Final Activity Report Summary - CAVEOLINANDNUTRIENTS (Identification of key regulators of the caveolar-endocytic pathway from cell surface to lipid droplets: ... intracellular storage of nutrients)
The ERG project aimed at characterising the caveolar-endocytic pathway to lipid droplets in fat cells. Indeed, adipocytes are full of caveolae formed by the association of caveolin proteins but little is known what they are good for. We have shown that these caveolins were able to be targeted to lipid droplets when cells were exposed o lipids such cholesterol and fatty acids.
In order to determine the key molecular determinants regulating this caveolin-endocytic pathway to lipid droplets, we designed a visual screen assay by using a stable cell line overexpressing fluorescent proteins (cav-1GFP and Adipophilin-RFP, lipid droplet marker). During the ERG funding, we established a human cell line expressing these two fluorescent proteins which will be used for a siRNA high-throughput screen for kinases. This screen will be performed in collaboration with the Max Planck Institute of Dresden.
Besides this high-throughput screen, we further wanted to understand the physiological importance of caveolins around lipid droplets. We therefore optimised a protocol of lipid droplet isolation to be able to obtain pure lipid droplet fractions from adipose tissues from WT and cav-1 KO mice. Our goal is now to characterise these lipid bodies' preparations in terms of lipid composition. Interesting results came out from a first analysis of steroids content by mass spectrometry which now need to be repeated. Additionally, general classes of lipids are going to be screened. This will be part of our work in the application proposed by the consortium 'lipidomicnet' focusing on the biology of lipid droplets. Altogether, comparison of lipid droplet preparation from WT mice tissues versus cav1-KO ones will allow us to have insights in the physiological importance of the presence of caveolin around lipid droplets. Such a large comparison of lipid classes will allow us to have an overview of lipid droplets content and the potential role of caveolins.
Finally, in order to get more insights on the role of caveolins around lipid droplets, we constructed a fusion protein caveolin-perilipin-GFP specifically targeted to lipid droplets. The subcloning of this construct in an adenovirus will allow us to infect adipocytes and will bring us new insights on the role of caveolins aound lipid droplets of fat cells.
In order to determine the key molecular determinants regulating this caveolin-endocytic pathway to lipid droplets, we designed a visual screen assay by using a stable cell line overexpressing fluorescent proteins (cav-1GFP and Adipophilin-RFP, lipid droplet marker). During the ERG funding, we established a human cell line expressing these two fluorescent proteins which will be used for a siRNA high-throughput screen for kinases. This screen will be performed in collaboration with the Max Planck Institute of Dresden.
Besides this high-throughput screen, we further wanted to understand the physiological importance of caveolins around lipid droplets. We therefore optimised a protocol of lipid droplet isolation to be able to obtain pure lipid droplet fractions from adipose tissues from WT and cav-1 KO mice. Our goal is now to characterise these lipid bodies' preparations in terms of lipid composition. Interesting results came out from a first analysis of steroids content by mass spectrometry which now need to be repeated. Additionally, general classes of lipids are going to be screened. This will be part of our work in the application proposed by the consortium 'lipidomicnet' focusing on the biology of lipid droplets. Altogether, comparison of lipid droplet preparation from WT mice tissues versus cav1-KO ones will allow us to have insights in the physiological importance of the presence of caveolin around lipid droplets. Such a large comparison of lipid classes will allow us to have an overview of lipid droplets content and the potential role of caveolins.
Finally, in order to get more insights on the role of caveolins around lipid droplets, we constructed a fusion protein caveolin-perilipin-GFP specifically targeted to lipid droplets. The subcloning of this construct in an adenovirus will allow us to infect adipocytes and will bring us new insights on the role of caveolins aound lipid droplets of fat cells.