Membrane Dynamics and Intracellular Trafficking
Eukaryotic cells are characterized by their internal membrane compartments (organelles), which allow different cellular processes, such a protein folding or degradation, to take place in a well-adapted environment. However, in order for the cell to function as a whole, efficient communication must exist between the various organelles, including the endoplasmic reticulum (ER), Golgi apparatus and lipid droplets (LDs), as well as the plasma membrane. Proteins are transported from one compartment to another, and to the exterior of the cell, largely through vesicular trafficking. Lipids can be transported by both vesicular and non-vesicular routes, the latter often occurring at sites of close contact between organelles (membrane contact sites, MCS). Disruption of lipid trafficking and metabolism leads to human pathologies such as obesity, diabetes, cancer and neurodegenerative diseases. Lipid and vesicular trafficking pathways are also subverted for the propagation of numerous viruses, including Hepatitis C and enteroviruses.
Vesicles are formed by deformation of the membrane by coat complexes, a process regulated by small G proteins such as Arf1. Our group has been studying the Arf1 activator GBF1 and its role in the early secretory pathway. We have also discovered that GBF1 and its downstream effectors regulate lipid droplet metabolism and targeting of proteins to the surface of this organelle (Figure 1A). Lipid droplets are the major energy storage depots in cells, and play a central role in lipid trafficking and metabolism.
Figure 1. Vesicular trafficking and lipid metabolism: a multidisciplinary approach. A) GBF1 localizes to both the Golgi and lipid droplets (LD) in numerous cell types, 3T3 L1 adipocytes shown here. B) Molecular dynamics simulations to predict biophysical properties of phospholipid surfaces. Shown is a bilayer of phosphatidylcholine (colored spheres) in water (blue lines). C,D) Targeting of proteins to organelles by amphipathic helices C) α-synuclein, a synaptic vesicle protein, has a long amphipathic helix consisting of repeats of the structure shown. NBD-labeled purified protein requires anionic charge and high curvature in order to bind to liposomes. POPS, palmitoyl-oleoyl-phosphatidylserine. D) Perilipins, targeted specifically to lipid droplets (LDs), have amphipathic helices of the structure shown that are sufficient for LD targeting. Bottom panel shows localization of a perilipin-mCherry probe in HeLa cells.
Each organelle has a unique membrane lipid composition, appropriate for its specific functions. For example, the endoplasmic reticulum membrane is made up of lipids conferring flexibility, thus facilitating protein translocation across it. The plasma membrane, in contrast, has high levels of cholesterol and saturated phospholipids that make it an excellent barrier to protect the cell from the external environment. Proteins also exploit the unique lipid compositions of organelle membranes in order to identify the compartment they need to associate with. We are studying targeting of proteins with amphipathic helices to organelles through direct protein-lipid interactions (Figure 1). We are also studying how organelle lipid compositions are maintained through lipid trafficking at membrane contact sites (Figure 2).
Figure 2. Characterization of membrane contact sites. A) Schematic diagram of a mammalian cell highlighting contact sites between the endoplasmic reticulum (ER) and organelles including the Golgi (orange), endosomes (violet), mitochondria (red), lipid droplets (yellow) and the plasma membrane (PM). B) Electron tomography of a HeLa cell showing an ER-PM contact site (boxed region enlarged in inset). C) Immuno-labeling of a HeLa cell showing ER (labeled with 5 nm gold particles) and Golgi, with a contact site visible. D) Yeast cells have numerous contacts between the ER (green) and the PM. E) The ER SNARE protein Sec22 functions at ER-PM contact sites. A wild type yeast strain and a temperature-sensitive sec22-3 mutant expressing a GFP-labeled PI4P-specific probe. The majority of PI4P in wild type cells is Golgi-localized, but after inactivation of sec22-3 at non-permissive temperature, PI4P accumulates at the PM. See Petkovic et al. Nat Cell Biol 16:434-44.
We are using a multidisciplinary approach consisting of advanced imaging (light and electron microscopy), biophysical, and in silico approaches, to explore the following research questions:
- Identification and characterization of components of membrane contact sites (MCS)
- coordination of vesicular trafficking and lipid transfer at MCS to maintain the unique lipid composition of intracellular organelles
- targeting of peripheral proteins to lipid droplets and bilayer-bounded organelles
Selection of publications
Moser von Filseck J*, Čopič A*, Delfosse V*, Vanni S, Jackson CL, Bourguet W, Drin G. (2015) Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate. Science 349(6246):432-6. *co-first authors
Maja Petkovic, Aymen Jemaiel, Frédéric Daste, Christian G. Specht, Ignacio Izeddin, Daniela Vorkel, Jean-Marc Verbavatz, Xavier Darzacq, Antoine Triller, Karl H. Pfenninger, David Tareste, Catherine L. Jackson & Thierry Galli. 2014. The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion. Nature Cell Biology 16, 434–444 (2014)
Gibson KH, Vorkel D, Meissner J, Verbavatz JM. 2014. Fluorescing the electron: strategies in correlative experimental design. Methods Cell Biol. 124:23-54.
Bouvet S, Golinelli-Cohen MP, Contremoulins V, Jackson CL. 2013. Targeting of the Arf-GEF GBF1 to lipid droplets and Golgi membranes. J Cell Sci. 126:4794-805.
Vamparys L, Gautier R, Vanni S, Bennett WFD, Tieleman DP, Antonny B, Etchebest C, and Fuchs PFJ. 2013. Conical lipids in flat bilayers induce packing defects similar to that induced by positive curvature. Biophys Journal 104: 585-593.
Pranke IM, Morello V, Bigay J, Gibson K, Verbavatz JM, Antonny B#, Jackson CL#. 2011. α-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding. J Cell Biol. 194:89-103.
Last modified 18 January 2017