Advanced flourescence microscopy of lipid membranes in polarized cells

The plasma membrane acts as a dynamic interface between the cell and its environment, where its biophysical properties, such as lipid packing and protein mobility regulate essential cellular processes. These properties are especially vital in epithelial cells, which rely on membrane organization and polarity to maintain their specialized functions. Disruptions in these factors can alter signaling pathways and contribute to disease progression. Understanding the relationship between lipid packing, protein diffusion, and their regulation in polarized and nonpolarized cells provides crucial insights into cellular dynamics and signaling. Notably, the loss of epithelial polarity is a hallmark of cancer, influencing cell proliferation, invasion, and metastasis. This thesis explores these aspects through different advanced fluorescence microscopy techniques, which must balance the trade-off between spatial and temporal resolution. These include spectral imaging, confocal and super resolution microscopy such as stimulated emission depletion (STED) microscopy, fluorescence correlation spectroscopy (FCS), (STED-FCS), and fluorescence recovery after photobleaching (FRAP). To investigate epithelial cell polarity, we optimized and used methods to culture MDCK (NBL- 2) epithelial cells in 2D monolayers and 3D cysts, which are spherical structures that mimic the architecture of epithelial tissues. During spectral imaging experiments, we encountered the photo selection challenge, which affects fluorophore excitation and detection in fluorescence microscopy. To address this issue, we proposed and implemented three solutions: culturing cells in 3D cysts, utilizing lightsheet microscopy, and employing a radial polarizer (Z-polarizer). These strategies enabled accurate characterization of lipid packing in polarized cells. Further, we analyzed the effect of RAS proteins (Rat Sarcoma virus proteins), a family of small GTPases that include HRAS, NRAS, and KRAS.