Lipid Rafts from Olfactory Ensheathing Cells: Molecular Composition and Possible Roles

Abstract

Olfactory ensheathing cells (OECs) are specialized glial cells of the olfactory system, believed to play a role in the continuous production of olfactory neurons and ensheathment of their axons. Although OECs are used in therapeutic applications, little is known about the cellular mechanisms underlying their migratory behavior. Recently, we showed that OEC migration is sensitive to ganglioside blockage through A2B5 and Jones antibody in OEC culture. Gangliosides are common components of lipid rafts, where they participate in several cellular mechanisms, including cell migration. Here, we characterized OEC lipid rafts, analyzing the presence of specific proteins and gangliosides that are commonly expressed in motile neural cells, such as young neurons, oligodendrocyte progenitors, and glioma cells. Our results showed that lipid rafts isolated from OECs were enriched in cholesterol, sphingolipids, phosphatidylcholine, caveolin-1, flotillin-1, gangliosides GM1 and 9-O-acetyl GD3, A2B5-recognized gangliosides, CNPase, α-actinin, and β1-integrin. Analysis of the actin cytoskeleton of OECs revealed stress fibers, membrane spikes, ruffled membranes and lamellipodia during cell migration, as well as the distribution of α-actinin in membrane projections. This is the first description of α-actinin and flotillin-1 in lipid rafts isolated from OECs and suggests that, together with β1-integrin and gangliosides, membrane lipid rafts play a role during OEC migration. This study provides new information on the molecular composition of OEC membrane microdomains that can impact on our understanding of the role of OEC lipid rafts under physiological and pathological conditions of the nervous system, including inflammation, hypoxia, aging, neurodegenerative diseases, head trauma, brain tumor, and infection.

Keywords: Cell motility; Gangliosides; Glial cells; Membrane microdomains.

Fig. 1

S100 and CNPase immunoreactivity of isolated OECs. Cultures were fixed in paraformaldehyde (4%). a OEC immunoreactivity for the S100 protein, b the same field showing OEC immunoreactivity for CNPase, c cell nuclei labeling (DAPI), and d merged image. All cells, including bipolar cells (arrowhead) and tripolar cells (arrow), displayed immunoreactivity for S100 and CNPase (a–d). Scale bar: 10 µm

Fig. 2

Sucrose gradient and density fractions. a Sucrose gradient tube containing 10⁹ cells after 20–22 h of centrifugation. A floating material was observed in the 5–30% sucrose interface (arrow). b Thirteen fractions of 1 mL from this gradient were collected and analyzed in the refractometer and the results converted to density. The density increased gradually along the gradient. Each fraction represents the mean and standard deviation of five different experiments

Fig. 3

Distribution of protein and cholesterol in Triton X-100 rafts. Triton X-100 extracts of OECs were run on a sucrose gradient and the separated gradient fractions were assayed for the distribution of total protein and cholesterol. In detergent-resistant membrane fractions, cholesterol was enriched in fractions 4 to 6, which showed a low protein concentration

Fig. 4

Characterization of Triton X-100 lipid-raft markers. OEC membranes were extracted in 1% Triton X-100 and separated on a density gradient composed of 40, 30, and 5% sucrose. Thirteen fractions were collected from the top of the gradient tube downward and submitted to dot-blotting analysis, where 500 µL from each fraction was spotted directly on the nitrocellulose membrane. Flotillin-1, caveolin-1, and G_M1 ganglioside (cholera toxin labeling) were found enriched in fractions 4 to 6, as expected for lipid-raft domains

Fig. 5

Lipid analysis in sucrose density gradient fractions of OECs. OECs were extracted with Triton X-100, submitted to a sucrose density gradient and ultracentrifuged. Total lipids from all fractions were determined gravimetrically and the extracted lipids were analyzed. a The neutral lipid composition of gradient fractions was analyzed by high-performance thin-layer chromatography (HPTLC). CHO: cholesterol. b Densitometric analysis of HPTLC for the cholesterol fractions, using the program ImageMaster, Pharmacia Biotech Inc., San Francisco, CA, USA. Each fraction represents the mean and the standard deviation of 3 different experiments. c Analysis by one-dimensional HPTLC for phospholipids. d Densitometric analysis of HPTLC for phospholipids, using the program ImageMaster, Pharmacia Biotech Inc., San Francisco, CA, USA. Each fraction represents the mean and the standard deviation of 3 different experiments. PC phosphatidylcholine, SM sphingomyelin, UP undetermined phospholipids

Fig. 6

Electron photomicrographs of lipid raft structures isolated using Triton X-100. a Electron micrographs of lipid raft fraction. b High-density fraction isolated from OECs using Triton X-100. OECs were fractionated by ultracentrifugation on a sucrose gradient, and fractions containing lipid rafts were pooled and dialyzed with TNE buffer. High-density fractions were treated in the same way as a negative control. All samples were negatively stained with uranyl acetate. Scale bar: 0.2 µm

Fig. 7

Characterization (dot-blot) of actin-related proteins in sucrose density gradient fractions. OEC membranes were extracted in 1% Triton X-100 and separated on a sucrose gradient. Thirteen fractions were collected from the top of the gradient tube downward and submitted to dot-blotting analysis. The analysis shows the immunoreactivity of actin-related proteins (CNPase, β1-integrin, and α-actinin), especially in raft fractions

Fig. 8

Sucrose density gradient profiles of 9-O-acetyl GD3 and other gangliosides recognized by the antibody A2B5 in Triton X-100 rafts. OECs were lysed in 1% Triton X-100. This solution was fractionated by ultracentrifugation on a discontinuous sucrose gradient and 13 fractions were collected from the top of the gradient tube downward. Dot-blotting analysis was performed for 9-O-acetyl GD3 (Jones antibody) and other gangliosides recognized by the antibody A2B5. Note the higher intensity in fraction 5. This fraction co-localizes with markers of lipid rafts