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Final Report Summary - PACKNCROWD (Lipid Packing and Protein Crowding in Lipid Droplets and the ER Membrane)

All cells of our bodies are composed of compartments, termed organelles, which are separated by hydrophobic membranes. Membranes are complex mixtures of many different lipid species and proteins. The specific lipid and protein composition determines organelle identity and enables cells to spatially organize cellular reactions. In addition, membranes can change their shape and protein or lipid composition. The objective of the project PACKNCROWD was to study how both the protein and the lipid component of cellular membranes contribute to their specific function. Rather than focusing on one specific problem or one specialized approach, my ambition was to take a more synergistic look at three poorly understood phenomena, as detailed below. Because these questions necessitate an interdisciplinary approach, I have combined my previous expertise in biochemistry, cell biology and genetics with development of novel biochemical and in silico approaches. In addition, I have established collaborations with scientists in France, Slovenia and elsewhere. Since the start of this project, I have obtained a permanent position at the CNRS as well as more funding to continue this research.

I. The effect of protein crowding on cell membrane deformation
Corresponding publications: Manni MM, Derganc J, Čopič A (2017) Crowd-Sourcing of Membrane Fission: How crowding of non-specialized membrane-bound proteins contributes to cellular membrane fission. Bioessays 39, 1700117 (7 pages). corresponding author
Derganc J, Čopič A (2016); Membrane bending by protein crowding is affected by protein lateral confinement. Biochim Biophys Acta. 1858:1152-9. corresponding authors
Protein targeting in the cell depends on the lipid and on the protein component of membrane-bound compartments. Proteins, which represent two thirds of membrane mass, can affect membrane properties in a non-specific manner through protein crowding. Extending my postdoctoral studies on COPII vesicle formation from the endoplasmic reticulum, I have performed a theoretical analysis of the parameters that influence how protein crowding affects membrane bending. Whereas this effect was previously thought to require asymmetric distribution of protein mass across a membrane, we have shown that when the bending patch of a membrane is surrounded by a diffusion barrier, even a membrane with completely symmetric distribution of protein mass becomes highly impacted by protein crowding. These findings have a wide range of implications becuase membrane protein crowding may affect diverse cellular processes.

II. Targeting of proteins to lipid droplets
Corresponding publication: Čopič A, Antoine-Bally S, Giménez-Andrés M, La Torre Garay C, Antonny B, Manni MM, Pagnotta S, Guihot J, Jackson CL. (2018). A giant amphipathic helix from a perilipin that is adapted for coating lipid droplets. Nat Commun 9(1):1332. corresponding author
Eukaryotic organelles are separated from their surroundings by a barrier consisting of a phospholipid bilayer. The one prominent exception is lipid droplets (LDs), whose function is the storage of fats and maintenance of cellular lipid homeostasis: LDs consist of a neutral lipid core surrounded by a phospholipid monolayer. To understand how proteins specifically target LDs, we used as a model an abundant adipocyte protein perilipin 4. We have shown that this highly unusual protein contains an extremely long and repetitive amphipathic helix, which localizes to LDs. We used fluorescently tagged helical probes to assess their localization in cellular models. Using purified helices and synthetic lipids, we could test in vitro how LD-associated proteins interact with lipid surfaces. We have determined that the combination of high length and low hydrophobicity permits the perilipin 4 helix to specifically target the lipid surface of LDs. We could demonstrate that perilipin 4 can directly coat the neutral lipid core when phospholipids are limiting, suggesting a new mechanism for how cells regulate their lipid droplet function depending on their metabolic needs.

III. Transport of lipids between cellular membranes via lipid transport proteins
Corresponding publications: Wong LH, Čopič A, Levine TP (2017) Advances on the Transfer of Lipids by Lipid Transfer Proteins. Trends Biochem Sci. 42, 516-530.
Drin G, Moser von Filseck J, Čopič A (2016): New molecular mechanisms of inter-organelle lipid transport. Biochem Soc Trans. 44, 486-492.
Moser von Filseck J*, Čopič A*, Delfosse V*, Vanni S, Jackson CL, Bourguet W, and Drin G (2015): Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate. Science 349, 432-436. *co-first authors
An important means of controlling membrane lipid composition is via lipid transport proteins that selectively transfer lipids from one compartment to another. I have studied how one such protein, Osh6p, present in yeast, transports phosphatidylserine from the endoplasmic reticulum to the plasma membrane by exchanging it with phosphatidylinositol 4-phosphate. Using a combination of yeast mutants, lipid probes and real time imaging of cells in a microfluidics device, I could demonstrate that Osh6 is capable of transporting PS to the plasma membrane, provided that PI4P is counter exchanged and ultimately hydrolyzed in the ER. This study indicates that lipid exchange permits transport of a lipid species against its concentration gradient, which is necessary for establishment of cellular lipid asymmetries.

Conclusion. Using model systems like budding yeast, cell culture lines and purified proteins and lipids, I have resolved mechanistic questions that lie at the basis of the regulation of lipid metabolism. Human population worldwide is currently experiencing a huge rise in metabolic disorders such as diabetes, obesity and heart disease, which is directly related to changes in our diet and life-style. In spite of an increasing awareness and ubiquitous recommendations for healthy living, these conditions remain surprisingly difficult to treat and prevent, underscoring the complexity of underlying metabolic processes. Dissecting the mechanistic details of these processes is therefore essential for improving our approaches towards treatment and prevention of these prevalent conditions.
With the support of the CIG grant, I have been able to work with students of different levels in a real research setting, including PhD and master’s students. Last but not least, I present a figure that we used as cover for our paper published in the journal Bioessays, discussing the influence of protein crowding on membrane fission. This figure was drawn by hand by a 13-year old student, Martin Tomazic, with whom I collaborated to construct a more realistic representation of a cellular membrane than what I could easily draw by computer.

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