Dendritic cells (DCs) play critical roles in directing innate and adaptive immune responses against infections and cancer. Understanding the mechanisms that control DC development and function may reveal new ways to alter the course of complex human diseases such as cancer. Despite their importance in immunity, some open questions remain in regards to DC basic biology. Two of these questions, in particular, are the subject of this application: 1) DCs are a heterogenous population, which can be subdivided into conventional DC type 1 (cDC1) and 2 (cDC2) subsets. Although their development depends on distinct transcriptional programs, cDC1 and cDC2 descend from a common precursor under the influence of the same growth factor cytokine. What determines cDC1/2 differentiation? 2) Various loss-of-function studies demonstrate that cDC1 are key antigen-presenting cells for initiating CD8+ T cell responses to tumours and some viruses. This primarily relies on a process termed cross-presentation. How is cross-presentation regulated in cDC1? Recent studies indicate that profound changes in cellular metabolism are coupled to immune cell function and may fundamentally underpin cell-fate decisions. Based on previous observations and our own preliminary data, we hypothesise that glycolysis programs cDC1 development and activation, whereas fatty acid metabolism controls the ability of the same cells to cross-present antigens to CD8+ T cells. We propose to define the metabolic programs that drive DC formation and that underlie cDC1 and cDC2 identity and complement this approach with loss and gain-of-function experiments that will allow specific testing of our hypotheses. Globally, these studies will identify novel mechanisms of immune cell control with implications for antiviral and anticancer immunity.

Myocardin-related transcription factors (MRTFs) are G-actin binding proteins which act as transcriptional co-activators Myocardin-related transcription factors (MRTFs) are G-actin binding proteins which act as transcriptional co-activators in association with the transcription factor SRF. MRTF-SRF target genes encode numerous proteins involved in actin dynamics, cell adhesion, migration and contractility. The subcellular localization of the MRTFs is controlled by actin binding which inhibits their nuclear import and promotes their nuclear export. Extracellular signals which activate Rho-family GTPases induce actin polymerization and G-actin depletion, which induces MRTF shuttling to the nucleus and transcriptional activation of MRTF-SRF target genes. The MRTF-SRF pathway activation via Rho plays an important role in cancer cell invasion and metastasis. In addition, in MRTF-SRF signalling inhibits cell senescence in hepatocarcinoma cells which present high Rho activity, but this has not been investigated in other cell types or cancer models. Cell senescence is a process of cell-cycle arrest which typically occurs in ageing, cancer, development or tissue repair, and can facilitate recruitment of immune cells. In the tumour microenvironment, senescent cells can direct events such as therapeutic resistance or metastasis that support malignant progression. In this project we will determine the molecular mechanisms by which MRTF-SRF signalling inhibits cell senescence in MEFs. We also aim to investigate the possibility for it to modulate melanoma progression in the BRafV600E mouse model, where senescence is an initial step prior tumour transformation. Increasing evidence suggests that anti- and pro-senescent therapies can be beneficial also in other pathologies, such as fibrosis, by limiting cell proliferation and allowing clearance of damaged cells. These studies have the potential to reveal new approaches to the modulation of senescence pathways for therapeutic benefit.

Inflammation is a host response that evolved to counteract noxious stimuli that result from infection or tissue injury, and serves to return the affected tissue to homeostasis. Cell death-associated sterile inflammation is a major contributor to secondary tissue damage associated with multiple conditions such as myocardial infarction, transplantation, and stroke. Damaged tissues are thought to elicit their inflammatory effects through the sudden release from cells of endogenous Damage-Associated Molecular patterns (DAMPs) that serve to recruit and modulate the function of immune cells. In vertebrates, a diversity of molecules have been implicated as DAMPs, including ATP, uric acid, and F-actin. In mammals, F-actin is recognised as a DAMP by the C-type lectin receptor DNGR1, expressed on CD8+ Dendritic cells (DCs), that signals to favour the cross-presentation of dead-cell antigen to CD8+ T-cells. Independently of its work on DNGR-1, the host laboratory discovered that extracellular actin elicits a JAK-STAT-dependent inflammatory response in the fruit fly (Drosophila melanogaster). DNGR-1 does not have a functional homolog in fly, therefore the actin sensor remains obscure. In order to identify the molecular sensor of extracellular actin we have conducted an in silico-based screen to identify a candidate list of potential sensors. To functionally evaluate these candidates, we will conduct in vivo RNAi and in vitro gain-of-function screens in Drosophila. We will validate the role for this novel sensor in mediating sensing of extracellular actin through multiple genetic and biochemical approaches. We expect our proposal to give novel insights into the signalling transduction and immunobiology of host responses to evolutionary conserved DAMPs. We anticipate that by understanding cytoskeletal-mediated innate inflammatory responses in fly, it will provide important insights into the evolution of similar damage sensor response pathways in higher organisms

Genome duplication is essential for cell proliferation. Errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. How changes in protein structure regulates function during key events in origin activation and replisome progression, such as melting of the DNA duplex upon CMG helicase assembly, or the mode of binding of Pol alpha during primer synthesis remain unknown. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. To address this issue, I will integrate single-particle cryo electron-microscopy (cryo-EM) with sophisticated biochemical approaches to image origin-dependent DNA replication reactions in vitro on native DNA substrates. To do so, I will establish short origin-containing DNA substrates that permit loading of a single bidirectional replication fork, allowing large numbers of protein bound origins to be visualised within a single field of view. Initially, I will investigate how the structure of duplex DNA changes upon CMG formation during origin activation. Next, I will examine the molecular mechanisms of primer synthesis after origin activation using both cryo-EM and in vitro DNA replication reactions. Finally, I will capture and image synthesising intact replisomes at near atomic resolution. By visualising entire DNA replication reactions instead of isolated replication complexes at high resolution, we will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.