Molecular genetics architecture laid foundations to many biological disciplines. However, we know today that biological functions and activity of DNA in our genomes cannot be predicted by linear sequence information alone. The local and global structure of chromatin determines the accessibility and activity of genomic DNA in the nucleus. A fundamental question in biology is what the order of DNA assembly as chromatin in the nucleus and how this order is influencing biological activities. Visualizing chromatin structure in the nucleus requires high resolution and could only be achieved by using electron microscopy (EM). Unfortunately, conventional EM stains do not enable the complex mixture of proteins and nucleic acids in the nucleus to be distinguished from each other. Thus, our current understanding of chromatin structure is based largely on in-vitro reconstitution experiments of purified nucleosomes and DNA and pre-extracted nuclei. O’Shea lab has developed ChromEM, which exploits a cell-permeable fluorescent dye that binds specifically to DNA and enables the high-resolution structure of chromatin in intact nuclei to be determined using correlated light and EM (CLEM). I propose to use Hi-C technique together with ChromEM to illustrate chromatin ultrastructure and 3D genome organization in vivo in the mouse lung. Furthermore, I will exploit state of the art genetically engineered mouse models (GEMMs) of the different subsets of human lung to visualize how chromatin ultrastructure, 3D genomic interactions and nuclear organization are transformed by oncogene/tumor suppressor mutations upon CRE driven tumor initiation and clonal progression.

Cell division is essential to organism growth and repair. The spindle, a dynamic machine made of microtubules, drives chromosome segregation during cell division. Segregation errors can lead to birth defects and diseases such as cancer. Animal spindles contain two poles where microtubules focus and where segregated chromosomes go. Spindle poles need to be robust to oppose segregation forces, and yet flexible and dynamic to remodel themselves as microtubules turnover – how do they achieve this? Many of the molecules focusing poles are known, but the lack of mechanical and spatially controlled approaches inside cells has left us with a poor understanding of how molecules engender the mechanics, architecture and function of poles.
The goal of this project is to define the basic mechanical architecture of the mammalian spindle pole and how it gives rise to its function. To this end, I will: (Aim 1) Use targeted laser ablation to spatially map the distribution of pole focusing forces and unravel basic pole mechanical design; (Aim 2) Use optogenetics to control dynein- and NuMA-based pole focusing molecular mechanisms in space and time to probe their contributions to spindle mechanics; (Aim 3) Determine how the robustness of pole focusing to external challenges changes across cancer cells with different spindle architectures. These new “optomechanics” approaches and framework will be applicable to other cytoskeletal architectures, and will provide insight into how nm-scale parts self-organize into mechanically specialized micron-scale structures such as spindle poles. In the long run, pole mechanical differences could be harnessed to selectively target cancer cells for therapy.

Germinal centers (GC) are specialized anatomical sites where B cells mutate their antibody-encoding genes, clonally expand, and compete for T follicular helper cell (Tfh) help signals, as part of the antibody affinity maturation process. Tfh select B cells bearing high affinity antibodies for proliferation and differentiation into antibody secreting cells by forming short-lived synapses. These transient dynamic interactions support a gradual increase in antibody affinity, as they permit Tfh cells to constantly scan B cells for improved antibody variants. Although the synapse between naïve T cells and innate cells is well characterized, less is known about the Tfh-B cell interactions and how they control the B cell fate in GCs. In the proposed study, we will utilize intravital multiphoton imaging techniques in combination with novel reporter mice for investigation of Tfh-B cell synapses. We will examine GCs in vaccinated peripheral lymph nodes and in the small intestine Peyer’s patches (PP), distinct lymph nodes exposed to a unique immunological environment. By using novel mice bearing fluorescent reporters, we will define the molecular machineries, cytoskeletal changes, adhesive machineries and signaling events that take place during transient T-B cell interactions within the popliteal lymph nodes and PP. Furthermore, we will study how these events control the frequency of B cell departure from the GC and the molecular machineries that regulate this process and their ultimate fate. This study will provide novel insights into the affinity maturation process, and may have implications for vaccine design.

Infectious diseases are a leading cause of mortality and morbidity in sub-Saharan Africa. Despite the burden of viral infections in this region, little is known about the virome of African populations or the role of microbial and host genetic factors in susceptibility to infection. In this project we will integrate microbial metagenomics, human genomics and functional genetics to identify and characterize the causes of acute febrile illness in Senegal and to provide novel insights into host and microbial variants that may be involved in susceptibility and pathogenicity. To do so, we will first collect blood samples from a large cohort of healthy and febrile individuals from Senegal and use next generation sequencing to comprehensively identify circulating RNA viruses present in the blood. Next we will use this metagenomic data to characterize the genetic diversity of circulating pathogens and gain insight into their origin, transmission and on-going evolution. We will also determine the causes of febrile disease, by comparing the frequency of viruses between febrile patients and healthy controls, and evaluate the importance of co-infection on disease outcome in Senegal. Lastly, we will profile the genetic variation of patients and controls and use computational and experimental approaches to predict and functionally dissect the viral and host genetic factors influencing susceptibility to and outcome of infection. Together the findings of this study aim to benefit both research into and the care of acute febrile infections.

The vagus nerve is a key part of the neuroendocrine axis that controls feeding behavior and metabolism. Within the gastrointestinal tract, vagal sensory neurons detect ingested nutrients and mechanical stretch of the stomach, although underlying sensory transduction mechanisms are not understood. Basic questions remain about how ingested food is sensed, and how inputs are relayed centrally to coordinate systemic responses. Unraveling the functions of different vagal sensory neuron types in feeding behavior and metabolism control would provide a basic understanding of gut-to-brain communication mechanisms, and perhaps provide new therapeutic targets to control appetite and help treat metabolic disorders like diabetes, obesity and anorexia. Here, I will characterize a subpopulation of vagal sensory neurons that express cholecystokinin receptor type- A (CCKAR), a receptor for the gut satiety hormone cholecystokinin (CCK). I will generate Cckar-ires-Cre knock-in mice, and will use powerful genetic techniques for anatomical mapping, in vivo imaging, and remote control of vagal afferents. I will (1) map neuron projections within the central nervous system and peripheral organs using Cre-based anatomical tracing, (2) measure neuron responses by real-time in vivo calcium imaging in vagal ganglia, and (3) determine neuron function using vagus nerve optogenetics. These studies will enable long-term efforts to study the sensory biology of the vagus nerve- from understanding signal transduction mechanisms in the periphery to determining the organization of central inputs that orchestrate behavioral and endocrine responses.

Stochastic variability is a property that is inherent to gene expression, and which can have profound consequences for cellular phenotypes, disease and development. One form of stochastic gene expression is random monoallelic expression, where - within a given cell - only transcripts from one parental allele can be detected. Despite growing interest in random monoallelic expression, many questions remain unresolved, including the molecular underpinnings of the process or if memory of allelic choice persists through mitosis.
The goal of my project is to apply novel RNA FISH methods (SNP FISH and iceFISH) to quantify expression of mRNAs in an allele-specific manner in single cells from mouse tissue or primary cell lines. Using these measurements I will infer to what extent random monoallelic expression can be explained by transcriptional bursting, and whether variability is due to intrinsic or extrinsic sources of noise. Studying allelic choices of neighbouring cells in tissue sections and in monoclonal cell lines will indicate if allelic choice is inherited during mitosis.
I will also test the potential mechanisms governing monoallelic expression: I will use circular chromosome conformation capture (4C) to map the topological environment of candidate genes and to identify their putative enhancers. Subsequently, I will test the role of these enhancers and the DNA topology by engineering rearrangements using CRISPR-Cas9 and Cre-mediated recombineering.
Collectively, these experiments will deepen our mechanistic understanding of stochastic processes in gene expression, and will help create a probabilistic framework to explain complex genotype-to-phenotype relationships.

This project will develop a microscope capable of high-speed and high image quality, which will be used to image neural activity while simultaneously applying cutting-edge approaches sensory stimulation to study multisensory integration in larval zebrafish. Neural activity in larval zebrafish is regularly studied via calcium fluorescent indicators, imaged with light-sheet microscopes. These can allow brain-wide imaging of neural activity at cellular resolution. However, it is very challenging to image large portions of the brain at suitably high speeds. We will introduce new strategies in 3D imaging to improve the image speed, without compromising the cellular resolution. We will combine this with fictive vestibular stimulation, a new class of sensory stimulation in which an optical trap is used to apply forces to the vestibular system, such that the larva perceives an acceleration. The vestibular system is extremely difficult to study using other methods, as it is activated by accelerations or rotations which are usually not permissible in a neuroscience experiment. We will combine fictive vestibular stimulation with fluorescent neural activity recording, a visual virtual reality screen, and a secondary imaging path to monitor behavior. We will investigate the neural processing of vestibular sensations, and how this is combined with the visual perception of movement. This could shed light on the more general question of how brains synthesize multiple sensory inputs into perception of the surrounding world.

An estimated one-third of the world’s population is latently infected with Tuberculosis (TB). Here, the bacteria exist in a poorly characterized host-pathogen equilibrium within granulomatous lesions in the lung. Recently, the McKinney lab used single-cell microfluidics and time-lapse microscopy to reveal a sub-population of non-growing but metabolically active (NGMA) M. tuberculosis cells in vitro and in vivo using murine models. Another sub-population of cells in the lungs of chronically infected mice resumed growth when re-introduced to salubrious conditions.
Building on this work, we aim firstly to explant thin tissue slices from the lungs of infected mice into custom-made microfluidic devices. This will be a significant advance over studying single cells extracted by homogenizing lung tissue. We will image individual bacterial cells within granulomas in a semi-native environment and investigate the spatial distribution and metabolic activity of NGMA and other cell phenotypes within the granuloma.
We then propose to reconstitute the murine granuloma environment entirely in vitro using a custom-built lung-on-a-chip microfluidic device. These novel approaches will allow real-time imaging of the roles played by NGMA and other cell types in granuloma formation and and their role in entering and exiting the latent state; challenging questions of immense significance. It will also lead to refinements of systems biology computational models for the host-pathogen equilibrium. In the long term, such a system can be extended to mimic the human lung and provide a leap forward in our understanding of latent TB, which is vital to tackling this global health challenge.

To beat wings 50-60 times per second to hover a ruby-throated hummingbird (Archilochus colubris) must burn calories faster than any other vertebrate. To provide these calories, hummingbirds ingest little more than simple sugars (glucose and fructose) found in floral nectar. Thus, their incredible flight can be powered by the simplest fuels at rates 55× those of mice, rats, or even aerobic dogs. When a tiny hummingbird stops feeding at night, it must keep itself warm by burning energy dense stored fats accumulated during evening hours, as opposed to sugars that are too heavy to carry. Similarly, when a hummingbird heads south in the fall on a long migratory journey to Central America, its journey is fueled by fat stores accounting for ~30% of its mass, built from the sugar in ingested nectar over just a few days prior to departure. Just what adaptations of hummingbird metabolism permit these extreme rates of sugar breakdown and fat deposition are not well understood. Our understanding of the function and regulation of enzyme pathways that catabolize sugars and produce fatty acids comes largely from studies of mice, rats, and humans, energetic sloths compared to hummingbirds. What we know from studies of these and other model organisms fails to explain how hummingbirds do what they do. For example, insulin plays a central role in control over blood sugar and the mixture of fuels our muscles burn, but birds are unresponsive to this hormone. Further, the metabolic pathways at work in hummingbirds are not simply faster versions of those in mice. Instead, the same metabolic machinery must operate with increased efficiency at the molecular level. Our team will use a variety of approaches, from studies of fuel use in freely-behaving hummingbirds (Welch), to studies of their enzymes (Wong), the genes that encode them (Timp), and the atomic structure of this molecular machinery for an improved understanding of its function (Valle) to build a fundamental understanding of the unique aspects of hummingbird physiology and behavior that enable their superior performance. In doing so, we will establish the hummingbird as a new model organism in which to understand the limits to physiological performance and function.

During tissue formation, interacting cells generate spatiotemporal patterns of gene expression to create multicellular structures. Traditional approaches are increasing our knowledge of molecular players and their interactions for tissue formation. Nonetheless, the principles of how such molecular players work together to achieve complex multi-cellular structures remains unclear. Synthetic “bottom-up” approaches can help reveal the logic of how self-organizing multicellular systems are constructed. Here, I will build artificial communication circuits between cells to investigate their potential to build complex structures. I focus on two types of communications that we postulate to be most important for pattern formation; direct contact communication mediated by transmembrane proteins like Notch-Delta and short-range paracrine signaling by lipid-modified secreted proteins like Wnt. In preliminary unpublished work, we have designed synthetic Notch receptors that recognize artificial ligands on membrane to induce specific gene expression. Synthetic receptor/ligand pairs avoid crosstalk with native Notch/Delta and can build multiple sets of orthogonal Notch-like communication channels. I will design an analogous synthetic Wnt ligand which keeps its spatial diffusion property and is recognized by a synthetic Notch receptor. I will explore how circuits with multiple synthetic receptor-ligand combinations can generate structures by screening a library of synthetic Notch and Wnt combinations. The comprehensive elucidation of relationships between simple intercellular circuits and self-organized patterns could be useful in guiding tissue engineering and regenerative medicine.

Serum cytokines induce dimerization of receptor extracellular domains (ECDs) to enable the central orchestrators of cytokine signaling, Janus family tyrosine kinases (JAKs), nexus inside the cell. This event is responsible for initiating signaling cascades associated with an extensive range of functional outcomes, and therefore dysregulation of the signaling causes many types of cancers. However, the structural link between JAKs and cytokine receptors remains poorly understood. The proposed work will advance our knowledge of this essential process, combining multifaceted structural and functional approaches to visualize the entire interleukin (IL)-6 signalosome as a model for cytokine-induced JAK signaling. In the first aim, we will determine an X-ray crystal structure of a receptor intracellular domain (ICD) bound to JAK1 N-terminal FERM domain. Given that the JAK domains are highly conserved, the binding mode elucidated from our structure should be widely applicable to all JAK-receptor pairs. In the second aim, we will explore JAK activation from an ultrastructural perspective. Cryo-electron microscopy (cryo-EM) single-particle reconstructions of a transmembrane holocomplex will yield an unprecedented 3-dimensional view of the orientation of signaling molecules in the working state. Taken together, our work will provide a thorough mechanistic view of JAK activation and inform design of novel molecular therapeutics for cancer.

The validity of Jean-Baptiste de Lamarck’s hypothesis of inheritance of acquired traits has long been a matter of great debate. The research plan proposed here, will utilize technically and conceptually innovative approaches to explore the molecular, physiological and mechanistic basis of transgenerational inheritance of acquired traits in C.elegans. First, I will determine transcriptomic changes in the nervous system in the F5 generation of starved ancestors. To detect subtle, but potentially biologically-important adaptations in highly specialized neurons, I will make use of INTACT that allows for tissue-specific mRNA isolation, which will then be subjected to next-generation sequencing and bioinformatical analyses. Second, I plan to identify stress-dependent physiological and behavioral adaptations in locomotory behavior, metabolism and sensory perception. Importantly, to decipher the relative contribution of small RNAs in mediating molecular and physiological behavioral changes, the previously described experiments will not only be performed in descendants from N2, but also from neuron-specific Dicer-1-deficient ancestors. Dicer-1 is critical for the generation of distinct classes of small RNAs, i.e. miRNAs and endo-siRNAs. Finally, upon identification and subsequent GFP-tagging of genes that are transgenerationally deregulated upon ancestral starvation, I will perform unbiased forward genetic screens using EMS. Mutagenesis prior to or after starvation will allow for the identification of novel signals involved in the initiation of the original stress response, as well as for the maintenance and inheritance of adapted gene expression across generations, respectively.

There is growing evidence that acute myeloid leukemia (AML), a cancer of the blood, arises through the accumulation of mutations that originate in self-renewing hematopoietic stem cells (HSC). At diagnosis, patients harbor pre-leukemic (pre-L) HSCs that possess a subset of the mutations that leukemic cells contain with DNMT3a being the most prominent example. These HSCs have a competitive growth advantage that result in their clonal expansion. However, the molecular mechanism explaining how mutations in DNMT3a or other pre-L genes confer clonal expansion in HSCs and whether there are synergistic effects between pre-L mutations is not known. My research plan provides a framework to identify pre-L driver events and determine how these changes disrupt normal HSC function leading to pre-L expansion. I am proposing to utilize the CRISPR/Cas9 system to conduct loss-of-function studies of DNMT3a and other newly identified pre-L genes. For this, I will screen individual and combinations of pre-L genes in HSCs and assay them for clonal expansion and differentiation phenotypes. Subsequently, I will characterize the molecular mechanism of functionally validated pre-L genes by comparing the transcriptional profile of pre-L HSCs to wild-type HSCs. I will investigate the consequence of loss of pre-L genes in differentiated cells as opposed to HSCs to gain insight into why HSCs are the cell of origin for these pre-L mutations. By identifying patterns and modes of action of pre-L genes, we will advance our knowledge of leukemia initiation. Because pre-L cells persist during remission and may participate in relapse, this study could aid in the development of targeted therapy against AML.

The RNA exosome is an essential multi-protein machine providing ribouclease activity in degradation and processing of a vast array of different cellular RNAs. It is composed of two multimeric protein rings forming a catalytically inert, barrel-like structure. Ribonuclease activity of the complex is conveyed by two additional proteins, Rrp6 and Rrp44, associated with the top and bottom end of the barrel, respectively. These two subunits contain three separate active sites and, to date, mechanistic understanding of how their activities are coordinated during a multitude of cellular functions ranging from precise 3'-end maturation of precursor RNAs to processive degradation of aberrant transcripts remains limited.
Using an in vitro reconstitution system, I propose to study how access of substrates to the different catalytic sites of human exosomes is regulated through a number of auxiliary factors, including the RNA helicase Mtr4. The specific aims of my project are:
A) Structure determination of hitherto unresolved human exosome complexes. I will combine X-ray crystallography and innovative electron microscopy (EM) to study interactions and conformational dynamics upon cofactor binding. I will further investigate paths for substrate engagement using tagged RNAs in EM imaging and biochemical RNA crosslinking experiments.
B) Biochemical analysis of activities of exosome complexes on a range of substrates mimicking physiological targets, such as RNA/DNA hybrids and structured RNAs.
This study will constitute an important step towards understanding how the diverse activities of human RNA exosomes are coordinated and controlled as part of larger multi-modular complexes in vivo.

Schizophrenia (SCZD) is a complex disorder of the human brain regarded as a ‘disease of synapses’, with reduced connectivity and synaptic vesicle release affecting neuronal networks of SCZD patients. However, how synaptic defects perturb the formation and activity of neuronal networks in SCZD is not understood. Neuronal networks are commonly characterized using graph theory and neuronal activity data. Neuronal activity can be recorded across large neuronal tissues using functional multineuron calcium imaging (fMCI). 3D neuron cultures, termed cerebral organoids, recapitulate human cortical development and disease and generate various types of neurons, long-range axon projections and neuronal activity, making them an ideal model for studying neuronal network development. I propose to establish a cerebral organoid based model to characterize neuronal network formation during cortical development using fMCI and graph theory and apply this model to analyze abnormal neuronal networks in SCZD. I will genetically introduce the fluorescent calcium indicator GCaMP6f into wild type and SCZD patient-derived, induced pluripotent stem cells and grow them into cerebral organoids. I will record neuronal activity in the developing cortices of cerebral organoids using high-speed fluorescent calcium volume imaging and identify, characterize and compare the present neuronal networks using graph theory. I will then test different antipsychotic drugs for their ability to restore normal neuronal network formation in SCZD organoids. This will reveal important insights into the pathology of SCZD. I envisage this model to become a valuable tool for future cognitive disease research.

Protein misfolding has been linked to numerous neurodegenerative diseases in humans. Decades of biomedical research have illustrated how cellular stress-response pathways evolved to reprogram the biochemical landscape of the cell to avert the deleterious effects caused by protein misfolding. However, previous studies focused on the protein-based stress response pathways with little attention on the role of metabolism in such response. It is the hundreds of thousands of metabolites that constitute an integral part of the physiological environment inside the cell, yet their contribution to the overall cellular robustness remains largely unknown. Sessile plants produce a variety of chemically diverse specialized metabolites as means to cope with a myriad of abiotic and biotic stresses. I hypothesize that a subset of such chemodiversity may be dedicated to resolve protein-misfolding problems. To test that, I will generate a series of Arabidopsis transgenics overexpressing those human neurodegenerative proteins prone to misfold. Using this system, I will investigate how the plant proteostasis system buffers against the toxicity of neurodegenerative protein aggregates, how the tipping point is reached when the system eventually collapses, and what biological functions these protein misfolding-induced metabolites play in mitigating proteolytic stress. I seek to understand the molecular interplay between metabolic regulation and protein misfolding stress, which may yield new insights for developing new treatment for human neurodegenerative diseases.

Plant growth depends on meristems, groups of pluripotent stem cells. The shoot apical meristem (SAM) is responsible for indeterminate growth and establishing vegetative and reproductive architecture. The size of the SAM is tightly regulated during development by balancing stem cell proliferation with the incorporation of daughter cells into primordia. This balance is maintained by the well-known CLAVATA-WUSCHEL (CLV-WUS) feedback signaling pathway between the stem cells and the underlying niche cells in the organizing center. However, nothing is known on signaling between stem cells and differentiated primordia. Studies in the host lab on the FEA3 (FASCIATED EAR 3) gene in maize, which encodes a novel CLV-related receptor-like protein, has led to a fundamentally new hypothesis, in which signals from differentiating cells in leaf primordia can provide feedback to the stem cell niche. Here, I propose a detailed analysis of FEA3 and its potential ligand, ZmFCP1, to test this novel signaling hypothesis. The functional study of FEA3 interactors and deciphering of its network by combining IP-MS (immunoprecipitation-mass spectrometry), transcriptomics and reverse genetic approaches, will shed light on its significance and molecular mechanism. In addition, forward genetics combined with genomics approaches will be used to clone new meristem regulation mutants in maize, to identify novel components in FEA3 or different meristem signaling pathways. By the proposed experiments, I will test the hypothesized novel signaling paradigm and investigate FEA3 function, which will shed light on plant development and stem cell regulation.

Animals learn from experience and experiences during early development have a profound influence on adult behavior. The goal of this proposed study is to understand, at the anatomical and synaptic level, how early experiences impact the development of the brain and influence behaviors at a later time. I will use a genetic tool developed in my host lab, combined with a newly developed high-volume whole brain imaging technique, to construct, for the first time, a longitudinal map of neuronal ensembles that are activated by experiences during early development in the mouse. I expect to discover the order in which different brain regions are activated by experiences during early development and how this sequence of events determines the functionality of the mature brain. I will then use post-weaning social isolation, a well-established early behavioral perturbation known to cause anxiety behaviors in the adulthood, to investigate how functional neuronal ensembles are perturbed and how the perturbation leads to anxiety behaviors. Finally, I will use mouse models of autism spectrum disorder (ASD), a neurodevelopmental disorder, to examine how disease conditions affect the response of the brain to experiences during early development.

Studies in developmental neurobiology focus on identifying factors involved in determining the fate and role of each neuron and how these neurons function together to constitute the nervous system. Each neuron differs with respect to their morphology and function, established during development by differential expression of select genes. We propose to perform a systematic approach, where we will identify differentially expressed transcripts in sets of isolated neurons using RNA-seq and couple ectopic expression experiments and traditional genetics to convert the identity and function of neurons. We will utilize the model organism C. elegans to convert PDE, a simple ciliated sensory neuron, into its sister neuron, PVD, a multi-dendritic sensory neuron, at the level of both morphology and function. This systematic approach of neuronal transformation has not been previously attempted and will be a new direction towards studying neural development.

Protein secretion is a fundamental process in which selected proteins within the cell are translocated to the extracellular environment by specialized secretion systems. The type 1 secretion system (T1SS) is widespread in Gram-negative bacteria and plays a critical role in nutrient acquisition, infection, and adaptation. The T1SS consists of three membrane proteins: two in the inner membrane, which belong to the ATP-binding cassette (ABC) transporter and membrane fusion protein (MFP) families, and an outer membrane protein (OMP). Through decades of characterizations, we have an overall understanding of this pathway. However, structural details of substrate recognition and translocation remain unknown. I propose to investigate this structure using cutting-edge cryo-electron microscopy (cryo-EM) techniques congruent with biochemical and electrophysiological approaches. First, I will establish methods to purify a stable T1SS complex. Next, I will reconstitute the T1SS complex into lipid bilayers. Extensive functional studies will be conducted to analyze the dynamic changes of ATPase activity and the properties of translocation channel formation. Cryo-EM single particle analysis will be used to determine high-resolution structures of the intact translocator complex. These studies should lead to unique insights into not only the structural basis of the translocator assembly but also the mechanisms of protein secretion.