The objective of this project is to elucidate the molecular mechanisms underlying a novel pathway for the endocytosis and intracellular trafficking of caveolae. It has recently become clear that molecules partitioning in lipid rafts do not always follow the classical clathrin-mediated pathway but can be internalized via a distinct caveolae-mediated route. First, I will develop fluorescence microscopy-based assays to analyze the intracellular trafficking and dynamics of caveolae upon internalization from the plasma membrane. Second, I will design a screening assay to identify key molecules involved in caveolae-mediated endocytosis by targeted, genome-wide RNA interference. Complementary, proteomics will be performed on isolated endocytic intermediates. We have the following aims:
1. To develop assays, using new light microscopy techniques that allow us to visualize and quantify the internalization and trafficking of individual caveolae in living cells.
2. To obtain a list of genes functionally involved in caveolae-mediated endocytosis, using genome-wide RNA interference screening.
3. To isolate caveosomes, to determine their constituents by proteomics analysis, and to design in vitro membrane traffic assays.
4. To obtain putative functions of the molecules identified in 2) and 3) with systematic database mining, sequence alignments and protein domain searches, and to use in vitro and in vivo systems established in 1) and 3) to test these predicted functions directly. This will provide complementary training and will generate data on which an independent research career in the field of endocytosis can be based. The screens will provide a proof of concept for the research community and novel insights into yet uncharacterised membrane traffic events. A better understanding is important because these pathways are involved in basic cell signaling, lipid homeostasis and are used by several viruses, pathogenic bacteria and bacterial toxins.
Fields of science
Call for proposal
See other projects for this call