal amplitude, photostability, live-cell compatibility, and fast response to voltage variations. Existing optical voltage sensors do not match all these requirements. Recently, semiconductor nanorods (NRs) with asymmetric structures have been shown to exhibit prominent voltage sensing capabilities. Their high luminescence and photostability will also allow for nanoscale resolution and reduction in photodamage of live cells. Therefore, here I aim to explore ways for functionalizing these particles with peptides of DNA origami for membrane insertion. I will evaluate the insertion mechanisms by measuring their orientations and accurate positions with respect to the lipid bilayer using single-molecule Metal-Induced Energy Transfer (smMIET) imaging. After stable insertion, I aim at characterizing their voltage sensing properties and performance with fast optical imaging techniques. In the end, I will explore their application in neuronal cultures by measuring action potentials and subthreshold events in individual cells and across the network. Membrane potentials in subcellular sections such as dendrites and spines will be monitored with high spatial resolution as a function of their morphology and structural dynamics, which has not been achieved to this date.

My project aims to identify neuronal circuits that are spared with severe spinal cord injury and function as motor command relay pathways during reactivation of the lumbar spinal cord. I will use a neuroprosthetic platform that supports bipedal stepping and implant an electro-chemotrode over the lumbar spinal cord of mice. First, I will optogenetically activate motor cortex projections in severely contused mice and assess whether immediate will-powered motor control is regained over completely paralysed hindlimbs during spinal stimulations (Aim 1). Next, I will use a combination of conventional tracers, such as Fast blue, and viral tracers that are regulated by a Cre promoter in vGluT2 Cre or 5-HT Cre transgenic mice to label and identify brainstem projections to the lumbar spinal cord. Moreover, I will prove these brainstem projections are required to rely motor cortex commands by chemogenetically silencing them with the DREADD technology, which should abolish any leg motor function (Aim 2). Mice will undergo 8 weeks of actively engaging neurorehabilitation with spinal electrochemical stimulation. I will assess recovery of will-powered voluntary motor function and remodeling of spared pathways (Aim 3). I will laser-microdissect cell bodies of motor command relay neurons in the brainstem and assess the transcriptional profile with RNA sequencing to identify which molecular cues are involved in circuit reorganization (Aim 4). Taken together, these results will establish the causal relationship between circuit reorganisation of residual descending projections and voluntary motor control with electrochemical neurorehabilitation in severe spinal contusion injuries.

Cell division and differentiation are strictly coordinated during development. Although it is known that differences in S-phase length are essential for embryogenesis our understanding of its causes and consequences is extremely poor. The second cell division of C. elegans is stringently asynchronous and is well-suited to study developmental control of replication regulation in a multi-cellular organism.
Studying replication initiation is limited by the availability of specific assays. I will develop a novel live assay to visualise the recruitment of replication factors towards origins in single cells within the C. elegans embryo. With the LacO/LacI system I will generate functional origins and measure the binding of fluorescently tagged replication factors by confocal imaging dissecting consecutive steps of DNA replication. This assay will permit to follow the dynamics of replication factors in real-time and to reveal changes in S-phase control during development. On this basis, I will perform an RNAi-based high-throughput screen for novel regulators of genome duplication in the C. elegans embryo. This unique screen implemented in a whole organism will reveal novel interactions between DNA replication and developmental signalling pathways. In a complementary approach I will generate mutants bypassing the normal cell cycle regulation in the embryo. I will examine potential changes in cell fate adoption through transcriptional analysis and will measure differences in their sites of replication. This approach will clarify if replication control plays a direct role in differentiation and illustrate a physiological role for S-phase differences in development.

It has been postulated that cancer stem cells (CSC), an identifiable subpopulation of cancer cell that can differentiate and repopulate cancer, exist. In this context, cancer cells follow strict hierarchy with CSC being at the apex of it. However, depending on the subtype and malignancy of cancer, cell fate plasticity had been observed where non-CSC dedifferentiates to acquire CSC function. However, the nature and regulation of CSC formation and plasticity has been difficult to understand in vivo. For example, what kind of niche signaling is responsible for cancer cell plasticity? What is the epigenetic or transcriptomic changes in cancer cell plasticity? In this project, I aim to address these questions in vivo using highly novel technique involving transgenic mice carrying recombinase-based state machine (RSM), which enables each individual cell within cancer to keep a memory of its history and sequence of changes in cell fate. The proposal will involve generation and validation of mammalian RSM that can label each cell with different cell fate history (e.g. original CSC and non-CSC that had dedifferentiated to CSC) with different fluorescent proteins. I will then cross the transgenic mice harboring RSM with mouse models of breast cancer and colon cancer can accurately identify heterogeneity in individual cancer cell fate in vivo and in situ. By examining the relative locations of cancer cell fate plasticity in intact tumor mass, and FACS sorting of cells with different fate history for transcriptomic or epigenetic analysis, I aim to dissect the extracellular factors and intracellular signaling events that govern cancer cell plasticity in vivo.

Spatial organization of chromatin and related genes is of key importance for genome expression and maintenance. Unlike stem cell differentiation, for T-cell activation gene regulation happens on a much faster timescale as the immune response is critical for the bodies defense mechanism. The chromatins’ three-dimensional organization as well as its interaction with nuclear lamina and the consequential impact on gene regulation is poorly understood. This proposal will in its first step focus on how mechanical forces can directly influence cellular functions such as the T-cell activation and furthermore elucidate how they ultimately change gene expression. We hypothesize that mechanical force triggers structural/conformational changes of the nuclear lamina network; hence genes encoded in chromatin proximal to lamina can be up/downregulated. Here, we propose to develop 3D and 4D Super Resolution Microscopy (SRM) technologies to enable us to determine the 3D structure of lamina associated chromatin domains, resolve how such domains are organized in 3D during the fast process of T-cell activation. We specifically aim to:
1. Establish imaging methods to measure tension and the dynamic structural re-organization of cytoskeleton, nuclear envelope and chromatin during T-cell activation.
2. Study the effect of perturbations of the mechanobiological pathway that links external cues to activation.

3. Link these perturbations to gene expression and spatial location of genetic loci and transcripts.
This project requires synergistic interplay between the two team members, which brings together early career researchers in T-cell biology, single molecule biophysics, structural biology and SRM.