Endosymbiotic organelles, like plastids and mitochondria, have a very small genome and rely heavily on import of cytosol-synthesised proteins (>95% of the proteome). During evolution, these organelles have evolved unique transmembrane protein complexes, named translocons, capable of recognising and importing precursor proteins in an efficient and selective way. In plastids, two translocons, TOC (Outer membrane) and TIC (Inner membrane), function in series to transport proteins across both membranes. Past research has identified many of the components and some mechanistic information in mature organelles. Yet, the synthesis/assembly of these multi-subunit complexes during development remains elusive, as does the mechanistic detail of their operation. Plastid biogenesis is a critical first step during rapid greening upon seed germination or leaf re-emergence in spring. To understand this, I am proposing a combination of 1. proteomics characterisation, to study the expression and assembly of translocon subunits, and 2. super-resolution imaging (cryo EM), to provide structural information on translocon biogenesis during different plastid developmental stages, in a higher plant, Arabidopsis thaliana, and an early-diverging land plant, Physcometrium patens. The results will elucidate structural heterogeneity and the intrinsic plasticity of TIC-TOC complexes in different types of translocons that transports both photosynthetic and non-photosynthetic pre-proteins. This research will broaden our understanding of organelle development and open new ways to engineer and enhance the targeting of proteins to plastids in different cell types.

Type 1 conventional dendritic (cDC1) cells play a major role in orchestrating anti-viral and antitumor immunity by cross-presenting antigens on major histocompatibility complex class I (MHCI) molecules to CD8+ T cells. cDC1 cells in both mice and human express dendritic cell natural killer lectin group receptor-1 (DNGR-1, encoded by Clec9a), which detects dead cells and supports cross-presentation of dead cell-associated antigens. Recent studies have shown that DNGR-1 favors cross-presentation by promoting phagosomal rupture upon ligand engagement, allowing the escape of phagosomal contents into the cytosol, where they access the MHC class I antigen processing pathway. Although DNGR-1 was shown to destabilize the phagosomal membrane, it is unclear how phagosomal rupture is regulated downstream of DNGR-1 signaling. Here, we will employ a genome wide high throughput screening approach to identify novel regulators of phagosomal rupture and integrity and examine their role in antigen cross-presentation and antitumor immunity. We will utilize multiple cellular assays to mechanistically evaluate the contribution

of potential regulators, followed by generation of in vivo mouse models to study their function. Using a multidisciplinary approach that combines cell biology and functional immunology, we aim to uncover important players in DNGR-1-mediated phagosomal rupture and study their roles in various cancer models. Our approach will provide new insights into the basics of antigen crosspresentation and open new avenues in the development of therapeutic strategies manipulating cross-presentation in cancer.

Cancer cells present altered metabolism to meet the bioenergetic and biosynthetic demands of high proliferation rates. Even though the mitochondrial genome (mtDNA) encodes key components of cellular energy homeostasis, and somatic alterations in mtDNA have been observed in many cancers, the functional significance of mtDNA mutations and alterations in the ETC remains incompletely understood. Previous research from Dr. McFadden’s lab has shown that the vast majority of the mtDNA in Hürthle cell carcinoma of the thyroid (HTC) harbored mutations in the complex I subunit of the electron transport chain (ETC). This loss of complex I function represents an early event in the progression of the disease and is maintained even during metastasis. This suggests that mtDNA mutations are a central driver in the pathogenies of this disease and opens the counterintuitive and exciting possibility that certain cancer types benefit from an impairment of mitochondrial respiration. Thus, I hypothesized that HTC represents an ideal disease outlier in which to interrogate the role of mtDNA alterations and ETC function in cancer. By using highly complementary approaches, I will first interrogate whether mtDNA mutations in humans predict impaired respiration and lead to altered metabolism. Then, I will study the mechanism by which mtDNA mutations confer a metabolic advantage in HTC in vitro, and finally, I will use novel genetically engineered mouse models to interrogate the role of complex I in thyroid tumorigenesis. To address these aims, I will use metabolomics, 13C tracer analysis, exome sequencing analysis, and in vitro and in vivo models of HTC. With this project, I will deliver a mechanistic and functional understanding of mtDNA mutations and complex I loss in HTC which will lead to insights more broadly applicable to tumors harboring mutations in mtDNA such as kidney and colorectal cancers.