Dynamic Targeting of Proteins to the Lipid Droplet Surface – LDsurfDynamics

Properties of the monolayer droplet surface and association of amphipathic helices in silico and in vitro
• Molecular dynamics simulations of the lipid droplet surface. Here we apply both all atom and coarse grained approaches, using a system in which a thick layer of neutral lipid separating two phospholipid monolayers. • Molecular dynamics analysis of AH association with droplets. We use a simulation strategy similar to our previous work (Vanni et al., 2013), in which we used an all-atom approach to model the association of an ALPS motif with the surface of bilayers of different phospholipid composition.
• Biochemical characterization of AH and LD bioprobes (binding to artificial droplets). The MD approaches, in particular for association of AHs with the droplet surface, are being carried out in parallel with binding studies using artificial droplets.

Targeting of proteins to lipid droplets in cells
• Characterization of LD bioprobes in cells. We are expressing AHs used in the biochemical analyses, but fused to a fluorescent protein, to compare localization in cells, binding to artificial droplets in vitro, and MD simulations.
• Analysis of perilipin wild type and mutant probes in cells

Role of GBF1 and its substrate Arf1 in lipid metabolism and trafficking
• Cell biological approaches to understanding the role of GBF1 in lipid trafficking and metabolism in mammalian cells

1. Identification of the mechansim by which perilipin is targeted to the surface of lipid droplets.
2. Characterization of lipid packing defects on the surface of LDs as a function of core lipid content and phospholipid density.
3. Novel lipid binding properties of perilipin AH.

Numerous proteins acting on lipid droplets contain large regions that are predicted to fold as amphipathic helices at the droplet surface, but what controls this adsorption / folding event is unknown.

1. Alenka Copic, Cesar la Torre Garay, Joachim Moser von Filseck, Romain Gautier, Bruno Antonny, Guillaume Drin, Cathy Jackson. Gordon Conference “Lipids, Molecular & Cellular Biology of”, July 26-31, 2015, Waterville Valley, NH, USA. Selected for an oral presentation.

2. Alenka Copic, César La Torre Garay, Bruno Antonny and Cathy Jackson. Jacques Monod Conference “Molecular basis for membrane remodelling and organization”, November 15-19, 2014, Roscoff, Brittany, France. Poster.

3. Amélie Bacle, Stefano Vanni, Cathy Jackson and Patrick Fuchs. Jacques Monod Conference “Molecular basis for membrane remodelling and organization”, November 15-19, 2014, Roscoff, Brittany, France. Poster.

4. Jackson CL and Bouvet S. (2014) Arfs at a Glance. J Cell Sci. 127:4103-9.

5. Jackson CL. (2014) GEF-effector interactions. Cellular Logistics 4(2):e943616

6. Jackson CL. 2014. Arf proteins and their regulators: at the interface between membrane lipids and the protein trafficking machinery, Chapter 8, in “Ras Superfamily Small G Proteins: Biology and Mechanisms 2”, Alfred Wittinghofer, editor, Springer International Publishing, Switzerland.

7. A. Copic, S. Bouvet, C. la Torre-Garay, C. Jackson. ASCB 2014 Annual Meeting, Philidelphia, USA. Invited Speaker.

8. Samuel Bouvet, Alenka Copic, César La Torre-Garay, Cathy Jackson. Jacques Monod Conference “Molecular basis for membrane remodelling and organization”, November 15-19, 2014, Roscoff, Brittany, France. Invited Speaker.

9. Cathy Jackson. IUBMB Annual Meeting, 21-26 octobre 2014, Taipei, Taiwan. Invited Speaker.

10. Bruno Antonny. IUBMB Annual Meeting, 21-26 octobre 2014, Taipei, Taiwan, Invited Speaker.

11. Bruno Antonny. Gordon Research Conference “Molecular Membrane Biology”, New Hampshire, USA, July 2015, Invited Speaker.

12. Stefano Vanni, Romain Gautier, Hisaaki Hirose, Mathieu Pinot, Helene Barelli and Bruno Antonny. Jacques Monod Conference, November 15-19, 2014, Roscoff, Brittany, France. Poster.

Eukaryotic cells contain a mysterious organelle: the lipid droplet. By mysterious, we do not mean that the function of this organelle is unknown; lipid droplets are the most efficient way for cells to store energy. What makes lipid droplets puzzling entities from the cell biological point of view is their very nature: a core of pure hydrophobic molecules (triglycerides and sterol esters) encircled by a monolayer of phospholipids and specific proteins. As such they differ fundamentally from other organelles, which are surrounded by a lipid bilayer.
Lipid droplets have triggered a revival of interest in recent years. One obvious reason is their importance for understanding metabolic diseases, including lipodystrophies and obesity. In addition, the realization that the frontier between lipid droplets and other cellular organelles is less clear-cut than previously thought stimulates new lines of research. For example, it is now relatively well established that lipid droplets emerge from the endoplasmic reticulum. Although the precise mechanism remains to be established, the most widely accepted model is a mechanism whereby a lens of triglycerides bulges between two phospholipid monolayers before becoming a distinct entity. In addition, it has been shown that some families of proteins that are well known for their association with classical organelles, also associate with lipid droplets. One intriguing example is the small G protein Arf1, best known for its critical function at the Golgi apparatus. Last, cell biology studies have also revealed that lipid droplets exhibit a very dynamic life cycle, undergoing phases of expansion and shrinkage in phase with the recruitment of specific proteins.
The laboratories of Cathy Jackson and Bruno Antonny have been collaborating for many years on the GTPase cycle of the small G Protein Arf1 and on the recognition of membrane-bound organelles by amphipathic helices. It turns out that both aspects are particularly relevant for studying lipid droplets. First, because an exchange factor for Arf1 named GBF1 seems to have the remarkable property to ‘jump’ from the Golgi apparatus to the surface of lipid droplets. In this way, it could coordinate Arf1-mediated functions on the surfaces of both organelles. Second, numerous proteins acting on lipid droplets contain large regions that are predicted to fold as amphipathic helices at the droplet surface, but what controls this adsorption / folding event is unknown.
To understand the dynamics of lipid droplets, it is necessary first to describe the unique properties of the LD surface, and how proteins associate with and modify this surface. What are the lipid packing properties of the LD phospholipid monolayer compared to a bilayer membrane? Are amphipathic helix-containing LD-associated proteins recruited to the phospholipid monolayer by direct protein-lipid interactions? If so, what are the properties of the monolayer (lipid composition, lipid packing) that mediate binding of specific amphipathic helices? How is GBF1 recruited to the lipid droplet surface? Does GBF1 activation of Arf1 alter the properties of the LD surface? The goal of the current proposal is to address these questions in the context of LD maturation, using a combination of approaches including molecular dynamics, reconstitution of protein binding to artificial lipid droplets in vitro, and imaging techniques at both light and electron microscopy levels.