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.

The generation of human cerebral organoids promises to profoundly change our understanding of human brain development by enabling its detailed study. However, current protocols have several limitations and give rise to organoids characterized by low reproducibility, small size, lack of vascularization, limited neuronal maturation, and reduced cellular diversity. These limit the use of organoids as a model system to study human brain development. Here, I propose to integrate existing 3D culturing protocols with synthetic biology and tissue engineering approaches in order to push the field beyond these limitations and study basic mechanisms of human brain development. In so doing, I expect to both generate cerebral organoids that better resemble the human brain and study basic mechanisms in an “understanding by building” framework. Specifically, I plan to (i) increase neuronal maturity by integrating the brain organoid with a smart functional vasculature system (ii) increase organoid size and cell diversity by modulating the gene expression dynamics of Hes1 (iii) increase organoid reproducibility by building an artificial notochord producing a sonic hedgehog (SHH) gradient in a time regulated fashion in order to pattern brain structures. By integrating live cell imaging, massive single-cell profiling, and electrophysiology, I will be able to study the overall effects of the three specific goals on organoid development.

Mitochondria are a fundamental component of eukaryotic cells that provide ATP through oxidative phosphorylation and play important roles in the regulation of metabolism and cell death. Accurate homeostasis of mitochondria is important for cell function, and misregulation of mitochondria in humans causes a variety of severe diseases. To achieve homeostasis, cells have to ensure that the amount of mitochondria doubles with each cell doubling period. Because mitochondria maintain their own genome encoding for essential mitochondrial proteins and RNAs, this requires a quantitative coordination of mitochondrial DNA replication, production of mitochondrial proteins, and cell growth. However, how cells couple mitochondrial biogenesis with cell growth remains unknown. Here, I propose to use budding yeast as a model organism to identify the molecular mechanism underlying the coordination of mitochondria biogenesis with cell growth and cell cycle. I will use an interdisciplinary approach, combining the microscopy based protein concentration and cell size measurements established during my postdoc, a LacO-LacI based live-cell imaging approach, and qPCRs to unravel how concentrations of mitochondrial components as well as their production rates are linked to cell cycle and cell growth. I will then use mathematical modelling to establish a quantitative model for mitochondrial homeostasis that I will test by measuring the dynamic response of mitochondria maintenance to transient perturbations. I expect that our work will then catalyze our knowledge of mitochondria maintenance in human cells and will thereby lead the way to a better understanding of mitochondrial diseases.

DNA methylation is essential for normal mammalian development. While seminal work has provided tremendous insight into the dynamic regulation of DNA methylation throughout embryogenesis, comprehensive understanding of how cell-specific methylation programs are established and maintained, and how are they involved in defining cell states in vivo through regulation of target genes, remains a formidable task. Revolutionary technologies now offer unprecedented opportunities for understanding the function of DNA methylation in specifying, memorizing and modulating embryonic programs. These powerful tools motivate further development of novel experimental systems, integrating single cell monitoring with flexible engineering of markers, reporters and perturbations. This will enable to precisely target key rare embryonic cell populations for in-depth analysis. Here, combining cutting-edge methods for single cell mapping of DNA methylation and gene expression, and by developing a novel approach for inferring spatial information from single cell genomic data, we propose to comprehensively chart the post-implantation embryo, at unprecedented resolution. To move to functional studies, we will implement our recently established reporter system that enables monitoring and isolation of cells based on endogenous locus-specific changes in DNA methylation. Specifically, we will study the developmental potential of rare epiblast cells that we identified to exhibit lower-than-expected genome-wide methylation levels. Our combined approach will open new avenues for elucidating the contribution of cell-specific DNA methylation changes to cell-state and function following implantation.

With the increasing plethora of microbial genomes, the importance of complex microbial communities in nature has been recognized. The human microbiome has particularly attracted researchers because of its direct link to health and disease. Despite their great diversity, which is apparent from the available genomic information, little is known about the bioactive small molecules secreted by the human microbiome. Few studies have investigated the presence and ecological roles of these metabolites in the human microbiome at physiological conditions, because of the lack of appropriate methodologies.
This project attempts to address uncharacterized peptide metabolites encoded in the genomes of human-associated anaerobic bacteria, and elucidate how they contribute increasing the fitness of the host microbes. These peptides are likely to have a novel scaffold made by a unique class of oxygen-sensitive enzymes. I intend to perform multidisciplinary research composed of two approaches: A) Production of the peptide metabolites in a heterologous microbe; B) A metagenome-based assay to evaluate the population changes in the cultured microbial community upon peptide treatment. The former approach has the potential to generate natural products independent of the strain; the latter enables us to decipher the biological activities of small molecules in a complex microbial community. Innovation in both concepts will increase our understanding and the availability of microbiome-derived compounds that could improve human health. Furthermore, given the broad distribution of target peptides in anaerobes, this research will also elucidate the breadth of anaerobic chemistry inside humans.

Synaptic diversity is crucial for neuronal function. The heterogeneity of individual synaptic proteomes in a neuron underlies its input integration, compartmentalization of function, and neuronal plasticity during learning. The canonical classification of synapses based on neurotransmitter systems (e.g. the generic excitatory or inhibitory synapses) has become increasingly inadequate to explain the diverse synaptic responses in electrophysiology and plasticity. Without quantifying the variability of molecular combinations in synapses, our basic understanding of synapses is incomplete. Recently single-molecule localization microscopy has created an opportunity to interrogate individual synapses across a whole neuron. DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) is ideally suited for localizing and quantifying protein copies of interest compared to conventional stochastic optical reconstruction microscopy (STORM) because of its well-defined blinking kinetics and absence of photobleaching issues. This proposal aims to quantify the proteomic heterogeneity of synapses in individual neurons using DNA-PAINT and to interrogate the relationship between plasticity and synaptic diversification using multiple approaches. It will address the following:
1. The differential localization, variability, and stoichiometry of five representative synaptic proteins in the synapses of a neuron
2. The correlation between synapse activity and their proteomes
3. A potential change in synaptic diversity after single-spine plasticity
4. The effects of global homeostatic scaling on synaptic diversity
5. The remodeling of synapse diversity by newly synthesized proteins

Our eye movements constantly generate motion patterns on our retina, yet our perception of objects in space remains stable. For the brain to recognize self-generated visual motions to construct an image that is spatially invariant of one’s own movement, retinotopic visual information has to be integrated with eye position. Primate studies have shown cortical areas containing neurons responding to different combinations of these two reference frames, and perturbations of these areas suggest a role for space perception. However, little is known about the cellular and circuit mechanisms by which reference frame transformations underlying visual stability occur. I propose to fill that void by bringing this field into mouse research where methods to monitor, perturb and dissect neurons and circuits in much more precise and targeted ways are possible. Using preparations in which visual inputs, eye positions and eye movements are experimentally tracked and controlled will allow me to isolate visual, proprioceptive and motor components. Brain-wide functional ultrasound imaging will reveal areas activated by each component alone or in combination. Calcium imaging will then provide large population responses with single cell resolution. Finally, single-cell-initiated rabies tracing and whole-cell recording will be used to delve into the circuit and synaptic mechanisms underlying the functional properties we find among different neuronal populations. This project will accelerate the field of visual stability, and more generally sensorimotor integration, by providing a new niche where cellular and circuit mechanisms can be thoroughly dissected.