Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcore TM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (k on ‫؍‬ 3.2 ؋ 10 4 M ؊1 s ؊1 ) and dissociated extremely slowly (k off ‫؍‬ 1.7 ؋ 10 ؊4 s ؊1 ), giving a low dissociation constant (K D ‫؍‬ 5.3 ؋ 10 ؊9 M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.Sphingomyelin, N-acylsphingosine-1-phosphorylcholine, is a major lipid constituent of animal cell membranes, accounting for up to 60 mol % of the total phospholipid content of some cells, such as sheep erythrocytes (1). In addition to its role as a structural component of membranes, recent studies have suggested the roles of sphingomyelin in various intracellular signaling pathways (2-6). A sphingomyelin cycle has been proposed in which extracellular agents such as tumor necrosis factor ␣, ␥-interferon, and interleukin 1 activate sphingomyelinase, resulting in the production of ceramide which serves as a second messenger mediating the action of these extracellular ligands (4 -6). Sphingomyelin was also proposed to be enriched in an organized membrane domain, caveolae, which contains cholesterol, glyco...

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S U M M A R Y Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/ MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence. (J Histochem Cytochem 54:375-384, 2006)

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