Author(s): Sarmento, Maria João Martins
Date: 2011
Persistent ID: http://hdl.handle.net/10451/8859
Origin: Repositório da Universidade de Lisboa
Subject(s): PI(4,5)P2; Distribution; Exocytosis; Protein palmitoylation; Calcium; GUV immobilization; Teses de mestrado - 2011
Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the plasma membrane of eukaryotic cells that is essential for several cellular mechanisms, including organization of the actin cytoskeleton, membrane trafficking, endocytosis and exocytosis. The regulation of these processes is thought to involve the local enrichment of PI(4,5)P2 in the plasma membrane at particular timings. In the first part of this work, we aimed to better characterize the mechanisms responsible for PI(4,5)P2 clustering in the plasma membrane of neuronal-like (PC12) and non-neuronal (HEK293) cells. Using FRET microscopy carried out with PH-domains labelled with fluorescent proteins (PH-CFP and PG-YFP), we detected a highly clustered distribution of PI(4,5)P2 in PC12 cells and some indication of clustering below the confocal resolution for HEK293 cells. Moreover, FRET efficiency decreased after inhibition of protein palmitoylation, emphasizing the importance of the interaction between palmitoylated proteins and PI(4,5)P2 for its lateral membrane organization. Additionally, since we were also interested in studying PI(4,5)P2 distribution in giant unilamellar vesicles (GUVs), we optimized a GUV immobilization method based on the interaction of biotinylated lipids with an avidin-coated surface. Three different biotinylated lipids were studied: DOPE-Cap-biotin, DPPE-Cap-biotin and DPPE-biotin. Since PI(4,5)P2 has been shown to partition into lipid rafts, studies with POPC:Chol:PSM (1:1:1) GUVs presenting a coexistence of liquid ordered/liquid disordered phases were performed. Our results showed that the immobilization conditions and the type of immobilizing lipid significantly influence the distribution of lipid domains within GUVs, potentially generating artefacts in lipid phase coexistence studies. By imaging GUVs with a fluorescent analogue of PI(4,5)P2, we observed a Ca2+ induced clustering of the fluorescent lipid at concentrations (>100 μM) known to occur in the proximity of calcium channels after synaptic stimulation. Then, PI(4,5)P2 can potentially act as a lipidic calcium sensor, regulating not only local plasma membrane charge and curvature but also synaptic protein organization.
