Identification of lipid-specific proteins with high-density lipid-immobilized beads.

In biological membranes, lipids often interact with membrane proteins (MPs), regulating the localization and activity of MPs in cells. Although elucidating lipid-MP interactions is critical to comprehend the physiological roles of lipids, a systematic and comprehensive identification of lipid-binding proteins has not been adequately established. Therefore, we report the development of lipid-immobilized beads where lipid molecules were covalently immobilized.

Assembly formation of minor dihydrosphingomyelin in sphingomyelin-rich ordered membrane domains.

The lipidome of mammalian cells not only contain sphingomyelin (SM) but also, as a minor component, dihydrosphongomyelin (DHSM), in which the double bond at C4-C5 in the sphingosine base is reduced to a single-bond linkage. It has been indicated that DHSM forms ordered domains more effectively than SM due to its greater potential to induce intermolecular hydrogen bonds. However, direct information on partition and dynamic behaviors of DHSM in raft-like liquid-ordered (L) and non-raft-like liquid-disordered (L) phase-segregated membranes has been lacking.

Emphatic visualization of sphingomyelin-rich domains by inter-lipid FRET imaging using fluorescent sphingomyelins.

Imaging the distribution of sphingomyelin (SM) in membranes is an important issue in lipid-raft research. Recently we developed novel fluorescent SM analogs that exhibit partition and dynamic behaviors similar to native SM, and succeeded in visualizing lateral domain-segregation between SM-rich liquid-ordered (L) and SM-poor liquid-disordered (L) domains. However, because the fluorescent contrast between these two domains depends directly on their partition ratio for the fluorescent SMs, domain-separation becomes indeterminate when the distribution difference is not great enough.

Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs.

Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker.