Single-molecule analysis of phospholipid scrambling by TMEM16F

Abstract

SignificanceMembrane proteins that translocate various compounds across biological membranes play important roles in maintaining intracellular homeostasis in cells. Scramblases are membrane proteins that translocate phospholipids between the inner and outer leaflets of the plasma membrane. However, the mechanisms through which these scramblases translocate phospholipids carrying a hydrophilic residue across the hydrophobic membranes are still unclear. Here, we developed a microsystem containing lipid-bilayer membrane arrays with asymmetrically distributed fluorescent phospholipids. Using this system, we measured phospholipid transport mediated by the TMEM16F scramblase at the single-molecule level, thereby enabling characterization of the biophysical features of phospholipid transport.

High-throughput single-molecule bioassay using micro-reactor arrays with a concentration gradient of target molecules

“…Finally, to encapsulate target molecules into micro-reactors, a lipid solution containing 0.3 mg mL −1 POPC and 0.001 mg mL −1 fluorescent lipid (TopFluor-TMR-PS) in chloroform and the third buffer solution were successively infused. The time interval between the injection of the second buffer and that of the lipid solution was shorter than 1 s. After infusion, lipid-bilayer membranes formed on the orifice of individual reactors, as previously reported, 3,23 resulting in the encapsulation of target molecules. Chloroform is a suitable organic solvent for the formation of a lipid membrane on microarrays.…”

“…The orifice of the micro-reactor is suitable to mechanically support the lipid-bilayer membrane owing to its hydrophobicity. 3,[21][22][23] First, CYTOP-A was spin-coated on a glass cover slide (32 mm × 24 mm) and baked for 1 h at 180°C . Photolithography was then performed using a positive photoresist (AZP4903) to pattern mask structures on the CYTOP layer.…”

“…Chloroform is a suitable organic solvent for the formation of a lipid membrane on microarrays. 3,23,25 Its volume is easily managed owing to its lower viscosity than those of other organic solvents for lipids, such as decane or hexadecane. Lipid bilayer formation was confirmed from the activity of a membrane transporter, α-hemolysin (see below), 26 which is not functional unless the lipid membrane becomes as thin as the membrane-spanning structure of α-hemolysin.…”

“…3b displays the time course of the fluorescence signals of Alexa 488 after the formation of lipid-bilayer membranes where the fluorescence intensity decays in a single-exponential fashion, representing the passive transport of fluorescent dye molecules through α-hemolysin pores, as previously reported. 3,23,27 The fluorescence intensity in the array became heterogeneous over time (inset, Fig. 4a), indicating the stochastic formation of the α-hemolysin complex in the lipid-bilayers, i.e., the active fraction (dark reactor in inset of Fig.…”

Digital enzyme assay using attoliter droplet array

“…Such quantized behavior has offered deep insights into the mechanisms of biochemical reactions. 1 – 9 Due to the small size of biomolecules, micro/nanofabrication technology, which has been sophisticatedly developed for the semiconductor industry, provides useful tools for single-molecule analysis, 10 – 20 allowing us to approach the discrete natures of biomolecules.…”