Abstract
Plasma membranes are organized into functional domains both by liquid-ordered packing into "lipid rafts," structures that resist Triton extraction, and by attachments to underlying cytoskeletal proteins in assemblies called "membrane skeletons." Although the actin cytoskeleton is implicated in many lipid raft-mediated signaling processes, little is known about the biochemical basis for actin involvement. We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H; d approximately 1.16 g/ml). By using matrix-assisted laser desorption/ionization time of flight and tandem mass spectrometry, we identified 19 major DRM-H proteins. Membrane skeleton proteins include fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, alpha-actinin 1, alpha-actinin 4, vimentin, and the F-actin-binding protein, supervillin. Other DRM-H components include lipid raft-associated integral membrane proteins (stomatin, flotillin 1, and flotillin 2), extracellular surface-bound and glycophosphatidylinositol-anchored proteins (IgM, membrane-type 6 matrix metalloproteinase), and intracellular dually acylated signaling proteins (Lyn kinase, Galpha(i-2)). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Galpha(i-2) also resist extraction with 0.1 m octyl glucoside. Supervillin, myosin-IG, and myosin-IIA resist extraction with 0.1 m sodium carbonate, a treatment that removes all detectable actin, suggesting that these cytoskeletal proteins are proximal to the DRM-H bilayer. Binding of supervillin to the DRM-H fragments is confirmed by co-immunoaffinity purification. In spreading neutrophils, supervillin localizes with F-actin in cell extensions and in discrete basal puncta that partially overlap with Galpha(i) staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains.
Highlights
Plasma membranes in eukaryotic cells are organized into domains of specialized function
We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H; d ϳ1.16 g/ml)
To examine signals from proteins closely associated with the plasma membrane, the ventral membrane skeleton was exposed by mechanical shear of spreading neutrophils [51, 52]
Summary
Plasma membranes in eukaryotic cells are organized into domains of specialized function. Pelleted neutrophil plasma membranes (Fig. 3A, fraction 1) were resuspended to 1–2 mg of membrane protein per ml of Triton X-100 extraction buffer (TEB: 1% Triton X-100, 25 mM Tris, 250 mM NaCl, 2.5 mM MgCl2, 1 mM EGTA, 1 mM ATP) containing protease inhibitors and were incubated for 60 min on ice. Samples (6 ϫ 2.7 ml) were underlayered with 0.3 ml of 48% (v/v) OptiprepTM in TEB and centrifuged at 100,000 ϫ g for 60 min at 4 °C in a Beckman TLA 100.3 rotor.
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