Growing evidence on the indispensability of microbiome in human-health suggests human development is intimately linked with microbial-symbiosis. However, when and where human-body encounters its first microbial-species remains elusive. The task of differentiating between the commensal and pathogenic-microbes lies with the immune system and recent reports show that its developmental programming starts quite early during fetal-growth. Advances in the field made the picture more complex, evincing early embryonic origin of tissue-specific immune cells, that are independent of bone marrow. We hypothesize that during fetal-development, selective microbial species may cross the placental-barrier and colonize fetal-tissues where they interact with the tissue-resident immune cells, selectively priming them for future functionality/tonicity. This project will identify the fetal microbial-colonizers in different organs, their spatiotemporal dynamics and crucial interactions with the developing immune-system, in human fetuses. Notably, we will also investigate the fetal brain microbiome and gut-brain axis to capture the role of microbes in defense mechanisms in brain. We will employ single-cell RNA-seq and microbial-genomics to identify unique fetal-determinants that condition the immune-system for establishing tissue-specific microbiome. This could be a key determinant in human immune-system development, with life-long implications for immune-homeostasis, memory and microbial-symbiosis. These investigations will be transformative for research in fetal development, host-pathogen interactions, and immune-modulatory interventions in various aspects of human health and disease.

Colonization of land by plants about 450 million years ago was one of the major evolutionary breakthroughs of the history of Earth. During terrestrialization, plants developed sensory systems to detect friends and foes in their new environment. In flowering plants, emerging evidence suggests that the subfamily XII of leucine-rich repeat receptor kinases (LRR-RKs) specialized in the innate immune perception of bacteria. Notably, this subfamily is not present in algae, and likely appeared in the common ancestor of all land plants. The liverwort Marchantia polymorpha has recently emerged as an ideal model plant for evolutionary studies. In this project, I will characterize the Marchantia LRR-RK XII subfamily through a combination of genetics, biochemistry and proteomics. In addition, I will use Marchantia as a tool to assess the impact of innate immune receptors on microbiome dynamics. Microbiome profiling of Swiss Marchantia accessions will be employed to design synthetic bacterial communities (SynCom). Inoculation of immunity-related Marchantia mutants with SynCom followed by phenotypic characterization and microbiome profiling will demonstrate how host immunity modulates bacterial populations. I will thus unveil the molecular basis of innate immunity against bacteria in early-divergent land plants and provide insights into the evolution of plant immunity, and more generally in RK signalling in land plants. The conclusions of this work will ultimately expand our knowledge on the evolution of plant innate immunity as well as on the interplay between plants and their microbiomes.

Humans spend approximately one third of their lives asleep, but the genetic and neural circuits underlying this state are poorly understood. Sleep and wake are two disparate physiological and neural states. Therefore, arousing- and sleep-promoting circuits must interact to ensure that the correct state is efficiently established and maintained. To better understand sleep and arousal regulation in vertebrates, I will use two approaches to identify circuits and genes underlying these processes in zebrafish.

Aim 1: Restless leg syndrome (RLS) is a sensorimotor disorder that causes leg movements especially during periods of rest or sleep and is a leading cause of insomnia. Genes linked to RLS provide an entry point into the circuits that promote arousal and disrupt sleep. Zebrafish homolog knockouts of the RLS-linked genes Meis1 and Skor1 induce hyperactivity and sleep fragmentation. I will identify what circuit changes underlie these phenotypes.

Aim 2: I will develop a novel and non-invasive neuronal activity recording system to identify sleep- or wake-active cells. This system will convert neuronal activity into patterns of CRISPR/Cas9-mediated editing of a DNA barcode during inducible time windows. I will combine single-cell RNA-sequencing and activity recording to identify neurons that regulate sleep and wake.

Together, these aims will identify sleep and arousal circuits and provide the foundation for my long-term goal of understanding how sleep and wake states are generated and maintained.

The quantum leap in achievable resolution using cryo electron tomography (cryo-ET) in recent years has led to a stunning advancement of the field of structural biology. The precise knowledge of the structure enables the inference of the function of whole protein complexes within large cellular machinery.
One example of such a large cellular machinery is the cilium. These elongated flexible organelles extend from the cell body of most eukaryotic cells and serve a stunning plethora of functions within the cell, from actuation, mechanosensing to signaling.
Defects of these organelles are at the root of a whole class of devastating diseases, known as Ciliopathies, including Karthagener Syndrome, certain kidney diseases or retinitis pigmentosa.
The assembly and maintenance of the cilium requires constant transport of ciliary building blocks. As there is no protein synthesis in the cilium all these components need to be transported from the cell body to the tip of the cilium. As diffusive transport in this extremely high aspect ratio, crowded and confined structure is limited, mass transport along the cilium relies on active transport, known as intraflagellar transport (IFT). Despite the fact that the genetics and biochemistry of IFT have been extensively studied, the molecular mechanisms of assembly, cargo association, dissociation and anterograde-retrograde conversion at the tip of the cilium are largely unknown.
We aim to decipher the complex inner workings of IFT machinery with a combination of correlated cryo-EM, light microscopy and high-speed atomic force microscopy. Together, the three microscopy techniques span the whole spectrum from resolution dynamic function.

Islands present great natural laboratories, and are considered as “simpler” habitats. Due to their relatively low numbers of species and limited areas, we can study evolutionary hypotheses in a somewhat controlled framework. Studying adaptation and speciation process in island species helps us understand how species adapt to novel environments. Island trait evolution has been studied at local and macroecological scales, yet the genetic basis for this variation is less understood. Combining phenotypic study with cutting-edge genomic analyses would enable shed light on the fundamental mechanisms driving evolution. A long-term evolutionary study on the Italian wall lizard (Podarcis siculus), on the islands of Pod Kopiste and Pod Mrcaru gives us a great opportunity to explore the role of genetic variation in species adaptation. Historical introduction of this lizard to Pod Mrcaru has resulted in rapid morphological changes, notably divergent from the origin population on Pod Kopista. This experimental framework is excellent for identifying the genetic components responsible for the witnessed morphological adaptations. I will contrast genomic samples of P. siculus from the two islands and then compare them to samples from the mainland. I will apply Next Generation Sequencing (NGS) technology to sequence whole-genomes of the lizard populations, and use it for genome-wide association study (GWAS) to identify the regions correlated with the morphological adaptation. I will use bioinformatical methods to characterize the genome participation in the adaptation process. This novel approach to studying island adaptation provides a baseline for more genomic island evolution studies.

A large part of an animal’s behaviour involves responding appropriately to stimuli in the environment. However, responses are highly dependent on internal states (a slight mishap on a good day can be a catastrophe on a bad day). This means that neuronal computations for action selection take environmental stimuli as inputs, as well as an animal’s homeostatic and affective internal states. Furthermore, it is clear that action selection also has an effect on the internal state of an animal: feeding leads to satiety, or finding shelter can reduce anxiety. I seek to understand how neuronal activity mediates the bidirectional interplay between action selection and internal state.

To this end, I propose to combine the quantification of home-cage behaviors in freely-moving mice together with deep brain calcium imaging to investigate the neuronal dynamics underlying state-dependent action selection. I will focus on the central amygdala, given its privileged position as a highly interconnected node in a network that integrates external and interoceptive stimuli, and that orchestrates motor and physiological responses.

In their natural environment and during infections, bacteria are commonly organized in surface-attached communities termed biofilms, which are held together by a self-produced extracellular matrix. These biofilms can develop from single cells into macroscopic three-dimensional communities with characteristic morphology and cellular differentiation, reminiscent of eukaryotic multicellular development. Recently, single-cell level live-imaging of complete biofilm development has become possible, which now permits the experimental testing of detailed simulation predictions for biofilm development, and places the grand challenge of a quantitative and predictive understanding of biofilm development within reach. The key ingredients of the required simulations are the cell-cell interaction mechanisms and behavioral states of cells, yet both are generally unknown. To overcome this barrier, I will develop techniques for spatiotemporal transcriptome data generation and analysis during Vibrio cholerae biofilm development, which will allow me to obtain spatiotemporal maps of cellular interaction mechanisms and cellular states. These spatiotemporal maps will then be used as input for individual-based simulations I will develop, to identify which of the vast possibilities of cellular interactions and properties are necessary and sufficient for biofilm development. This interplay of experiments and simulations based on spatiotemporal transcriptome data and single-cell microscopy will ultimately not only identify the key cellular interaction mechanisms, but also the cellular interaction principles that are required irrespective of the underlying molecular mechanisms.

The act of making a decision is a complex cognitive process, where the brain integrates new information with an internal representation of the world to select an anticipated outcome among several choices; usually based on reward expectations. During decision-making several cortical and subcortical specialized networks within the cortico-striatal-thalamic loop route and integrate information to produce a choice. But how do our brains learn to make decisions in a novel environment? How do these networks evolve based on reward expectations and decision-making outcomes? It is still unclear how signals that encode reward expectations are routed through specialized networks, and whether these networks can directly influence each other through what is known as transfer learning. Here, under the supervision of Dr. A. Benucci, I propose an integrative theoretical-modeling approach, based on Recurrent Neural Networks (RNN), in close-loop with experimental work at the host lab, to reveal the neural underpinning of learning and decision-making in specialized networks within the cortico-striatal-thalamic loop. We will implement biologically-feasible RNN, directly trained using large-scale population dynamics experimental data from mice performing vision-based decision-making tasks. By reverse engineering the RNN computations we will implement causal, experimentally dynamical predictions, testable via optogenetic perturbative experiments, to reveal the neural basis of learning and decision-making. Only by considering the collective orchestration and interaction of these circuits will it be possible to highlight how the brain learns to make decisions.

Ion channels are important for all living organisms due their involvement in several essential processes such as synaptic transmission, muscle contraction and cell signalling. Better understanding of how ion-channels function will aid in the development of new drugs against pathologies related to those processes. Ion-channels are embedded into to the cell membrane, making them difficult to study using conventional structural biology approaches. Solid state NMR offers the possibility to investigate membrane proteins in a native-like environment under physiological buffer conditions and at room temperature. NaK is a bacterial non-selective ion-channel that conducts both sodium and potassium ions. Due to its similarity to human cyclic nucleotide-gated ion channels, NaK has become an important model system for non-selective ion channels. We will use proton detected solid state NMR to investigate the mechanisms of non-selectivity, gating and ion permeation in NaK. Better understanding of the dynamics involved in these processes is essential to understand how non-selective ion-channels function. Gating is expected to depend on protein-membrane interactions, but the mechanism is unknown. There are currently different models for how ions permeate through the selectivity filter. Solid state NMR can detect very subtle changes in local environments which will allow us to characterize the ion permeation process and investigate how lipids in the membrane and water molecules interact with the protein.

A major goal of regenerative research is to restore human tissues, such as limbs, following injury. Salamanders, including the axolotl (Ambystoma mexicanum), are the only tetrapods able to regenerate fully patterned limbs. Amputation induces fibroblasts to migrate from the stump to the wound site, where they de-differentiate, form a morphologically unpatterned blastema, proliferate and eventually regenerate a patterned limb. The mechanisms enabling pattern restoration are not clear.

Grafting experiments have shown that fibroblasts in anterior, posterior, dorsal and ventral parts of the steady state axolotl limb exhibit different properties, which are maintained even after transplantation. This indicates that fibroblasts harbour stable positional identities (knowledge of location in the body). However, the purpose of these identities and underlying mechanisms are not known. I hypothesise that positional identities are determination states that instruct fibroblasts on where, and when, they should express patterning genes after amputation. This would enable correct pattern restoration during regeneration.

This proposal addresses the transcriptional and epigenetic bases for positional identity. I will test the role of positional identity in limb regeneration using CRISPR/Cas9-mediated transgenesis. I will establish new genetic tools to manipulate positionally distinct fibroblasts, and test their contributions to regeneration. In sum, this work will shed light on how new pattern is formed from existing pattern during regeneration. As human fibroblasts also harbour positional identity, this work may provide insights into re-patterning potential in human cells.

Cancers arise in cell types that turnover by proliferation and death at highest rates, likely because these rates become unbalanced. Our group found that mechanical forces control both processes in epithelia: stretch activates proliferation whereas crowding activates cell extrusion and death. However, it was unclear what causes crowding and stretching forces in epithelia. I will investigate if cell migration from sites of proliferation drives the conveyor belt forces that control stretch-induced cell division and crowding-induced death. If so, the rate of cell migration could drive the rate of cell turnover and, hence, the propensity for a tissue to become cancerous. While my host lab has already identified roles for the stretch-activated ion channel Piezo1 in controlling proliferation and extrusion, based on preliminary compelling findings, I will determine if it also controls cell migration from sites of division. To do so, I will use established models in cell culture and mouse gut and develop an in vivo zebrafish gut model for homeostatic epithelial cell turnover. Additionally, I will test if frequent Piezo1 mutations in colon cancer impact cell proliferation, migration, and death. If my hypothesis is correct, I will reveal a new, unexpected role for cell migration in not only normal epithelial cell turnover but also in carcinogenesis. Should Piezo1 act as a central transducer of mechano-chemical coupling, it could provide a new target for therapeutics.

How our genomes are activated during the early stages of embryonic development has been a long-standing unsolved mystery for decades. Zygotic genome activation (ZGA) has been the focus of extensive studies, however, no clear understanding of the overall regulatory mechanism has been achieved.
This is mostly due to the maternal deposition of inactive regulatory proteins in the egg, and on the lack of a technique able to distinguish active from inactive proteins. Thermal profile proteomics (TPP) is a recently developed technique that addresses this need, using heat to build a denaturation – or melting – profile of a protein, and mass spectrometry detection to expand the throughput proteome-wide. Changes in the biochemical status of proteins – such as association into complexes or post-translational modifications – change protein resistance to denaturation, inducing a shift in the melting profile. In vivo TPP will identify proteins whose melting profile shifts upon ZGA, qualifying them as potential regulators. This will also provide a technological advancement to TPP, paving the way for applications that will greatly benefit from in vivo settings, such as drug screening.
Candidate ZGA regulators will then be functionally validated through the generation of germline-specific mutants, lacking both maternal and zygotic expression of the candidate proteins. The effect on ZGA will be tested by GRO-seq to identify nascent transcripts, by ChIPseq for RNA polymerase II to distinguish the effect on recruitment and activity of the transcription machinery, and by ATAC-seq to identify difference in chromatin accessibility and pioneering activity of the protein in study.

Plants tightly adapt their architecture to the environment. However, they also have to balance energy and resource expenses for plastic growth with those required for other processes that are also relevant for fitness. Even though plasticity has been extensively studied, how organ function interplays with its plasticity remains an important question to elucidate. The root system is plastic since it produces multiple architectures from a single genotype, depending on environmental conditions. However, the resulting architecture must act as an efficient transport network to achieve its function. Root growth and transport are costly and concurrently decrease the transport performance, thus root system architecture (RSA) must integrate these two competing objectives: cost and performance. Recently, the shoot system architecture has been shown to resolve this tension by finding an optimize tradeoff between cost and performance. Here, I challenge the idea that RSA plasticity relies on this design principal. To this end, I will set up a multi-disciplinary approach that integrates, phenomics, modeling, genetics, cellular and molecular biology. I will build a comprehensive phenotypic dataset of RSA for more than 300 Arabidopsis thaliana ecotypes grown in 10 different nutrient conditions. Using modeling, I will analyze the optimal cost-performance tradeoff according to the RSA and based on these data, I will perform GWAS to identify cost-performance and plasticity-related variants and genes to decipher how cost-performance tradeoff relates to plasticity. Taken together, this project will decipher the developmental and molecular framework which control the root system plasticity.

Breast cancer is one of the most common cancers worldwide. It is also one of the most heterogenous ones, both in the genetic background of susceptible individuals as well as between the developing tumors. While studied extensively, molecular mechanisms leading to breast cancer progression and the interplay between the immune and cancer cells remain elusive. While Immunotherapy protocols revolutionized cancer treatment, those are still lacking in breast cancer. Thus, there is a need understand early events leading to breast cancer progression and illuminate the curtail cancer-immune interactions during this process. I will use seqFISH methodology developed in the lab of Dr. Cai, which allows investigation of hundreds of mRNA transcripts on single cells within intact tissues. I will combine seqFISH with multiplex antibody staining, to study changes in transcription and in protein expression and localization. I will use this approach to study cancerous and benign biopsies, as well as serial breast aspirates extracted from patients at high risk to develop cancer. These samples will provide comparison between individuals as well as within the same individual over time. This methodology will allow me to (1) gain a comprehensive spatial characterization of the tumors at different stages and identify networks of interacting cells; (2) identify changes in the composition of the tissue which predict aggressive cancer progression and (3) illuminate important cancer-immune interactions. These measurements will provide valuable insight on signaling events and cellular populations predicting breast cancer progression and reveal the functional role of immune cells within breast tumors.

Robust T cell migration is essential in their search for foreign antigen within lymphoid organs. Importantly, the movement of rare antigen-specific T cells occurs in an environment densely packed with other T cells. Moreover, lymphoid tissue is under constant flux, with large numbers of T cells both entering and leaving at any given time. From studies of human crowd behavior, we know that the movement of elements in crowds can give rise to unpredictable behaviors affecting both the dynamics of individual elements, as well as population level phenomena (e.g. leading to dangerous crushes at exits). Static images of lymphoid tissue display T cells jammed together with no apparent room to maneuver, yet microscopy studies of T cell trafficking have largely followed only individual labelled cells - giving the impression that they roam freely in space. Here, we will investigate how T cell crowding alters their motility and direction choice in order to begin to understand how specific defects in T cell migration may alter their function in unexpected ways. We will use specifically designed microfabricated channels mimicking key aspects of lymphoid structures, imposing bottlenecks or barriers, to compare individual T cell direction choice and velocity with that of cells embedded in a crowd. Overall, we will bridge a fundamental knowledge gap in our understanding of how T cells operate effectively in vivo without impeding each other, and establish critical tools to investigate T cell-intrinsic and extrinsic structural features of lymphoid tissue which facilitate the flow of T cell crowds.

Biological molecular machines such as rotational or translational motor proteins are intriguing systems that can interconvert mechanical and chemical energy. The translational motors myosin, kinesin, and dynein have been studied extensively and in great detail. However, fundamental principles are difficult to derive due to the complexity and diversity of these systems. I propose to re-engineer cytoskeletal translational motors from de novo designed proteins to achieve a better understanding of the principles behind directed motion in natural systems. De novo designed filaments will serve as tracks, and the motors will be constructed based on a minimal model for directed movement that involves a conformational equilibrium and a binding equilibrium that are connected via a fuelling reaction.
While the Rosetta software has enabled the de novo design of a variety of stable rigid proteins, designing dynamic proteins that can switch between multiple conformations remains challenging. I will combine existing multistate-design approaches with repetitive positioning of hydrogen-bond networks to design proteins that can switch between multiple well-defined, rigid conformations. To enable motor binding to the track, I plan to screen previously designed as well as novel heterodimers for increased solubility and reduced promiscuity of the corresponding monomers. I will use a fueling reaction that is coupled to both conformational switching and track binding to construct first a monomeric, nonprocessive motor and then a dimeric, processive motor. The proposed project will shed light on the fundamental mechanisms of motor proteins and advance computational design of dynamic proteins.

Accurate telomere replication is essential for genome maintenance and cell proliferative potential. However, telomere structure challenges the progression of replication forks, increasing the chance of fork stalling within the telomere. Previous work has focused on understanding how Shelterin promotes recruitment of enzymatic activities to telomeres that prevent replication stress by dissolving secondary structures. However, little is known about how cells respond to replication stress within the telomere once it has already been generated and replication forks have irreversibly stalled. I propose that dormant telomeric origins are essential for ensuring telomere maintenance upon induction of replication stress, by rescuing telomere replication in the vicinity of collapsed replication forks. To directly address the problem, I will first develop experimental approaches to specifically inhibit activation of telomeric origins in different cells lines. I will then characterize these cells and their telomere phenotypes and compare their behavior in unchallenged and replication stress induced conditions. If my hypothesis is correct, I would observe defects in cell proliferative potential and telomere dysfunction phenotypes upon induction of replication stress when telomeric origins are compromised. Since cancer cells are known to experience high levels of replication stress, tumor cells might depend more on dormant telomeric origins for telomere maintenance and survival. I will therefore study the effect of inhibiting telomeric origins on cell proliferation in cancer cells and test potential synergistic effects with replication stress inducing drugs.

Phylogenomic data suggests that eukaryotes arose from the symbiosis of an archaeal cell with a bacterial partner. However, this does little to explain the origins of eukaryotic cell organization, since the closest living relatives of these two partners that can be studied at the cellular level (TACK family archaea and alpha-proteobacteria) lack internal membranes. Still, TACK archaea generate exosomes, whose scission is thought to depend on ESCRTIII, as it does in eukaryotes. Thus, there appear to be striking parallels between archaeal and eukaryote trafficking, which I aim to explore during my postdoctoral fellowship. I will aim to answer the following questions: How exactly are archaeal exosomes formed and shed? How is this regulated in space and time? How does cytoplasmic and membrane cargo loading occur? In the proposed project, we will use Sulfolobus acidodaldarius to study membrane trafficking in archaea. To investigate cargo loading we will initially use Sulfolobicin as a model cargo. We will also investigate the functions of proteins found in isolated Sulfolobus exosomes with structural similarities with eukaryotic proteins involved in cargo loading (e.g. flotilins) as well as ESCRTIII proteins homologues (CdvB1, CdvB2 and CdvB3). We will follow cargo loading and vesicle formation by immunofluorescence, live cell microscopy (using a special microscope), electron microscopy and CryoEM, to observe protein complexes formed at the neck of vesicles. This work will not only help to elucidate the cell biology of archaea, but could also have a wider impact across biology by shedding light on mechanisms and evolutionary origins of key aspects of eukaryotic cell biology.

How do organs attain and maintain their size and complexity during development, repair and regeneration? This question is one of the last biological frontiers, and thus most of the basic mechanisms involved are yet to be elucidated. I started to address this topic during my postdoctoral studies. I uncovered that cells use both internal and external information to coordinate aspects of this process, but the molecules involved remain mostly unknown. The goal of this proposal is to identify and characterise the mechanisms involved in cell communication within and between tissues during the regulation of organ growth. I hypothesise that the compensation of developmental insults is a whole-organ response that emerges from the local interaction between injured and spared cells, within the overall control from the surrounding tissues. To test this, my lab will study catch-up growth: the recovery of normal growth after a transient perturbation during development. The long bones are especially amenable to explore this response, which we will study in transgenic mouse and quail models where developmental insults can be transiently triggered with very precise control of space and time. For the analysis, we will focus on the cell interactions and molecules involved, both within the growing bone and between the bone and other tissues. In summary, part of the difficulty of studying organ growth is that extrinsic and intrinsic mechanisms may have opposite effects. By dissecting how the internal and external regulators of organ growth operate and interact, the outlined experiments will provide a new framework to studying and eventually understanding this centuries-old biological question

Basic knowledge is lacking to understand immunological tipping points, where the same stimulus can cause paradoxical immune responses. For example, immune activation after injury or infection is required for downstream tissue repair and pathogen removal, but may also cause tissue damage; immune tolerance or rejection may occur after similar type of tissue transplant, but it is difficult to predict the outcome a priori; inflammation is needed to form blood vessels connecting mother and embryo upon implantation, but too much inflammation can lead to immune disorders during pregnancy. In contrast to these critical clinical complications caused by immune overactivation, cancer cells can bias the tipping point towards the opposite direction to suppress the immune response, but the underlying mechanism still remains unclear. Driven by a series of recent breakthroughs made in our laboratories, we propose to establish planarian flatworms as a novel ideal model to delineate these paradoxical effects: with their simple immune system, we will quantify and manipulate immune cell behaviors both directly in live animals and in engineered ex vivo reconstituted cell systems; with their unsurpassed regenerative ability, we will test the response of immune cells to immunological challenges during allogeneic tissue transplantation. Integrating quantitative experiments, genetic manipulation, and mathematical modeling, we will connect gene functions, cell feedback circuits, and organismal phenotypes, to provide for the first time a multi-scale systems-level mechanism by which the immunological tipping point balances the system sensitivity and robustness. This mechanistic and predictive understanding will also allow synthetically rebuilding immune cell circuits ex vivo for bioengineering and therapeutic purposes. Our work will establish a mechanistic footing to understand more complex immune systems, and can help optimize outcomes in cancer immunotherapy, tissue transplantation, autoimmune diseases, and pregnancy disorders. This work is only achievable through a unique international collaboration that bridge several distinct fields: cellular immunology, functional genomics, and quantitative biology.