Metazoan development relies on tightly regulated gene expression patterns. Hence, the regulatory landscapes of developmental genes are complex and often contain several simultaneously active enhancers. Recent studies reported that the combined enhancer activities in such loci can either be additive, super-additive or sub-additive compared to the sum of the individual activities. Yet, the principles that might govern enhancer cooperativity remain elusive. To fill this gap, I propose measuring the activity of large collections of systematically paired developmental enhancers using STARR-Seq (500x500 enhancers), which was developed in my host lab to map individual enhancers genome-wide. I expect to identify an unprecedented number of additive, super- and sub-additive enhancer pairs and to reveal the sequence features associated with these distinct behaviors. To test all the genomic elements that could potentiate and/or interfere with developmental enhancer activity, I will use another STARR-Seq variant in which selected developmental enhancers will be individually paired with a comprehensive library of genomic DNA fragments (1xall elements). This will address how housekeeping enhancers, which form a functionally distinct class of enhancers, and other types of genomic elements impact developmental enhancer activities. Finally, I will validate my results in situ using CRISPR/Cas9 genome editing and test whether the genes’ endogenous regulatory landscapes influence enhancer cooperativity. Overall, this original approach will address a key aspect of developmental gene regulation which, despite its relevance for the study of development and pathogenesis, remains elusive.

The neonatal period is witness to fast physiological changes. The, up until birth, largely sterile environment becomes colonized by microbes, crucial for imprinting durable and specific immune responses, a period named “window of opportunity”. Postnatal immune imprinting involves extensive inter-organ trafficking of immune cells, and hence their interaction with endothelial cells of blood and lymphatic vessels. Still, to date virtually nothing is known about immune-vascular cross talk during postnatal period and the role of endothelial cells in immune imprinting. By using a mouse model that expresses a photoconvertible fluorescent protein, I will first characterize the immune subsets trafficking from gut, an essential organ for postnatal immune education, to other organs. Second, I will study the molecular changes in endothelial cells in this neonatal period to understand how this affects inter-organ communication and the pre-programming of long-lasting immune responses. This will be done by flow cytometry, high resolution 3D imaging and single cell RNA sequencing. Moreover, while there is a significant knowledge on the impact of environmental factors such as nutrition, exposure to antibiotic and farm animals on future health, much less is known about postnatal education of blood and lymphatic vasculature. Accordingly, I will investigate the impact on diet and microbial colonization in the neonatal period and their effects on both immune and vascular components. Altogether, I wish to unveil how early life events impact the immune-vasculature crosstalk and future health imprinting, thus helping to unlock intricate disease mechanism and their prevention in the future.

T cells are key effectors of adaptive immunity that are constantly moving, can enter most tissues, and operate in large crowds. Millions of densely packed T cells continuously roam through lymphatic tissue in search of foreign antigen; activated T cells divide vigorously, flock to tissues, mount local immune responses to pathogens or cancer cells, and remain there to guard against further intrusions. Crowding is normally expected to deter motion and lead to jamming, an effect confirmed in very diverse systems ranging from pedestrians and vehicular traffic to cellular monolayers and granular material. Yet T cells defy this general principle and are capable of fluid motion even in extremely dense environments such as lymphatic tissue and the epidermis, where there is no apparent room to maneuver. How they achieve this is unclear: although novel microscopy modalities can visualize migrating T cells in living tissue, studies of T cell movement have primarily focused on individual or few cells and have not addressed potential crowding effects. We hypothesize that (1) T cells have evolved to cooperate effectively in large groups and avoid crowding issues, such that (2) impaired T cell crowd operation is largely confined to anomalous tissue environments such as tumors. Here, we aim to unravel the mechanisms that facilitate fluid, “jam-free” motion of densely packed T cells, and determine in which situations T cell traffic might nevertheless be disturbed by emerging detrimental crowding effects. We will study T cell crowds in a wide range of conditions in silico, in vitro and in vivo: we will (1) conduct simulations to predict how T cell crowds operate in challenging conditions and which emerging crowd behavior is expected; (2) design microfluidic devices to expose T cell crowds to hallmark crowding scenarios, identify molecules involved in cellular crosstalk within crowds, and identify transcriptional signatures associated with efficient crowd operation; (3) examine T cell crowds in human and mouse tissues to test whether dynamic or static patterns indicating jamming can be confirmed in physiological or pathological conditions in vivo. Thus, by combining our unique joint expertise in crowd dynamics, immunology, and computational biology, we will shed light on how T cell crowds, a system quite unlike any other many-particle system studied so far, maintain motion with such remarkable efficiency in many conditions and what it would take to disrupt their smooth flow.

Cell motility is an integrated process implicated in many cellular functions such as embryogenesis, immunological response, wound healing, and growth cone path finding. For a long time, the study of cell migration has mainly focused on the observation of molecular components responsible for the formation of cell protrusion: actin networks and actin regulatory proteins. Recent studies propose that membrane biophysics, in particular membrane tension, could control actin network formation and cell migrations. Thus, in this project I propose to study the interplay between membrane tension and actin networks during cell migration. To do so, I will use a tension-sensitive fluorescent probe (FliptR probe) to measure membrane tension, together with a cell permeable fluorogenic F-actin label (SiR-actin) suitable for live labelling of the actin cytoskeleton. Using these probes, I will measure the local membrane tension variations during cell migration and monitor how these affect and/or are affected by actin cytoskeleton. By combining these approaches with the cutting-edge technique of adhesion protein micro-patterning, I will create a controlled and reproducible environment to finely tune cell migration. This will allow us to understand how the cellular environment affects the interplay between membrane tension and actin cytoskeleton. All together, I propose an innovative approach that will enable us for the first time to study the impact of local membrane tension on cell migration. The findings of this project will shed light into the cell migration, actin and lipid membrane fields.

The intestinal epithelium is a mucosal tissue reliant on its regenerative capacity as it is perpetually exposed to cell death whether it be during homeostasis or insult. Defective regeneration of the epithelium is associated with multiple diseases such as Intestinal Bowel Disease. Macrophages can act to sense damage and enhance tissue repair in the gut. Whether they can integrate the recognition of dying cells with intestinal epithelial regeneration remains unknown. We previously reported that the recognition of apoptotic cells by macrophages enhances their capacity to promote tissue repair. However, depending on the nature of intestinal injury, intestinal cells can undergo divergent cell death modalities including apoptosis, necroptosis and pyroptosis. Thus, we propose a model wherein macrophages directly participate in the integration of cell death with tissue repair. We aim to characterize cell death modalities, including that of stem cells, in models of intestinal injury. We additionally plan to identify the relevant cell death receptors for macrophage detection of apoptotic, necroptotic or pyroptotic cell death and correlate these findings with regeneration/repair or its failure during disease. Current treatment strategies focus on resolving inflammation during intestinal disease instead of enhancing regenerative responses of the epithelium. Thus, uncovering the nature and consequence of cell death in the gut may provide valuable insight into the treatment of gastrointestinal diseases.

Sex determination through the X and Y chromosomes is among the novelties that characterise mammals. The genetic dimorphism generated by the different content of sex chromosomes between males (XY) and females (XX) had led X-dosage compensatory mechanisms to arise. One of the two X chromosomes in females needs to be silenced in order to avoid a double dose of X-related genes in females as compared to males. The way to achieve X-chromosome inactivation (XCI) has evolved differently between two mammalian infraclasses: eutherians (placentalia) and metatherians (marsupialia). A series of epigenetic modifications and the coordinated activity of lncRNAs led by the master regulator Xist, are key for XCI in eutherians. In marsupials, Xist is not conserved and little is known about how this process occurs. James Turner’s group found that another lncRNA, namely RSX, is specifically expressed from the inactive X in the opossum Monodelphis domestica and may drive XCI in metatherians. In this project, I will investigate the epigenetic mechanisms responsible for achieving XCI in the opossum during embryonic development. Specifically, I will focus on the role that repressive histone modifications play in the germline and in the early embryo to regulate imprinted XCI. I will also interrogate if the link between pluripotency and biallelic expression of X-linked genes is maintained in the opossum and how the regulatory landscape of Xist and RSX has evolved between eutherians and metatherians. This project will be important to gain insight into how genomic imprinting and the epigenetic landscape is established in metatherians and how it has evolved since the divergence from eutherian mammals.

Stem cells face various challenges in their differentiation process including external perturbations, such as cell-to-cell signaling and internal perturbations, such as genetic mutations. To robustly differentiate, stem cells must integrate and respond to cues, yet be tolerant of destructive ones. Previous work shows that chromatin landscape is determinant of cellular activity, hence, it may infer the cellular sensitivity to perturbations as well. However, how the dynamics of the chromatin landscape reflects the cell’s sensitivity, is still missing. My hypothesis is that sensitivity to perturbations during differentiation is facilitated by a more accessible chromatin landscape. Here, I set to (a) define the temporal sensitivity of differentiating cells to various perturbations, and (b) define how the chromatin landscape reflects the sensitivity and robustness of the cells. First, we will grow and differentiate stem cells along several trajectories while characterizing their differentiation in-depth, utilizing sequence-based technologies such as chromatin accessibility and conformation, methylation and gene expression. Second, we will perturb the several differentiation trajectories, imposing external and internal perturbations, defining the perturbations’ effect on differentiation. Finally, we will perturb the cells at dense time points in the differentiation process, generating sequence-based time-specific profiles of perturbed vs normal differentiation. This work will characterize several differentiation trajectories in great details and provide the necessary high-throughput data to investigate the causality between chromatin landscape and sensitivity to perturbations.

Liquid-liquid phase separation is key for organising cellular content dynamically without the need for membrane confinement. Phase separation is an intriguing concept to explain as yet puzzling observations in chromatin organisation. It is known to be involved in the condensation of nucleoli, heterochromatin and long-range chromatin-chromatin contacts such as super-enhancers. The cellular mechanisms that control these phenomena are still poorly understood. Studies so far were focussing mainly on biophysical characterisation rather than function or regulation. Preliminary data in the host lab points to the dual-specificity kinase DYRK3, a known regulator of condensation, to be a regulator of novel nuclear condensates containing the chromatin remodeler Set1/COMPASS complex and other factors involved in gene expression control. On this basis, I will combine proteomics, chromatin and transcriptome profiling, cell biology and high-throughput multiscale imaging to determine the composition of transcriptional condensates and how they are controlled by DYRK3. Targeted perturbations will reveal the impact of these condensates on gene expression. The mechanisms discovered for DYRK3 will form a basis for understanding kinase-controlled transcription regulation more generally. Understanding the regulation of these condensates will not only shed light on the basic biology of cellular organisation but may enable therapeutic intervention in protein-aggregation diseases and cancer.

Isopeptide bonds - covalent amide bonds between amino acid side chains - stabilize and crosslink protein folds, notably in gram-positive bacterial pili, or fibrin clots. In contrast to disulfide bonds, far less is known about the generation of isopeptides. Here, I propose to de novo design and characterize proteins that can autocatalytically form both intra- and intermolecular isopeptides. To study a family of pili subunits in gram-positive bacteria that forms intramolecular isopeptides without enzymes, corresponding autocatalytic conformations will be constructed de novo. By controlling the orientation and hydrogen bonding of catalytic amino acids in the protein, I will dissect and optimize the mechanism of isopeptide formation. Resulting protein designs will serve as minimal models to investigate the stabilizing biological functions of isopeptides. Pathogenic bacteria use proteins containing thioester bonds to attach to their host via intermolecular isopeptides - a poorly understood process that I propose to elucidate with de novo design. A reactive Cysteine-Glutamine thioester will be confined onto a designed simplified protein scaffold. Docking de novo binders into proximity of the thioester will enable me to isolate key variables that govern autocatalytic intermolecular isopeptide formation in a controlled environment. Computational design of autocatalytic reactions in proteins enables radical new ways to covalently stabilize and crosslink them, currently lacking in the Rosetta software suite. Fundamentally, I aim to reveal what drives autocatalytic isopeptide formation and what constitutes their biological function by deconstructing their de novo design principles.

Although growing evidence suggesting that typical immune molecules contribute to shaping the synaptic plasticity and regulating the neuronal activity in the brain, the cellular and molecular mechanisms of these molecules, remains unclear. Moreover, the spatial and temporal expression of these molecules in the brain during development as well as in adults remains unresolved. Here I aim to use the most advanced approaches and techniques to investigate the role of such immune molecules for regulating the synaptic plasticity, neuronal circuits, and brain function. For that, I am interested in tackling fundamental and unsolved questions in the field: Does neuron in the brain express typical immune cytokine receptors or another immune molecule? If it so, does such receptor express within the synaptic cleft? Does it express in pre or post-synaptic or both? What is the molecular function of such receptor? Does it bind their classical cytokine and mediate intracellular signaling? What is the molecular and cellular consequence of such receptor activation? Does it affect synaptic transmission, plasticity, and strength? Does it have any impacts on the neuronal function or circuits? Could the activation of such receptors influence the animal behavior? What are the molecular mechanisms regulating such receptor and cytokine expression? Tackling these fundamental questions could help us better understand the regulation of neuronal function by the immune system in the brain which could be critical for processes such as learning, memory, and behavior during development as well as in pathophysiological conditions.

The intestinal microbiome is a species-rich, spatially heterogeneous ecosystem and core determinant of overall host health. An explosion of metagenomic studies interrogating the functions of microbiome composition in health and disease has revolutionized our view of intestinal microbial communities, but many important gaps remain in our understanding of these ecosystems. Chief among these are the combined contributions of viruses - particularly bacteriophages - to microbiome homeostasis, and of the spatial and temporal structure of the microbiome. Spatially structured communities, or biofilms, are the norm rather than the exception among microbes in the wild, including the intestinal tract, qualitatively altering their ecological and evolutionary dynamics across species but also with their host or predators. Here, we propose to fill these gaps by performing a mechanistic characterization of the physical and ecological forces driving host-microbiome-phageome ecosystem stability and dynamics. To achieve this, we will use an interdisciplinary approach combining tools and concepts form ecology, physics and engineering. First, we will develop a novel experimental framework built around the miniGut, a tube-shaped organoid that recapitulates critical biological and physical features of the intestine, including mucus secretion. We will use miniGuts to identify and quantify the critical factors governing the steady states and stability of a model microbiome, including the presence of a lysogenic phage engineered to allow us to track bacteria-phage-intestinal spatial dynamics in live samples at single-cell resolution. Underlying all aims will be the development of a dynamical systems perspective on the two- and three-way interactions where we will record ecosystem equilibrium points and trajectories in phase diagrams. Finally, using this knowledge, we will engineer lambda phage to manipulate ecosystem steady-states within miniGuts. To do so, we will transmit new functions to host microbial species via lysogeny, for example altering the bacterium’s ability to adhere to mucus, to tolerate antibiotics, and to either cooperate or antagonize other members of the model microbiome. Altogether, our interdisciplinary study will set the stage for mechanistic explorations of the role of phage and microbiome spatial structure in host physiology.

In host-microbe interaction studies, genomic technologies have significantly advanced our understanding of host responses and structure of host-associated microbial communities (microbiota). However, spatial and physiological heterogeneity in host-microbe interactions has been more challenging to study. Plants are comprised of various cell types, each of which may interact with a distinct set of microbes and, at the same time, accomplish other tasks such as growth and nutrient acquisition/utilization. To date, interactions between individual plant cell types and the microbiota are poorly understood due to our lack of knowledge about cell type-specific responses of plants and the composition of associated microbes at the cellular level. Here, I employ high-throughput single cell trasncriptomics to analyze plant cell type-specific responses to the microbiota under nutrient (phosphate) replete and starvation conditions. I will mainly investigate immune response and phosphate starvation response and crosstalks between them in individual cell types. I will also develop a method for in situ microbiota community profiling by imaging-based sequencing of bacterial 16S rRNA, which provides a spatial map of the plant microbiota at the strain-level of resolution. By linking plant gene expression and microbiota distribution at the single cell resolution, I will investigate the roles of plant genes and microbiota members that show plant cell-type specificity. This project attempts to reveal plant cell type-specific responses to biotic and abiotic environmental signals and microbiota assembly that are significant and biologically meaningful.

The proposed project aims to implement a new technology based on functional ultrasound (fUS) imaging and Ca2+ imaging using acoustic sensors of calcium (ASC). Functional ultrasound imaging is a new neuroimaging modality providing highly sensitive functional imaging of the brain by measuring cerebral blood flow. Ultrasound has excellent penetration depth, providing whole-brain coverage. However, the reliance of existing fUS methods on imaging blood flow results in an indirect readout of neuronal activity. Recently, it has become possible to connect ultrasound more directly to cellular function using genetically encoded acoustic reporters based on air-filled proteins derived from buoyant bacteria. In this project, I will use new versions of these proteins engineered to serve as dynamic sensors of Ca2+, which I will express in neurons and perform functional calcium imaging in the mouse brain using ultrasound. In addition to the ultrasonic detection of ASCs during task-evoked and resting state acquisition, I will complement the technology with simultaneous hemodynamic measurements to understand how the two signals relate to each other. This Whole-brain Acoustic Tracking of Calcium and Hemodynamics (WATCH) will provide valuable insights on neurovascular coupling and allow the interpretation of hemodynamics signals when models cannot be modified genetically with ASCs.

Because of its ancient origins, little is known about the first steps in the transition to multicellularity. Perhaps the largest gap in our knowledge concerns the origin of cellular differentiation. Our proposed research will examine how aging (a nearly-universal feature of cellular life) can be co-opted by simple multicellular organisms to drive the emergence of novel multicellular development. The proposal combines the PIs’ complementary expertise in aging and protein homeostasis (Saarikangas) and evolutionary biology (Ratcliff). Our research uses a unique approach: we evolve multicellularity from a single-celled yeast in the lab, examining how, over thousands of generations of evolution, they become more complex. Following our preliminary results, we focus on the role of aging and the chaperone protein Hsp90, mechanistically examining their role in the origin of simple cellular differentiation in our model system. We contextualize these results with mathematical modeling, examining the potential for age-dependent development to arise in different lineages of multicellular organisms. By showing how multicellular development can arise de novo, our research illuminates a problem of profound biological importance, and removes one of the last conceptual hurdles remining in our understanding of the origin of multicellular life.

Biomolecules in cells can self-organize into immiscible fluid droplets via liquid-liquid phase separation (LLPS). These liquid-like domains act as organizational hubs and play important physiological functions, including facilitation of biochemical reactions, transport, and signaling. Alteration of their size, internal structure, and coalescence dynamics have been linked to neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and ALS. Despite the continuous discoveries of new liquid-like compartments in cells, a biophysical understanding of their formation and resulting properties is still missing. Here, I will describe experiments designed to study: i) how intermolecular interactions control the assembly, material properties such as viscosity and surface tension, and dynamical arrest of liquid-like droplets; and ii) how non-equilibrium processes in cells, such as mechanical stresses exerted by the cytoskeleton, affect LLPS and regulate the size distribution and fluidity of the liquid domains. I will develop an in vitro system based on phase separating mixtures of DNA oligomers suspended in an active cytoskeletal network composed of purified filamentous proteins that will model the liquid condensates and cell interior. DNA oligomers are an ideal system to relate molecular structure to phase behavior because of the specificity and determinism of the base pairing interactions. Active cytoskeletal networks will mimic the non-equilibrium features of the cell interior. By exploiting the unique features of these systems, I will build a biophysical framework to decipher the roles of non-equilibrium pathways to LLPS and irreversible protein aggregation in living cells.

The ribosome which functionally establishes all proteomes across all life kingdoms, has historically been considered as a homogenous molecular machine that only passively contributes to gene expression. In contrast, there is growing evidence that ribosomes are heterogeneous and actively regulate gene expression through selective mRNA translation. In humans, there are hundreds of ribosomal DNA (rDNA) copies spread across multiple chromosomal loci, with recent studies showing copy number and sequence variations with unknown effects. In this project, I will characterize for the first time how variant rDNA alleles give rise to heterogeneous ribosomes that contribute to human physiology and disease. Using full genome sequencing data for 120,000 healthy and diseased individuals, I will (1) map population-wide rDNA variability in sequence and copy number variation and (2) identify rDNA variation that predicts human disorders. To get to a mechanistic understanding of rDNA variability I will (3) employ reporter assays to characterize the impact of ribosomal RNA (rRNA) variation on ribosome functionality and (4) use super resolution microscopy to test if rRNAs display expression specificity within a tissue and between different tissues including cancer samples. rDNA sequence variants reflect a hidden facet to our genomes and human health with the potential to change our understanding of cellular physiology at a global populations level. By studying the genetic variability of human rDNAs and its role in health and disease we aim to find how this diversity controls gene expression at the translation level and detect disease associated variants for early disease prognostics.

The regeneration of the optic nerve in the adult mammalian brain is limited [1,2]. During embryonic development different guidance molecules direct retinal ganglion cell (RGC) axons from the retina to the dorsal lateral geniculate nucleus (dLGN) [3]. Within the dLGN certain receptor/ligand gradients [4,5] and spontaneous patterned neural activity [6] assure correct retinotopic mapping. However, little is known on how these developmental mechanisms could be exploited to functionally reconnect RGC axons to the adult brain. Thus, we will ask: 1) How could axons of in vitro grown RGCs reinnervate and functionally reconnect to the dLGN in the adult rodent? 2) How does the axonal innervation pattern in the adult dLGN adapt in response to stimulation or genetic modification of the RGCs? To tackle these challenging questions, we will build a novel biohybrid platform in which RGCs are cultured on an implantable stretchable multi-chamber multielectrode array (MEA) [7] that is connected to a bioabsorbable axon-guiding nanofiber tube. In contrast to traditional organoid or explant based axonal rewiring approaches the new biohybrid system offers three unique advantages: First, we can modulate gene expression in a spatially defined subpopulation of RGCs. Second, we can spatiotemporally control the activity patterns of the RGCs. Third, we can target axons to deep brain regions and stimulate at an almost single cell resolution. This is significantly better than the mm3 size stimulation of any conventional deep brain stimulation electrode and has the potential to restore functional vision in blind patients without eyes.

A number of transformative molecular tools for biomedical research and therapeutics have been developed. However, a lack of safe, efficient delivery methods has precluded the widespread therapeutic application of these tools. One promising route to overcoming this limitation is to investigate the diverse biomolecule exchange systems found in nature and develop delivery tools based on these systems. Recently, a novel membrane vesicle (MV)-mediated horizontal gene transfer system was found in archaea. In this system, a relatively large 50 kbp plasmid is enclosed in a MV and delivered to other archaea. These plasmid vesicles (PVs) use a viral-like plasmid, pR1SE, that encodes the proteins required to assemble PVs and transfer the plasmid to other cells. As intercellular communication by MV is a universal process among the three domains of life, these systems may be exploited to deliver tools in human cells or the human microbiome. However, the mechanisms of archaeal MV and PV formation are largely unknown. I propose to identify the key proteins in the PV system. I will narrow down the candidates by remote homology detection among pR1SE and related viruses, and validate them by biochemical experiments. Furthermore, I will use genome-wide CRISPR screening to identify key proteins of host archaeal cells. Ultimately, I aim to reconstruct the PV system towards programmable synthetic vesicles production. This proposal will elucidate basic archaeal biology and provide a hint to the origin of viruses with a long-term objective of harnessing archaeal vesicle systems for delivery tools. Finally, this approach will serve as a paradigm for engineering archaea for molecular technologies.

Some animals can regenerate lost organs and tissues with high efficiency. In many such species, cellular dedifferentiation plays a major role in this process. How dedifferentiation is induced in vivo in the correct context is unknown and represents a major question in regenerative biology. The cnidarian Hydractinia can regenerate any lost body part in a tissue-specific manner. Regeneration of some body parts is mediated by adult, migratory stem cells called i-cells that are absent from the intact head. Surprisingly, host lab found that isolated heads can, nevertheless, regenerate a fully functional animal including i-cells that arise by dedifferentiation. Following amputation, isolated heads transiently display features consistent with cellular senescence. Here, I propose to study the mechanisms accompanying injury-induced senescence in Hydractinia dedifferentiation-dependent whole-body regeneration. My approach includes the mechanistic characterization of the molecular/cellular events that occur between decapitation and the appearance of new i-cells through dedifferentiation. For this, I will perform transcriptomic analyses, gene function experiments, and in vivo imaging. The strength of my proposal rests upon the usage of a unique animal model that is highly regenerative, displays natural dedifferentiation, and allows performing in vivo experiments that are impossible to conduct in other animals.

Many bacteria have evolved protein machineries to inject proteins into eukaryotic or prokaryotic cells. The type III secretion system (T3SS), widely distributed among gram-negative bacteria, is made up of a needle complex, an export apparatus and a sorting platform (SP). Although, the whole injectosome was characterized in a recent study, the assembly process of the SP is poorly understood. It has been shown that SpaO (a core component of the SP) localizes in two spatial populations, one at the bacterial membrane and another one located within the bacterial cytosol. This observation of the presence of SpaO-associated clusters away from the bacterial membrane suggests the presence of fully or partially assembled SPs in the bacterial cytoplasm. Furthermore, there are two existing versions of SpaO, a short (SpaOS) and a long (SpaOL) version. It has been postulated that SpaOS plays a role in chaperoning SpaOL during the assembly process of the SP but is not a structural part of the SP itself. If this holds true, SpaOS should only be localized in the (partly assembled) SPs located in the bacterial cytosol. In order to understand the assembly of the SP and the role of SpaOS in this process, I will image all proteins of the SP using recently developed 4Pi microscopy together with DNA-PAINT super-resolution microscopy. This novel combination, will enable me to image all proteins of the SP with molecular resolution (5 nm isotropic resolution). This will be an ambitious step towards understanding the sequence of events leading to the assembly of the SP in the T3SS. I anticipate that the tools developed for this work will bring new technologies to the field of bacterial physiology.