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.

Plants are the primary producers in land ecosystems, a central position that give them a fundamental ecological importance. In the wild, plants interact with multiple organisms, including bacteria and insects, to form rich interconnected networks. In particular, plants are colonised by communities of microbes, including both beneficial and pathogenic bacteria. For these microorganisms, it is crucial to spread between plants, which they often do by recruiting herbivorous insects as vectors. Plant pathogens may even manipulate the behaviour of the insect that carries them. However, the underlying mechanisms and evolutionary dynamics of this important behavioural component of plant-insect-bacteria systems remain unknown.

My work will address how bacteria attract and alter the behaviour of their insect vectors by combining three fields of biology: plant physiology, insect behaviour and bacterial genomics. This project follows two independent questions: 1) how pathogenic bacteria attract insect vectors and 2) whether bacteria that have colonized a vector can manipulate vector behaviour. To address these questions, I will
use the Sacptomyza-Arabidopsis-Pseudomonas system as a model of bacteria-facilitated herbivory
and build high-throughput experimental assays to score the behaviour of the drosophilid Scaptomyza. This quantitative approach will allow me to compare the effect of dozens of bacterial stains on the behaviour of their vector and ultimately to find the bacterial genes required for vector recruitment and manipulation. This approach could to transform a largely descriptive area of plant-insect-bacterial interactions into a high-throughput and mechanistic field.

Humans have an intimate bond with their gut microbes. These minute organisms, six orders of magnitude smaller than us, populate our gastrointestinal tract to form one of the densest ecosystems on earth. The gut microbiota, often referred to as ‘the forgotten organ’, has been shown to play important roles in human health; however, we still understand little on the molecular interactions among microbes and between microbes and the host. During my HFSP-supported postdoctoral training,I studied the role of gut microbes in complementing our immune system. We found that each microbe plays a unique melody on the host’s immune system and together the wealth of immune-modulatory gut microbes creates an exquisite harmony that complements our immune system. I also introduced a novel fluorescent-labeling method to enable, for the first time, to visualize live gut microbes, at real-time, in their natural habitat. For my CDA, I propose to combine my systems biology background with my postdoctoral training in immunology and microbiology, to study how the microbiota actively and mechanistically network and interact with the mammalian host to maintain homeostasis and confer protection from disease. My postdoctoral studies identified redundant immunomodulatory capacities of gut microbes across distant phyla.To me, these findings emphasize the importance in gut microbiota diversity. I hypothesize that the gut microbiota diversity is vital and physiological relevant, and that this diversity represents an intricate, delicate, evolutionarily balanced host–microbiota relationship that occupies a central role in health and disease. During my CDA, if granted, I would like to test this hypothesis.

Mitosis is key to the cell cycle, as it guarantees the accurate segregation of replicated chromosomes to daughter cells. This process relies on the mitotic spindle, a microtubule-based, dynamic, and bipolar structure. Spindle morphology varies greatly among species and cells to optimise its function. Size and architecture, in particular, are both essential for accurate chromosome segregation, cell division and cytokinesis. However, despite decades of study and the investigation of hundreds of proteins involved in spindle assembly, it remains unclear how spindle microtubule subpopulations organise into complex assemblies. Especially, how correct spindle morphometrics, at both the size and architectural levels, are established is poorly understood. Using the egg extracts of two Xenopus species of different spindle sizes and architectures, X. laevis and X. tropicalis, we will reconstruct microtubule structures assembled independently from the two major organising sites that contribute to spindle assembly, the spindle poles and chromatin. By combining cutting-edge fluorescence microscopy and electron tomography analyses, we aim to reveal the dynamic and ultrastructural bases of spindle substructure size and architecture. Our goal is then to extract quantitative parameters and combine them into physically realistic simulations to decipher the biophysical basis of the different architectures and scaling properties, and ultimately their implication for the regulation of spindle morphology. Altogether, this study at the frontier between cell biology and biophysics will not only unravel key mechanisms of microtubule organisation but also fundamental principles of spindle assembly.

The aerial courtship “dance” of mosquitoes has fascinated entomologists for over 150 years. This dance involves highly controlled variations in the frequency and intensity of flight-tones (i.e. sounds generated by the flapping wings) with concurrent changes in flight speed and direction, and enables recognition of conspecifics, display of fitness and transmission of mating interest. However, despite over a century and a half of research, significant knowledge gaps continue to exist in our understanding of this behavior. To decipher this courtship dance, entomologists have to integrate acoustic, energetic and flight information for untethered, free-flying mosquitoes, but the tools that can provide these data have, so far, not been available. In the current project, the two investigators combine their respective expertise in computational biomechanics and acoustics, and behavioral entomology, to generate unprecedented data and insights into the biomechanics and physics of courtship-associated acoustic communication in mosquitoes. In particular, by combining computational modeling with biological assays, the team will generate six-dimensional soundscapes of free-flying mosquitoes engaged in courtship and determine how these soundscapes are actively modified during courtship. We will also estimate for the first time, the energetic costs of courtship and mate-chasing, and the potential constraints this places on courtship behavior. Finally, the team will characterize the degree to which, carefully tailored exogenous sounds can alter and even disrupt courtship. The success of this novel approach could be transformative for future research into comparative auditory mechanisms of communication across a wide range of flying insects. In addition, the insights gleaned here could form the scientific foundation for novel insecticidal/surveillance traps and also lead to environmentally friendly strategies for diminishing mating success in mosquito species that are vectors for malaria, Zika fever and other devastating mosquito-borne diseases.

Mosquitoes rely on their exquisitely tuned sense of smell to efficiently home in on humans to blood feed. This epidemiologically important behavior is intricately gated by the internal physiological state of the mosquito. For instance, upon starvation the African malaria mosquito Anopheles gambiae reflexively exhibits heightened attraction towards human scent. Such state-dependent shifts in sensory perception are often evoked by neuromodulation of olfactory circuitry mediating attraction to food. Here, I propose to apply genome engineering coupled with multiphoton imaging in the mosquito nervous system to identify key olfactory circuits and neuromodulatory networks that control An. gambiae attraction to humans. To achieve this goal, I will initially characterize how whole human scent and its constituent odorants are represented in the primary olfactory processing center of the An. gambiae brain, the antennal lobes. Subsequently, I will determine if this pattern of neural representation is altered during fed and fasted states, as well as stages of malaria parasite infection previously shown to influence olfactory behavior in this mosquito species. Finally, I will evaluate a potential role for neuropeptide signaling in altering synaptic physiology during state-dependent changes in mosquito food search behavior.

Life requires that the brain accurately measure both internal (body core) and external (environmental) temperature. Environmental temperature detection allows animals to find suitable thermal climes and to perceive and learn to avoid painful stimuli. The mechanisms of environmental temperature detection are increasingly well understood, however the mechanisms by which the brain measures internal body temperature remain poorly defined. The vagus nerve is the dominant sensory system that monitors the state of the viscera, and vagal afferents are critical for commanding unconscious processes essential to life, such as keeping heart rate constant and controlling food digestion. But how the vagus nerve contributes to thermoregulation remains a mystery, and almost nothing is known about the specific molecules, cells, and pathways by which these afferents can trigger physiological and behavioural responses to temperature. I propose to identify the molecular and cellular mechanisms underlying thermodetection by vagal afferents innervating the gastrointestinal tract (GI) including the oesophagus, stomach and intestine. I will determine the molecular identity of vagal afferent cell types that measure core body temperature, determine the neurochemical signals that these thermosensitive vagal neurons use to communicate with the brain and test the hypothesis that these vagal cell types are essential for regulating temperature-dependent autonomic and behavioural functions in awake, behaving animals.

The redundant and long-distance activity of regulatory sequences such as enhancers controls tissue-specific gene expression during development. Enhancers are brought in proximity with their target genes through the 3D folding of chromatin in domains of specific interaction. Several factors including CTCF have been shown to be important for this process, but the role of repetitive sequences remains unknown. Here, I propose to investigate the impact of transposable elements (TEs) on gene regulation and the 3D organization of the genome. Focusing on one type of evolutionary young retrotransposon in mice, the MusD elements, I will address this question through three lines of research. (1) a locus-specific approach focused on a model of TE-associated limb malformation to investigate the impact on 3D chromatin folding and gene expression; (2) a genome-wide approach to gain insight into the impact of active TEs on gene regulation and (3) a targeted approach to identify a TE trans-acting factor responsible for silencing such elements. This will be achieved by combining cutting-edge genomic technologies with mouse embryo analysis and the generation of mutants through CRISPR/Cas9. These investigations are expected to reveal specific mechanisms by which transposable elements are able to influence chromatin folding and the expression of developmental genes. My work will shed light on the role of repetitive sequences in shaping the 3D architecture of the mammalian genomes thereby bringing new mechanistic insight into basic mechanisms of gene regulation and retrotransposon biology.

The overarching goal of this proposal is to investigate the spatiotemporal coding of acetylcholine (ACh) and dopamine (DA) with high-resolution and precision in the striatum using state-of-the-art genetically encoded biosensors combined with modern optics in awake animal imaging. The striatum is crucial for movement, learning and flexible behavior, with striatal DA and ACh both playing key roles in these functions. While the role of DA is relatively well established, the role of ACh in natural behavior still remains enigmatic. Cholinergic interneurons (CINs), the major source of striatal ACh, are involved in processing contextual information that guides flexible behavior. Locally, CINs also exert control over striatal DA release, hijacking DA axons and making them release ACh by activating nicotinic receptors near their terminals. We propose to image the spatiotemporal dynamics of striatal DA and ACh using two-photon microscopy and endoscopy in awake mice engaged in tasks requiring behavioral flexibility. To image DA and ACh simultaneously during behavior we will extend the color-spectrum of DA and ACh biosensors. We will also further optimize the performance of these biosensors to make them suitable for robust in vivo application. Our combined interdisciplinary but complementary expertise – in biosensor engineering, imaging, modelling and behavior – is essential for our aims. We will ensure a coherent, interactive approach by sharing procedures, behavioral tasks, and biosensor technology, with regular planning sessions and feedback of results. A successful outcome of this program will reveal, for the first time, the spatiotemporal coding of neuromodulatory signaling by DA and ACh and how it shapes the function of striatal circuits during flexible behavior. We will also obtain a mathematical understanding of the genesis of the spatiotemporal dynamics. The newly engineered sensors developed in the program will have further broad applications in various biological systems of interest, which will ultimately pave the way toward a more complete understanding of brain function at synaptic, microcircuit, and behavioral levels.

The evolution of animal sociality has been driven, in part, by the balance of cooperation and conflict. Extensive studies of dyadic (one-on-one) conflicts across taxa have revealed how opponents achieve safe resolution by assessing competitive ability. While conflicts between groups of social animals are equally important, little is known about how competing groups assess ability. Studying intergroup assessment can reveal principles that extend across taxa, influencing our understanding of the evolution of sociality.
I will adapt the theoretical and experimental framework of dyadic assessment to test principles of intergroup assessment. I will first use a database of over 600 banded mongoose contests to test central components of conflict dynamics: how group composition predicts competitive success, how territory ownership confers an advantage, and if groups of similar ability have more dangerous contests. I will also experimentally test how banded mongooses use scent markings and collective “war cry” calls in intergroup assessment, including if and how “leaders” of conflicts differ from other group members. Finally, working in the extremely tractable wood ant system, I will use automated tracking and network analysis to test how individual-level behavioural variation influences overall group contest dynamics.
This project integrates big data, behavioural ecology, and state-of-the-art individual tracking to reveal principles of intergroup assessment that extend across taxa and levels of social organisation. The outcomes of this work can influence research in fields like animal contests, social evolution, and human psychology, among others.

Mitochondria are dynamic, multifunctional organelles that are a defining feature of eukaryotic cells. Maintaining mitochondrial homeostasis is essential for normal cellular and organismal physiology. Mammalian cells have roughly ~1,500 mitochondrial proteins, however the vast majority are encoded in the nuclear genome. Therefore, in response to stress, mitochondria must signal their internal states to the nucleus, which can mount a compensatory transcriptional response. Despite the fundamental importance of mitochondrial regulation, the full range of perturbations that disrupt homeostasis, the subsequent signalling cascades and the resultant nuclear responses remain poorly defined in higher eukaryotes. One consequence of mitochondrial stress is the activation of the integrated stress response (ISR), though the mechanism of this remains obscure. I propose to address these aspects of mitochondrial homeostasis in human cells using complementary genetic and biochemical strategies. i) First, I will use a CRISPRi screen to identify factors involved in activation of the ISR. ii) I will use Perturb-seq technology to objectively and systematically establish the full spectrum of stress induced transcriptional responses needed to maintain mitochondrial homeostasis. I will then use the approaches in (i) to explore the exact mechanism for key responses. iii) Finally, I will dissect the role of a new factor TMA7, identified through a previous genetic screen aimed at uncovering genes that sensitize mitochondria to proteotoxic stress. In all cases, the ultimate goal is to delve into the biochemical mechanism of how promising hits are involved in maintaining mitochondrial homeostasis.

Proteins emerging from previously non-coding DNA regions are becoming increasingly appreciated as an important path to creating completely novel functions. However, their path of emergence is very poorly characterized. Comparative genomics studies are finding numerous genes that have emerged from non-coding sequences, providing deep insight into gene evolution. However, these studies provide no insight into the transition from non-coding to coding because these genes have already been shaped by selection. For instance, de novo gene emergence could be frequent but mostly deleterious, or rare but mostly advantageous. In our project, we propose to go beyond classical comparative genomics and force novel proteins to emerge to measure their fitness effects and biochemical properties, and establish the relationship between the two. We will measure the fitness effect of these proteins, confirm their existence and localization, as well as their propensity to interact with other proteins. We will identify protein properties such as length and intrinsic disorder that define the fitness effects and thus the likelihood for a novel protein to emerge. Because many of these biochemical properties are largely defined by nucleotide sequences, our findings will lead to models that directly link de novo gene sequences to fitness effects, allowing us to model gene emergence from sequence composition alone. This will be the first project that is poised to answer the major question of how novel proteins can emerge from previously non-coding sequences.

Neuronal circuits store information through the mechanism of synaptic plasticity, a process where synaptic transmission is strengthened or weakened. Long-term potentiation (LTP) is a major form of synaptic plasticity. It requires both activation of CaMKII and subsequent trafficking of receptors and other proteins to the postsynaptic site. Despite extensive research, the causative relationship linking these two processes is still unknown. Here, Hayashi (live imaging and electrophysiology), Zhang (structure biology), and Lucic (cryoelectron tomography) will team up and take a unique minimalist approach to reconstitute synaptic plasticity from purified proteins. We will reconstitute postsynaptic density (reconstituted PSD or rPSD) on a glass substrate using a group of key scaffold proteins (PSD-95, SynGAP, SAPAP, Shank, and Homer) and receptor such as NR2B. Once a key process is found in minimal system, we will test if the same mechanisms work in intact neurons. Finally, we will investigate the persistent modification of the rPSD induced by the activation of CaMKII, which is expected to act as a hub for trafficking of various proteins. The network organization of the resulting complexes in vitro and in situ will be determined by cryo-electron tomography. The final goal of this proposal is to understand the minimum essential machinery for activity dependent delivery of postsynaptic proteins.

Is evolution possible in the absence of template-based replication?
Evolution, in life as we know it, relies on the copying of DNA with errors, providing the basis of reproduction with heredity and variation. Likewise, in the hypothetical RNA world, copying of RNA with errors is thought to have played the same role. However, the high complexity of RNA polymerases suggests that replication must have been preceded by reproduction and evolution based on simpler catalysts. Reproduction can occur by autocatalytic synthesis of single ribozyme species from RNA fragments, or by collective autocatalysis of multiple ribozyme species, all denoted AutoCatalytic Systems (ACS). For ACS to evolve in an open-ended way, theory indicates that there must exist a large diversity of such ACS throughout the sequence space. Further conditions for evolution are that ACS must amplify within compartments (enabling reproduction with heredity), propagate to other compartments as a function of their differential amplification (selection), and that rare events trigger the appearance of novel ACS (variation).
We aim to test the hypothesis that RNA reproduction based on ACS is widespread in the sequence space, and from this diversity, demonstrate that evolution in ACS is indeed possible.
For this, we will generate a large landscape of self-reproducing molecules using the natural group I intron family as an input for statistical inference methods. Self-reproducers will be constructed by fragmentation of these ribozymes by generalizing the strategy formerly applied to a ribozyme from the Azoarcus bacterium, and developing innovative in vitro screening methods with droplet microfluidics. We will show how the autocatalytic dynamics of individual self-reproducers, and their propensity to catalyse the formation of other self-reproducers or self-reproducing networks, allow to implement the properties of reproduction with heredity and variation underlying evolution.
Evolution will be tested in bulk and in compartmentalized populations, by submitting ACS to cycles of incubation and propagation. Finally, the probability of emergence of evolution in ACS and its open-ended character will be assessed based on the density of functional reproducers and their evolutionary accessibility in the sequence space.