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 ϳ1.16 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, ␣-actinin 1, ␣-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, G␣ i-2 ). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and G␣ 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 G␣ i staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains.
Compartmentalized signaling involving cholesterol-rich, liquid-ordered membrane domains occurs during cell activation triggered by receptor cross-linking, growth factors, or other extracellular stimuli (1-3). The redistribution of similar liquidordered domains, called lipid ''rafts,'' accompanies and is required for cell polarization and directed migration (4 -8). Although we do not know the precise molecular mechanisms by which the redistributions of plasma membrane domains occur, an active role of the actin-based membrane skeleton has long been postulated (reviewed in Ref. 9).Redistributions of activated or cross-linked receptors are accompanied by corresponding changes in the localizations of actin, nonmuscle myosin II, spectrin, and associated cytoskeletal proteins (9). Furthermore, disruption of actin filament integrity inhibits many lipid raft-mediated processes, including epidermal growth factor receptor capping in A431 cells (10), insulin receptor capping in lymphocytes (11), activation of fibroblasts (12), polarization of T lymphocytes (5), and downregulation of Fc⑀RI-mediated signaling in mast cells (13). A requirement for myosin II is shown by the greatly diminished receptor redistributions and/or developments of cell polarity that have been observed in cells that either lack myosin II (14, 15) or express a dominant-negative mutant of myosin II function (16 -18). Erythrocyte spectrin (19) and the nonerythroid spectrin called fodrin 1 (20, 21) also have been implicated in lipid raft-mediated processes. Taken together, these observations suggest that actin filaments, perhaps as components of a spectrin-based membrane skeleton, are required for the
M2-type TAMs are increasingly implicated as a crucial factor promoting metastasis. Numerous cell types dictate monocyte differentiation into M2 TAMs via a complex network of cytokine-based communication. Elucidating critical pathways in this network can provide new targets for inhibiting metastasis. In this study, we focused on cancer cells, CAFs, and monocytes as a major node in this network. Monocyte cocultures with cancer-stimulated CAFs were used to investigate differentiation into M2-like TAMs. Cytokine array analyses were employed to discover the CAF-derived regulators of differentiation. These regulators were validated in primary CAFs and bone marrow-derived monocytes. Orthotopic, syngeneic colon carcinoma models using cotransplanted CAFs were established to observe effects on tumor growth and metastasis. To confirm a correlation with clinical evidence, meta-analyses were employed using the Oncomine database. Our coculture studies identify IL6 and GM-CSF as the pivotal signals released from cancer cell-activated CAFs that cooperate to induce monocyte differentiation into M2-like TAMs. In orthotopic, syngeneic colon carcinoma mouse models, cotransplanted CAFs elevated IL6 and GM-CSF levels, TAM infiltration, and metastasis. These pathologic effects were dramatically reversed by joint IL6 and GM-CSF blockade. A positive correlation between GM-CSF and IL6 expression and disease course was observed by meta-analyses of the clinical data. Our studies indicate a significant reappraisal of the role of IL6 and GM-CSF in metastasis and implicate CAFs as the "henchmen" for cancer cells in producing an immunosuppressive tumor ecological niche. Dual targeting of GM-CSF and IL6 is a promising new approach for inhibiting metastasis. .
The villin-type "headpiece" domain is a modular motif found at the extreme C-terminus of larger "core" domains in over 25 cytoskeletal proteins in plants and animals. Although headpiece is classified as an F-actin-binding domain, it has been suggested that some expressed fusion-proteins containing headpiece may lack F-actin-binding in vivo. To determine the intrinsic F-actin affinity of headpiece domains, we quantified the F-actin affinity of seven headpiece domains and three N-terminal truncations, under identical in vitro conditions. The constructs are folded and adopt the native headpiece structure. However, they show a wide range of affinities that can be grouped into high, low, and nonspecific-binding categories. Computer models of the structure and charged surface potential of these headpiece domains suggest features important for high F-actin affinity. We conclude that not all headpiece domains are intrinsically F-actin-binding motifs, and suggest that the surface charge distribution may be an important element for F-actin recognition.
SummaryArchvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton
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