Asthma is a clinically heterogeneous disorder caused by genetic and environmental influences that remain poorly understood. Promoter variations that induce over-expression of the Orosomucoid-like 3 gene (ORMDL3) in humans are the strongest genetic associations with severe childhood asthma. Orm-family proteins are a component of an essential enzyme complex, the SPOTS complex, for Serine Palmitoyl-transferase, Orm, Tsc3, and Sac1 complex, which catalyzes the rate-limiting step for de novo sphingolipid synthesis. Orm-family proteins act as inhibitors of Serine Palmitoyl-Transferase (SPT) and play an integral role in sphingolipid homeostasis. Elevated expression of ORMDL3 inhibits the synthesis of sphingolipids that appear to be protective against asthma and other inflammatory diseases. These observations raise the exciting possibility that sphingolipid synthesis may be a point of intervention that is orthogonal to existing therapies. However, the molecular architectures of the SPOTS complex and the Orm proteins, as well as the details of their functional roles in sphingolipid metabolism, are entirely unknown. I propose to determine the structure of the SPOTS complex in different functional conformations using cryo-electron microscopy (cryo-EM) and to characterize the mechanisms governing sphingolipid homeostasis with biochemical methods. These results will improve our understanding of sphingolipid homeostasis and the pathogenesis of asthma and other inflammatory diseases.

Preservation of genome integrity is fundamental to the maintenance of life. Double-strand breaks (DSBs) are a particularly lethal form of DNA damage and cells have evolved conserved and dedicated pathways to repair the same. Although important for cell viability, recent studies have shown that DSB repair can also be a potent source for mutagenesis. While cells in all domains of life are capable of faithful repair via homologous recombination, eukaryotes and some bacteria also employ an alternative, end joining pathway for DSB repair. This pathway is error-prone, but it avoids the lethality of a DSB, making it important. End joining has been well studied in eukaryotes. However, this pathway was relatively recently discovered in bacteria and its mechanism of regulation remains unclear. The objective of this proposal is to study the regulation of error-prone DSB repair in the opportunistic pathogen, Pseudomonas aeruginosa, which encodes for an active end joining repair pathway, and assess the role of such repair in bacterial growth and survival during stress. Using single-cell and single-molecule fluorescence microscopy in combination with genome-scale assays, this study aims to comprehensively elucidate the mechanism of end joining repair in vivo and understand how cells regulate pathway choice between homologous recombination and end joining-mediated DSB repair. Given that end joining repair is conserved across several pathogenic bacteria as well as eukaryotes, this study should provide novel insight into general principles of mutagenic repair and its likely impact on antibiotic and stress resistance.

In this project, we will provide new insights into the behavioral strategies, biomechanics and motor control animals use to perform complex versatile and adaptive functions and provide novel bio-inspired robot technology for solving complex motor control problems.
Despite the constraints imposed by their miniature brains and bodies, dung beetles are capable of the most impressive
feats of motor control and navigation. Beetles of the species Scarabaeus galenus are able to move large objects – dung balls that can exceed 10 times their own weight – accurately between a food source and their burrow by combining celestial compass orientation with step-counting. In addition, they are able to use their specialized body and legs to dig burrows, manipulate different dung types and move them over different types of terrain (e.g. sand or grass) using a variety of locomotor patterns. How do they achieve these complex versatile and adaptive behaviors when moving over difficult terrain? Our project aims to address this through our integrative approach which combines behavior (EB), biophysical, kinematic and biomechanical analyses (SG) of S. galenus, and computational motor control and robotic modeling (biorobotics, PM).
Research on behavior and neuro-biomechanical functions of insects has, until now, largely dealt with few behavioral modes – like walking, climbing, or navigating – in isolation. We will launch a major research effort aimed at uncovering behavioral strategies, biomechanics, and computational motor control that enable the miniature brains and bodies of insects to effectively generate multiple adaptive behavioral modes. Our behavioral experiments, both in the field and in the lab, will investigate locomotion, navigation, food transportation, and their combination in S. galenus. The data from these experiments will lay the foundation for novel biophysical, kinematic and biomechanical analyses, and computational motor control development. Simultaneously, biomechanical principles underlying the different behavioral modes will be used to shape the behavioral experiments and guide the structural design of robotic modeling. We will develop a biorobotic model that will enable us to test our hypotheses about the relationship between structural and mechanical features and how they interact with motor patterns and neural mechanisms to form complex behaviors.

The ribosome is the universal translation machine, which makes all the proteins in the cell, including its own. Much is known about ribosome structure and assembly pathway. However, cell-free synthesis and assembly of ribosomal proteins and rRNA into functional ribosomes has not been reconstituted, which would be essential for establishing a protein-based self-replicating model system of artificial cells, and provide a new means to develop antibiotics. We will address the challenge of cell-free ribosome biogenesis by combining the reconstitution of E. coli translation from purified components developed by Shimizu and Ueda (PURE system), with the miniaturized on-chip DNA compartments introduced by Bar-Ziv's lab. We address following three questions: i) What are the minimal requirements for cell-free ribosome biogenesis?; ii) How is ribosome biogenesis affected by excluded volume interactions, segregation, and macromolecular gradients in the DNA compartment?; and iii) Can DNA brushes coding for rRNA and ribosomal proteins mimic spatial ‘operons’ and promote efficient assembly coupled to synthesis? For these questions, we will attempt to 1) develop a methodology to label and localize cell-free synthesized ribosomal proteins and rRNA; 2) tailor the PURE system to support cell-free ribosome biogenesis reactions; 3) tailor DNA compartments geometrically to direct ribosome biogenesis and measure synthesis and assembly or ribosomal proteins and rRNA using time-lapse fluorescence microscopy; and 4) develop approaches to resolve, physically and biochemically, new from old ribosomes. The combination of molecular biology and biochemistry with soft matter physics and materials science are expected to provide favorable conditions for in vitro ribosome biogenesis and novel means to separate new from existing ribosomes.

A fundamental challenge in biology is to understand how brain cells change in response to experience and how these changes contribute to memory and establishment of other long-lasting behaviors. Changes in neuronal gene expression are important for memory formation, maintenance and retrieval. Recent advances in genome biology have found that three-dimensional (3D) genome organization—genome topology—is critical for transcriptional regulation. We hypothesize that neuronal genome topology provides special “topology codes” for activity-driven transcriptional modulation in neuroplasticity, and abnormal topology codes contribute to cognitive disorders. This project will examine genome topology and transcriptomics in mouse hippocampus and explore how the dynamics of genome topology contribute to the rapid and highly coordinated transcriptional response during learning and the long-lasting changes in gene expression that underlie memory, epilepsy and other enduring forms of neuroplasticity. Our multidisciplinary team with complementary skills in neuroscience, genomics and bioinformatics will take an integrated approach that includes a novel 3D genome mapping strategy for small numbers of cells (Ruan), advanced genetic labeling and biochemical isolation techniques for transcriptional and epigenomic profiling of neuronal ensembles (Barco), and state-of-the-art, multimodal microscopy for nuclear imaging (Wilczynkski). We will develop and optimize our platform in cultured neurons subjected to different stimuli (Aim 1), and we will examine the 3D epigenomic dynamics of the nuclear response to synaptic activity in two well-established in vivo paradigms: epileptic seizure (Aim 2) and the formation of associative memory (Aim 3). The large volumes of high-quality and multiplex genomic data representing dynamic changes in neurons in response to activation will be integrated by comprehensive computational analysis (Aim 4). Predicted genetic loci that have important roles in chromatin topology and transcriptional regulation will be validated structurally by super-resolution nuclear imaging and functionally by genome editing (Aim 5). The results of this effort will open new chapters in learning and memory and epilepsy research by laying a foundation for understanding the dynamic and topological mechanisms of genome regulation in neuronal plasticity in health and disease.

When hearing a sound, structures of the cochlea vibrate. These mechanical vibrations are transformed by sensory inner hair cells (IHC) into electrical activity that travels to the brain where it is decoded. Sounds of different frequencies resonate at different locations along the cochlea. The resonant properties of the cochlea are enhanced by the activity of outer hair cells (OHC) that use metabolic energy to amplify the faintest sounds and sharpen the frequency selectivity. Because of a coupling between cochlear mechanics and neuronal processing, it is still unclear how a complex sound containing multiple frequencies is integrated by the brain and what is the role of the cochlear amplifier.

Here, we will deconstruct cochlear vibrations using optogenetics. We will express channelrhodopsin in IHC and stimulate them with single cell resolution to isolate the neuronal processing. The electrical activity of neurons will be monitored at key locations along the auditory pathway. First, we will evoke auditory response in the brain with optical stimulation of IHC and reproduce the response to a single tone using the appropriate pattern of light stimulation. Second, we will decipher the role of spatial and temporal information in auditory processing. Third, we will use vocalizations and understand how cochlear mechanics shapes the integration of complex sounds at the periphery of the auditory system.

This proposal will provide important information about the role of both cochlear mechanics and neuronal processing in shaping the sensation of sounds. These results will be of particular importance in the development of a new generation of cochlear implants based on optogenetics.

This work leverages cutting-edge genomic and shape analysis methods to study the evolution of mammalian cooperative societies. Current approaches to studying vertebrate cooperative behavior work in the classical, but mechanism-free, frameworks of behavioral ecology and life history theory. Thus, while cooperation itself is of long-standing interest, we know little about how animals that occupy distinct roles in cooperative societies differ at the molecular level.
Here, we propose to integrate new molecular and computational approaches with a 24-year field study of the most cooperative nonhuman mammal yet described, the meerkat. Like cooperative insects, meerkat societies are characterized by a division of social roles: adults are dominant breeders or morphologically and physiologically distinct helpers, who feed and guard the breeders’ young. However, all helpers retain the capacity to transition to breeder throughout life. Meerkats thus present an exceptional opportunity to study alternative phenotypes in cooperative societies. We will first investigate how helpers and breeders are differentiated at the gene regulatory level, including whether steroid hormone signaling generates these differences. Second, we will test social role-driven differences in growth and immune defense. We will track growth by developing computational geometry-based approaches to perform 3D skeletal reconstructions from X-ray data and immune defense using experimental pathogen challenges. Finally, we will test the hypothesis that the competing demands of growth, reproduction, and immune defense create “competition” at the transcriptional level, which is resolved differently by helpers and breeders. Our experiments will not only quantify phenotypic transition-associated trade-offs, but also identify the genes and pathways that mediate them.
The methods required for these analyses either do not exist or will need to be generalized to field studies for the first time. Thus, this study requires collaboration across behavioral ecology, genomics, immunology, and computational image analysis. Together, it will contribute a powerful model for applying modern tools to long-standing puzzles in evolution and behavior. It will also yield new insight into the molecular changes involved in the evolution of cooperative societies—a subject of fascination and controversy for almost two centuries.

Pre-implantation development in mammals involves complex processes at the level of genomic organization, DNA methylation and transcriptional activity, leading to embryonic genome activation. How different epigenetic markers interact in single mouse blastomeres at early stages of development to influence the transcription of genes, and to what extent these processes vary from cell to cell and between embryos, is not fully understood. Although, in recent years a number of methods that enable single-cell omics measurement have been described, their integration to measure more than one entity in a single cell has remained challenging. Here, I propose a novel strategy for the integrated measurement of different epigenetic markers in combination with the measurement of nascent transcripts in single cells. The strategy relies on the use of a combination of specific restriction enzymes, for the measurement of 5-methylcytosine (MspJI), 5-hydroxymethylcytosine (AbaSI) and accessible chromatin (MseI and/or NlaIII), as well as 2’-fluoro GTP analogues in combination with specific RNases for the measurement of nascent transcripts. The application of these technologies to pre-implantation mouse embryos will shed light into the relationships of different epigenetic markers and their influence on transcription at the single cell level, and how these processes result in cell differentiation during early development. I further propose to use new deep-learning libraries, such as TensorFlow or Caffe, to quantitatively model and gain understanding of these dynamic processes in single blastomere cells.

The poor regenerative capacity of central nervous system (CNS) neurons leads to devastating and irreversible outcomes after injury or disease. The CNS is an immune privileged site, which maintains specialized crosstalk with the immune system. While the immune response to acute injury in the CNS is very similar to the wound healing process in other parts of the body, this response is oftentimes insufficient to achieve significant repair and restoration of neuronal function. One of the key challenges in analyzing this response is the diversity of the types of cells that participate in the process, which change with time and some of which may be unknown. Cutting-edge advances in massively parallel single-cell genomics, many pioneered by the Regev lab, will now enable me to analyze the entire injured tissue, at high, single-cell resolution, that takes on its complexity, heterogeneity, and temporal variation. Using this approach and comparing low- and high-regeneration states could help identify junctions where the immune response is insufficient to support regeneration, new cell types involved in the process, and targets for therapeutic intervention. Emerging cellular and molecular candidates will be perturbed experimentally in vitro and in vivo, using CRISPR/Cas9 technology and cell augmentation and depletion experiments, to validate their significance for neuronal regeneration. Human samples will be incorporated to establish validity in the clinical setting. The findings could be relevant for different types of CNS trauma, inflicted though mechanical or biochemical injury, autoimmune, neurodegenerative or vascular disease, at the brain, eye and spinal cord.

For individual genes, the number of transcripts per cell typically increases with cell size, yet how this is achieved is unknown. Recent image-based analysis suggests that both cell volume and surface area contribute to this regulation, with distinct classes of genes showing tighter correlations with either one of these two features. Here, I aim to go beyond these correlations – dissecting the mechanism by which cell geometry shapes the transcriptome. First, using quantitative RNA-sequencing and RNA metabolic labelling in cells with a range of defined shapes and sizes, I will define genome-wide classes of transcripts that scale with cell volume and surface area, and analyze the extent to which regulation of transcription and RNA stability contribute to this scaling. High-resolution mapping of changes in chromatin accessibility and modification state in these constrained cells will also be used to reveal the basis of transcriptional control and to suggest DNA-binding factors involved. Second, high-throughput image-based quantification of specific transcripts in single cells will be combined with genome-wide genetic screening. For each single-gene knockdown, this will generate a multivariate dataset containing the absolute abundance of a panel of selected transcripts in addition to hundreds of single-cell and population-context features. I will specifically identify and pursue genetic perturbations that disrupt the scaling of transcript abundance with either cell volume or area. Following these unbiased approaches with hypothesis-driven experiments and mathematical modeling will provide insight into how single cells precisely adapt genome expression to cell size and shape.

Human mobility is made possible by skeletal muscles, a voluntary tissue under the control of the central nervous system. Upon injury or insult, skeletal muscle regenerates to restore its form and function. Muscle regeneration requires an adult stem cell population termed satellite stem cells (SCs) that reside in a specialized ‘niche’ atop the sarcolemma of multinucleate myofibres and ensheathed by a proteinaceous matrix. Most of the time SCs are quiescent; a state characterized by inactivity. Injury induces SCs to activate and produce committed progenitors called myoblasts that fuse into multinucleate myofibres. In the present state of our knowledge, SC activation is an essential and intriguing process that is not well understood. Although SC activation is tightly controlled in the body, when SCs are removed from the body and studied in two-dimensional culture, activation is the default state. This observation points to the enticing possibility that physical or structural aspects of the three-dimensional tissue microenvironment might contribute to the spatial and temporal control of SC activation. In particular, we hypothesize that injury rapidly alters the SC niche mechanical stress profile and that this serves to engage intracellular mechanisms that tip the balance in favor of SC activation over quiescence by directly altering transcriptional activity in a highly localized manner. The Gilbert (Canada) and Betz (Germany) labs share a common interest in resolving the impact of mechanical stresses on cellular fate, and the Darzacq (USA) is driven by a curiosity as to how the nuclear environment imposes on the fundamental rules of gene regulation. By joining efforts in an international collaboration, we aim to (a) define the mechanical stresses (e.g. compressive, shear) exerted on SCs in resting and regenerating niches and (b) determine the molecular implications of niche mechanics as they relate to the SC transcriptome in the native niche, for the first time. We will integrate our three distinct experimental approaches – SC cell and molecular biology (Gilbert), quantitative biophysics (Betz), and high resolution single molecule imaging of transcription factor binding and nascent transcript activity (Darzacq), to conduct a collaborative study and achieve a holistic understanding of the earliest events that evoke SC activation in the native niche.

Sleep takes approximately one third of our lives yet its functions remain elusive. Extensive efforts have been made and hypotheses raised to link sleep with synaptic plasticity and memory consolidation. The sleep/wake pattern and sleep architecture evolve as development proceeds, and so does cortical connectivity, as featured by spinogenesis in early postnatal life and spine pruning during later adolescence. Several subcortical neuromodulators, including hypocretin, norepinephrine and dopamine, play critical roles in promoting arousal and thus control the sleep/wake cycle. We hypothesize that these arousal circuits help to shape the synaptic connections in cortex during development and the disruption of coherent sleep/wake cycle at certain developmental stages may cause defects in brain functions in adulthood. Mechanistically, synapses “tagged” by the arousal inputs during the previous wake phase may be selectively strengthened while other “untagged” synapses weakened in the following sleep. We will combine a series of state-of-art techniques including optogenetic and/or chemogenetic manipulations, viral tracing, two-photon live imaging as well as fiber photometry to interrogate these hypotheses, which will broaden and deepen our understanding towards sleep and brain plasticity in a developmental point of view and provide potential clinical insights on sleep and arousal disorders.

Living cells organize their interior in a non-random, polarized manner to carry out specialized biological functions. Cell polarity arises from a complex interplay between the plasma membrane, associated proteins and cytoplasmic molecules. In eukaryotic cells, symmetry is in most cases initially broken at the level of membrane-bound molecules such as phosphatidylinositol lipids and Rho-type GTPases. These conserved signaling hubs synergistically control cell morphogenesis and movement through the actin cytoskeleton. While many of the components of these intimately linked systems have been defined, very little is known about the biochemical mechanisms underlying cell polarity and shape changes. Instead of studying intracellular pattern formation and actin assembly in the complex cellular environment, I want to establish the minimal requirements for these processes by re-building them from purified components. To this aim, I will combine multiprotein reconstitution on artificial membranes with advanced fluorescence imaging techniques and employ synthetic biology methods. Specifically, I aim to understand (i) how membrane-associated signaling systems self-organize into macroscopic spatial patterns and (ii) how membrane polarity can be harnessed by the actin cytoskeleton to construct distinct structures at opposing ends of the cell. As a whole, this work has the potential to advance our understanding of the mechanistic foundations of cell polarity and morphogenesis at the systems biochemistry level.

The current Zika virus epidemic attracted public attention to the problem of mosquito-borne viral infections. Vector control is an essential element to prevent disease transmission due to the absence of arbovirus-specific drugs and limited availability of vaccines. Historical vector control methods such as the use of insecticides and environmental control are facing challenges due to the wide spread of insecticide resistance throughout natural mosquito populations and the complexity of breeding site elimination in the modern urban environment. Novel genetics-based strategies are emerging as promising complement to historical mosquito control methods. One idea is to genetically-manipulate the vectors so that they become unable to support pathogen infection, replication or transmission. The development of these novel transmission-blocking interventions requires in-depth insights into how mosquito vectors interact with and transmit arboviruses. It is thought that the immune system influences the efficiency by which mosquitoes transmit specific viruses. Recently, a novel immune response was identified that recognizes viral RNA and breaks it down into small fragments. In this project, the investigators will study whether mosquitoes from different locations across the globe differ in this immune response. Moreover, they will analyze how these differences influence transmission of the epidemic Dengue and Zika viruses. The multi-disciplinary research team includes experts in mosquito evolution and genomics, entomology, and virology, allowing a complementary approach to address the research aims. The proposed project will have immediate and profound implications for public health and may lead to novel mosquito control strategies.

The nucleolus is a large, complex, and dynamic nuclear body, whose structure is directly related to its role in producing ribosomes. It consists of three morphologically and compositionally distinct sublayers with liquid like properties, which hierarchically self-organize around transcriptionally active rRNA genes in a process that is poorly understood. Moreover, despite the significance of ribosome biogenesis, little is known about the order and loci of ribosome assembly, and the role ribosomal intermediates play in shaping nucleolar organization. A major difficulty in studying these processes lays in the absence of stable physical boundaries in between the sublayers, which facilitate a continuous flow of pre-ribosomal intermediates and hampers single layer isolation for in vitro analysis. Here I propose to recapitulate immiscible liquid phases of purified nucleolar components onto core particles with tethered rRNA in order to seed nuclei with isolated nucleolar sublayers. I will monitor the recruitment of sublayer specific components from the nucleoplasm into the synthetic layers and study ribosome biogenesis using the immobilized rRNA. The ability to seed nucleolar sublayers will be used to study nucleolar self-organization and to probe compositional changes in each sublayer separately, and to link them to concomitantly occurring ribosome production steps. Furthermore, it offers a means for studying ribosome biogenesis in vivo while restricting ribosome production steps only to those occurring within a specific sublayer, synchronizing the initial rRNA level of assembly, and sequestering ribosomal intermediates.

What underlies the ability to count and where did it come from? This project tests the broad hypothesis that the ability to represent the number of objects in a set (numerosity) has an evolutionarily conserved neural basis, and identifies the cell and molecular processes involved using multidisciplinary analysis in zebrafish. Although a wide range of species are able to estimate numerosities, only in primates has a neural mechanism homologous to humans’ been demonstrated and the underlying cellular processes are unknown. Using automated operant conditioning we train zebrafish to perform numerical tasks and identify lines of fish with differential abilities (e.g. mutants in candidate genes identified from human studies generated using CRISPR). We use 2 photon light sheet imaging of neural activity as wildtype and mutant larvae discriminate numerosities to identify circuits involved. Behavioural analysis will establish the ontogeny and extent of zebrafish’ numerosity. Genetic analysis tests the hypothesis that numerosity has an evolutionarily conserved basis. Neural imaging will test the hypothesis that “number neurons” exist in fish as in primates and indicate the circuits involved.

Our proposal is aimed at discovering the molecular mechanisms underlying the remarkable force-sensing and responsive properties of cellular attachment to the extracellular matrix. Many proteins contribute to the intracellular machinery that links the cytoplasmic domains of the transmembrane integrin adhesion receptors to the actomyosin contractile apparatus within the cell. These integrin adhesion complexes (IACs) provide a paradigm for a distinctive class of subcellular protein complex. Rather than assembling a structure of fixed stoichiometry (e.g. ribosome, centriole) via exclusive interactions, evidence is emerging that IACs engage a dynamic set of heterogeneous interactions that evolve from IAC initiation through maturation to achieve their signaling and mechanical functions.
Thus, we hypothesize that a key feature of IACs is their ability to exchange multivalent interactions between components, so changing their composition in response to diverse inputs, including force, developmental history and location within the organism. We have selected a few pivotal components of the IACs as the focus for our project: namely talin, kindlins, the IPP sub-complex (integrin-linked-kinase (ILK), PINCH, parvin), and vinculin.
To test this hypothesis we will combine Giannone's expertise in live single protein tracking and super-resolution microscopy with Brown's expertise in Drosophila developmental genetics. First we will advance existing methods to achieve the challenging task of quantitative super-resolution imaging within IACs in living tissues. Second, we will develop new tools to image interacting proteins, study their dynamic behavior and alter the interactions. Discovering the regulation of IAC rearrangement will greatly improve our understanding not only of mechanisms mediating Integrin adhesion but also of dynamic macromolecular protein complexes.
By bringing together the contrasting approaches of the two applicants we will gain an exceptional view of how the molecular machinery at integrin adhesion sites has evolved to be able to respond diverse environments and activities within the organism. We anticipate that this will lead to an understanding of general principles directing the progressive formation of macromolecular complexes.

The proposed project will investigate the origin of animal cell contractility through both comparative and experimental evolution approaches in the choanoflagellate Salpingoeca rosetta. The project will address three scales of biological organization: (1) Gene expression. The differential transcriptomic profile of contractile and non-contractile S. rosetta cell types will be determined by single-cell RNAseq. In parallel, I will investigate the function in S. rosetta of the three key myogenic transcription factors of animals (Mef2, SRF and Myocardin) by ChIP-seq and CRISPR/Cas9-based genome editing, and I will determine their interaction partners by pulldown/mass spectrometry. (2) Cellular phenotype. Choanoflagellates show an intense contractile activity during settlement. I will test whether this contractility is controlled by membrane depolarization and calcium influx by live imaging of choanoflagellates expressing calcium/voltage sensors, pharmacological assays, and microelectrode voltage measurements. (3) Multicellular phenotype. S. rosetta develops into spherical colonies of up to 50 cells. In an experimental evolution approach, I will assess whether simple (actomyosin-mediated) departures from this spherical shape can be brought about by consistently applying, over hundreds of generations, selection pressures thought to have favored the early evolution of animal morphogenesis: selection for larger colonies or for benthic feeding from bacterial biofilms.

Due to its high prevalence among birds and high human fatality rate, avian influenza represents a serious and continuing pandemic threat, in particular via mutations that facilitate human infection, resulting in pathogenic strains. Mutations associated with human infection are concentrated in the PB2 subunit of influenza polymerase. This subunit is imported into the nucleus, where viral replication and transcription occurs, via a selective interaction with importin-alpha. Once in the nucleus, interaction of PB2 with protein ANP32A has been proposed to play an essential species-specific regulatory role.

The behavior of PB2 in solution reveals a high level of conformational flexibility that is essential to function. In addition intrinsically disordered domains of both ANP32A and importin-alpha are thought to play important roles in the interaction with PB2. This uncommonly high level of disorder presents particular challenges for structural studies, requiring development of state-of-the-art NMR-based technology.

My aim is to investigate the molecular basis of avian influenza adaptation to nuclear import and replication in human host. I will characterize structurally and dynamically key interactions between human-adapted avian influenza polymerase and human host proteins to contribute to a deeper understanding of viral replication, providing the basic information to facilitate design of innovative drugs.

Viruses of the positive-sense RNA ((+)ssRNA) type are a major class of pathogens. In humans they cause diseases ranging from hepatitis C to viral myocarditis, Zika, Dengue fever and Chikungunya. The extracellular stage of these pathogens, the virus particles, have been studied in great detail often resulting in 3D structures at atomic resolution. Here, we propose to study an intracellular stage of (+)ssRNA viruses which has been more elusive to structural and mechanistic studies. Upon entering a target cell, (+)ssRNA viruses create dedicated organelles called replication complexes (RCs) , in which the viral genome is copied and hidden from detection until it is packaged into new virus particles. Since RCs form on cellular membranes as inseparable parts of the infected cell, they have largely resisted detailed characterisation by established methods of structural and molecular biology.

To overcome this challenge, we will study (+)ssRNA virus RCs using cryo-electron tomography (cET). cET can create 3D images (tomograms) of the interior of cells with a resolution high enough to identify individual macromolecular complexes. These tomograms will reveal in unprecedented detail how replication complexes are constructed from virus proteins and membranes inside the infected cell.

In parallel, we will create synthetic replication complexes from their individual protein-, RNA- and lipid components. The formation and subsequent RNA synthesis by such in vitro reconstituted replication complexes will be monitored by fluorescence microscopy, allowing us to make detailed observations about their molecular mechanisms.