Cancer cells heavily rely on the proteasome-ubiquitin system mediated tuning of proteins stability and proteome composition. Proteasome and ubiquitin ligases targeting drugs are already in use against certain haematological cancers, and similar inhibitors are currently explored as a possibility for autoimmune diseases and parasite infestation treatment. E3 ubiquitin ligases are the ultimate target for these therapies as they determine specificity in substrate recognition. It is thus of utmost interest for both basic and translational research to find new ubiquitin ligases essential for cell proliferation, cell migration or cancer progression and couple them with their substrates. In the proposed LIGER project, we aspire to fuse cutting-edge methods to identify new ubiquitin ligase – substrate pairs. In particular, we will focus on E3 ubiquitin ligases involved in cancer progression and more specifically, in its two crucial aspects - cell growth and cell migration. To pre-select the ubiquitin ligases for further analysis, we will perform an unbiased screen for ubiquitin ligases that are involved in cell growth and cell migration. To achieve this goal, CRISPR library screening methodology will be employed. Consequentially, we will purify proteins associated with the selected ubiquitin ligases and identify their potential substrates. Standard biochemical and molecular biology methods will allow us to study in detail the protein interface between the ubiquitin ligases and their novel substrates and describe the biological function of their degradation. LIGER project will increase our understanding of ubiquitin pathway in physiological and pathological conditions and identify new potential targets for cancer therapy.

Cilia and flagella are evolutionary conserved organelles indispensable for vital processes in eukaryotic organisms, such as environment sensing, cell motility, signaling and development. Broad spectrum of ciliary functions, together with omnipresence of the cilium throughout human body, explains the range of symptoms associated with congenital ciliary disorders called ciliopathies. On the other hand, cilia are essential for survival of parasites, such as trypanosomatids, in the host. Therefore, cilia are of great interest as a potential therapeutic target. The ciliary tip is an essential ciliary domain; it provides capping and mechanical stabilization of the ciliary cytoskeleton, it is a turning point of the intraflagellar transport trains, a sole place of cilium growth and a place of budding of signaling vesicles. Yet the tip is the most enigmatic of all ciliary domains, with structures constituting the ciliary tip largely unknown. This hampers our understanding of how are the tip-related processes brought about and orchestrated. To gain insight into the dynamic ultrastructure od the ciliary tip, I will develop a novel technique for cryogenic correlative light and electron microscopy based on solid immersion lens (SIL) optics. I will integrate the resulting imaging data with the tip proteome project project carried out by the host lab and provide mechanistic understanding of the resulting tip model by employing top-down synthetic biology and in vitro reconstitution approaches. Key achievements of this project will include: (i) development of the cryo-SIL technique and (ii) unraveling the functions of the the ciliary tip domain, which will broaden our knowledge of the principles of self-organization of biological systems.

T cells have a central role in most adaptive immune responses, including immunity to infection, cancer, and autoimmunity. Increasing evidence shows that even resting steady-state T cells form many different subsets with unique functions. Variable level of self-reactivity and previous antigenic exposure are most likely two major determinants of the T-cell diversity. However, the number, identity, and biological function of steady-state T-cell subsets are still very incompletely understood. Receptors to ligands from TNF and B7 families exhibit variable expression among T-cell subsets and are important regulators of T-cell fate decisions. We hypothesize that pathways triggered by these receptors substantially contribute to the functional diversity of T cells.The FunDiT project uses a set of novel tools to systematically identify steady-state CD8+ T cell subsets and characterize their biological roles. The project has three complementary objectives. (1) Identification of CD8+ T cell subsets. We will identify subsets based on single cell gene expression profiling. We will determine the role of self and foreign antigens in the formation of these subsets and match corresponding subsets between mice and humans. (2) Role of particular subsets in the immune response. We will compare antigenic responses of particular subsets using our novel model allowing inducible expression of a defined TCR. The activity of T-cell subsets in three disease models (infection, cancer, autoimmunity) will be characterized. (3) Characterization of key costimulatory/inhibitory pathways. We will use our novel mass spectrometry-based approach to identify receptors and signaling molecules involved in the signaling by ligands from TNF and B7 families in T cells. The results will provide understanding of the adaptive immunity in particular disease context and resolve long-standing questions concerning the roles of T-cell diversity in protective immunity and tolerance to healthy tissues and tumors.

Antigen-stimulated naïve CD8+ T cells proliferate and differentiate into effector and memory cells. Whereas effector T cells remove infected or cancerous cells, memory T cells protect the organism from re-infections. Despite decades of research, the challenging central questions of how naïve T cells form diverse progeny and what drives the differential response of naïve and memory T cells to infection remain unanswered, largely because of lacking experimental tools. The goal of this project is to generate a comprehensive model of cell-fate choices of naïve and memory CD8+ T cells in vivo. We will achieve this by addressing three complementary specific objectives: 1) To understand the early and late fate choices in naïve T cells. 2) To uncover differences between naïve and memory T-cell responses and fates. 3) To identify the role of proximal protein kinases LCK and FYN in T-cell fate choices. We will pursue these aims using a combination of experimental immunology and systems biology. We used the synergy between novel genetic models and single cell atlases (i) to characterize an unprecedented transient stage of activated T cells, (ii) to determine the early gene expression signatures and fate choices of in vivo activated naïve and memory T cells, and (iii) to observe that LCK secures memory T-cell formation. These tools and findings offer us novel perspectives to tackle the challenging objective in its full complexity. We will develop additional unique experimental models coupled with innovative in-silico techniques to uncover the cellular and molecular mechanisms underlying diverse fate choices of particular T-cell subsets and to narrow the gap between mouse and human immunology. Overall, this project has the ambition to resolve long-standing fundamental questions in immunology to open new avenues for targeting and modulating T-cell fates in vivo for efficient vaccine design and for promoting beneficial cytotoxic responses to chronic infections and cancer.