Lipid Biology Laboratory

Outline

Whereas the bilayer organization of biomembranes can be reconstituted in artificial liposomes with a simple lipid composition, biomembranes contain thousands of different lipid species. The relative abundance of these lipids moreover varies from one organelle to another. Also from one leaflet to another, within the same bilayer, the distribution is asymmetric. Even more striking is the lateral segregation of lipids within the same leaflet.

This complex distribution of lipids suggests that the targeting of lipids is highly regulated and that cells require complex supramolecular lipid organization within membranes. One attractive proposal is that the formation of membrane domains is promoted by purely physical interactions between lipids and proteins. In this context, the idea of transient raft-like microdomain formation by clustering of sphingolipid and cholesterol has drawn much attention.

The aim of our laboratory is to elucidate the molecular mechanisms of the assembly and the dynamics of lipids and lipid domains via visualizing lipid molecules and to uncover the principle of the establishment of biomembranes.

Recent Research Topic

Revisiting lipid rafts

Fig. 1 Mode of interaction of lysenin to lipid membranes

Liposomes were prepared using sphingomyelin (SM) and “fluid” phosphatidylcholine (dioleoyl phosphatidylcholine, DOPC) (upper row) or “rigid” phosphatidylcholine (dipalmitoyl phosphatidylcholine, DPPC) (lower row) and the binding of GFP-lysenin (Venus-lysenin) was examined (right). Venus-lysenin binds SM in fluid but not in rigid phosphatidylcholine membrane. In fluid membrane, SM forms clusters (upper row, center). In the center photo, red represents rigid membrane containing SM whereas green shows the fluid membrane rich in DOPC. The left picture schematically indicates the formation of sphingomyelin-rich cluster in orange. In rigid DPPC membrane, SM and DPPC are mixed well and therefore SM is dispersed in the membrane (lower row, left and center).

Fig. 2 Heterogeneity of plasma membrane lipid domains

Jurkat cells were doubly labeled with non-toxic lysenin (HmV-NT-Lys) and cholera toxin B subunit (CTxB). Plasma membranes were uniformly labeled under a fluorescence microscope (upper left). However, under an electron microscope, lysenin domain (at left, labeled with 5 nm gold; colored red at right) and cholera toxin domain (labeled with 10 nm gold; colored blue) were differently distributed. (Below) Ripley's K-function indicates lysenin (labels sphingomyelin) and cholera toxin (labels ganglioside GM1) form domains, whereas a co-cluster of GM1 and sphingomyelin is not formed.

Biomembranes contain thousands of lipid species. These lipids are not randomly distributed in the membrane; rather, it is speculated that nanometer scale domains that contain specific lipids play a crucial role in cellular function. One (and in many cases only one) of the rationales of this hypothesis is the observation that the nanoscale distribution of specific proteins is affected by altering cellular cholesterol content. A few successful examples of direct observation of the lipid distribution by electron microscopy using lipid-specific proteins support the heterogeneous distribution of lipids on the cell membranes. However, the molecular events which underlie “cholesterol sensitivity” are not well known. Studies on red blood cells revealed the asymmetric lipid distribution between the outer and inner leaflets of the plasma membrane. However, the biochemical analysis of lipid asymmetry, which has been used to study lipid asymmetry in red blood cells, is applicable to only a limited number of membrane samples.

In order to understand lipid nanodomains, we need to clarify the dynamics of lipids in the outer and inner leaflets of the membrane at the nanometer scale. Our knowledge of lipid nanodomains is limited, and the elucidation of lipid domains relies only on the careful observation of lipids. The development of new tools and new methods is required for this purpose.

We have been studying lipid specific antibodies, toxins, peptides and low molecular weight compounds in order to visualize lipids. Interactions of these molecules with lipids are not simple. The recognition of lipids is dependent on different factors such as the organization of lipids, membrane curvature, etc. The earthworm-derived toxin, lysenin, specifically binds to sphingomyelin clusters (Fig. 1). In Fig. 2, we showed that there are two different lipid domains on the plasma membrane using lysenin and cholera toxin, which binds glycolipid GM1.

Lipid rafts are well-studied lipid domains which are composed of sphingolipids and cholesterol. Cholesterol plays a crucial role in the formation and maintenance of lipid rafts. We found that poly(ethylene glycol) cholesterol ether (PEG-Chol) is a low molecular weight, low toxicity probe for cholesterol. Although cholesterol is distributed both in the outer and inner leaflets of the plasma membrane, PEG-Chol is restricted to the outer layer because of the bulk PEG moiety of the molecule. Thus, PEG-Chol is used to follow the internalization cholesterol-rich membrane domains on the outer leaflet of the plasma membrane.

In addition to PEG-Chol, the antibiotic filipin and sterol-binding toxins are known as cholesterol tracers. The cholesterol concentrations of the membrane required for filipin binding and toxin binding are different. This makes it possible to estimate cholesterol concentration in the biomembranes using these two cholesterol probes.

Only a limited number of molecules are known to bind specific lipids. The discovery of new probes for lipids or lipid domains is undoubtedly useful to understand the detailed structure of lipid nanodomains. It is also important to develop methods to follow lipid dynamics without using lipid probes.