Stomatocyte–discocyte–echinocyte sequence of the human red blood cell: Evidence for the bilayer– couple hypothesis from membrane mechanics

Abstract

U nder physiological conditions, a normal human RBC assumes a biconcave discoid (discocyte) shape Ϸ8 m in diameter. It has been known for more than 50 years (1) that a variety of agents can modify this shape systematically and reversibly at constant area and volume § (Fig. 1 Left) (refs. 2 and 3, and ref. 4 and references therein). One set of agents, including anionic amphipaths, high salt, high pH, ATP depletion, cholesterol enrichment, and proximity to a glass surface, induces a series of crenated shapes, called echinocytes, characterized by convex rounded protrusions or spicules. Under further loading, the spicules become smaller and more numerous and eventually (in a process that we shall not discuss further) bud off irreversibly, forming extracellular vesicles composed of plasma membrane materials and leaving behind a more or less spherical body with reduced area and volume (the spheroechinocyte). Another set of agents, including cationic amphipaths, low salt, low pH, and cholesterol depletion, induces concave shapes called stomatocytes. On further loading, multiple concave invaginations are produced, which eventually bud off to form interior vesicles and leave a spherostomatocyte. This ''main sequence'' is universal in the sense that the shapes seen and their order of appearance do not depend on which echinocytogenic or stomatocytogenic agent is used. Other shapes outside of this main sequence are also seen under certain conditions (Fig. 2 Left).The RBC consists of a composite membrane [plasma membrane plus membrane skeleton (MS)] surrounding a fluid interior, so it is natural to search in the membrane properties for an explanation of these shape changes. The first explanation was provided by Sheetz and Singer (5), who proposed that the mechanism involves small changes in the relaxed area difference ⌬A 0 between the two leaflets of the plasma membrane. Thus, any effect that expands the outer leaflet relative to the inner one (increasing ⌬A 0 ) produces a tendency to form convex structures on the cell surface (e.g., echinocytic spicules) to accommodate the extra area; conversely, an expansion of the inner leaflet relative to the outer one (decreasing ⌬A 0 ) favors concavities (e.g., stomatocytic shapes). This so-called bilayer-couple hypothesis explains the universality of the main, stomatocytediscocyte-echinocyte sequence by postulating that all shapechanging agents (chemical, biological, or even physical) act solely or mainly through their effect on ⌬A 0 . Biochemistry comes in only to explain how and to what extent each agent modifies ⌬A 0 . For example, added amphipaths partition differentially between the two bilayer leaflets because of the known asymmetry in composition: The inner leaflet contains a significant fraction of negatively charged lipids (6), thus making it a more attractive environment for cationic amphipaths. Cholesterol, on the other hand, is known to prefer the outer leaflet. Thus, cholesterol addition tends to expand the outer leaflet, whereas addition of cationic amphipaths tends to...

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