CAVEOLINANDNUTRIENTS · Identification of key regulators of the caveolar-endocytic pathway from cell surface to lipid droplets : Physiological importance for intracellular storage of nutrients
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2006-01-01 → 2006-12-31
- EU contribution
- €40,000
- Participants
- 1
- Scheme
- ERG
Lines connect the coordinator with its partners.
Results in brief
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.
Data: CORDIS, © European Union
Project objective
Nutrients overload leads to adipocyte hypertrophy and excess of adipose tissue causing deleterious consequences known as the metabolic syndrome. During my post-doc, we showed that the fat cell surface and the lipid droplet can communicate through an ER-ind ependent endocytic caveolar pathway of critical importance in targeting cholesterol to lipid droplets. This caveolin-dependent route could therefore function more generally to supply lipids to adipocyte lipid droplets. In order to identify key regulators o f this pathway, we will design a screen assay based on microscopy imaging. We will use caveolin fused to the green fluorescent protein (GFP) and the lipid droplet marker adipophilin fused to the red fluorescent protein (RFP). In lipid-loading conditions, c aveolins associate with fat bodies, resulting in the co-localization of the two proteins and yellow colour (merge of green and red emitted fluorescence). By combining this assay with RNAinterference of kinases genes, we hope to identify knock-downs which r everse this phenotype by screening for distinct red and green fluorescence patterns. An important issue is to compare these kinases requirements with the ones involved in caveosomes and clathrin-coated pathways.This project also aims at testing the physiol ogical importance of this caveolar-endocytic pathway in lipid droplet storage in vivo. Thus, we will identify lipid species able to stimulate association of caveolins with lipid bodies (starting from unstimulated conditions where caveolin and adipophilin d ont co-localize). We will in this case screen for induction of yellow colour. The physiological contribution of this pathway in nutrients intracellular storage will be evaluated by measuring concentrations of these lipids candidates in fat bodies isolated from animal models for metabolic syndrome. Identification of keys regulators of such a pathway can thus provide new perspectives for the control of lipid storage, a key feature in the battle against obesity.
Original text from CORDIS.
Participants
- INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE · PARISCoordinatorFrance
Links
Data: CORDIS, © European Union