Current and future research in our institute will focus on two thematic areas:
The yeast cell cortex as a model system for the study of lateral membrane segregation, endocytosis and secretion
This project will build on our extensive experience with TIRF-based imaging of cortical structures in yeast. We will automate image acquisition by using microfluidics and micro-patterns to facilitate high-throughput imaging of cells with various markers and mutations. Using pattern-analysis algorithms developed for the study of lateral membrane segregation, we can then exploit to the full the powerful tools available for budding yeast, such as yeast knock-out collections or arrays of temperature-sensitive mutants. The planned imaging workflow can also be easily adapted to the study of libraries of chemical compounds. Important target molecules for our studies include a large number of channels and transporters in the yeast membrane as well as signal transduction modules. In addition, we have already identified important new steps in cargo-mediated endocytosis, and we have started to systematically characterize late steps in secretion by monitoring exocyst assembly and vesicle fusion with high temporal resolution. One focus will be to understand the physical and regulatory interactions between different cortical actin structures (endocytic patches and cables) that are found in close proximity to each other but fulfill distinct functions.
This line of research will build on our initial characterizations of novel acto-myosin structures inmammalian cells. First, we will expand our analysis of a dynamic apical acto-myosin network in epithelial cells. We have started to examine changes in network organization after mechanically perturbing cells by means of fluid flow or AFM (atomic force microscope). Our initial findings revealed rapid actin reorganization upon mechanical stimulation, with net disassembly at the cell cortex and assembly in the cytosol and around the nucleus. This reorganization occurred within seconds and was caused by rapid calcium influx. Currently we are investigating the precise role of calcium in global actin reorganization and the consequences of perinuclear actin assembly for transcription regulation. We will also identify other factors that interact with cortical actin and characterize effects on acto-myosin organization upon drug treatments or knock-downs by small interfering RNAs. In a separate set of experiments we have identified a prominent cytoplasmic acto-myosin meshwork that plays an important role in organizing the nucleus during interphase and in chromosome positioning during early mitosis (prophase-metaphase). We will evaluate a potential link between these structures and the still enigmatic spindle matrix.