BFLDs · Direct Imaging of Budding and Fusion of Lipid Droplets Mediated by Proteins in Emulsion Droplets Based on Microfluidics - Dynamics of Proteins Interactions, Assembly and Metabolism Energy
FP7 — People (Marie Curie Actions)
- Duration
- 2012-04-01 → 2015-03-31
- EU contribution
- €280,018
- Participants
- 1
- Scheme
- MC-IOF
Lines connect the coordinator with its partners.
Results in brief
Direct Imaging of Budding and Fusion of Lipid Droplets Mediated by Proteins in Emulsion Droplets Based on Microfluidics - Dynamics of Proteins Interactions, Assembly and Metabolism Energy
Lipid droplets (LDs) are cellular fat droplets at the core of cellular energy metabolism. Their regulation is crucial for human health. A malfunction of LDs has direct consequences such as the development of cardiovascular diseases and type II diabetes, and many other lipid pathologies including liver steatosis or lipodystrophy. LDs perform many other functions, distinct from the basic regulation or cellular energy metabolism, and serve as hosts for the proliferation of Hepatitis C and Dengue viruses. The fate of LDs and their consequences on human health is based on the specific binding of proteins to their surface. During the three years term of the BFLD project, I tackled important questions of how proteins localize to lipid droplets (LDs) and how is their binding regulated. This is the main question of the LD field. 1) I contributed to unveil the role the vesicular trafficking machinery COPI on controlling LD proteins targeting to LDs. We showed that COPI increases the surface tension of LDs to favor either the direct binding of proteins to LDs, as their triglyceride oil phase become exposed, or the establishment of bridges between the ER and LDs to enable the passage of proteins. 2) We have demonstrated that for such process, COPI provides and energy of 1500-2000 kBT to bud nano particles from membranes, especially from LD monolayers. Knowing this energy allows better understanding how COPI budding is mechanically regulated in vivo by a simple remodeling of membrane properties. 3) The binding of proteins to LDs determines LD protein composition and fate. We demonstrated that proteins compete for binding LD. Proteins have different binding motifs. We found that amphipathic helices such as of CCT1, an important enzyme of phosphatidyl choline synthesis, are competed off from LDs by other strongly binding proteins, e.g. GPAT4 having a hairpin-binding motif. This competition for binding is best illustrated by lipolysis during energy remobilization. Indeed, LDs get consumed, shrink and diminish their surface. The compression of the LD surface during this process leads mainly to expel of amphipathic helix-binding proteins.
Data: CORDIS, © European Union
Project objective
Lipid droplets (LDs) were believed many years ago to be mere passive reservoirs of lipids, which role was limited to supply lipids to cell membranes when needed and to avoid the accumulation of fat in adipocytes leading obesity. Since this last decade a newly focus on LDs has led to understand their dynamic organelles behavior and their implication in many diseases proliferation in cells. Indeed, the virus or proteins linked to them are located on LDs and therefore the understanding of the biogenesis of lipid droplets (by budding from a membrane) and their pathway (fusion between LDs) became of great interest. Unfortunately, a little is understood about that but many Biology labs start now to converge on the topic. However, all the approaches proposed to study the budding and fusion of LDs are done in in vivo systems mostly by knocking down or over expressing a list of proteins, presumable responsible of the effect, and watch the consequences. We think it doesn’t guarantee their direct implication and there is additionally a lack of a clear visualization of the processes and their energy characterization. We propose to use emulsion droplets based on microfluidics so study them. COPI (GTP, Arf1 and coatomers proteins) which is involved in the budding of vesicles is likely implicated in the budding of LDs. To prove that, we will form water drops in an oil containing phospholipids that will cover their interface. The water drops will contain the COPI; oil droplets will be therefore budded from the phospholipid monolayer at the interface: we have preliminary results confirming that. Regarding the fusion process, the way SNARE proteins assembly (v and t-SNAREs) leads to fuse vesicles is believed to provoke the merge of LDs. We will investigate that by forming in microfluidics oil drops decorated separately with t and v-SNAREs that will be approached together by using the micropipette technique and fusion events will be watched.
Original text from CORDIS.
Participants
- CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS · ParisCoordinatorFrance
Links
Data: CORDIS, © European Union