Phagocytic podosomes in uptake and processing of biologically relevant targets

• Applicant: Professor Dr. Stefan Linder
• Subject Area: Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565062662

Cells use phagocytosis to internalize and degrade particles with a diameter >0.5 µm, enabling elimination of pathogens, apoptotic cells or debris. Dynamic remodelling of the actin cytoskeleton is crucial for growth and closure of the phagocytic cup that enables attachment and engulfment of particles. This phagocytic actin network has conventionally been seen as uniform and continuous. However, recent evidence using artificial beads shows that this network can be structured differently, in the form of micron-sized, F-actin-rich dots, so-called phagocytic podosomes. This project investigates phagocytic podosomes for the first time in the context of pathophysiologically relevant targets such as the emerging pathogen Candida auris, a yeast associated with a heavy disease burden. We could already show that uptake of C. auris by primary human macrophages is associated with the formation of numerous phagocytic podosomes. that contain typical components of matrix-associated podosomes and also share a similar architecture, consisting of several substructures, that is necessary for podosome function. We aim to unravel the molecular mechanisms that regulate phagocytic podosomes, and their contribution to internalization and intracellular processing of C. auris by human macrophages. The work programme is structured in 3 work packages, with WP1 focusing on composition, regulation and dynamics of phagocytic podosomes, WP2 investigating potential functions such as uptake efficiency, barrier function, phagosome maturation, recruitment of lytic enzymes, and particle disruption, while WP3 investigates formation and regulation of phagocytic podosomes in vivo, in zebrafish embryos. To investigate this, we will use a combination of techniques: i) molecular biological techniques such as siRNA-mediated knockdown, expression of fusion or mutant constructs, or pharmacological inhibition, ii) microscopical techniques such as confocal live cell imaging, fluorescence recovery after photobleaching, and superresolution techniques including 3D STED, SoRA and MINFLUX, iii) software analysis of podosome architecture, iv) phagocytosis-oriented assays such as inside-out staining, dequenced BSA, acrylamide beads of tunable stiffness, as well as v) infection experiments in zebrafish embryos. For this, we have teamed up with national (Christian Gorzelanny, chitinases) and international (Daan Vorselen; forces in phagocytosis; Maria Forlenza: zebrafish analysis) experts. Results gained from this study will reveal the regulatory mechanisms of phagocytic podosomes as well as their functions during uptake and intracellular processing of particles. These data will also highlight for the first time biologically relevant targets, such as the emerging pathogen C. auris, whose uptake into immune cells is mediated by phagocytic podosomes.

• DFG Programme: Research Grants