CRC 629 A16 - The mechanism of primary endocytic vesicle formation in papillomavirus endocytosis

Basic data for this project

Description

Endocytosis is essential in eukaryotic cells to internalize extracellular particles, fluid, and ligands generally termed cargo with crucial implications in development, the immune response, neurotransmission, intercellular communication, signal transduction, as well as cellular and organismal homeostasis. Phagocytosis, the uptake of large particles, is typically restricted to specialized cells. In contrast, the uptake of small particles and solutes (pinocytosis) occurs virtually in all eukaryotic cells. Endocytosis starts with the formation of primary endocytic vesicles (PEVs), endocytic vacuoles generated by pinch-offs from the plasma membrane. This multistep process involves activation, cargo capture/sorting, induction of membrane curvature, dilation of curvature, and scission. PEVs are targeted to endosomal organelles. From there, cargo is further sorted to destination organelles. To further elucidate the mode of PEV formation, we address the following questions: What is the itinerary of events during the formation of PEVs in this novel endocytic pathway, i.e. when do factors associate with receptor bound virus during internalization? High resolution live cell microscopy (total internal reflection (TIRF) and spinning disc confocal microscopy) will help to define a hierarchy of interactions, which we will use to hypothesize their functional interdependencies. How do the identified factors contribute to the various steps in PEV formation? Using the hierachical interaction information, we will test the involvement of several proteins in cargo immobilization, membrane curvature formation, scission, intracellular targeting. For this we will use live cell TIRF microscopy combined with computational tracking of viruses, thin section electron microscopy, and cryo electron tomography combined with specific host cell perturbations (siRNA-mediated knockdown, small compound inhibitors, and dominant mutants). With this approach, we hope to describe primary endocytic vesicle formation of this novel pathway, both in terms of tempo-spatial regulation and in terms of morphological alterations of the plasma membrane required for PEV formation.