Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis

Abstract

Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinasedeficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.-Makino, A., Abe, M., Murate, M., Inaba, T.,

Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle

“…The distribution of SM on the plasma membrane has been investigated with a variety of fluorescently labeled pore-forming toxins derived from marine organisms that bind directly to SM (12,13), and we sought to construct a SM biosensor using one of these, equinatoxin II (Eqt), as a scaffold. Eqt is a member of the actinoporin family, a group of small (∼20 kDa), exceptionally stable proteins that are secreted as inactive monomers and undergo conformational changes to oligomerize into a cytotoxic pore in membranes containing SM (14-20) (Fig.…”

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

“…GFP, mCherry, mKate, Venus or Dronpa) or small organic molecules ( e.g. Alexa Fluor), have proved useful in confocal or super-resolution microscopy analyses [22, 23, 26, 114] (see Table 1). For further general information on lysenin, please see [110, 111, 115].…”

Transbilayer lipid distribution in nano scale

“…Previously, we have shown that the binding of lysenin to SM was inhibited by the presence of glycolipids (Ishitsuka et al, 2004;Makino et al, 2015). To examine the possibility that the low signal of MBP-lysenin in the inner leaflet was due to the high concentration of glycolipids, we studied the distribution of the main glycosphingolipid of RBCs, GM3 (Larson and Samuelsson, 1980).…”