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To whom correspondence should be addressed: Dept. of Cell Biology, University of Massachusetts Medical School, Biotech 4, Ste. 306, 377 Plantation St., Worcester, MA 01605. Tel.: 508-856-8661; Fax: 508-856-8774; E-mail:
* This work was supported by Grant GM33048 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org ) contains information for Fig. 4. § Present address: Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
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 ∼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, α-actinin 1, α-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, Gαi-2). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Gαi-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 Gαi staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains.
detergent-resistant membrane fragments
DRMs with higher buoyant densities (1.15–1.18 g/ml)
DRMs with lower buoyant densities (1.09–1.13 g/ml)
antibodies against the amino-terminal 340 amino acids of human supervillin
the amino-terminal 340 amino acids of human supervillin
matrix-assisted laser desorption/ionization time of flight
tandem mass spectrometry
membrane-type 6 matrix metalloproteinase
Plasma membranes in eukaryotic cells are organized into domains of specialized function. Many of these domains are relatively stable on the cell surface and involve a series of linkages between cytoplasmic cytoskeletal proteins, integral membrane proteins, and extracellular attachments to the substrate or to other cells. For example, intermediate filament anchorages to membranes at desmosomes and hemidesmosomes distribute mechanical stresses over epithelial surfaces and maintain the integrity of skin and other tissues (
). In each of these domains, interactions between cytoskeletal proteins provide resistance against disruption by nonionic detergents, such as Triton X-100 and octyl glucoside, permitting the transmembrane protein assembly to be isolated as a “membrane skeleton” (
Lipid-lipid interactions also can organize membranes into detergent-resistant domains. Such lipid “rafts” are stabilized against disruption by cold Triton X-100 through liquid-ordered packing of component cholesterol, (glyco)sphingolipids, and gel-phase glycerolipids (
). When analyzed in vivo by spectroscopic methods, plasma membrane lipid rafts consist of clusters of ∼20 proteins and ∼3500 sphingolipid molecules and are ∼50 nm in diameter, a size well below the resolution limit of light microscopy (
). These dimensions can be increased into the submicron to micron range through aggregation of raft components, e.g. by receptor activation or addition of bivalent antibodies against raft-associated surface antigens (
). After treatment of cells or membranes with cold Triton X-100, the coalescing detergent-resistant remnants of the lipid rafts are called detergent-resistant membranes (DRMs).1 DRMs, which represent a subset of the endogenous raft lipids and proteins (
). Selective recruitment of proteins from the cytoplasm or protein diffusion within the plane of the membrane can regulate signaling events, such as those downstream of growth factor addition, ion channel activation, or changes in cell adhesion. Because many signaling proteins, including heterotrimeric Gα proteins and members of the Src family of protein tyrosine kinases, are dually acylated, lipid rafts have been proposed as platforms for the controlled interaction of substrates and signaling enzymes (
). Compartmentalized signaling at lipid rafts may be especially important for hematopoietic cells, which form dynamic attachments to substrates and other cells and lack many types of stable cell surface adhesions. Although signaling downstream of lipid raft-associated adhesive stimuli requires filamentous actin (
). They engulf microbes coated with complement proteins and/or immunoglobulins and kill the ingested microorganisms by releasing proteases, antimicrobial proteins, and oxygen metabolites into phagolysosomal compartments. Although polarity is maintained by cytoplasmic microtubules (
Despite its importance in early immune defense, the neutrophil membrane skeleton is largely uncharacterized. In agreement with previous observations of a spectrin-, actin-, and band 4.1-containing membrane skeleton in neutrophils (
). We also have used F-actin blot overlays and immunoblotting to show that neutrophil plasma membranes contain ezrin, moesin, and a 205-kDa F-actin-binding protein that we named supervillin. Molecular cloning of supervillin cDNA indicated that this protein contains a carboxyl terminus with predicted similarities to villin and a novel amino terminus (
), implying a role during physiological activation of neutrophils.
In this paper, we present proteomic and microscopic evidence for a DRM-associated membrane skeleton from neutrophil plasma membranes (Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7). This lipoprotein complex is enriched in cholesterol (Fig.2) and exhibits a higher buoyant density than do DRMs with less associated membrane skeleton material (Figs. 3 and 7, DRM-Hversus DRM-L). Cytoskeletal proteins characteristically enriched in this fraction include fodrin, supervillin, myosin-IIA, myosin-IG, and α-actinins 1 and 4 (Figs. Figure 3, Figure 4, Figure 5, Figure 6, Figure 7). Immunoaffinity purification of supervillin-associated DRM complexes confirms that these proteins are in the same lipoprotein complexes (Fig. 5). Octyl glucoside extraction demonstrates that most of the hydrophobically associated lipid raft proteins in this complex are relatively stabilized against extraction by this detergent (Figs. 5 and 6). Carbonate extraction demonstrates that the peripheral membrane proteins, supervillin, myosin-IIA, and myosin-IG, are very tightly associated with DRM lipids and/or integral membrane proteins, remaining bound even in the absence of detectable actin (Fig. 6). As supervillin and Gαi-2 immunoreactive proteins appear to preferentially co-localize in actin-rich areas of cells engaged in active spreading (Figs. 1 and 7D), the DRM-H fraction may represent a transiently induced, membrane skeleton-associated subset of neutrophil signaling domains (Fig.8).
The buoyant density of the DRM-H fraction (d ∼1.16 g/ml) is probably the reason it has not been described previously. DRM purification methods typically involve the collection of lipoprotein complexes with densities within the range of 1.02 to 1.16 (5–35% sucrose) (
). Presumably, attachment of DRM-associated proteins to the cytoskeleton causes an increased density due to the increased ratio of protein to lipid in the detergent-resistant fraction, resulting in equilibrium banding at intermediate sucrose concentrations. DRMs with densities in the range of our DRM-H fraction (d ∼ 1.15–1.18; 33–40% sucrose) are likely to sediment, or to be spread throughout, the 30–35% sucrose step gradient used in many DRM purification methods (
). Also, the isolated DRM-H fraction is not necessarily homogeneous with respect to the relative ratios of the co-isolating proteins. For instance, the small amount of DRM-H clathrin (Fig. 4) may be caused by a few contaminating coated pits or it may represent a limited association between coated membrane and a subset of DRMs. Other components of the DRM-H fraction, especially proteins present in minor amounts, remain for future analysis. The determination of which proteins interact through direct interactions and which are co-associated through a common affinity for the gel-phase DRM lipids is similarly beyond the scope of this investigation. However, the silver-stained profile of proteins that co-sediment as the DRM-H fraction (Figs. 3, 4, and 7) is similar to the pattern of proteins that are co-purified using antibodies directed against supervillin (Fig. 5). This result suggests that supervillin directly or indirectly associates with lipoprotein complexes containing the other proteins in Fig. 4 and may therefore serve as a marker protein for DRM-H rafts in neutrophil plasma membranes.
Our results are consistent with electron microscopic observations of a subplasmalemmal filament complex associated with Triton X-100-extracted neutrophil ghosts (
). In the latter studies, high resolution scanning electron micrographs revealed that the cytoplasmic surfaces of adherent, spreading neutrophils contain interlocking networks of globular centers and radiating branched filaments. Morphologically similar networks are observed in cross-sections of purified plasma membranes (Fig. 3D,a) and in negatively stained DRM-H samples (Fig. 3D,b andc). Given our observations of myosin-IIA and myosin-IG bound to DRMs lacking detectable F-actin, these domains may represent regions where actin and myosin are recruited and organized at the membrane, as originally implied (
). The partial superimposition of the supervillin and Gαi-2immunofluorescence signals (Fig. 7D) suggests that at least some of these areas of overlap may correspond to domains involved in the active signaling between chemotactic receptors and the actin cytoskeleton.
Actin, spectrin, or fodrin, and/or associated proteins have been co-purified with lipid raft components from other hematopoietic cell types. For example, erythrocyte DRMs are enriched in stomatin, flotillins 1 and 2, β-actin, and a loosely bound member of the spectrin gene family (
). Among a total of ∼70 DRM proteins, including markers for mitochondria, rough endoplasmic reticulum, the Golgi complex, and the nuclear envelope, were sequences representative of fodrin, myosin-IIA heavy and light chains, supervillin, myosin-IG (GenBankTM accession numberAI089291), vimentin, β-actin, flotillin 2, Gαi-2, and the Src-related tyrosine kinase Lck. Although it is not known whether these proteins co-associate with each other in T cells, as opposed to being present in different DRMs, it is striking that 12 of the 25 polypeptides identified in our study are also present in total Jurkat T cell DRMs. Thus, lipid raft-associated membrane skeletons similar to DRM-H may be present in hematopoietic cells of different origin.
The neutrophil DRM-H fraction also exhibits some compositional similarities to the microdomains associated with the multichain receptor for IgE (FcεR) in mast cells (
). Either type of Fcμ receptor would be hard to detect in the presence of the more abundant, co-electrophoresing IgM decamer, leaving open the question of whether the IgM that co-sediments with the neutrophil DRM-H fraction is bound through a specific Fc receptor.
Although not documented previously, the actin-independent association of myosins and other F-actin-binding proteins with DRMs is consistent with reports (
) indicating that lipid raft domains are intrinsically linked to active movements of the cortical actin cytoskeleton. After priming by receptor occupancy with IgE, for example, the FcεR is associated with Lyn in lipid rafts on mast cells (
). Following receptor cross-linking by multivalent antigen, the structure and organization of these microdomains are changed both by actin-based lateral movements of signal transduction proteins within the plane of the membrane and by recruitment of signaling proteins from cytoplasmic stores (
We hypothesize that fodrin, myosin-IIA, myosin-IG, and supervillin are components of a mobile, actin-based membrane skeleton that regulates the organization and/or transport of cholesterol-rich signaling domains at the neutrophil plasma membrane (Fig. 8). Associations with membrane skeleton components also could differentially stabilize and/or localize these domains within the membrane. Whereas the intracellular localization of myosin-IG is unknown, fodrin and myosin-II both localize throughout the cortex, as well as at the plasma membrane (
), suggesting that supervillin may be a characteristic component of this type of lipid raft-associated membrane skeleton. The tight association between supervillin and DRM-H fragments, in conjunction with the high affinity binding of supervillin for F-actin and its ability to reorganize cytoskeletal structures (
), suggests that supervillin may be involved in recruitment of the actin-based cytoskeleton to such liquid-ordered regions of the plasma membrane. The tight association of myosin motors with the DRM-H fragments suggests the possibility of a role in membrane transport or directed motility at the cell surface.
We gratefully acknowledge Dr. Gregory Hendricks for assistance with the electron microscopy, Drs. Mark Mooseker and Martin Bähler for their generous gifts of antibodies against myosin I sequences, and Dr. Silvia Corvera for providing anti-clathrin heavy chain antibody. We also thank Megan Staples for help with immunoaffinity purification, Louise Ohrn for the megaliters of solutions used for plasma membrane purification, and Ernestina Bernal and Donna Castellanos for expert glassware washing.