Advanced fluorescence microscopy
In central project Z2, we will make a wide range of imaging techniques available to other subprojects within the SFB1324, and we will also further develop quantitative fluorescence microscopy. Besides conventional confocal and widefield microscopy, we will employ super-resolution methods including STED microscopy, structured illumination microscopy (SIM) and localization microscopy (PALM/STORM) to observe cells, tissues and entire model organisms in all three spatial dimensions. The Z-project is located at two sites (Institute of Applied Physics in Karlsruhe and Bioquant in Heidelberg), providing complementary expertise and a wide range of imaging infrastructure to the consortium. Quantitative microscopy will be further advanced to enable measurements over extended periods of time under minimally invasive, near-physiological conditions. Specifically, we will set up a new multi-modal fluorescence microscope with light-sheet excitation especially designed for fast three-dimensional (super-resolution) imaging and particle tracking. We will utilize and further refine quantitative fluorescence microscopy data analysis tools such as image mean-square displacement (iMSD) analysis and fluctuation methods (FCS, RICS, N&B etc.) to measure fast (sub-millisecond) dynamics of biomolecules and to quantify protein-protein interactions. We will apply these powerful methods to shed light on key processes involved in Wnt signaling, i.e., secretion, transport, ligand-receptor interactions and downstream signaling. To study the intricate processes along the secretory pathway that enable cells to release Wnt proteins as morphogens into the extracellular space, we will resort to fast 3D imaging by using light-sheet microscopy in combination with localization microscopy (PALM) to achieve a spatial resolution of ca. 30 nm. Quantitative image analysis will reveal transport modes of secretory vesicles such as directed motion or diffusion. We will further image processes at the membrane such as vesicle fusion and clathrin-coated pit formation as part of exosomal excretion, also by using widefield/total internal reflection fluorescence (TIRF) techniques. By using targeted photoactivation, e.g., with EosFP, we will tag proteins and track their pathways through the secretion machinery. We will further study and quantify how Wnt proteins are transported to the receiving cells, e.g., by binding to carrier proteins, on lipoprotein particles or exosomes, or even via filopodia. We will also address the interaction of Wnt ligands, Wnt modulators such as Dkk and receptors in great detail by using fluctuation spectroscopy methods and fast imaging. Here the aim is to quantify ligand-receptor interactions, and to better characterize signalosome structure and dynamics including internalization and downstream effects. With these experiments, we aim to collect quantitative data on functional processes along the Wnt signaling pathway to contribute to further advancing our knowledge of this complex network of biomolecular interactions.