Supervillin modulation of focal adhesions involving TRIP6/ZRP-1.

Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV-TRIP6 interaction may regulate FA maturation and/or disassembly.

Supervillin reorganizes the actin cytoskeleton and increases invadopodial efficiency.

Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.

Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes.

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.

Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton.

The membrane skeleton protein supervillin binds tightly to both F-actin and membranes and can potentiate androgen receptor activity in non-muscle cells. We report that muscle, which constitutes the principal tissue source for supervillin sequences, contains a approximately 250 kDa isoform of supervillin that localizes within nuclei and with dystrophin at costameres, regions of F-actin membrane attachment in skeletal muscle. The gene encoding this protein, 'archvillin' (Latin, archi; Greek, árchos; 'principal' or 'chief'), contains an evolutionarily conserved, muscle-specific 5' leader sequence. Archvillin cDNAs also contain four exons that encode approximately 47 kDa of additional muscle-specific protein sequence in the form of two inserts within the function-rich N-terminus of supervillin. The first of these muscle-specific inserts contains two conserved nuclear targeting signals in addition to those found in sequences shared with supervillin. Archvillin, like supervillin, binds directly to radiolabeled F-actin and co-fractionates with plasma membranes. Colocalization of archvillin with membrane-associated actin filaments, non-muscle myosin II, and--to a lesser extent--vinculin was observed in myoblasts. Striking localizations of archvillin protein and mRNA were observed at the tips of differentiating myotubes. Transfected protein chimeras containing archvillin insert sequences inhibited myotube formation, consistent with a dominant-negative effect during early myogenesis. These data suggest that archvillin is among the first costameric proteins to assemble during myogenesis and that it contributes to myogenic membrane structure and differentiation.

F-actin and myosin II binding domains in supervillin.

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.