Anionic lipid domains: correlation with functional topography in a mammalian cell membrane.

Abstract

Polymyxin B was used to explore distribution of anionic phospholipids in sperm plasma membranes by electron microscopy of freeze-fracture replicas. After exposure to Hepes/Tris-buffered polymyxin at 4 mM, phosphatidylcholine liposomes showed no perturbations nor didthey fluoresce with dansylated incubation. When phosphatidylethanolamine was included in the liposomes, they became perturbed and fluoresced. Plasma membranes of Drosophila larval cells, containing or lacking cholesterol, were also disrupted by polymyxin.The cell membranes of guinea pig sperm were likewise disrupted but in specific functional areas. Fusional membrane domains showed protrusions; the stable membrane of the flagellum revealed diffuse bubbling. Regions of well-defined particle arrays and the postacrosomal segment maintained smooth contours. By fluorescence microscopy, we detected the same heterogeneous binding of the polymyxin dansyl derivative.By electron microscopy of freeze-fracture preparations, the cell membrane is a tapestry of different textural domains. In the guinea pig sperm plasma membrane, particle arrays (proteins) observed in freeze-fracture replicas exhibit characteristic patterns on particle surfaces-e.g., the "quilt" of the head, and the zipper and annulus of the tail (1). Recently, labeling sperm cells with filipin, a polyene, has also demonstrated cholesterol in a heterogeneous distribution (2, 3). Like filipin, polymyxin B (PXB) disturbs the contour of membranes containing the lipids that it binds. PXB binds anionic lipids, revealing their concentration distribution. After exposure to PXB, the outer membranes of Gram-negative bacteria erupt in protrusions (4-6) discernable by electron microscopy of thin-section, freeze-fracture, and scanning preparations (7,8); the number of protrusions is correlated with the PXB concentration (9). Moreover, PXB has a proven affinity for anionic lipids (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).In this report, we demonstrate that PXB binds to a mammalian spermatozoon and insect larval cells, producing protrusions similar to those observed in prokaryotes. Unlike the uniformly disposed eruptions of bacterial membranes, however, various membrane perturbations emboss only specific regions of the sperm membrane. Tris buffer at pH 7.4. The vesicles were stored under argon at 4VC until used. We incubated treated and untreated vesicles in 25% glycerol for 1-2 hr before freezing, or processed them without glycerol for thin-sectioning as specified (2). Treatment of liposomes consisted of incubation at 370C in 2 mM PXB (Sigma) in 0.1 M Hepes/Tris buffer (pH 7.4). Additionally, one group of PtdCho/PtdEtn/cholesterol liposomes was incubated for 1 hr in 1.25% glutaraldehyde (pH 7.4, 1% sucrose) in the same buffer and processed for freeze-fracture.Cell Suspensions. Third-instar Drosophila melanogaster larval cells, provided by C. M. Havel (Department of Biochemistry and Biophysics, University of California, San Francisco), were grown in cholesterol-free medium until depleted of the ster...

Plasma-membrane diversity in a highly polarized cell.

“…Therefore, after capacitation, the "flip-flop" of anionic lipids alone could account for the emergence of these lipids in membrane regions where none could be previously observed . But biochemical data suggest that, in addition, concurrent anionic-phospholipid synthesis takes place in sperm (30) . And comparing the information derived from PXB binding, which recognizes outer-leaflet lipids, and adriamycin, which does cross the bilayer, we can but conclude that the cytochemically detected differences in anionic-lipid concentration in adjacent domains of the membrane are also real (14,27,30) (Fig .…”

“…These two macrodomains also contain microdomains. The band of membrane fronting the tip of the nucleus is less perturbable by PXB than the surrounding membrane (14,30) and susceptible to clearing of sterol/filipin complexes and intramembrane particles when glycerinated as a standard freeze-fracture cryoprotectant procedure (12,14,33) (34,35). Even more obvious are certain intramembrane-particle and lipid microdomains in the sperm tail (Fig.…”

“…Like the sterols, acidic phospholipids demonstrable by polymyxin-B (PXB) binding and membrane perturbation are also far more numerous over the anterior part of the acrosomal cap than over the post-acrosomal sector (14,30) (Fig . 12) .…”

Platelet membrane skeleton revealed by quick‐freeze deep‐etch

“…Thrombin (from John Fenton) injections were calculated to give a final concentration of 3 U/ml. A polymyxin B (Sigma, St. Louis, MO) stock solution was prepared and either injected with or without thrombin in the same manner to give a final concentration of 4 mM as described (Bearer and Friend, 1980). Frozen specimens were stored in liquid nitrogen until further processing.…”

Biochemical pharmacology of paradoxical sleep.

“…Several mechanisms by which microdomains of lipids differing in composition from their surroundings can be formed have been discussed by Nicolson (1976). The existence of these microdomains has been visualized by electron microscopy of freeze-fracture preparations using antibiotics which induce local membrane deformity as a consequence of their binding to lipids (Bearer & Friend, 1980). Even more important is the distinction between bulk lipids and boundary lipids.…”