The human red blood cell membrane is uniquely designed for its function. The lipid membrane and underlying protein cytoskeleton collaborate to create a characteristic biconcave shape and is flexible enough to allow the cell to traverse capillary openings a fraction of the cell diameter. Due to constraints imparted on the membrane by the cytoskeleton, amphipathic compounds induce cell shape changes consistent with the transbilayer location of the lipid. A well-characterized example that exploits this property is measurement of the activity of the erythrocyte flippase. Red cell membranes are comprised of four major classes of phospholipids that are organized non-randomly; the choline-containing phospholipids are concentrated on the outer membrane monolayer and the amine containing phospholipids, particularly phosphatidylserine (PS), are enriched on the cytofacial monolayer. This asymmetric distribution is maintained by the thermodynamic barrier to spontaneous diffusion and the action of lipid-specific flippases. The first evidence for PS flippase activity came from studies of exogenously incorporated synthetic lipids on red blood cell shape. The increase in outer monolayer surface area by PS induced the formation of spiky echinocytes, followed by reversion to discocytic and, subsequently, invaginated stomatocytic shapes, consistent with flippase transport of PS to, and relative expansion of, the inner monolayer surface. The lipid movements inferred from these shape changes have been verified with labeled lipids and have provided valuable information about flippase biochemistry. Using a microfluidic system to mix cells and lipids, we have recorded lipid-induced shape changes in real time to further examine flippase activity in individual cells. Our initial studies have revealed a non-uniform rate of lipid incorporation and transbilayer transport that might reflect age or metabolic differences. This technique is also being used to address the interaction of the membrane and the cytoskeleton, particularly at the site of formation of evaginations and invaginations in amphipath-induced shape changes.

Measurement of flippase activity in individual red blood cells

255th National Meeting (2018)

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