Numerous proteins target lipid droplets (LDs) through amphipathic helices (AHs). It is generally assumed that AHs insert bulky hydrophobic residues in packing defects at the LD surface. However, this model does not explain the targeting of perilipins, the most abundant and specific amphipathic proteins of LDs, which are weakly hydrophobic. A striking example is Plin4, whose gigantic and repetitive AH lacks bulky hydrophobic residues. Using a range of complementary approaches, we show that Plin4 forms a remarkably immobile and stable protein layer at the surface of cellular or in vitro generated oil droplets, and decreases LD size. Plin4 AH stability on LDs is exquisitely sensitive to the nature and distribution of its polar residues. These results suggest that Plin4 forms stable arrangements of adjacent AHs via polar/electrostatic interactions, reminiscent of the organization of apolipoproteins in lipoprotein particles, thus pointing to a general mechanism of AH stabilization via lateral interactions.

The reversible environmental changes around flaccid lipid vesicles represent a considerable experimental challenge, particularly because of remarkable softness of flaccid membranes, which can warp irreversibly under the slightest hydrodynamic flow. As a result, we have developed a microfluidic device for the controlled analysis of individual flaccid, giant lipid vesicles in a changing chemical environment. The setup combines the advantages of a flow-free microfluidic diffusion chamber and optical tweezers, which are used to load the sample vesicles into the chamber. After a vesicle is loaded into the diffusion chamber, its chemical environment is controllably and reversibly changed solely by means of diffusion. The chamber is designed as a 250 micrometres-long and 100 micrometres-wide dead-end microchannel, which extends from a T-junction of the main microchannels. Measurements of the flow-velocity profile in the chamber show that the flow rate decreases exponentially and scales linearly with the flow rate in the main channel. The characteristic length of the exponential decrease is 15 (1 ± 0.13) micrometres, meaning that a large part of the diffusion chamber is effectively flow-free. The diffusion properties are assessed by monitoring the diffusion of a dye into the chamber. It was found that a simple 1D diffusion model fits well to the experimental data. The time needed for the exchange of solutes in the chamber is of the order of minutes, depending on the solute's molecular weight. Here, we demonstrate how the diffusion chamber can be used for reversible environmental changes around flaccid, giant lipid vesicles and membrane tethers (nanotubes).

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant.