NANODROP

Lipid droplets (LDs) are the organelles regulating the energy balance in cells. They are the cell’s main lipid reservoirs, play a major role in membrane biogenesis – critical to cell growth and division – and are involved in a number of metabolic and infectious diseases. LDs are similar to oil-in-water emulsion droplets, and consist of a droplet of oil (i.e., non-polar lipids, such as triolein) surrounded by a phospholipid monolayer. The function of LDs is mostly decided at birth (LD biogenesis), in the endoplasmic reticulum (ER), where most lipids and proteins are loaded to the LDs. Therefore, elucidating the mechanism of LD biogenesis is the key step to understanding LD function. Despite their fundamental role in metabolism and disease, the mechanism of LD formation in the ER remain poorly understood. To form a LD, oil molecules must accumulate between the bilayer leaflets and induce deformation of the membrane. Membrane deformations imply a significant energy cost. What are the driving forces for LD budding? Knockdown of LD-associated proteins identified so far does not prevent LD formation, therefore it has been hypothesized that chemical composition and physical properties (namely surface tension) of the nascent LD play a major role in budding, but no definitive proof confirms this scenario. Another open question on LD budding regards its direction: LDs bud preferentially towards the cytosolic side of the ER. What determines directionality? Directional budding may result from asymmetric lipid or protein distribution, without a requirement for specific curvature-inducing proteins. Again, no proof confirms this hypothesis. The main objective of the present proposal is to determine the mechanism and driving forces for LD directional budding. More specifically, our goal is to understand how LD chemical composition and physical properties promote or hinder LD directional budding, in cells and in model systems. So far it has been extremely difficult to control and even identify the changes in LD composition and physical properties during the different steps of LD biogenesis in the ER of living cells. To break the current deadlock, in the first part of our project we will rely on model systems, which allow a fine control of chemical and physical properties. We will combine cutting-edge molecular-level computer simulations with experiments on artificial LDs. During the past years we have developed expertise in building and manipulating two types of artificial LD systems of different size, both in simulations and in experiments. In both cases, simulation and experimental systems are physically equivalent. Here we will use such model systems to explore the LD budding propensity as a function of LD size, chemical composition, and surface tension. Our integrated computational-experimental approach is novel in the field of LD biology. Molecular simulations will enable the interpretation of experimental data in terms of molecular structures and dynamics on short length and time scales, not accessible otherwise. The properties of model systems can be more easily controlled, but what is the precise correspondence between model systems and living cells? Whenever possible, we will test our findings from model systems on living cells. Preliminary results show that modulation of LD composition has an effect on LD budding propensity. Quantitative measures of LD size distribution in cells will clarify to what extent findings on artificial LD systems can be transferred to cells. Understanding LD biogenesis will have a tremendous impact on current understanding of cell metabolism and cell homeostasis. In turn, a better understanding of cell metabolism will be instrumental to the development of new technologies to modulate cell metabolism, with implications for biotechnology (crop improvement) and for the treatment of common metabolic diseases (e.g., obesity, diabetes, and atherosclerosis) and viral infections.