Cytoskeleton networks are the main vehicles of cell mechanics. Coordinated network dynamics is responsible to processes as diverse as cell motility and cytokinesis. Understanding the principles governing motion in these networks requires studying in vitro assemblies of purified components which can be subjected to finely tuned conditions. Recent works in purified systems have shown them to be unstable: either extremely contractile to the point of network collapse; or extensile and turbulent. This proposal delineates a research plan for studying active gels of microtubule filaments and kinesin motors as a prototypical cytoskeleton. Its main goal is to understand how asymmetric contractile and extensile modes of deformation emerge from inherently symmetrical network elements. My experimental approach will be to develop tools for modifying and controlling network dynamics on three length scales. At the filament scale, I will use the SNAP-BG crosslink to decorate filaments with motors. I expect that motors crosslinked at the minus end of a microtubule will encourage contractile motion and that plus-end crosslinked motors will encourage extensile motion. I will elongate the minus-end crosslink with photosensitive short DNA. Irradiating a mixed sample of minus-end and plus-end crosslinked motors with UV, I will be able to spatiotemporally tune contractility on the micron scale. Macroscopic stresses in the network will be measured and applied through a patterned soft substrate. The multiscale approach combined in a single set-up will allow us to uncover mechanisms responsible for contractility in cells and provide us with novel tools for designing biomimetic systems.

Direction selectivity is a feature of selected neurons in the visual cortex that is conserved in evolution. The emergence of cortical direction selectivity has been extensively studied but its circuit mechanism is still debated. My goal is to understand the circuit mechanism of cortical direction selectivity that is independent of retinal direction selectivity. I plan to approach this by using two animal models in which retinal direction selectivity has been disrupted. I will investigate the neuronal circuits of those cortical direction-selective neurons which retain their specificity in these animal models by performing single-cell-initiated functionalized rabies tracing and single-cell electrophysiological recordings. I will ask the three following questions: Do axon terminals of the principal cells of the lateral geniculate nucleus (LGN) fully lose direction selectivity after the abolishment of retinal direction selectivity, or could cortical feedback from layer 6 entrain direction selectivity in a subset of LGN cell axon terminals? What is the synaptic input profile underlying retina-independent cortical direction selectivity in pyramidal neurons? What is the contribution of the local presynaptic networks to the direction-selective tuning of pyramidal neurons with retina-independent cortical direction selectivity? This work will provide insights into how cortical selectively for environmental features arises.

Eukaryotic genomes are organized into a highly-compacted nucleoprotein complex known as chromatin. Hi-C and high-resolution microscopy studies reveal organization of chromosomes at multiple levels, from chromosome territories, to MB-scale functional domains that are spatially separated from each other, to shorter contact domains often called topological associated domains (TADs). While genetic studies in cell culture coupled with Hi-C or high-resolution microscopy reveal key roles for a variety of factors in higher-order chromatin folding, our understanding of chromatin fiber folding largely derives from biochemical studies in vitro. Mostly, these in vitro studies rely on artificial, homogenous chromatin templates that do not reflect the heterogeneous local folding properties of in vivo chromatin.
The aim of this project is to approach higher-order chromatin folding and chromosome organization biochemically. Micro-C detects internucleosomal interactions, which refer to local chromosome folding, by identifying nucleosomal DNA sequences and is therefore equally suitable for in vivo and in vitro studies. I will use chromatin prepared from cells (ex vivo) and in vivo-like in vitro reconstituted chromatin for chromatin compaction studies. With Micro-C I will be able to measure autonomous salt-dependent chromatin folding driven by all endogenous factors (ex vivo chromatin) or solely by histone-DNA interactions. Biochemical manipulation will allow to remove or test individual candidate factors.
This biochemical approach will help to decipher the regulatory role of higher-order chromatin organization on DNA templated processes, such as gene regulation.

Synthetic approaches toward redesigning cellular circuits and signaling networks have accelerated our understanding of sophisticated cellular behaviors. In particular, exploiting optogenetic approaches on neurons to manipulate neuronal activities have significantly expanded our comprehension on the nature of neuronal circuitry, whereas those approaches were applied much less in immune cells. Indeed, repurposed and/or engineered immune cells have been successfully used as cancer treatments, but they are only sometimes successful and can have harmful side effects. We hypothesize that these attempts have been limited because they have been restricted to a limited number of wild-type like signaling inputs. Here, through evolution-based approaches we intend to engineer chimeric antigen receptors (CARs) in T cells as a model system to answer a simple, yet, fundamental question regarding finding the maximal potency and unique phenotypes of T cell-based immune responses. Also, we offer a generalizable design scheme for safely controlling CARs through an optogenetic module. Newly engineered CARs could be of great use in eradicating cancer, and unique combinations and sequences of T cell signaling components could provide unexplained mechanisms and further understanding of the evolutionary nature of designed immune system. In order to achieve the goal, we build CAR intracellular domain libraries, and with a properly designed activity-based selection strategy we anticipate to find novel classes of CARs with extraordinary potency of immune responses.

It has been a long-standing question what mechanisms counter antibiotic resistance in nature. Are there any widespread multidrug strategies in soil bacteria for avoiding, inhibiting, or bypassing resistance mechanisms against antibiotics, whose biosynthetic pathways evolved a hundred million years ago, but still exist across natural microbial communities? One of the well-known examples of naturally occurring combination strategies is the co-production of beta-lactam antibiotics and beta-lactamase inhibitor in Streptomyces spp. which has led to the development of a potent drug combination for clinical use known as Augmentin. However, our knowledge on this issue is extremely limited due to the shortage of systematic investigations. It is important to emphasize that the extraordinary capacity of soil bacteria to produce multiple natural products may reflect an ecological advantage for them. Therefore, I hypothesize that a systematic investigation of the combinatorial biosynthesis of secondary metabolites, including antibiotics and non-ribosomal antimicrobial peptides, could reveal novel drug combinations. First, using state-of-the-art computational approaches I will explore the co-occurrence of biosynthetic genes of known secondary metabolites and antibiotics in the genome sequence of soil microorganisms which presumably co-evolved under strong selection pressure. Second, I will explore the capacity of novel candidate combinations i) to restore susceptibility of the resistant bacteria, ii) to synergistically inhibit bacterial growth or iii) to invert the selective advantage of resistance mutations.

Plants have evolved sophisticated mechanisms to recognize non-self molecules, allowing them to deploy effective immune reactions against a myriad of pathogens. Sometimes, however, plants mistakenly identify their own molecules as foreign and induce autoimmunity, causing severe growth defects including plant death. Autoimmunity is a hallmark of a syndrome that is seen in certain intra- and interspecific hybrids and that is known as hybrid necrosis. A particularly interesting case in Arabidopsis thaliana is that of the RPP7 and RPW8 loci, where three different pairs of alleles from different wild strains interact in F1 hybrids to cause hybrid necrosis. RPP7, which confers resistance against oomycetes, encodes members of the well-studied family of NLR immune receptors. The biochemical function of the RPW8 coiled-coil domain proteins that primarily confer resistance to fungi is less well understood than that of NLRs. I propose to reveal the mechanisms by which interactions between RPP7 and RPW8 proteins activate immune responses, taking advantage of the allele-specificity of these interactions. By interpreting these findings in the context of normal immunity, my work will not only advance our understanding of genetic incompatibility and autoimmunity, but will also contribute to optimizing combinations of favorable immune alleles in crop breeding.

Cells are constantly making millions of proteins that need to be synthesized correctly, folded, trafficked, and assembled to their functional state. All of these steps are monitored by the cells for mistakes or failures, and these failed products are degraded to avoid their accumulation. These quality control (QC) pathways are emerging as major contributors to neurodegeneration. The newly discovered ribosome-associated quality control (RQC) pathway represents one of the most cleverly imposed QC pathways to allow cotranslational surveillance of erroneous translation products. Recent studies have identified the factors involved in RQC pathway and defined its primary steps: recognition and splitting of stalled ribosomes into subunits, signaling of the heat shock response, ubiquitination of nascent chains, and extraction of polyubiquitinated nascent chain from the 60S subunit for degradation. I propose to dissect the mechanism of two unresolved steps of the RQC pathway and define its endogenous clients. Aim1 will investigate an essential step in signaling the heat shock response: the addition of C-terminal Alanine and Threonine (CAT) tails to stalled nascent chains. I will reconstitute this unusual mRNA-independent polypeptide elongation reaction in vitro to identify the minimum required components, define the energy source, and order the primary steps of this process. Aim2 will reconstitute nascent chain extraction in vitro to determine how a stalled polypeptide is delivered from the ribosome to the proteasome. Aim3 will identify the physiological clients of the RQC by trapping in vivo intermediates and identifying associated mRNAs or partially synthesized nascent chains.

Many sexually reproducing microorganisms are self-fertile, but in-breeding and out-breeding strategies have different outcomes on genetic diversity. Recent work from the Martin lab showed that self-fertile fission yeast S. pombe cells exhibit an exploratory polarity patch at their periphery, which allows them to preferentially pair with non-sister cells. However, the mechanism of this preference and its prevalence in nature are unknown. Here, I will study whether out-breeding is a general feature of fission yeast cell mating and dissect the molecular mechanism and genetic basis of this phenomenon. I will first develop a zygote FACS sorting method, which I will use together with high-resolution live-cell microscopy to study how pheromone concentration, cell size and transcriptional timing influence exploratory polarization and mate choice. I will then investigate whether out-breeding is a general feature amongst the recently sequenced collection of natural variants. If I detect pre-zygotic barriers amongst these strains, I will use pedigree analysis to probe for possible causative mutations. Finally, I will combine FACS sorting with experimental evolution to isolate strains favoring in- or out-breeding and uncover the genetic basis of the mating preference via comparative genomics/transcriptomics. Along the evolutionary experiment, I will also follow the exploratory polarization patterns by live-cell microscopy to relate the molecular mechanism of mating preference alteration with genetic changes. Finally, I intend to integrate the identified mutations into wild type lab strain to reconstruct the altered Cdc42 dynamic patterns and breeding preference.

To understand how cellular machineries work, we typically rely on reconstituted systems that often do not represent the complexity existing in vivo. We lack innovative methods to describe protein-protein interactions at high-resolution, specially the very transient ones, in their physiological environment. Chromatin is a good example of a system whose complexity cannot be fully reconstituted in vitro. Indeed chromatin is regulated by hundreds of chromatin remodelling enzymes and hundreds of possible combinations of histone post-translational modifications (PTMs) and variants. This complexity cannot be fully reconstituted in vitro.
We propose a novel combination of synthetic biology, mass spectrometry and high-resolution imaging to define the molecular details of how proteins function on chromatin in their physiological environment at high resolution. The challenge is to use photochemical traps installed by genetic code expansion in histones of living cells to “freeze” interactions of proteins bound to chromatin, especially the very transient ones that could be disrupted or missed by conventional purification or bulk chemical cross-linking. The trapped protein-chromatin complexes will be analysed by cryo-electron microscopy. By mass spectrometry we will map the interactions between remodelers and histones in vivo and we will quantitatively describe all chromatin PTMs associated to specific remodelers. This way, we will be able to analyse the spatio-temporal activity of chromatin-bound complexes at high-resolution at specific time points or upon specific stimuli.

Adult mammalian tissues are composed of heterogeneous cell types generated during development. To maintain homeostatic function, the identity of each of those cell types remains stable over time, and tissue renewal is normally sustained by dedicated stem cells. After injury, however, differentiated cells in many tissues can exit their quiescent state, acquire stem cell features and cross developmental identity boundaries to participate in the repair process. In the adult mammalian brain, injury-induced cellular plasticity (i.e. the acquisition of a new identity) remains poorly understood.
Here I plan to identify which brain cells exit their normally quiescent state upon stroke and study the molecular regulation of this process in situ. To this end I propose a novel strategy combining unbiased genetic fate mapping of stroke-generated cells with single-cell and spatially-resolved gene expression analyses. Specifically, stroke-generated cells will be marked with a genetic tag, identified using single-cell RNA-seq, and their contribution to repair traced over time. Next, we will use spatial transcriptomics to identify molecular triggers of cell plasticity, as well as their cellular source within their native environment. Finally, the identified molecules will be functionally tested for their capacity to enhance endogenous recovery after stroke by increasing cellular plasticity. This study will shed light on the cellular and molecular mechanisms governing the self-repair capacity of the brain.

Amino acid and peptide radicals have been implicated in a wide variety of biochemical processes and physiological disorders, including Alzheimer’s disease, arteriosclerosis and aging. At a molecular level, they are associated with protein damage and enzyme function, with the glycyl radical enzyme (GRE) family as important yet mostly unknown contributor. Using the high reactivity of peptide backbone radicals, GREs enable strict and facultative anaerobic organisms to catalyze a diverse range of chemically difficult reactions, including anaerobic toluene metabolism, the conversion of choline to trimethylamine (TMA) via C–N bond cleavage or glycerol fermentation. In taking advantage of the reactivity of the protein-based radical intermediates, tools will be developed to characterize novel GREs within intact microbial communities. For the first time, the concept of activity-based protein profiling will be expanded to include an entirely new class of enzymes. Furthermore, assigning functional and interactive relationships is suspected allow for the development of specific small molecules inhibitors to improve host health thereby opening up new opportunities in the way medicinal chemistry approaches certain disease. An additional key obstacle in studying GREs is the posttranslational mechanism of activation, which involves separate activating enzymes (AEs). The reconstruction of activase activity can be problematic and therefore, unnatural amino acid mutagenesis using designed glycine radical precursors to install a radical in the enzyme active site in a separate chemical step will facilitate studying GREs.

Some animals have great regenerative capability to regrow whole bodies even from small fragments. This ability relies on their pluripotent adult stem cells, which are capable of self-renewal and differentiating into any missing body part. During regeneration, positional signals such as Wnt signaling are crucial for stem cell regulation and specification. In planarians, inhibition of Wnt signaling promotes formation of head-specific cell types. However, the molecular control of stem cell fate by Wnt signals is unclear. In part, this is due to the limitation of lineage tracing in the well-established planarian system. Here I propose to use the acoel, Hofstenia miamia, as an alternative system to dissect this process. Hofstenia is an early diverging bilaterian and evolutionarily distant to planarians, and yet possesses whole-body regenerative ability and similar patterning pathways. In particular, Hofstenia produces large amounts of accessible embryos that are injectable, making lineage tracing of stem cells possible. I will first identify conserved Wnt signaling components by generating spatial transcriptomes along the anterior-posterior axis. Next, I will test the role of Wnt signaling in stem cell fate specification by using RNAi of Wnt pathway genes together with single-cell RNA sequencing. These data will reveal distinct transcriptional landscapes and identify Wnt-responsive cells within stem cell populations. Finally, I will develop lineage tracing methods for live imaging of stem cell differentiation and behavior. Together, this study will provide a basic understanding of how stem cell fate is regulated by positional signals during regeneration.

Cellular signalling pathways usually involve cascade processes. For some of these processes, ensemble average analyses cannot answer biological questions originating from stochastic biomolecular interactions. Though single-molecule analyses are sometimes helpful but still the oligomeric forms of many membrane protein complexes remain controversial. Protein or polymer scaffold lipid discs have emerged as platforms for monodispersed proteins. It is difficult to manipulate them to study protein-protein stoichiometric interactions. To overcome this, and motivated from DNA origami approaches, I propose to use DNA scaffolds to form lipid nanodiscs (DSN). Using DSN I will develop a novel programmable platform to study membrane protein-protein interactions in an isolated environment. First, I will create DNA nanorings, functionalised along their inner circumference with hydrophobic groups to hold a lipid membrane. Computational approaches will be used to optimise the designs along with experimental characterisation. The functional nature of DSNs will be established with an embedded P450 oxidoreductase enzyme. To develop a programmable platform, embedded proteins will be tethered at the edges of DSN with DNA linkers. Using DNA strand-displacement methodology proteins will be released to diffuse freely and interact in the membrane. As a proof of concept, an experiment will be set up using two formyl peptide receptors. Interactions will be analysed using AFM, single-molecule spectroscopy etc. Such platform, if achieved, could prove revolutionary for single-molecule analysis of protein oligomerization for studying, e.g., signal pathways, interaction energetics, metabolism etc.

Traditionally, model systems in experimental and theoretical condensed matter physics have provided a simplified/stylized version of target problems enabling fundamental advances. Over the years, soft condensed matter physics has undertaken the goal of explaining basic properties of living matter phenomenologically. This led to tremendous insights into specific phenomena such as behaviour of individual biological macro-molecules or mechanochemistry of biological forms, but the fundamental goal of understanding what the exact boundary is between living and non-living active matter remains elusive. Based on recent advances in soft-matter physics, here we propose an alternative approach to this question via developing an experimental abiotic (non-biological origin) physical model system capable of exponential self-replication and evolution (Aim 1a). With this framework in place, we intend to write down the fundamental properties of self-replicating and evolving matter in conjunction with the physical environment, laid out in terms of laws of physics (Aim 2), linking genotypes and phenotypes (Aim 3). These microfluidic experiments will be compared to robust hydrodynamic Molecular Dynamics (MD) simulations and theoretical Stokesian fluid mechanics, where hydrodynamic interactions and thermal noise are accounted for (Aim 1b). Together, these techniques could help formulate a theory for the essential ingredients of directed evolution and reproduction subject to physical constraints. We thus aim to discover new robust, abiotic architectures capable of self-replication of physical matter and information (Aim 4) and further exploit these findings in exponential manufacturing.

Sleep has roles in memory consolidation. Memory consolidation is thought to include systems consolidation which reflects communication among different brain regions, as well as synaptic consolidation at the local circuitry level. I found systems consolidation during sleep among separated regions in cerebral cortex (Miyamoto et al., Science, 2016), however, synaptic consolidation pattern is still unknown. Net cortical synaptic strength changes homeostatically through wake/sleep cycle: average synaptic strength increases during wakefulness and decreases during sleep (Tononi & Cirelli, Neuron, 2014). While skill learning is known to potentiate subsets of synapses in motor cortex for encoding skill memory (Hayashi-Takagi et al., Nature, 2015), plasticity of those synapses through sleep is unknown. If sleep depresses memory-encoding potentiated synapses, it may result in impairing memory consolidation. If sleep depresses other non-potentiated synapses relatively, it may enhance signal/noise ratio and memory consolidation. Here I will use complex-wheel task for studying synaptic mechanisms of sleep-dependent skill memory consolidation in mice. Through skill learning and sleep, I will perform in vivo two-photon imaging in motor cortex to visualize fluorescence tagged AMPA receptors, since the expression of these excitatory glutamate receptors is an established index of synaptic strength. To visualize plasticity in synaptic population, I will analyze time-course of fluorescence intensity change at single-synapse resolution. This study will help clarifying how sleep favors both memory consolidation and synaptic homeostasis.

The formation of higher order chromatin structure is crucial for DNA replication, transcription, recombination, repair and chromosome segregation. The cohesin-complex is thought to contribute to formation of chromatin loops and of topologically associating domains (TADs) by entrapping two different regions of the same chromatids. Its binding along genomic DNA is thought to be controlled by two of cohesin’s cofactors, CTCF and Wapl. Cohesin has been proposed to regulate the long-range chromosomal cis-interaction by a “loop extrusion” mechanism, in which cohesin can regulate chromatin loop formation by translocating along genomic DNA. However, how cohesin controls chromatin structure remains unclear. To address this question, I will first establish assays based on FISH, Cas9-FISH in living cells, oligopaint strategy and super resolution microscopy (SRM) to visualize chromatin loops and TADs. Second, I will employ these assays and Hi-C to analyze chromatin structure in cells in which the genomic distribution of cohesin is experimentally altered. For this, I will use knowledge from my host lab which recently discovered that the distribution of cohesin in mammalian genomes depends on the DNA binding protein CTCF, the cohesin release factor Wapl and transcription. Third, I will monitor the transition of cohesin re-location and of conformational change of chromatin induced by rapid degradation of Wapl and CTCF in real time. I will then test prediction of the loop extrusion model by comparing my experimental data with the simulations recently described in the literature.

How genetic programs regulate mammalian morphogenesis is one of the central problems in developmental biology. Digit patterning, a representative model to study mechanisms of morphogenesis, is regulated by multiple factors including cytokines and Homeobox-containing genes, or Hox genes, particularly Hoxa13 and Hoxd13. Recently, it has been illustrated that a reaction-diffusion model can explain this patterning in which the interactions of Sox9, a transcription factor, and WNT and BMP, cytokines, generate the periodic digit pattern. Interestingly, this periodic wave is modulated by FGF signaling and Hox genes. However, little is known about the molecular basis how Hox genes contribute to the proper digit patterning by regulating these interactions. In this project, to elucidate the role of Hox genes in mammalian digit patterning and autopod tissue identity, I will conduct integrative analyses including genetic, biochemical, and computational approaches. Firstly, single-cell RNA sequencing will be performed on both of wild type and Hoxa13 and/or Hoxd13 knockout mice to discover a gene network consisting the periodic wave pattern and analyze dosage effects of Hox genes on it. Secondly, HOXD11/D12/D13 will be epitope-tagged with genome editing tools. Thirdly, they will be immunoprecipitated to comprehensively analyze their binding specificity to the genome and other proteins. Lastly, these data are integrated with the reaction-diffusion model and investigate mechanisms of the modulation of BMP and WNT signaling by Hox genes to generate the periodic digit pattern, and consequently, establish the identity in autopod regions.

Controlling the direction of cell expansion is one of the key aspects of environmental and developmental signaling responses in plants. The direction of cell expansion largely relies on the microtubule network just beneath the plasma membrane. These cortical microtubules are dynamic and their organization is rearranged by a wide range of signals such as gravity, mechanical stress and light, but the molecular mechanisms that carry out these signal transmissions and microtubule array rearrangement are poorly understood. Our recent studies have demonstrated that Phototropin signaling regulates microtubule nucleation and severing activities. However, it is not clear how Phototropin regulates these activities. Phototropin is thought to have kinase activity and control wide range physiological responses. While our preliminary data with minus-end regulator suggest microtubule reorientation in response to blue light also requires the regulation of minus end dynamics, we have almost no information on how the remarkable minus end dynamics are orchestrated in plant cell. To tackle this linking question between plant physiology and cell biology, I propose to study about how the microtubule organization are regulated by transmitted blue light signaling through Phototropin. Specifically, I will propose following 3 projects: 1) identify factors being downstream of Phototropin kinase and upstream of microtubule organization, 2) investigate the molecular mechanism of the microtubule minus ends, and 3) introduce a new technique enabling us to conditionally manipulate target proteins to understand their spatiotemporal function during live-cell imaging.

A myocardial infarction in mammals typically displays limited regeneration and instead produces significant cardiomyocyte death. This loss of contractile cells is instead replaced with a permanent non-contractile fibrotic scar. In contrast, the pro-regenerative zebrafish displays increased cardiomyocyte proliferation and fibrotic scar resolution following injury. In order to find candidate genes, other labs have RNA sequenced whole mammalian hearts prior to and after injury. The spatial resolution however is lost, and other unrelated genes may mask candidate genes responsible for regeneration. This project will use a cardiac injury zebrafish model and at critical stages during regeneration, apply a novel genomic technique “RNA Tomography (Tomo-seq)” to spatially resolve gene expression at a genome-wide level. In addition, Tomo-seq concepts will be applied to develop a new genomic technique to examine the regulatory regions surrounding genes. Bioinformatic analysis will identify candidate genes and their regulatory regions associated with different phases of regeneration, and spatially correlate them with different areas in the injury site. Finally potential targets are functionally validated in zebrafish. By identifying pro-regenerative candidate genes at a spatial-temporal resolution in zebrafish, we can further understand and possibly modulate signals to increase the limited regenerative capacities in mammals.

The gut is under constant surveillance by immune cells, which ensures the homeostasis of commensal gut microbiota whilst conferring protection from invading enteric pathogens. B lymphocytes are central to maintaining balanced responses through production of huge quantities of the Immunoglobulins IgA and IgG. Ig production is the outcome of specialized germinal center (GC) responses, which form spontaneously in gut-associated lymphoid tissues in response to chronic exposure to microbiota. Despite being the precursor to Ig secretion, gut GC responses are entirely uncharacterized. Recently, when mesenteric lymph nodes (mLNs) were visualized using multicolor fate mapping (MCFM) large proportions of spontaneous GCs became monoclonal, an unexpectedly specific outcome in the face of exposure to such a diverse range of antigens. We intend to combine MCFM with in vivo models of commensal colonization to track the kinetics and clonal diversity of GC and Ig responses in mLNs through multiphoton microscopy. After GC isolation, we will use sequencing approaches to determine which bacterial species predominantly induce clonal GCs and map subsequent Ig specificity. Following this, we will investigate GC responses during enteric infection. We are especially interested in whether newly activated pathogen-specific B cells utilize pre-existing spontaneous GCs for their own affinity maturation, and how this is altered by composition of the current or past bacterial community. These data could have wide-reaching implications for both the fundamental biology of gut immunology as well as universal principles of GC dynamics in the presence of multiple antigens.