Coral reefs are one of the most biologically diverse ecosystems worldwide. They are home to about one third of all known marine species and sustain livelihoods of over 1 billion people through fisheries, tourism and more. However, they are increasingly threatened by climate change, leading to significant ecological, economic, and societal problems. In particular, ocean warming disrupts the essential yet vulnerable symbiotic relationship between corals and small, photosynthetic algae called dinoflagellate. Living inside coral tissues, they exchange essential nutrients with the coral while receiving safe harbour. This “coral bleaching” prevents the transfer of key nutrients, often resulting in the death of the host and eventually the collapse of the dependent ecosystem. This proposal aims to deepen our understanding of this essential partnership between corals and dinoflagellates at a molecular level. To do so, I will particularly focus on a process called protein glycosylation, a biochemical mechanism in which sugars attach to proteins and as a result influence their properties and functions. I hypothesise that protein glycosylation plays a holistic role in reef coral symbiosis, however, due to its complexity, has thus far evaded detailed investigations. To address this, I will leverage novel technological approaches to identify, track and quantify dynamic protein glycosylation features that occur during the different stages of symbiosis lifetime - from the initial recognition between corals and symbionts to the maintenance and finally breakdown of this partnership during bleaching events. I will examine how symbiosis progression is affected by these changes and how they might serve as a “switch”, indicating successful symbiosis establishment. These experiments will be conducted in laboratory settings on the anemone Aiptasia, a model organism for coral symbiosis. To assess whether the findings are of ecological relevance, I will compare them to Great Barrier Reef coral samples curated by the Reef Restoration and Adaptation Program (RRAP) during natural bleaching events. These samples will offer an unprecedented view into the environmental reality of corals in the Great Barrier Reef. My research will provide critical insights into the molecular foundations of coral symbiosis biology and will reveal new strategies to protect and restore reefs, thereby addressing an urgent environmental problem of global importance. Efforts to engineer bleaching-resistant corals or dinoflagellates could be advanced and innovative approaches, e.g. promoting efficient re-establishment of symbiosis, could be developed. Beyond these direct implications, the results of this project will have a translatable impact on other areas of science, including the symbiotic human microbiome, central to human health.

The mammalian cerebral cortex, made up of neurons and glial cells, is crucial for functions like thought, memory, and sensory processing. As the brain grows, the cortex expands more than the rest of the forebrain, forming characteristic folds known as gyri (outward ridges) and sulci (inward grooves between the gyri). While it was once believed that these folds formed simply because the cortex grew too large to fit within the skull, research over the last several decades has revealed that cortex folding is an intrinsic and highly regulated process, not just a byproduct of growth. These folds play a key role in brain organization, with primary folds aligning with critical functional areas, such as those involved in movement or sensation. Folding patterns are across individuals within a species and even among different species, following specific patterns dictated by genetic and cellular processes. In fact, researchers have identified a genetic ‘map’ that predicts where gyri and sulci will form during brain development. In species with folded cortices, like humans, primates, and certain carnivores, these maps are divided into regions or modules in the developing brain, each playing a role in the emergence of the folds. Additionally, particular types of progenitor cells—the cells that give rise to neurons—are especially active in the regions where folds form, indicating that the behavior of these cells may be closely linked to the development of cortical folds. Despite these discoveries, many questions remain about how these genetic, cellular, and metabolic processes work together to guide the organization of cells and the formation of folds in the cortex. For example, while we know that specific genes and progenitor cells are involved, it’s still unclear how their activities are coordinated to control the folding process across the developing brain. Our research aims to shed light on this by exploring how multiple biological processes interact to shape cortical folding. To do this, we will use the ferret as an animal model. The ferret’s brain, unlike that of mice or chicks, has simple but consistent folds across individuals, making it ideal for studying this process. Mice and chicks, whose brains do not fold naturally, will also be used to help manipulate or induce folding patterns, providing insights into how these processes may be triggered in species that don’t typically exhibit folds. The goal of our project is to understand how early molecular signals, metabolic activity and cellular processes come together to promote the formation of gyri and sulci. This involves studying the activity of genes, the behavior of progenitor cells and the migration of neurons into regions where folds will form. By creating a detailed, publicly accessible atlas of cortex folding across a variety of species—from non-mammals to mammals—we hope to offer valuable insights into the evolutionary principles behind brain development. The research may also uncover links between gene mutations, cell behavior, and neurodevelopmental disorders tied to abnormal cortical folding in humans, offering new pathways for diagnosis and treatment.

Structural complexity mediates species richness and diversity across ecosystems. Yet how fine-scale structural complexity shapes animal populations and communities is unknown. Structural complexity is typically measured on broad spatial scales, leaving a fundamental knowledge gap, as fine-scale variation in complexity may mediate fine-scale variation in animal distribution, behaviour and interspecific interactions. Two such interspecific interactions are those between predator and prey species and cleaning mutualisms, both of which have been shown to be affected by structural complexity. However, how fine-scale structural complexity mediates these relationships, and how these effects cascade throughout the wider interaction network, remains less understood. This project will employ highly novel and cutting-edge techniques to challenge our current understanding of how changing habitats mediate animal behaviour across ecological and spatial scales. Using coral reefs as a study system, we will test the central hypothesis that fine-scale structural complexity drives significant variation in animal space use, interspecific interactions and cognition. Objective 1 will use cognitive tests to determine the extent to which fine-scale structural complexity drives spatial cognition in individuals. Objective 2 will explore how fine-scale structural complexity and cleaning mutualisms mediate predator-prey interactions in a natural, but controlled, environmental setting. Finally, Objective 3 aims to incorporate predicted changes to structural complexity, based on climate-derived predictions of reef change, into regional species distribution models. Understanding how fine-scale structural complexity shapes animal behaviour under human-induced environmental change is necessary to accurately predict and manage our impacts. This project will address this fundamental knowledge gap. As an integrated whole, this project will draw links across ecological scales, from individual-level behaviour to regional species distributions, offering insight into how fine-scale structural complexity mediates behavioural cascades in changing habitats.

Insects are the most diverse group of animals on the earth. One of the major factors that has enabled the great prosperity of insects is the evolution of metamorphosis. Metamorphosis manifests in two primary forms: hemimetaboly and holometaboly. Hemimetabolous insects, such as cockroaches and crickets, possess juveniles (nymphs) that resemble adults and mature through several molts. In contrast, holometabolous insects, such as flies and butterflies, have larvae that are distinctly different from the adult form and undergo a complete transformation during the pupal stage. It is still not understood how these metamorphosis abilities have evolved. So far, several transcription factors, often called master regulators, have been shown to play essential roles in metamorphosis. One of these master regulators is an adult specifier called E93, which is required for adult differentiation. E93 transcripts are highly expressed at the pre-adult stages and induce adult differentiation in both hemimetabolous and holometabolous insects. However, E93 transcripts also exhibit peak expression levels at the early embryonic stages in hemimetabolous insects, or ancestral insects, suggesting an ancestral role of E93 in insect development. Previous work also showed that E93 induces adult differentiation by modifying chromatin remodeling in the model fruit fly Drosophila melanogaster. Given the expression pattern of E93 in early embryos and its function in adult differentiation by modulating chromatin accessibility, I hypothesize that the ancestral function of E93 is as a maternal-to-zygotic transition (MZT) pioneer factor. The MZT is an essential process in animals during which the maternal contributions to the egg are degraded and the embryonic (zygotic) genome is activated. During this process, several pioneer transcription factors modify chromatin structure and bind many regulatory elements, thereby driving zygotic genome activation. First, I will explore the ancestral role of E93 and other MZT pioneer factors by studying its embryonic function in crickets (a hemimetabolous insect) by conducting functional, transcriptomic, and chromatin accessibility analyses. Next, I will generate gene-edited strains in which epitope tags are introduced into the genomic loci of these endogenous transcription factors by targeted gene knock-in. Lastly, using these gene-edited strains, I will analyze pioneer factor-specific binding regions comprehensively. Then, I will integrate all the resulting omics data to elucidate the independent roles of these transcription factors and their interactions. Finally, I will compare these omics data obtained from crickets with those of Drosophila to offer a new evolutionary model regarding insect metamorphosis. The goal of this research is to elucidate the relationship between MZT and insect metamorphosis, providing new insights into the evolution of insect metamorphosis, and macroevolution. Insects have colonized a wide range of environments, from the tropics to polar regions, by evolving diverse adaptive mechanisms, such as phase polyphenism, caste systems, diapause, and seasonal adaptations. This research will serve as a prime example of how understanding the molecular mechanisms and evolutionary origins of these diverse insect phenomena can yield significant benefits. The results of this study will greatly advance our knowledge and capabilities in insect science.

The evolution of antibiotic resistance in bacterial communities presents a formidable scientific challenge with profound implications for human health, which led the World Health organization to include it in the top 10 global health threats. Antibiotic treatments have favored the evolution of different mechanisms of antibiotic resistance across a diverse range of bacterial taxa, including deadly pathogens that recently developed multi-drug resistance. The arsenal of antibiotic resistance mechanisms that have been identified since the discovery of the penicillin is alarmingly diverse. A major threat in the fight against pathogens relies on an evolutionary mechanism that makes such arsenal potentially available to all pathogens: Horizontal Gene Transfer. Antibiotic resistant genes are often carried in small, mobile DNA molecules called plasmids. These plasmids can be transferred during bacterial \"mating\" via a process known as conjugation, even between species that are otherwise unrelated. This constant genetic sharing happens in natural environments, including the human gut, where bacteria frequently exchange resistance genes. Bacterial communities, or microbiotas, are incredibly complex ecosystems where species interact in multiple ways. Some bacteria cooperate by sharing nutrients, while others compete for resources or even release antibiotics to suppress rivals. These interactions create a dense network of relationships, and while we are actively studying these ecosystems due to their importance for human and environmental health, much remains unknown. A key question is how these interactions influence the spread of antibiotic resistance through horizontal genetic transfer. Beyond its health implications, this is also a fundamental question in the fields of evolutionary biology and microbial ecology. My project aims to answer this question with an interdisciplinary approach combining microbial community ecology experiments with theory and concepts from statistical physics. In the lab, I will cultivate bacterial communities that include multiple species and various environmental conditions, such as different types of nutrients and antibiotics. I will then observe how different interactions between species affect the transfer of resistance genes from one species to another. Specifically, I will examine how the frequency of horizontal transfer changes based on the intensity of species interactions and the concentration of antibiotics. In highly interactive ecosystems, bacteria can exhibit complex and emergent behaviors, such as strong fluctuations of species abundances or dramatic shifts in composition in response to different initial states. Statistical physics, typically used to explain how macroscopic properties like temperature emerge from molecules, will provide powerful tools to model these complex bacterial dynamics. Just as water can shift between liquid, solid, and gas, bacterial communities display distinct \"ecological phases\" that can affect gene transfer in different ways. I will also take a closer look at the problem from the perspective of plasmids themselves, which can be thought of as genetic \"parasites\" in a symbiotic relationship with their host bacteria. In particular, I’ll investigate how bacterial immune systems, like the well-known CRISPR/Cas, affect the exchange of plasmids. By exploring the interplay between these immune defenses and the surrounding ecological environment, I will understand how plasmids, and the resistance genes they carry, manage to persist and spread among bacteria. Ultimately, the project aims to develop a predictive framework that explains how antibiotic resistance spreads through ecological interactions and gene transfer in bacterial communities. This research will help lay the groundwork for new therapies based on microbial ecology to combat antibiotic resistance, offering new strategies in the fight against this growing threat.

The regulation of gene expression is essential for development, stress responses, and homeostasis. During this process, the information encoded in the genome is turned into functional proteins. Transcription of a deoxyribonucleic acid (DNA) segment creates a ribonucleic acid (RNA) molecule, which acts as a blueprint to make a protein, a functional component of the cell. The transcription of genes into RNA is regulated by proteins called transcription factors. Transcription factors have two main functions: they bind to specific DNA sequences and either activate or repress transcription. Traditionally, it was believed that transcription factors worked alone or in pairs. However, recent research has unveiled a surprising twist: many transcription factors often join forces, forming larger assemblies of molecules that together navigate DNA to identify specific regions. The nature of these assemblies is poorly understood. Our project focuses on one particular category of these assemblies called filaments. Filaments are open-ended, rod-like structures composed of multiple copies of the same protein. Based on our preliminary analysis, we propose that many human transcription factors might have unreported abilities to form filaments, which would allow them to simultaneously bind to long DNA sequences. To study how filament formation affects the function of transcription factors and how general this mechanism is, we will rely on various approaches. First, we will use experiments, computational analysis, and machine learning to study known examples of filamentation. We will also use cryo-electron microscopy, an imaging technique suited for tiny objects, to study structures of filaments formed by transcription factors in the presence or absence of DNA. Finally, we will employ a transformative software called AlphaFold to predict new human transcription factors with a propensity to form filaments. Overall, the project will establish the prevalence and importance of filamentation as a mechanism by which transcription factors regulate expression of human genes.

Liver is a major organ for detoxification in human body. How the liver pushes the toxin out of the liver into gall bladder remains vague. We hypothesize that the toxin accumulated in micro-bile duct in the liver contracts and generates pressure to push the toxin into the larger bile duct, and liver-bile duct connection serves as a pressure valve. We will study what constitute the pressure valve and how it works in normal and diseased liver. Understanding exactly how the pressure is regulated will enable therapies to treat diseases such as cholestasis, evaluate supplements claiming to cleanse liver, and help to establish fundamental understanding of how the liver detoxify the human body. The cells in the pressure valve are pre-mature cells that divide very fast. Upon liver injury, these putative pressure-sensitive cells are the first cells involved in liver regeneration. Thus, this study paves the way to understand the earliest signals and events leading to liver regeneration.

Reactive oxygen species (ROS) are highly reactive molecules produced in various biochemical reactions from oxygen in our bodies. Accumulation of ROS regulates cellular behavior and plays a crucial role in adaptations against environmental perturbations. In particular, ROS-induced protein modifications are critical for T cell activation, as the antigen recognition process drives ROS accumulation and is required to expand and differentiate into effector T cells with anti-viral and anti-tumor roles. Despite the widely appreciated importance of ROS over cellular response, the molecular basis through which ROS regulates T cell activation remains obscure. Recent reports demonstrated that ROS regulates protein function by selectively oxidizing cysteine residues. Given that the T cell activation process is controlled by an array of transcription factors (e.g., NFAT, JUN, and NF-kB), I hypothesize ROS could modify their cysteine residues, thereby driving T cell activation. My proposed research aims to elucidate a redox-sensitive transcriptional factor in T cells. My host lab recently developed a new mass spectrometric approach that allowed us to comprehensively quantify cysteine modifications on a proteome-wide scale. Leveraging this method, I will perform quantitative profiling of oxidative cysteine residues in T cells stimulated with Anti-CD3/28, a known activator of TCR signaling. This will provide the first comprehensive map of redox-sensitive protein cysteines with functional importance in T cell biology. Among them, I will prioritize functional validation of cysteines on transcription factors with well-established roles in activating T cells. I will induce point-mutation of these residues to establish a causal link between these redox signaling nodes and phenotypes of T cell activation, such as proliferation and effector function. Next, I will establish the molecular and structural basis of how oxidation of these cysteine residues controls T cell activation. Given that those transcription factors are enriched in cysteine residues on their DNA binding domains and regulatory sites, I predict that oxidation introduces a structural remodeling, which may alter the mode of DNA binding to their cis-regulatory elements. I will perform ChIP-seq on wild-type and cysteine-mutant T cells to examine the genome-wide consequences of mutations over DNA binding. Lastly, I will use X-ray crystallography to establish the structural basis of how the cysteine mutation leads to conformational changes in the target protein and enhances/abolishes DNA binding. My proposed research provides the first molecular insights into how physiological redox signaling governs acquired immunity. Importantly, redox-reactive cysteine residues, such as proposed herein, are prone to be targets of small molecule compounds that covalently engage the cysteine thiol group through the exact chemical mechanisms that ROS modify these cysteines. Given the central role of T cells in various diseases, such as cancer, viral infections, and autoimmune diseases, my research will pave the way for discovering small-molecule compounds for those diseases by targeting the newfound redox signaling nodes in T cells. Beyond clinical application, this study will provide the first solid evidence that the physiological oscillation of ROS can directly control transcription factors in mammalian immune cells. The emerging new concept of the interplay between transcription factors and redox signaling may be a universal mechanism by which ROS guides differentiation in various physiological contexts. Ultimately, this work may provide important clues about how organisms have adapted to the advent of oxygen by utilizing ROS as a signal.

Mammalian development requires the extremely precise control of gene expression in space and time, ensuring that each cell adopts their correct identity and function. This tight control of transcriptional programs begins with the embryonic genome activation (EGA). EGA is a critical step in early development following oocyte fertilization, in which the embryo undergoes dramatic (epi)genomic remodeling starting its own transcription for the first time. EGA is commonly described as a burst of transcription throughout the genome, characterized by an initial wave in which the transcription of specific transposable elements (TEs), widespread integrated in our genome during evolution, is crucial for embryo progression. These elements, which include sequences like Endogenous Retroviruses (ERVs) and Long Interspersed Nuclear Elements (LINEs), are embedded in our DNA due to evolutionary processes and their transcription, rather than its transcribed products, is essential for early development. Despite our progress in understanding how TEs influence early development, the precise molecular mechanisms underlying TEs activation and the effect of such a vast and unique transcriptional wave remain elusive. One intriguing aspect of TE function and EGA is the way early embryos deal with the topological stress that results from these transcriptional waves. Transcription generates topological stress in the DNA double-helix, leading to twists and turns known as DNA supercoiling, which have profound effects in virtually any aspect of genome dynamics. Traditionally, DNA supercoiling has been considered as a mere byproduct of transcription, but recent works suggest it plays an active role in controlling processes like 3D genome architecture or transcription process itself. However, how this specific DNA topology is established in early developmental stages or its influence on EGA remains unexplored. Here, I hypothesize that TE-derived supercoiling during EGA might provide the initial wave of DNA supercoiling in the embryonic genome, actively contributing to developmental complexity. From a broader view, this proposal will explore the contribution of DNA supercoiling and DNA topology in early embryo development. To test the previous hypothesis, I will apply a range of cutting-edge methods to systematically characterize and map DNA supercoiling in mouse embryo models from fertilization to blastocyst stage, with a particular focus on EGA and TE dynamics (Aim 1). The project will generate maps of DNA supercoiling in a developmental process in vivo for the first time. Additionally, I will disrupt DNA supercoiling using the CRISPR-Cas technology during EGA to establish causality between DNA supercoiling and early developmental processes (Aim 2). While DNA supercoiling is well-established as a key regulator of transcriptional and replication programs in prokaryotes, its potential role as a regulatory mechanism in mammalian development remains unexplored. This research will address this question by identifying a potential connection between DNA topology and the regulation of transcriptional and epigenetic programs during early mammalian development, increasing our understanding of the mechanisms driving embryogenesis and early development.

All living organisms, including plants, are in constant contact with a myriad of microbes, and must respond appropriately to avoid diseases. While promoting beneficial partnerships, they must prevent pathogenic associations. This project aims to explore this delicate equilibrium using the symbiosis of the plant Medicago truncatula and arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis as a model. AM fungi form a vital partnership with most land plants. Plants provide organic carbon to the fungus, and fungi supply essential minerals like phosphorus and nitrogen to the plant. This partnership not only boosts plant growth but also helps plants to thrive under adverse environmental conditions. For successful AM fungus engagement, plants must initiate symbiosis signaling that helps form a beneficial partnership while also preventing them from pathogen attack. Once AM symbiosis is established, plants switch their immune system back on, which allows them to defend themselves against pathogen attack without disrupting the beneficial fungus. However, the mechanisms of how plants perceive multiple inputs, “decide” and transduce specific signals to trigger appropriate responses, remain largely unknown. We aim to uncover new signaling pathways by which plants dynamically modulate their responses to beneficial and pathogenic microbes. Recent studies have shown that plants control AM symbiosis through specific signaling molecules called CLE peptides and their respective receptors like CLV1, CLV2 and CRN. CLE peptides are small signaling molecules produced by plants, that regulate growth and development. Besides plants, it has been found that both symbiotic AM fungi and parasitic nematodes produce CLE-like peptides to manipulate plant signaling to their advantage. However, CLE-like peptides from other pathogens remain elusive. Our data suggest CRN is a key player in symbiosis and defense, but we do not know how CRN specifically responds to different CLE peptides. We will investigate two main areas. First, we aim to identify and characterize CLE peptides from both plants and microbes that are involved in the interplay of symbiosis and immunity. Using bioinformatics, we will mine genomes and proteomes of various pathogens, to identify pathogenic CLE-like peptides, and then we will study their role during AM symbiosis and during infection by pathogens. Second, we will examine how the CRN protein, which is involved in plant immune responses and AM symbiosis, interacts with these CLE peptides. CRN is thought to be a key player in integrating signaling pathways that manage both symbiosis and immunity. We will use advanced biochemical techniques like to map CRN interactors and identify novel signaling pathways that mediate the interplay of beneficial and pathogenic interactions. The project aims to elucidate the molecular mechanisms underlying plant-microbe interactions. identify new targets for engineering crops with enhanced disease resistance and symbiotic efficiency. This will lay the groundwork for the creation of crops that are more efficient in forming beneficial symbiotic relationships and fighting against pathogens. Such advances will not only enhance agricultural productivity but also contribute to environmental sustainability by reducing the need for fertilizer or pesticide inputs.

One of the most profound questions in biology is how simple single-celled organisms evolved into the complex eukaryotic cells that make up plants, animals, and humans. This transformation, which occurred billions of years ago, set the stage for the diversity of life we see today. This research project aims to shed light on this pivotal moment in life's history by exploring a group of enigmatic microbes known as Asgard archaea. Asgard archaea are fascinating because they possess features that are remarkably similar to those of eukaryotic cells, making them the closest living relatives to our earliest ancestors. Despite their significance, we know very little about how these microbes regulate their genes and how this might have influenced the evolution of complex life. Central to this project is the study of epigenetics—the mechanisms that turn genes on or off without changing the underlying DNA sequence. In eukaryotes, epigenetics plays a crucial role in development, allowing cells to differentiate into various types despite having the same genetic code. However, the origins of these complex regulatory systems remain largely unknown. This research project focuses on enzymes called methyltransferases (MTases), which are essential players in epigenetic regulation. These enzymes add chemical \"tags\" to DNA, RNA, or proteins, influencing how genes are turned on or off. By investigating MTases in Asgard archaea, I aim to discover whether these enigmatic microbes possess complex epigenetic systems similar to those found in eukaryotes. If they do, it could mean that the roots of epigenetic regulation—and thus the foundation of cellular complexity—are much older than previously thought. To achieve this, I will use cutting-edge computational tools and laboratory techniques. First, I will employ advanced artificial intelligence models to scan the genomes of Asgard archaea for genes encoding MTases. This approach allows us to identify potential enzymes even in organisms that cannot be easily grown in the lab. Next, I will develop high throughput methods to produce and study these enzymes, overcoming the challenges of working with proteins from uncultivated microorganisms. By analyzing the structure and function of archaeal MTases, I hope to reveal similarities and differences compared to their eukaryotic counterparts. This could provide crucial insights into how complex epigenetic systems evolved. Additionally, mapping the patterns of chemical modifications introduced by these enzymes will help us understand their roles in the cell. The methodologies developed in this project—combining advanced computational tools with innovative laboratory techniques—will enable us to study proteins from uncultivated microorganisms effectively. This approach can be extended to investigate other elusive proteins from a wide array of organisms, enhancing our ability to explore the vast diversity of life. While we understand how DNA is stored, the regulation of the enzymes that modify genetic material remains elusive, particularly in enigmatic organisms like Asgard archaea.

Neuron are highly polarized cells with distinct protein distribution on its axon and dendrite. This research investigates how neurons organize and transport vital proteins to specific locations to function properly. A disruption in this process is linked to a variety of neurodegenerative and developmental disorders. The focus of the study is on calcium channels, which are ion channels that is essential for neurons to send signals to each other and to regulate brain activity. There are two key types of calcium channels examined in this research: CaV2 and CaV1. These proteins play distinct roles in different parts of the neuron. CaV2 channels are essential for releasing chemical signals at presynapsic active zone. CaV1 channels, on the other hand, are found in somatodendritic parts of the neuron and help control its electrical activity. Although both types of channels are made in the soma of the neuron and exhibit limited sequence difference, they need to be transported to their specific locations—CaV2 to the axon and CaV1 to the somatodendritic compartments. Understanding how neurons sort and transport these proteins to their correct locations is still a long-standing question, and this project seeks to uncover the mechanisms involved. The hypothesis driving the research is that specific sequences in the calcium channels help determine where they need to go. These sequences act like address labels, guiding the channels through the neuron’s complex transport system to their target locations. The project has two main goals: Investigating how CaV2 channels are transported: CaV2 channels are made in the neuron’s soma and must be moved to the active zone to help neurons communicate effectively. This process likely involves packaging the channels into small transport units within the cell, which are then carried along pathways inside the neuron. The study will explore the molecular machinery responsible for this transport, including the proteins that help direct CaV2 to the correct location. Additionally, the research will look at whether other active zone proteins involved in traveling together with CaV2 channels, helping guide them to their final destination. Understanding how CaV1 channels are sorted and transported: Similar to the investigation of CaV2, this part of the research will focus on CaV1 channels. The study will look at how specific sequences within CaV1 help guide it to the dendrites, where it regulates electrical activity. The research will test whether switching these sequences between CaV1 and CaV2 channels can redirect them to different parts of the neuron, further illuminating how neurons control the movement and placement of these important proteins. By studying how these calcium channels are sorted, transported, and anchored within neurons, this research aims to build a clearer understanding of the establishment of polarization that keep neurons functioning correctly. These findings will contribute to a broader understanding of how the brain maintains its complex communication networks and how disruptions in these processes may lead to neurological disorders. The insights gained could help identify new strategies for treating brain diseases, offering potential pathways for intervention where protein transport goes awry.

Bacteria are surrounded by a cell envelope that is essential for growth, integrity, and pathogenesis. The envelope and the synthesis pathways that build it are the target of many of our most effective antibiotic and vaccine therapies. In most bacteria, the envelope is composed of sugar polymers (polysaccharides) that are crosslinked together into a dense meshwork called the cell wall. The cell must constantly expand this meshwork as it grows. Interestingly, a subset of bacteria, including the human pathogens Bacillus anthracis and Clostridiodes difficile, assemble a crystalline shell on top of the cell wall. This shell is called the surface layer (the \"S-layer\"). It is made of approximately half a million copies of a single protein that form a crystalline monolayer. The therapeutic potential of disrupting this layer motivates a more holistic and mechanistic understanding of how the S-layer is assembled and a detailed picture of its function. In the proposed study, Dr. Chen Zhang, a postdoctoral fellow in the laboratory of Prof. David Rudner at Harvard Medical School, will investigate the assembly and function of the surface layer in the bacterium Bacillus anthracis. In Bacillus anthracis, the individual S-layer proteins are connected to the underlying cell wall. During growth, the rod-shaped B. anthracis inserts its cell wall material uniformly along the cell cylinder. Breaking the crystalline S-layer array throughout its length to incorporate new S-layer proteins as the cell wall grows would be difficult and energetically costly. In fact, evidence suggests that growth of the S-layer occurs in gaps or fissures in the crystalline array. It is unknown how these gaps are generated and what dictates where in the array they occur. Dr. Zhang's proposal seeks to figure out how the S-layer expands during growth. This fascinating and important protein monolayer is also important for envelope integrity but how it functions in this capacity is poorly understood. Dr. Zhang's second aim is to determine the function of this protein array. To study how the Bacillus anthracis S-layer is assembled and remodeled, newly synthesized S-layer proteins and the cell wall will be fluorescently labeled and imaged by high-resolution fluorescence microscopy. This analysis will establish the size and position of the gaps in the surface layer that enable its expansion relative to newly synthesized cell wall. Dr. Zhang hypothesizes that surface layer assembly is mediated by crack propagation, in which cracks or fissures are generated by imperfections in the two-dimensional crystalline layer. There are a small set of surface-layer-associated proteins in Bacillus anthracis and Dr. Zhang has proposed that some of these generate inhomogeneities in the crystal layer that nucleate the cracks. He will rigorously test this model. To explore S-layer function and the interplay between the synthesis of the surface layer and the underlying cell wall, Dr. Zhang will perform genetic screens to identify factors that become critical for cell viability when the surface layer is absent. Dr. Zhang envisions he will identify genes that are involved in cell wall synthesis or its modification and genes that coordinate the synthesis of the cell wall and the surface layer. Importantly, an unbiased genetic screen has never been used to investigate surface layer function and holds the promise of elucidating critical roles for this protein monolayer. Dr. Zhang will characterize the identified factors using the fluorescence imaging tools he develops to study S-layer assembly. Overall, the successful completion of this project will provide a deeper understanding of surface layer assembly and function in Bacillus anthracis, and will reveal both fundamental and biomedical insights with implications for how to combat this important human pathogen.

Project Overview: Unlocking the Secrets of Nature’s Tiny Compasses. Cryptochrome proteins play crucial roles in plant growth and circadian rhythms; and they are believed to help migratory animals (e.g., birds, turtles, fish) navigate using Earth’s magnetic fields. They are activated by transferring charges across a series of aromatic residues, a critical process also fundamental to broader biological systems including photosynthesis and respiration. This project, titled “Decoding Structural Dynamics of Charge Transfer in Cryptochromes by Time-Resolved Crystallography,” aims at uncovering how nature’s tiny internal compasses, avian cryptochromes, operate at the atomic level. Utilizing cutting-edge techniques in time-resolved serial crystallography at X-ray free-electron laser facilities, I intend to capture the hidden three-dimensional structural transformations from femtoseconds to milliseconds after energy input by a short light pulse. Aims and Objectives: Dynamic Visualization: I will observe how “protein quakes” (i.e., structural changes) emerge, propagate and eventually subside with the cryptochrome proteins, shedding light on how they manage electron flow—a crucial process for their activation and downstream biological capabilities. I will then interpret the observed structural changes with respect to charge transfer theory. I anticipate that the results may challenge conventional knowledge in electron transfer, which neglecting the intricate motions of proteins/solvents in biological environments. A particularly intriguing aspect of this study is the exploration of how these proteins may enable birds to detect geomagnetic fields. By contrasting cryptochromes from migratory and non-migratory species, I hope to reveal the structural basis that could underpin this fascinating magnetosensing ability. This research is poised to bridge gaps in our understanding of molecular dynamics and charge transfer within biological systems. This could impact a range of scientific fields, from revising existing theoretical mechanisms to deepen our comprehension of how birds utilize quantum mechanics to navigate over vast distances.

DNA packaging proteins called histones undergo covalent modifications causing epigenetic changes in gene expression. These changes are reversible leading to a switch between ON and OFF expression states without DNA sequence alterations. Such epigenetic switches must be robust during differentiation and yet susceptible to change in novel environments. We do not understand how epigenetic switches can be controlled to drive complex phenomena such as cellular differentiation and adaptation. THE GOAL OF MY PROPOSAL is to couple theory and experiments to build a synthetic epigenetic oscillator whose expression is tunable, reversible, and heritable with periodicities ranging from a few to many cell divisions. Developing this synthetic framework will reveal architectures that enable cells to maintain epigenetic memory that is robust and yet flexible to change. Histone H3 lysine 9 methylation (H3K9me) marks silent genomic regions called heterochromatin in fission yeast (S.pombe). Heterochromatin in S.pombe exhibits stochastic switching between ON/OFF expression states and serves as a versatile model system for my experimental goals. OUR CENTRAL HYPOTHESIS is that the H3K9me-dependent epigenetic switch relies on the activity and dosage of different chromatin regulators. By designing novel synthetic positive and negative chromatin-based feedback loops, we will transform a stochastic epigenetic switch into an oscillator. AIM 1: Develop a theoretical framework modeling epigenetic oscillations to identify the sources of noise that lead to stochastic switching between ON/OFF expression states. By modeling the interactions between chromatin modifiers and H3K9me, I will program the frequency of switching within cells and study their collective behaviors to determine how oscillations are coupled across cells. AIM 2: Implement theory-based predictions to build a synthetic epigenetic oscillator in S.pombe. Using microscopy and lineage tracing, I will visualize H3K9me dependent switching of a GFP reporter gene between ON/OFF states in individual S.pombe cells. My work is a unique synthesis of theory and experiments made possible by working with Jane Kondev (Physics) and Kaushik Ragunathan (Biology) at Brandeis University. The Kondev lab develops mathematical models centered on chromosome organization and gene regulation while the Ragunathan lab has expertise in using genetic and biochemical tools to inducibly control H3K9me epigenetic switching in S.pombe. Using precision engineered genetic tools, I will implement novel H3K9me dependent positive and negative feedback loops to produce oscillations in expression that will be further refined by additional theory. Building a tunable single cell epigenetic oscillator will break new ground in our understanding of epigenetic switches and would eventually lead to experiments in tissues where synthetic oscillators can program responses to an external stimulus in a coherent manner.

The developmental fate of each embryonic cell has been extensively studied with both classical lineage tracing and modern omics technologies. However, how cells are primed for distinct fates before acquiring their transcriptional identity is unknown. Here, we set out to identify the earliest mechanical signatures that mark fate separation while cells navigate the embryo along complex migratory paths. We posit that similar to complex atmospheric and oceanic large-scale flows, hidden material surfaces known as Lagrangian Coherent Structures (LCSs) represent a novel kind of embryonic boundaries organizing cellular flows and patterning. Using dynamical systems and control theory, we will develop a technique to identify LCSs of in-toto 3D zebrafish gastrulation from sparse cell tracks. Specifically, we will focus on multicellular repellers, LCSs that compartmentalize the embryo very early, setting cells apart before they acquire distinct transcriptional profiles. Repeller-mediated cellular flows are invisible to standard methods based on cell velocity and trajectories. Therefore, we propose an integrative approach to unveil the mechano-dependent epigenetic modifications instructing fate bifurcation. First, we will determine the viscoelastic properties that cells experience across the repellers. We will quantify this highly dynamic mechanical landscape in vivo and in situ with unprecedented spatiotemporal resolution using live microscopy, image analysis, and novel rheological techniques. Second, we will analyze how divergent mechanical properties along opposite repeller-mediated cellular flows impact chromatin accessibility and marks, resulting in transmissible changes in gene regulation that will dictate cell lineage differentiation. Third, we will devise a data-driven differentiation model for cells across repellers, accounting for epigenetic and mechanical cues--effectively providing a mechanics-mediated Waddington potential. This proposal will revolutionize the way we understand early embryo compartmentalization and elucidate its very first mechanical and epigenetic drivers.

Viruses kill up to 40% of ocean microbial cells daily, impacting global ocean food webs, carbon and nutrient cycling, and climate. Soil viruses likely play similarly essential roles in terrestrial ecosystems, yet their diversity and impacts are virtually unknown. Here we aim to quantify and predict the role of viruses in ecosystem methane flux in climate-significant peatlands from multiple continents. Viruses and their microbial hosts that produce and consume methane will be studied across scales from single cells to communities to ecosystems. We will test the overarching hypothesis: viruses in peatlands affect the growth, activity, interactions and death of methane cycling prokaryotes thereby changing the ecosystem methane flux. This will be achieved by characterizing the spatial structuring of virus-host interactions across different locations and within each location across the peat column with changes in water content, oxygen and hydrological connectivity, and develop novel approaches for quantifying the temporal dynamics and fluxes of carbon transfer between viruses and their hosts. Through novel experimental and computational approaches, we will investigate the strategies of virus infection, host specificity, and outcomes in terms of methane uptake or loss using microfluidics, single cell genomics, viromics, metagenomic- and protein- stable isotope probing. We will leverage deep learning-based tools to improve viral protein annotations and host predictions to advance viromics research in peatlands and beyond. At the community scale, we will quantify the contribution of viral-host interactions to ecosystem methane cycling in the lab and field by quantifying methane production and oxidation rates and identify sources and fate of methane using stable carbon isotope analysis. Finally, a population dynamics model will simulate virus-host interactions to create generalisable predictions of methane flux rates. This interdisciplinary project with complementary expertise from multiple countries will enable generation of novel understanding of virus-prokaryote interactions across temporal and spatial scales to link viruses to the global cycling of a potent greenhouse gas.

Bats are reservoirs for many viruses, including coronaviruses (CoVs). Their unique physiology and immunity help them maintain these viruses without disease. Limited evidence suggests periods of active infections in bats (increased viral replication and shedding) are triggered by physiological stressors that provide opportunities for transmission to humans. Food scarcity and reproduction are two such stressors that correlate with viral shedding in bats. However, the pathways through which they modulate viral replication and shedding are unknown. In this study, we will investigate the physiological manifestation of intrinsic and extrinsic stressors in fruit bats inhabiting intact and fragmented forests in Belize by integrating field studies, mathematical models, and in vitro experiments. Earlier studies have identified alphacoronaviruses (alpha-CoVs) in the bat subfamily Stenodermatinae, which includes genera Artibeus, Dermanura, Sturnira, and Uroderma. Using this host-pathogen system in Belize, we will test the role of physiological stress in viral pathogenesis. We include three complementary aims with testable hypotheses. First, we will sample bats in wet and dry seasons to quantify physiological stress via neutrophil to lymphocyte ratios as well as fecal and hair cortisol. We will also standardize a qPCR assay to quantify heat shock proteins in blood. We hypothesize bats inhabiting fragmented forests will have elevated physiological stress. Stress will also vary seasonally and be highest in reproductive females. Generalized linear mixed models (GLMMs) will test the effects of season, habitat quality, and reproduction on stress. Next, we will detect alpha-CoVs in bat saliva and feces using RT-PCR and correlate it with stress biomarkers using GLMMs. We hypothesize elevated physiological stress will increase viral replication and shedding, potentially mediated by immunosuppression. We will develop mathematical models capturing this relationship to test the sensitivity of various epidemiological parameters to physiological stress. Finally, we will develop cell line and organoid models for these bat species to validate our field observations and experimentally test our predictions. We hypothesize innate immune markers will have an inverse relationship with viral replication in cell lines and organoids and that both will respond to cortisol challenge in a dose-dependent manner. We will challenge our models with an alpha-CoV with high similarity to CoVs found in Belize bats (HCoV-229E) in the presence of cortisol. We will use gene expression analyses to discover bat cellular response to stress in the face of CoV infection. For the first time, we will elucidate how stressors affect bat-borne zoonoses. Our work will represent a unique integration of field studies, mathematical models, and in vitro methods to allow mechanistic understanding and provide novel, cutting-edge approaches to test critically important ideas in the field of disease ecology.

Sea ice is a major, at times ubiquitous, feature of the biosphere, where bacteria, archaea and eukaryota can survive and thrive at freezing and deep freezing temperatures. Although of fundamental importance to understand the origin of life and its occurrence elsewhere in the universe, the questions of how life colonizes sea ice while concentrating up to 20,000 times in this natural bioreactor, and adapts to drastic phase shifts during ice growth remain mysterious. Meshing biology, glaciology, bioengineering, biochemistry, and optics, this project will use ice-dwelling microalgae as models to characterize at both the submillimeter crystal size and textural subcentimeter scales the physical and biochemical processes that lead to colonization of growing sea ice, and the physiological and genetic controls underpinning the survival and plasticity of biota during phase changes. Purpose-built sea ice chambers will allow the growth of various types of sea ice and monitor interactions with microalgae during key phases including nucleation of ice crystals, scavenging of cells by ice crystals, and colonization of this porous medium filled with brine inclusions. To specifically address the possible role of nucleation in the colonization of sea ice by microorganisms, experiments will also be conducted using a novel microfluidic approach where individual diatom cells will be injected in pressure-controlled supercooled seawater.

Symbiosis with prokaryotes is a key adaptive strategy used by unicellular eukaryotes (protists) that live without oxygen. Instead of using oxygen as an electron sink, anaerobic protists rely on electron sharing with a prokaryotic partner. Although this process, known as syntrophy, has evolved many times independently, the metabolic adaptations and cellular integration underlying these partnerships are poorly known. This is despite the fact that protist-prokaryote syntrophies are ubiquitous in oxygen-depleted habitats, where they are key to biological food webs and the cycling of globally important compounds (e.g., the greenhouse gas methane). Here, we will combine evolutionary genomics, microbial physiology, geochemistry, and advanced cell biology methods to determine convergent metabolic strategies and symbiosis infrastructure across three distinct eukaryotic lineages (ciliates, jakobids, and breviates) separated by almost two billion years of evolution. We hypothesize that these distinct lineages will share electrons via the exchange of hydrogen using a combination of lineage-specific and conserved metabolic and structural adaptations, and such interactions will be sensitive to environmental factors. Objective 1: Using genomics, we will deliver high-quality genomes of these representative protists and their symbionts. These genomes will be used to infer metabolic interactions between different partners and, using phylogenetic inference, we will identify lineage-specific and conserved features of symbiosis. Objective 2: We will optimize growth conditions of each microbial culture and establish methods to quantify cell growth under different abiotic parameters such as temperature and oxygen concentration. Metabolic flux analysis will be used to discover which metabolites are shuttled between the protists and their symbionts. Objective 3: We will establish high-resolution single-cell imaging to map the ultrastructure of the protist:prokaryote interaction. This imaging will be coupled to spatial proteomics that will deliver a subcellular atlas of protein localization. Our results will deliver a holistic understanding of electron-sharing symbioses in unrelated anaerobic protists that can only be accomplished by combining distinct technology and organismal expertise.