Shedding light on endocytosis with optimized super-resolution microscopy

Super-resolution microscopy is a relatively new microscopy technique that is still under optimization. In this thesis we focus on the improvement of the quality of super-resolution images, to apply them to the study of the processes of cell signaling and endocytosis. First, we show that the use of a temporal median filter in super-resolution blinking movies can correct the background that comes from out-of-focus fluorescence or fluorophores with suboptimal blinking. We also explore fixation methods that are compatible with immunolabeling, to help achieve high resolution images of the actin cytoskeleton and its regulatory and binding proteins. Then, we apply our optimized super-resolution microscopy to further elucidate the role of actin polymerization in clathrin mediated endocytosis, and the difference between two clathrin coated structures: clathrin coated pits (CCPs) and flat clathrin lattices (FCLs). We show that FCPs are platforms of clathrin coated vesicle formation, controlled by actin polymerization activated via N-WASP and the Arp2/3 complex. Moreover, we show that FCLs recruit important membrane receptors such as EGFR and LPAR1, and that they are crucial in the regulation of the signaling of LPAR1. We also use our optimized super-resolution microscopy to study the effect of Perifosine, a synthetic lipid, on EGFR. We show that Perifosine induces the internalization of EGFR via an unconventional mechanism, specifically in cells that highly express the receptor.