This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm. Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific ‘electron-transparent windows’ in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.

The formation of crossovers during meiotic cell divisions is a crucial process to produce sperm and egg cells. This mechanism not only secures proper chromosome segregation but also enhances genetic diversity, playing an essential role in sexual reproduction and evolutionary adaptation. Disruptions in the regulation of crossover events can have detrimental effects on individual organisms and entire species populations. Thus, crossover formation is tightly regulated by both positive and negative pathways. Crossover assurance guarantees that each pair of homologs undergoes at least one crossover, facilitating proper segregation. Simultaneously, crossover interference prevents individual crossovers from occurring too closely within the genome, minimizing the risk of damage. However, the molecular mechanisms of these key regulatory processes and the functional coupling between them are still not understood. To dissect the regulatory mechanisms of crossover formation, we have achieved a groundbreaking visualization of this process in vivo, employing advanced imaging technology and AI-powered image analysis. Our approach includes real-time imaging and correlative super-resolution microscopy for temporal and structural analysis of key steps in crossover formation. In COntrol, I will now exploit these tools to acquire the quantitative data necessary to obtain a mechanistic understanding of crossover regulation. I will develop and rigorously test biophysical models through precisely targeted genetic perturbations, harnessing C. elegans' unique toolset of advanced real-time imaging and genetics. Furthermore, I will validate the uncovered mechanistic principles by examining their conservation in the vertebrate model system zebrafish. COntrol will thus shed light on one of the most fundamental questions in biology, namely how organisms distribute and shuffle genetic information among their progeny while maintaining the genetic integrity of future generations.

Influenza is a major public health burden, with seasonal outbreaks contributing significantly to mortality worldwide, and the emergence of pandemic strains remaining an ever-present threat. Influenza drug and vaccine conception efforts are aided by a thorough understanding of its molecular biology. A key aspect of the influenza lifecycle is the production of capped and poly-adenylated messenger RNA by the heterotrimeric influenza polymerase (FluPol). Ground-breaking work performed by the Cusack lab, has described with residue-resolution detail, the FluPol structures that form during transcription of short, non-nucleoprotein (NP) bound viral RNAs (vRNAs). However, influenza transcription in vivo occurs within the ribonucleoprotein (RNP) particle and does not utilise naked genome segments. The viral RNP (vRNP) is a super-helical complex composed FluPol bound at the conserved 3′ and 5′ ends of a vRNA, which is coated with NP. The current low-resolution structures provide little information about the molecular details of vRNP function, particularly, how NPs interact with FluPol and the vRNA template. Via an inter-disciplinary approach, I will utilise cryo-electron microscopy methods, transcription assays and single-molecule fluorescence, to obtain the first high resolution structure of a dynamic influenza vRNP, with a particular focus on the spatial organisation of NPs relative to FluPol. In addition to this work facilitating future influenza drug research, it will provide a basis to investigate the vRNP during other lifecycle stages and act as proof-of-principle for study of other viral protein-RNA complexes, such as those from corona-, arena- and bunyaviruses. Work will be performed in the groups of Stephen Cusack and Olivier Duss based at EMBL Grenoble and Heidelberg, respectively. Here, I will have access to world-leading facilities and training opportunities, supporting my growth as an independent researcher and an expert in RNA virus structural biology.