Phosphatidyl-inositol 3,4,5 triphosphoshate-dependent Rac exchanger (P-Rex) are a group of proteins involved in the activation of the small GTPase Rac, by acting as a guanidine exchange factor (GEF). Both Rac and P-Rex proteins are known to be overexpressed and altered in several cancers, consistent with their role in both cell mobility and their numerous interactions with both the mTOR axis and the PI3K/PTEN pathway. P-Rex1/2 specifically, are known to interact with mTOR and PTEN, among others, placing them at the nexus of regulatory pathways for cell metabolism, proliferation and mobility.
Despite their central role in cell signaling, very little is known about the interplay between such interactions. No structural information is available for the full-length P-Rex proteins and how different interaction partners affect its structure and function. I will therefore set out to better characterize the emerging properties of the multidomain P-Rex proteins by purifying both P-Rex1/2 and determining their structure using cryo-EM and orthogonal biophysical characterization methods. I then aim to define the interaction between P-Rex proteins and its regulatory partners, PTEN and the mTOR complexes 1 and 2. I will define their domains of interactions and determine the structure of the complexes, using a combination of cryo-EM and cross-linking and mass-spectrometry.
I expect to provide new insights into the functions of P-Rex proteins and their crosstalk with core cellular signaling pathways. Structures of full-lengths P-Rex alone or in complex with its interaction partners will serve as a platform for drug discovery, thereby providing new cancer therapeutic avenues.

The brain comprises billions of neural cells interwoven in a highly precise way and it is ultimately responsible for how we perceive, react to, and remember the world around us. We are fascinated by how the intricate structure of the brain is assembled step-by-step at an early stage of life (“embryonic development”), starting as a relatively shapeless mass within the nascent embryo and culminating in a highly complex organ in an adult. Specifically, we hypothesize that blood vessels that permeate the early brain have important roles in fostering brain assembly, and we further predict that they produce important cues that actively shape a specific early step of brain development. This is of great significance, because blood vessels were once thought only to passively supply oxygen and nutrients to various organs. We instead suggest an active role in which blood vessels function as a ‘signaling center’ to actively control the arrangement of neural cells during brain assembly.
We have assembled an intercontinental team to tackle this question via a three-part, integrated effort. First, we will create a map of where and when arteries and veins (two major types of blood vessels) first enter the nascent brain. Second, we will intermingle neural and blood vessel cells in a Petri dish and study their interactions. Third, we will block specific functions exerted by blood vessel cells and propose that this will have a commensurate impact on the spatial arrangement of nearby neural cells. These activities are innately highly collaborative and will involve interactions between the partner laboratories in each nation.
Taken together, we propose to explore how the brain is assembled during embryonic development and we will test whether blood vessels act not as passive support for, but rather as active executors of, this intricate process at early steps. If true, this has important ramifications. Not only will it shed light on how the early brain is assembled, but it adds to the emerging idea that neural cells and blood vessels share an intimate functional and spatial relationship, starting from the embryo and lasting into the adult — an idea pertinent to early brain assembly; our efforts to engineer this process in a Petri dish for regenerative medicine; and finally, the origin of certain neurological disorders that are caused in part by blood vessel dysfunction.

Sporadically, novel and potentially devastating pandemic influenza A viruses (IAVs) are generated through genome reassortment between human and animal co-infecting IAVs. Such pandemic viruses emerge as a consequence of the segmentation of IAVs genome into a bundle made of 8 distinct viral RNAs (vRNAs). However the molecular mechanisms of vRNA intracellular transport and assembly into vRNAs bundles, which are critical for reassortment, remain largely unknown. Our project aims to elucidate these fundamental aspects of IAV life cycle by developing innovative approaches.
We challenge the original model that newly synthesized vRNAs, in the form of viral ribonucleoproteins (vRNPs), are transported across the cytoplasm on Rab11-dependent recycling endosomes. Based on our recent work, we hypothesize that the concomitant transport and assembly of vRNPs is driven by their physical association with remodelled endoplasmic reticulum (ER) membranes and Rab11-dependent transport vesicles distinct from recycling endosomes.
We will set up a cellular system which resembles the natural respiratory tissue targeted by IAVs, while being amenable to simultaneous imaging of the endogenous Rab11 protein and tagged vRNAs. We will develop two cutting-edge and complementary imaging methods: dual-color single molecule fluorescence in situ hybridization (FISH) in live cells for the tracking of distinct vRNAs that diffuse concomitantly, and cryo-Focused Ion Beam combined with electron microscopy in situ hybridization (EMISH) to image individual vRNPs and their transport vesicles at molecular resolution. We will further assess the role of cellular ER-shaping proteins by performing CRISPR/Cas9-mediated knockdowns, and by monitoring changes in the viral-induced remodelling of ER and biogenesis of vRNP transport vesicles by live fluorescence imaging and cellular EM.
The proposed research will require the very close collaboration between three partners with distinct but complementary expertise. The approaches developed jointly are poised to revolutionize our understanding of IAV multi-RNA genome transport and bundling, and thus help in the broader goal of achieving better prevention and treatment of influenza disease. Additionally, the proposed technical developments in live cell FISH and cellular EM, will have impact on other fields of studies well beyond the scope of the proposed project.

Glycolysis is a fundamental energy metabolic pathway which consists of ten enzymatic steps. Unlike the mitochondrion, which is a membrane-bound organelle, glycolytic enzymes are soluble proteins in the cytosol. Based on biochemical evidence, glycolytic enzymes have long been hypothesized to form functional complexes to sustain the rates of glycolysis. This purported complex, called the glycolytic metabolon, was a subject of intense study and debate thirty years ago. Still today we do not yet understand how these purported complexes are organized in cells to sustain local energy metabolism, or their physiological importance. This gap in knowledge results from the fact that when glycolysis was being rigorously examined forty years ago, the techniques did not exist to conclusively answer these questions. The main challenge in addressing these questions lay then, as now, in the ability to both examine the localization of the glycolytic enzymes in living cells, while understanding the biophysical and biochemical mechanisms of their association, and its implications in cellular physiology. We have established a collaboration to address these fundamental questions by making use of our joint expertise in vitro reconstitution and C.elegans physiology, using the energy demands of the C.elegans synapse as a model system.

Plants live in an ever-changing environment which is not always compatible with their survival. A major life threatening condition is drought stress. While most plants can deploy an array of physiological countermeasures to endure remarkable levels of stress, there is a huge variability in the different strategies that plants choose to adopt to deal with drought stress. Many plants evade detrimental stress conditions by activating their reproductive development (flowering) earlier compared to non-stress conditions, a strategy known as drought escape (DE). Notably, even in the most water-rich environments plants face unpredictable dry periods, yet how this information affects the floral network at the molecular levels is unknown.
We will address this question with a blend of molecular and genetics-based approaches. We will leverage known mutants with altered DE to identify candidate mechanisms that are drought sensitive and can act as molecular switch to activate flowering. To comprehensively define DE mechanisms utilized under natural conditions we will assess the variability of the DE response in natural plant populations and in crops, which were selected by human intervention for the different field scenarios. Our targeted large-scale screens will allow us to identify naturally occurring variants in the DE process and decipher the molecular mechanism responsible for DE activation. This information will provide breakthroughs in our understanding of novel regulatory mechanisms that play a role in driving developmental adaptations across extremely variable environmental conditions, their natural genetic variation and the selective forces that maintain such variation in populations, an important aspect for predicting and dealing with the effects of climate change. Finally, because flowering time is a major component of yield potential in crops, the defined mechanisms will help us develop breeding strategies targeted for sub-optimal irrigation scenarios to produce crops with ameliorated performances under drought conditions.

The endocannabinoid system (ECS) is involved in a variety of brain functions, including reward and aversion, mainly through type-1 cannabinoid receptors (CB1). The Nucleus Accumbens (NAc) is a key brain region in the control of positive and negative reinforcement. ECS impairments in the NAc lead to negative emotional states. By responding to neurotransmitters with intracellular Ca2+ increases and releasing gliotransmitters that modulate synaptic transmission and animal behavior, astrocytes play active roles in neural information processing. While CB1-mediated astrocyte-neuron communication has been shown in several brain regions, it is unknown whether this communication exists in in the NAc. Mitochondrial CB1 receptors (mtCB1) directly regulate mitochondrial energetic activity, synaptic transmission and behavior, but the role of astroglial mtCB1 in these processes is unknown. Combining electrophysiology, subcellular-specific live Ca2+ imaging and behavior, this proposal aims at defining the contribution of astrocytic CB1 (plasmatic and/or mitochondrial) to synaptic plasticity in the NAc and their behavioral impact. The specific goals of this proposal are to define 1) whether astrocytes in the NAc express functional CB1; 2) how astrocytic CB1 regulate synaptic transmission and plasticity in the NAc; 3) how astrocytic CB1 impact reward and aversion and 4) how astrocytic mtCB1 influence astrocyte activity, synaptic plasticity in the NAc and behavior. The expected results will shed light onto the involvement of astrocytes in brain motivational systems through the ECS, and would reveal astrocytes as potential targets for treatment of motivational disorders.

Compartmentalization into functional units is the key principle of cellular life. In addition to conventional membrane-bound organelles, cells utilize membrane-less biomolecular condensates to locally concentrate proteins and nucleic acids. Prominent examples of such condensates are nucleoli, P granules or centrosomes, which play central roles in diverse cellular functions. Recent studies have demonstrated that membrane-less condensates assemble by liquid-liquid phase separation of proteins that are characterized by multi-valency, intrinsic disorder and low complexity sequences. The molecular mechanisms that control assembly and disassembly, shape and size of such membrane-less organelles remain key unanswered questions of modern cell biology. To address this fundamental gap in our knowledge, I propose an interdisciplinary research programme that combines powerful in vitro reconstitution with systematic screening technologies. Based on my discovery that the chromosome surface protein Ki-67 acts as a surface-active agent (surfactant) for one of the largest membrane-less assemblies, the mitotic chromosome, I will explore the radically new concept of surfactants for the global regulation of other membrane-less organelles. Starting from defining the molecular properties, mechanisms and regulatory basis for the action of Ki-67, I will systematically identify and characterize the surfactants of other membrane-less organelles in human cells. This ambitious research programme has the potential to provide novel paradigms of spatial and temporal control of membrane-less cellular assemblies and thereby substantially advance our understanding of cellular organization.

Organisms need strategies to survive when conditions are hard. For mammals, winter is particularly difficult - they have to invest large amounts of energy into keeping warm, while food availability is low. For this reason, many mammals migrate or hibernate. However, what to do if you are too small to migrate long distance, burn your energy fast, and cannot hibernate? The common shrew is such a mammal and has evolved an astonishing strategy: each individual shrinks in winter by up to 20% and then regrows in the spring by about 13%. This size change, thought to allow shrews to survive on fewer resources because of the smaller size and linked lower energy requirements, include not just overall size, but specifically organs that do not usually change size in fully grown animals, such as the brain, heart and liver.
The process of neurological degeneration and regeneration is of great interest, since many central nervous system diseases (e.g., Alzheimer’s, multiple sclerosis) involve degeneration, but ongoing research for therapies to reverse this process has been of limited success. As one of only a few recorded examples of mammalian brain regeneration, understanding how the shrew regrows its brain can accelerate research that leads to future therapies.
To answer the question of how the shrew shrinks and then regrows its brain, we will establish this unusual species as a new model, by studying the biological, molecular, biochemical and genetic processes behind this reversible size change. Besides establishing a database of information that can be mined and researched in years to come to discover the pathways that generate this cycle in the shrew, we will test a metabolic model of neurological change by artificially blocking molecular access to fats. Thus, the cross-disciplinary study of this wintering adaptation may help us understand more about regeneration in mammals in general, and the brain in particular.

A plausible explanation for why mitochondria have their own DNA (mtDNA) is to ensure colocalization of redox-genes and redox-regulation to the same membrane-bound organelle. While this distributed control system seems logical, very little is known about how such autonomous regulation takes place. A variety of methods that are widely applicable to study genomes fail to work on mtDNA, and sequence-to-function mapping of the mitochondrial genome is missing. I plan to create a modified, hypermutating DNA polymerase gamma (polG), the designated mtDNA replicase. polG is encoded in the nuclear genome and can therefore be easily manipulated. Hypermutator polG will be integrated into the nuclear genome of S. cerevisiae, which will introduce mutations in mtDNA without altering the nuclear genome. I will then use selection assays followed by next generation sequencing to study the ability of mtDNA yeast variants to adapt to respiratory conditions. Such experiment will highlight regulatory hotspots in mtDNA that will be mechanistically evaluated using genome-wide methods like native elongating transcript sequencing (NET-seq) and mitochondrial ribosome profiling that are the ‘bread and butter’ of the Churchman lab. Clear view of the fitness landscape associated with mtDNA will inform research into many deep questions in mitochondrial biology, ranging from ‘how selection shapes mitochondrial genomes?’ to ‘why do certain mtDNA mutations cause cancer?’.

Trypanosoma brucei is the causative agent of sleeping sickness. In the mammalian host, parasites exist in two major niches: the blood, and the brain. Recently, Dr. Figueiredo’s lab found a previously undiscovered third reservoir of parasites in the adipose tissue which were transcriptionally distinct from blood stream forms, and the genes upregulated included various markers relevant to parasite metabolism. My proposed work in Dr. Figueiredo’s lab is to implement in vivo imaging methods to a) investigate the parasite’s mechanisms of homing and crossing of the vascular endothelium into the adipose tissue, and to carefully define the adipose tissue niche that acts as reservoir for the parasites; and b) to elucidate host vasculature, and parasite factors involved in allowing such unique phenomenon, including generating parasite mutants deficient in adenylate cyclises, flagellar proteins, and mechano-sensing proteins among others. Using in vivo techniques and biophysical and molecular methods, I aim to investigate the biological significance of parasite residence in the adipose tissue – a location which could be clinically relevant for interventions in human and veterinary medicine.

Harmful algal blooms (HABs) have been long studied in marine and fresh water environments, yet most research to date has focused at the bulk scale. Despite the well-documented toxic ramifications of HABs on vertebrates and mammals, including humans, we still lack a biophysical understanding of the mechanisms by which toxins disperse during a bloom event. Using a combination of experiments and modelling, in this project I will explore the physico-chemical interplay underlying the release and transport of toxins at the scale of the microorganism. Towards this I will develop micro- and millifluidic experiments to generate ‘bloom-in-lab’ and visualize the dispersion of targeted toxins under three ecologically relevant cues: fluid flow, temperature and salinity. Specifically, I will study red-tide forming Heterosigma akashiwo, a globally distributed marine raphidophyte, known to release a range of toxins, including reactive oxygen-nitrogen species (RONS) during bloom conditions. Original data from high-speed and time-lapse microscopy on fluorescently labelled ‘target’ molecules will put forward first experimental quantifications of toxin release and transport under different micro-environmental cues, thereby informing a new mathematical model that will capture, at microscales, dispersion and auto-feedback dynamics during bloom events. Analysing the coupling between microbial biophysics, transport phenomena and biochemistry, this project will bring about a fundamental, mechanistic understanding of toxin transmission, and help develop accurate and comprehensive algorithms for predicting HAB formation, especially during the rapidly warming climate we encounter today.

Human medicine is on the verge of a new era where we face the possibility to precisely rewrite the genome to prevent, ameliorate and cure a wide range of immune-hematological diseases. Hematopoietic stem and progenitor cells (HSPC) have long been a preferred source for ex-vivo gene therapy, as gene correction in multipotent progenitors ensures a life-long supply of corrected progeny and polyclonal reconstitution of the bone marrow. Recently, programmable nucleases brought the possibility of genome editing (GE) within the reach of gene therapy by allowing locus-specific gene correction without the risk of aberrant transgene expression. However functional studies that accurately assess possible acute and long-lasting consequences of GE procedures in HSPC are needed to harness GE therapeutic potential. Indeed, despite recent advances in the generation of corrected HSPC by GE, the efficiency of the targeting process and the ability of edited cells to stably reconstitute the hematopoietic system upon transplantation remain limited, pointing to a loss of repopulating capacity of HSPC upon targeting. The goal of this project is to investigate the role of DNA damage response (DDR) pathways in GE strategies and uncover novel molecular players that could be exploited to increase the yield of long-term engrafting gene-edited HSPC. Our findings could be easily applied to gene correction strategies for a variety of inherited pathologies affecting the human immune-hematopoietic system and provide a foundation for more efficient modalities of editing HSPC at genomic sites of interest for both basic and translational research.

Areas of visual cortex integrate signals representing sensory input, ongoing motor actions, and internal states. While neurons in primary visual cortex (V1) were classically interpreted as feature detectors that signal the presence of a specific visual stimulus, recent evidence suggest that V1 circuits participate in the specific, topographical integration of sensorimotor signals. How sensory and sensorimotor computations are implemented in the circuitry of V1 and higher visual areas, and how these computations depend on the behavioral context, is unknown. I will combine novel circuit dissection tools, wide-field-of-view holographic stimulation, and virtual reality environments to approach these questions. I will selectively activate V1 ensembles with distinct functional properties during sensorimotor behavior using holographic stimulation and monitor the effects on the surrounding populations in V1 and higher visual areas. Furthermore, I will analyze the effects of holographic stimulation as a function of different behavioral parameters and during optogenetically induced variations in internal state. Finally, I will explore methods to target the expression of genetically encoded tools to distinct V1 ensembles, each with specific function during sensorimotor behavior, which will allow me to study if different ensembles are selectively connected to circuits throughout the brain, as well as to determine their selective impact on brain-wide dynamics. This project will shed new light onto the function of V1 during sensorimotor behavior, and yield fundamental insights into the mechanisms by which cortex can adapt its processing machinery to varying behavioral demands.

Transposable elements (TEs) are important drivers of genome evolution with the potential to rewire transcriptional networks but require constant surveillance to minimise deleterious effects. Major players in the transcriptional regulation of TEs are KRAB domain-containing zinc finger proteins (KZFPs) that co-evolved in response to new TE invasions and constitute the largest family of transcription factors encoded by the human genome.
Recent views have shifted from a simple arms race between TEs and their host genomes towards a more complex domestication process. This can involve the rewiring of gene regulatory networks using TE-derived regulatory elements, co-option of TE-derived genes, but also the exaptation of KZFPs to acquire new regulatory functions after the interaction with their target TE has become obsolete.
Such newly adapted functions for KZFPs can be mediated by novel protein interactors but can also involve the binding to, and regulation of RNA molecules, as zinc finger domains are not limited to interactions with DNA. Indeed, many of the evolutionary conserved KZFPs show weaker interactions with the TE-silencing machinery and instead display unique protein interactomes that indicate an RNA-based functionality.
This proposal lays out the experimental strategy to systematically identify RNA-binding KZFPs in the human genome and profile their corresponding RNA interactomes to elucidate the underlying regulatory mechanism. Exaptation of KZFPs to function as RNA-binding proteins extends their role from transposon control to post-transcriptional regulation of protein-coding and non-coding RNA molecules, thus providing intriguing new layers of gene regulation.

Cell division is a complex but fundamental life process. Among its many purposes, it is needed for a fertilized egg to develop into a human being, for our wounds to heal, for infections to clear and for our bodies to sustain life. Whenever a cell divides, its genetic information is duplicated and packaged into chromosomes which are then separated and equally distributed between the new daughter cells. For the newly formed cells and ultimately the body to be healthy, distribution of the chromosomes should be highly accurate. Accurate chromosome separation during cell division is driven by dynamic cellular cables that are connected to special chromosomal regions known as centromeres. Indeed, defects in centromere formation or function compromise chromosome separation and lead to daughter cells containing too many or too few chromosomes, a hallmark of cancerous cells.
When eggs are prepared for fertilization, a specialized form of cell division called meiosis separates the chromosomes. The accuracy of chromosome separation during meiosis determines whether a fertilized egg can develop into a healthy human being. Surprisingly, meiosis in humans and other mammals is highly prone to errors and often leads to eggs that contain the wrong number of chromosomes. Fertilization of such chromosomally abnormal eggs frequently leads to human embryo deaths and conditions such as Down’s syndrome. Complications arising from erroneous chromosome separation in eggs become even more frequent as women get older. Research in the field of meiosis has only scratched the surface of why chromosome separation in eggs is highly error-prone. Furthermore, the reasons behind the deterioration in the quality of this process as women get older largely remain unknown.
In this research proposal, we will test the hypothesis that defects in centromere function that accompany ageing may contribute to poor quality of eggs in older women. To achieve this, we will combine our unique but synergistic expertise in advanced light microscopy, genome editing and electron microscopy. Knowledge gained from this study will advance our understanding of why eggs of older women are often chromosomally abnormal. In the long-term, this work can potentially be exploited for treatments of human infertility.

To study how nervous systems arise and function, scientists use animal models in which it is possible to integrate research on fundamental processes across different levels of organization from genes to behavior, and from the zygote to the adult. The medicinal leech (genus Hirudo) provides one useful model, because its nervous system is much simpler and easier to work with than vertebrate or mammalian nervous sytems, even though it functions in a similar manner--Hirudo has been used to study phenomena of general importance such as: how glial cells function; how neurotransmitter are released; how synapses form and regenerate; and how neural circuits function to control behavior. Another leech (genus Helobdella) is used to study development and how development changes during evolution, giving rise to kinds of animals over hundreds of millions of years. These two leech species exhibit marked similarities of course, but also some differences. Technical considerations (small embryos for Hirudo; small adults for Helobdella) have made it difficult to integrate these two models, e.g. by applying molecular approaches in Hirudo or to study the adult nervous system in Helobdella. The goals of our project are: 1) to enhance the power of the Hirudo model by introducing newly-developed molecular approaches; 2) to implement approaches in Helobdella that will enable us to unite molecular and cellular approaches to developmental and behavioral neurobiology; 3) to develop new optical techniques for stimulating and recording neuronal activity without exogenous dyes or genetic manipulations.
The intellectual significance of the proposed work is twofold. First, it will enhance our abilities to answer fundamental questions regarding how nervous systems function and development by introducing cutting edge technical approaches to the cellularly simple, physiologically accessible leech models. Of equal importance, it will provide a new evolutionary perspective into neurobiology, by allowing us to examine similarities and differences between leech and other models, including arthropods, nematodes, and vertebrates which have all been evolving separately for more than half a billion years.

When an animal navigates in an environment, neurons located in the hippocampus fire selectively when the animal is in a particular place. Together, these ‘place cells’ form a map of the environment and report the position of the animal on this map. To construct its spatial map, the hippocampus relies on self-motion information (e.g. distance travelled or direction of motion) but also on sensory cues (e.g. visual, auditory or olfactory cues). The hippocampus thus gives special weight to sensory information, which is processed by cortical areas. Sensory areas of the cortex were classically thought to provide the hippocampus with purely sensory information, independently of the position of the animal in the environment. But we recently discovered that the activity of neurons in the primary visual cortex is influenced by the spatial context during navigation, suggesting that navigation signals modulate sensory processing in cortical areas. It is yet unknown which navigation signal influences visual cortex and how this phenomenon generalizes to other sensory modalities and other behavioral contexts where hippocampus is involved. By recording neurons in hippocampus and visual or auditory cortices while mice perform a task in a virtual environment, I will investigate three questions: 1) Are visual responses modulated by the route that the animal takes or by its absolute position? 2) Is there a similar modulation by spatial context in other sensory areas and how signals from different senses are integrated together during navigation? 3) Are sensory responses modulated in non-spatial contexts where the hippocampus still builds a representation?

Proteins have evolved to adopt many structures and perform diverse functions by exploring a sequence space spanned by twenty canonical amino acids (AAs). Whilst ten of the AAs were ‘obvious’ choices, as they abounded in the prebiotic world, the other ten were far less accessible prebiotically, thus provoking the question: Why (and how) were these AAs included in the genetic code, and was their inclusion prerequisite for protein evolution to be as successful as it has been? These seeming Gedankenexperiments are directly testable using an interdisciplinary approach we have devised.
Specifically, we propose to synthesize random protein libraries built from reduced (evolutionarily early) and alternative AA alphabets to compare the structure/function-forming potential of the proteinogenic and non-canonical yet prebiotically abundant AAs. The team of Stephen Fried will customize both commercial and home-made cell-free protein translation systems to express protein libraries composed of alternative AA alphabets. The team of Klara Hlouchova will use biophysical approaches to explore the structure-forming potential of the purified libraries. The capacity of the libraries to evince prebiotically-relevant functions will be assessed by the group of Kosuke Fujishima through selections that can relate genotype to phenotype (e.g., mRNA-display). Large sequence space (>10^12) will be analyzed in each experiment and because the same template libraries will be used, the outcomes of both the structural and functional studies will be directly comparable. This would not be possible without coordinated collaboration among the three teams.
Each team member enters the project with a key set of skills and scientific expertise. Klara and her team have a strong background in protein biochemistry, bioinformatics and experience with expression of protein libraries. Kosuke is an astrobiologist with strong experience in RNA molecular biology and his team takes a synthetic biology approach to studying peptide-RNA interactions. Stephen has experience in biophysics and synthetic biology and his newly started lab performs research in protein folding and engineering. This project relies on synergy of the above mentioned disciplines connected by our mutual interest in the origins of life, making it possible to address a broad fundamental biological question in a systematic way.

Non-canonical initiation of protein translation plays a central role during cellular stress, apoptosis and cell survival. Death-associated protein 5 (DAP5) acts as the major scaffolding initiation factor to promote cap-independent translation of cellular mRNAs such as p53, Bcl2, Apaf1 and XIAP. Potentially, translation of such transcripts can be initiated via internal ribosome entry sites (IRESs), an alternative to canonical cap-dependent translation. The structural and molecular mechanism of how DAP5 regulates IRES-driven translation is not understood. To resolve the inherent molecular processes, I will investigate IRES recognition by DAP5 in target mRNAs through novel NMR techniques, biophysical tools and mass spectrometry. My research is aimed to solve the first three-dimensional structure at atomic resolution of a cellular IRES along with its functional characterization. Based on this, the interaction between this IRES and DAP5 that is relevant for cell fate decisions will be studied structurally and biophysically. I will analyze not only the role of the structured MIF4G domain of DAP5, but also the so far unknown function of its disordered regions in IRES recognition. In order to shed light on regulatory principles of DAP5, I further aim to investigate structural effects on mRNA binding upon DAP5 phosphorylation. In a proteome-wide examination, I will identify additional factors of IRES-mediated translation to define a potential core complex associated with different mRNAs. My project thus comprehensively aims to decipher the mRNA recognition mechanism of DAP5 that controls IRES-driven, cap-independent translation linked to cell fate decisions.

Adaptive resistance is an emergent phenomenon in melanoma cells that allows tumor cells to adapt and escape treatment through a change in signaling state. The complex interplay between transcriptional and post-transcriptional regulation that gives rise to adaptive resistance is in large parts poorly understood. To unravel the underlying molecular mechanisms, in silico approaches involving statistical or kinetic models have to complement experimental analysis. Statistical models are well-suited to identify unknown molecular mechanisms but cannot describe emergent phenomena. In contrast, kinetic models intrinsically describe emergent phenomena but are challenging to apply when the underlying molecular mechanisms are unknown.
In this project, I propose a novel, integrated, data-driven approach to unravel the molecular mechanisms that give rise to adaptive resistance in melanoma. The approach combines kinetic and statistical modeling to harness the benefits of both approaches. The statistical modeling will be used to derive biological hypothesis to construct and extend kinetic models in an unbiased, data-driven, automated fashion. This will render the construction of kinetic models less dependent on prior knowledge. The constructed kinetic model will be able to quantitatively describe emergence of adaptive resistance and provide insight into underlying molecular resistance mechanisms.