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.

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, archos; '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.

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.

Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.

A recent application of optical tweezers has shown that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels control adhesion of the membrane bilayer to the underlying cytoskeleton, by regulated direct binding of PIP(2) to cytoskeletal proteins and/or indirect effects on cytoskeleton structure.