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Originally published In Press as doi:10.1074/jbc.M305311200 on August 12, 2003

J. Biol. Chem., Vol. 278, Issue 46, 46094-46106, November 14, 2003
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F-actin and Myosin II Binding Domains in Supervillin*

Yu Chen{ddagger}§, Norio Takizawa{ddagger}§, Jessica L. Crowley{ddagger}§, Sang W. Oh{ddagger}§, Cheryl L. Gatto{ddagger}§, Taketoshi Kambara§||, Osamu Sato§||, Xiang-dong Li§||, Mitsuo Ikebe§||, and Elizabeth J. Luna{ddagger}§**

From the {ddagger}Department of Cell Biology, the ||Department of Physiology, and §Program in Cell Dynamics, the University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication, May 20, 2003 , and in revised form, August 4, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (~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 ~280-342, ~344-422, and ~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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 liquid-ordered 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 down-regulation of Fc{epsilon}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 anchorage of many receptors to lipid rafts, or as tracks for myosin-driven lateral movements of membrane proteins during signaling, or as part of both processes.

Lipid raft components may be isolated by flotation into low buoyant density sucrose fractions after treatment of cells or membranes with cold Triton X-100 (3). During this procedure, the ~50-nm liquid-ordered domains present in unactivated cells (22, 23) coalesce into detergent-resistant membranes (DRMs)1 that represent a subset of the endogenous raft lipids and proteins (2, 3). Both lipid rafts and DRMs contain resident integral membrane proteins, such as caveolin, stomatin, and flotillin, and signaling proteins, including heterotrimeric G proteins and members of the Src family of protein-tyrosine kinases (24).

Raft-associated integral membrane and signaling proteins in bovine neutrophil plasma membranes sediment in sucrose gradients as both "light" (DRM-L) and "heavy" (DRM-H) fractions with buoyant densities of ~1.09-1.13 and ~1.15-1.18 g/ml, respectively (25). The neutrophil DRM-H fraction also contains a subset of cytoskeletal proteins, including actin, myosin II, fodrin, {alpha}-actinins 1 and 4, vimentin, myosin 1G, and the actin-binding protein supervillin. Most of the integral, signaling, and cytoskeletal proteins in the DRM-H fraction continue to co-sediment with each other after solubilization of raft lipids with octylglucoside, indicating that these proteins are associated through interactions exclusive of those with the bilayer. Supervillin, myosin II, and myosin 1G remain bound to the bilayer after a high pH carbonate extraction, suggesting that these proteins are more proximal to the membrane than are the other DRM-H cytoskeletal proteins. Thus, the DRM-H fraction consists of an actin- and fodrin-based membrane skeleton that is associated with a subset of lipid raft signaling domains, possibly through interactions with supervillin and myosin.

Similar raft-associated membrane skeletons may be present in many cell types. Supervillin is a constituent of total lipid rafts from Jurkat T cells (26) and is purified from HEK293 cells as part of a dodecyl maltoside-insoluble complex that also includes integrin {beta}2, {alpha}-actinin, {beta}-actin, P2X7 ATP receptors, laminin {alpha}3, phosphatidylinositol 4-kinase, and receptor protein-tyrosine phosphatase-{beta} (27). A muscle-specific isoform of supervillin, called archvillin, co-isolates with dystrophin and the raft-organizing protein, caveolin 3, in a low buoyant density fraction from skeletal muscle (28).

Supervillin, so-named because of C-terminal similarities to the microvillar protein villin, binds directly and specifically to F-actin (29, 30) and localizes to sites of cell-cell and cell-substrate adhesion in epithelial cells (30, 31). Archvillin localizes at costameres, specialized adhesion sites in muscle (28). The shared N terminus of supervillin and archvillin is novel, lacking any similarity to the S1 domain in villin and gelsolin that is involved in actin filament severing activity (32-34). Instead, the supervillin N terminus contains functional nuclear localization sequences and F-actin binding and bundling activities (31). Overexpression of supervillin or its N terminus disrupts stress fibers and vinculin-containing focal adhesions (31), suggesting a role in the regulation of cell-substratum interactions. Dysfunction caused by overexpression of supervillin is supported by the increased levels of this protein found in many carcinoma cell lines (35) and by the demonstration that increased levels of supervillin can activate signaling through the androgen receptor (36).

To understand better the role of supervillin at the membrane, we are mapping functional domains. Here we report the presence and localizations of a binding site for myosin II and three binding sites for filamentous actin within the supervillin N terminus. These F-actin-binding sites support filament bundling and cross-linking in vitro and thus can account for the observed aberrations in F-actin distributions documented in vivo (31). The myosin II-binding site, which is also present in archvillin, selectively recognizes nonmuscle and smooth muscle myosin II isoforms, as opposed to skeletal muscle myosin II. This selectivity is consistent with the co-localization of archvillin with nonmuscle myosin II at the sarcolemma in differentiating and mature skeletal muscle (28). Overexpression of the myosin II-binding sequence disrupts the co-localization of nonmuscle myosin IIB with actin filament bundles in COS7 cells, suggesting a role for supervillin, and by extension, archvillin, in the organization of actin and myosin II at liquid-ordered membrane domains.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Glutathione-SepharoseTM, PreScissionTM Protease, and DEAE-SephacrylTM were purchased from Amersham Biosciences. Chicken gizzards were from Pel-Freez Biologicals (Rogers, AR). Chemical reagents were from Sigma, Calbiochem-Novabiochem, Fisher, or VWR International Inc. (Grove, IL).

cDNAs
EGFP-Supervillin—Bovine supervillin cDNA (NCBI Nucleotide Database accession number AF025996 [GenBank] ) was used as a template to generate by PCR chimeric cDNAs from forward and reverse primers with 5' restriction enzyme sites for cloning into pGEM-T (Promega Corp., Madison, WI), pGEM-T Easy (Promega), or pCR2.1-TOPO® (Invitrogen) TA vectors. Primer and/or vector cloning sites were used to transfer supervillin sequences into the pGEX-6P-1 vector (Amersham Biosciences) for expression as fusion proteins with glutathione S-transferase (GST) and into pEGFP vectors (Clontech, Palo Alto, CA) for mammalian cell expression (Table I). The construction of EGFP-SV-(1-830) has been described previously (31).


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  		TABLE I
PCR primers for EGFP-tagged bovine supervillin constructs

 
GST-Supervillin—Supervillin sequences in pEGFP-N3 and pEGFP-C1 vectors were transferred in-frame by ligation of inserts removed by digestion with BglII and EcoRI to pGEX-6P-1 cut with BamHI and EcoRI. Constructs in pEGFP-N2 (Table I) were transferred similarly from TA vectors to pGEX-6P-1 after re-PCR with primers containing appropriately situated 5' BglII and EcoRI sites plus the gene-specific sequences shown in Table I.

DNA Sequencing—All PCR products in TA vectors were verified by sequencing at the Iowa State University DNA Sequencing and Synthesis Facility (Ames, IA) or at the University of Massachusetts Nucleic Acid Facility (Worcester, MA). Expression constructs were checked by sequencing through the cloning sites.

Proteins
GST Fusions—GST fusion proteins were expressed after induction in BL21 cells and purified with glutathione-SepharoseTM (37). After cleavage of GST with PreScissionTM Protease, the proteins were further purified by chromatography on DEAE-SephacrylTM and elution with 0-0.2 M NaCl. The supervillin fragments were dialyzed against dialysis buffer (100 mM KCl, 2 mM MgCl2, 1 mM DTT in either 40 mM PIPES, pH 7.0, or 40 mM MOPS, pH 7.5). Dialyzed proteins were frozen quickly in liquid nitrogen and stored at -80 °C until use.

Muscle Proteins—G-actin was prepared from an acetone powder of rabbit skeletal muscle (38). G-actin either was used directly in viscosity measurements or was column-purified (39) for use in co-sedimentation assays and 125I-labeled F-actin blot overlays (40, 41). G-actin was stored up to 10 days at 0-4 °C in dialysis against Buffer A (2.0 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT, 3.0 mM NaN3). Rabbit skeletal muscle myosin II and skeletal muscle heavy meromyosin (HMM) were isolated as described (42, 43). Smooth muscle myosin II was isolated from frozen turkey gizzards (44). Smooth muscle myosin rod domain and light meromyosin (LMM) were generated by papain digestion (45) and chymotryptic digestion (46), respectively. Smooth muscle HMM (Met1-Ser1110) and myosin subfragment-1 (S1, Met1-Gln856) were expressed from recombinant baculoviruses and purified (47). Constructs encoding HMM C-terminal deletions (HMM{Delta}1, Met1-Glu1058; HMM{Delta}2, Met1-Asn1030), and myosin subfragment-2 (S2, Leu851-Glu1106) also were made, and the recombinant proteins were expressed in Sf9 cells. Essential and regulatory light chains were co-expressed and co-purified in association with HMM, HMM{Delta}1, HMM{Delta}2, and S1.

Cell Culture and Transfection
COS7-2 cells were grown in Dulbecco's modified Eagle's high glucose media supplemented with 10% fetal calf serum and transfected by electroporation (48) or with calcium phosphate-precipitated DNA (49). For analysis of EGFP-tagged supervillin sequences by F-actin blot overlay and anti-EGFP staining, transfected cells were washed with PBS (pH 7.4) and harvested from 10-cm plates using 0.5 ml/plate of M-PER® Mammalian Protein Extraction Reagent, 3 µM pepstatin, 2 µM leupeptin, 0.2 µg/ml phenylmethylsulfonyl fluoride (PMSF), as described by the manufacturer (Pierce).

SDS-PAGE and Immunoblotting
Proteins were denatured by heating for 5 min at 95 °C in 2x SDS sample buffer and separated on SDS-polyacrylamide gels (50). Protein concentrations were determined by BCA Protein AssaysTM (Pierce). Gels were stained for protein with Coomassie Blue or electrotransferred to nitrocellulose (0.45-µm pore size; Schleicher & Schuell) for immunoblot analyses. Nitrocellulose blots were blocked with 5% nonfat powdered milk and probed with primary antibodies for 2 h at room temperature or overnight at 4 °C. Primary antibodies used in this study were diluted as follows: affinity-purified anti-supervillin ({alpha}-H340 (25)), ~1 µg/ml; a murine monoclonal antibody MMS-456S against most forms of myosin II (Babco-Covance, Richmond, CA), 1:1000; a murine monoclonal antibody 5F9F5 against all actin isoforms (Novus Biologicals, Littleton, CO), 1:10; and a murine monoclonal antibody FIL-2 against filamin (Sigma), 1:1000. Interacting antibodies were visualized using anti-mouse IgG, mouse IgM, or rabbit IgG antibodies conjugated to horseradish peroxidase, an ECL substrate kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and Biomax-MS x-ray film (Eastman Kodak Co.). For double labeling with radioactively labeled F-actin, anti-rabbit antibody conjugated to alkaline phosphatase was used with a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate kit (Kirkegaard & Perry Laboratories) for colorimetric detection.

Blot Overlays
F-actin—125I-Labeled actin was prepared, polymerized in the presence of rabbit gelsolin, stabilized with phalloidin, and used at a final concentration of 50 µg/ml in 5% nonfat powered milk (41). In some experiments, actin was labeled with [{alpha}-32P]ATP (51), using 1 mg of actin and 1 mCi of [{alpha}-32P]ATP. Nitrocellulose blots were exposed to film, or the signal was visualized with a Phosphor Imager SITM optical scanner and ImageQuant software (Amersham Biosciences).

35S-SV-(1-174)—In vitro transcription and translation of SV-(1-174) was carried out with the TNT T7-coupled reticulocyte lysate system (Promega Corp.) in the presence of ~20 µCi of [35S]methionine (PerkinElmer Life Sciences). 35S-Labeled SV-(1-174) was stored in 50-µl aliquots at -80 °C until use. For blot overlays with 35S-labeled SV-(1-174), proteins resolved on 8% SDS-PAGE gels were electrotransferred to nitrocellulose and blocked for 1 h at room temperature or overnight at 4 °C with 5% nonfat milk and 0.05% Nonidet P-40 in hybridization buffer (25 mM HEPES-KOH, pH 7.7, 25 mM NaCl, 5.0 mM MgCl2, 1 mM DTT) (52). Bound proteins were denatured and renatured by a series of 10-min incubations at room temperature in hybridization buffer, 0.05% Nonidet P-40 plus the following concentrations of guanidine hydrochloride: 6, 6, 3, 1.5, 0.75, 0.375, and 0.187 M. After two additional 10-min washes with hybridization buffer, nonspecific sites on the filter were blocked for 1 h at room temperature with 5% milk, 0.05% Nonidet P-40, hybridization buffer and for another hour at room temperature with 1% milk, 0.05% Nonidet P-40, 1 mM methionine, hybridization buffer. Filters were incubated with 35S-labeled SV-(1-174) (25-50 µl) for 2.5 h at room temperature in 1 ml of H Buffer (20 mM HEPES-KOH, pH 7.7, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40, 1 mM methionine). Filters were washed five times for 1-2 min per wash with H Buffer and then exposed to film or a PhosphorImager screen.

MALDI-TOF and MS/MS via MALDI Post-source Decay
Proteins specifically bound to GST-SV-(1-174) were identified from tryptic digests of Coomassie Blue-stained polypeptides (53) by Dr. John D. Leszyk, Proteomic Mass Spectrometry Laboratory of the University of Massachusetts Medical School (Shrewsbury, MA). PSD fragment ions were fitted to a generated curve, which was calibrated with PSD fragments from the synthetic peptide, P14R. Data base searches were performed using Protein Prospector (prospector.ucsf.edu). Average peptide masses were searched against the NCBI nonredundant data base using the MS-Fit program and 250-500 ppm mass tolerances with 1 or 2 missed cleavages. PSD fragments were searched against the NCBI nonredundant data base using MS-Tag and 0.5 Da as the parent tolerance and 1.0 Da as the fragment tolerance.

Actin Binding Assays
Co-sedimentation binding assays were performed either by adding supervillin fragments cleaved from purified GST fusion proteins to pre-polymerized F-actin (Fig. 2A) or by co-polymerizing actin with GST fusion proteins (Fig. 2C). All proteins and G-actin in Buffer A were clarified by centrifugation at 250,000 x g for 21 min at 4 °C immediately before use. In co-polymerization experiments, G-actin, GST, or GST fusion proteins were mixed on ice and co-assembled in a final volume of 250 µl of an actin polymerization buffer (100 mM KCl, 5 mM MgCl2, 1 mM ATP, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0). The final concentrations of actin, GST fusion proteins, and GST were 2.3, ~2, and 3.5 µM, respectively. Assay mixtures were incubated on ice for 1 h and then for 1 h at 18-20 °C. Actin filaments in 150 µl of each assay mixture were centrifuged as above through 50 µl of 10% sucrose in actin polymerizing buffer. Supernatants were collected as the top 100 µl in each tube, the rest of the liquid was carefully removed, and pellets were resuspended to 150 µl with Buffer A. Equal volumes (30 µl) of supernatant and pellet fractions were loaded onto SDS-polyacrylamide gels and immunoblotted with anti-GST antibody or stained with Coomassie Blue.



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  		FIG. 2.
F-actin binds independently and directly to three regions of supervillin (SV-(171-342), SV-(343-571), and SV570-830)). A, separation of individual purified fragments of supervillin (0.18 mg/ml), as shown in Fig. 1C, into supernatants (S) and pellets (P) after sedimentation in the presence (+) or absence (-) of pre-polymerized F-actin (1.0 mg/ml), pH 7.5. All proteins were detected by SDS-PAGE and staining with Coomassie Blue, except for SV-(1-342), which was detected with anti-H340 antibodies due to co-migration with actin. All fragments, except for SV-(1-174), sediment with actin filaments to some degree; no supervillin fragments sediment in the absence of F-actin. B, blot overlays with 125I-labeled F-actin of lysates from COS7 cells transfected with EGFP fusions with supervillin sequences. After protein resolution by SDS-PAGE and electrolytic transfer to nitrocellulose, identically loaded blots were probed with either antibody against EGFP (left) or 125I-labeled F-actin (right). C, anti-GST immunoblots showing that only nearly full-length GST-SV-(171-342) and GST-SV-(570-830), but essentially all C-terminal truncations of GST-SV-(344-571), co-sediment with F-actin at pH 8.0 (top panel). No sedimentation of any GST-tagged protein was observed in the absence of actin (bottom panel). FL, locations of full-length proteins; GST, location of GST with no C-terminal supervillin sequences.

 
Co-sedimentation experiments with pre-polymerized F-actin (1 mg/ml) and cleaved supervillin fragments (0.18 mg/ml) were performed similarly, except that the polymerization buffer contained 10 mM MOPS, pH 7.5, instead of Tris-HCl. Actin (1.25 mg/ml) was pre-polymerized for 30 min at 28 °C before addition of the supervillin fragments and continued incubation for 10 min at 28 °C.

Actin viscosity was measured using a low shear falling ball viscometer (54-56). Varying amounts of the supervillin fragments (0-200 µg; 12.5-17.5 µM)) were mixed on ice in 300-µl aliquots with 150 µg (11.6 µM) G-actin into a final assay buffer containing 26.6 mM PIPES, pH 7.0, 100 mM KCl, 10 µM CaCl2, 2.0 mM MgCl2, 1.0 mM ATP, 1.35 mM DTT, and 0.55 mM NaN3. Mixed samples were drawn into 100-µl capillary micropipettes and incubated horizontally at 28 °C for 2 h before the viscosities were measured at room temperature. The viscosities presented are means of triplicate determinations for each sample; error bars denote standard deviations (see figures).

Myosin Binding Assays
For initial binding experiments with GST fusion proteins, chicken gizzards were disrupted and extracted with a low salt extract containing 2 mM Tris, 1 mM EGTA, 0.5 mM PMSF, pH 9.0 (57). Extracts were clarified by centrifugation at 12,000 x g for 10 min at 4 °C, neutralized with acetic acid, and stored in aliquots at -80 °C until use in sedimentation assays with GST and GST-SV-(1-174) on glutathione-SepharoseTM beads. Thawed extracts were pre-cleared by centrifuging at 8000 x g for 15 min at 4 °C and then rotated for 5-6 h at 4 °C with glutathione-SepharoseTM beads (500 µl), 0.05% Triton X-100, 0.2 mg/ml PMSF and centrifuged at 500 x g for 5 min at 4 °C. The supernatant was divided, and aliquots were incubated separately with end-over-end rotation overnight at 4 °C with glutathione-SepharoseTM beads containing bound GST or GST-SV-(1-174). Beads were collected by centrifugation at 270 x g for 5 min at 4 °C and washed three times with 0.5 ml of PBS, 0.1% Triton X-100. In some experiments, beads were incubated at 4 °C for 20 min with higher stringency solutions containing 0.35 M NaCl, 0.5 M NaCl, 1% Nonidet P-40, or 1% Nonidet P-40 + 0.05% SDS. Beads were then washed two more times with 0.1% Triton X-100, PBS before elution with glutathione.

To investigate the interaction of double-headed myosin monomers with GST-tagged supervillin sequences, purified turkey gizzard myosin II or rabbit muscle HMM (0.5 mg in 0.5 ml) were incubated for 1 h at 4 °C with glutathione-SepharoseTM (50 µl) containing pre-bound GST or GST-supervillin proteins (100 µg) in binding buffer (10 mM MOPS, pH 7.5, 1 mM ATP, 5 mM MgCl2, 50 mM NaCl, 0.05% {beta}-mercaptoethanol). Beads were transferred to a column, and the columns were washed with 0.5 ml of binding buffer and then with 100-µl portions of binding buffer containing increasing concentrations (0.1, 0.15, 0.2, and 0.3 M) of NaCl. GST proteins and bound myosin were eluted from the washed columns with 250 µl of 5 mM glutathione in binding buffer. Fractions were collected and analyzed by Coomassie Blue staining of 6-20% gradient SDS-polyacrylamide gels.

For co-sedimentation experiments with purified myosin filaments or sedimentable myosin fragments, supervillin fragments (18 µg) were incubated for 10 min on ice with myosin II (100 µl of 1 mg/ml), rod (100 µl of 0.25 mg/ml), or LMM (100 µl of 0.4 mg/ml) in 50 mM KCl, 10 mM MgCl2, 20 mM HEPES, pH 7.1, 0.1% {beta}-mercaptoethanol, 0.2 µg/ml leupeptin, 0.01 mM EGTA. Myosin filaments were pelleted by centrifugation at 100,000 x g for 10 min at 5 °C. Precipitates were dissolved with 100 µl of 0.3 M NaCl. Supernatants (46 µl) and precipitates (46 µl) were loaded onto 6-20% SDS-acrylamide gels and stained with Coomassie Blue.

Immunofluorescence Microscopy
Cells were washed twice with pre-warmed phosphate-buffered saline, pH 7.4 (PBS), and immediately fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Unpermeabilized cells were rinsed three times for 10 min in PBS and permeabilized either by a 1-min incubation with 0.5% Triton X-100 in PBS (phalloidin staining) or by a 5-min incubation with 0.1% Triton X-100 in PBS (anti-myosin staining). After three 10-min washes with PBS, samples were blocked for 30 min in blocking solution (10% horse serum, 1% BSA, 0.02% sodium azide, PBS). Transfected cells were stained for 1 h at room temperature with a 1:30 dilution of rhodamine-phalloidin (Molecular Probes, Eugene, OR) in blocking solution and washed three times for 10 min in PBS. Other samples were stained for 1 h at 37 °C with a 1:500 dilution of rabbit polyclonal antibody against nonmuscle myosin IIB (Babco-Covance) and a subsequent incubation with Texas Red-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). Coverslips were mounted in Slow Fade Light Antifade solution (Molecular Probes) and sealed with nail polish. Slides were analyzed on a Zeiss Axioskop fluorescence microscope or a Bio-Rad MRC 1024 laser scanning confocal microscope (Bio-Rad) equipped with LaserSharp Version 3.2 software. Counts of morphological changes induced by various constructs were analyzed for significance by one-way analysis of variance with the Student-Newman-Keuls post test and InStat version 3.0a software (GraphPad Software, San Diego).

Electron Microscopy
Supervillin fragments were dialyzed against 40 mM PIPES, pH 6.9, 100 mM KCl, 2 mM MgCl2, 1 mM DTT. Dialyzed supervillin fragments and G-actin in Buffer A were clarified by ultracentrifugation, as described above. The supervillin fragments (4 µg) were mixed on ice in 100-µl aliquots with 15 µg of G-actin into a final assay buffer containing 100 mM KCl, 40 mM PIPES, pH 6.9, 2 mM MgCl2, 1 mM ATP, 1 mM DTT, 0.004% NaN3, 0.1 mM EGTA, and 10 µM CaCl2. Mixed samples were incubated at 28 °C for 2 h. Negative staining was performed according to published methods (58). Briefly, samples were deposited onto EM grids that had been coated with carbon and glow-discharged on the day of the experiment. Samples were allowed to adhere for 15-60 s, washed for 15 s with 100 mM KCl, 2 mM MgCl2, 10 mM PIPES, pH 7.0, 0.3 mM NaN3, and stained for 15 s with freshly filtered 1% uranyl acetate in PBS. Grids were dried slowly and examined on a model EM-301 transmission electron microscope (Philips Electron Optics Inc, Rahway, NJ) at an accelerating voltage of 60 kV.

Yeast Two-hybrid Screens
A bait plasmid encoding supervillin residues 11-174 was constructed and used to screen 1.62 x 108 clones from a library of 1.03 x 107 conditionally expressed target plasmids derived from HeLa S3 cDNA (Hybrid HunterTM Premade cDNA Library and Two-Hybrid System, Invitrogen). A cDNA encoding bovine supervillin amino acids 11-174 was PCR-amplified using primers containing an EcoRI or SalI site (forward primer, 5'-AGGGAATTCTTAGAAGGAATTGAAACCGACACGC-3'; reverse primer, 5'-ATCGTCGACTATAGCCCCGAGAGCTCAGTCCT-3'). The PCR product was cloned into pCR2.1-TOPO® and then into the bait vector pHybLex/Zeo, in-frame with the LexA DNA binding domain. The bait vector (pHybLex/Zeo SV 11-174)) was transformed (59) into yeast strain EGY48 containing the reporter plasmid pSH18-34. Transformants with HeLa cDNA target plasmid were screened for leucine prototrophy and for {beta}-galactosidase activity on filter lifts, according to the manufacturer's instructions. Plasmid DNAs were isolated from positively interacting colonies (60), amplified in bacteria, and sequenced at the University of Massachusetts Nucleic Acid Facility (Worcester, MA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To characterize sequences within the supervillin N terminus responsible for binding to F-actin (29, 31, 61), we generated constructs encoding subsets of the N-terminal 830 supervillin amino acids (SV-(1-830); Fig. 1). To identify likely boundaries between functional domains, we aligned predicted protein sequences from cDNA and genomic data bases, and we identified regions of low sequence conservation among vertebrates. These candidate regions were further analyzed for the presence of predicted secondary structures using the algorithms available on the Jpred server (www.compbio.dundee.ac.uk/~www-jpred/submit.html) (62). Bovine supervillin residues 171-174, 342-344, and ~570 were chosen as probable inter-domain boundaries because these sequences correspond to predicted protein loops with relatively low interspecies conservation. Together, they permitted the supervillin N terminus to be subdivided into four regions of approximately equal size (Fig. 1A). Each of these four regions and each combination of adjacent regions were generated by PCR, expressed in COS7 cells as fusions with EGFP (see below), expressed in Escherichia coli as GST fusions (Fig. 1B), cleaved, and purified (Fig. 1C). The four smallest fragments (SV-(1-174), SV-(171-342), SV-(343-571), and SV-(570-830)) could be purified to >90% (Fig. 1C, lanes 1-4). Larger fragments were isolated with variable amounts of truncated protein products (Fig. 1C, lanes 5-7). Bacterially expressed proteins containing more than ~60 kDa of supervillin sequence were mostly degraded (not shown).



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  		FIG. 1.
Supervillin constructs and purified proteins. A, schematic representation (bar at top) of the N-terminal 830 amino acids (aa) of bovine supervillin, residues shown previously to induce all major cellular morphological changes associated with overexpression of full-length EGFP-tagged supervillin (31). Also shown are the names and structures of the supervillin (SV) sequences used in this study as purified proteins and chimeric proteins with GST or EGFP. B, Coomassie Blue-stained 6-20% gradient SDS-acrylamide gel showing partially purified chimeras of GST with supervillin sequences, as indicated above each lane. These recombinant proteins exhibited increasing degrees of proteolysis and/or incomplete translation with increasing lengths of C-terminal supervillin sequences. C, Coomassie Blue-stained 6-20% gradient SDS-acrylamide gel showing purified supervillin sequences, as indicated above each lane.

 
Identification of F-actin-binding Sites—Three of the four subdomains of the supervillin N terminus (SV-(171-342), SV-(343-571), and SV-(570-830)) bound F-actin. Each of these purified proteins co-sedimented with actin filaments in vitro (Fig. 2A, left panels, lane 2). Binding was enhanced by the presence of two actin-binding sites. Proteins containing combinations of two smaller actin-binding domains (SV-(171-571), SV-(343-830)) were almost completely pelleted with F-actin in this assay (Fig. 2A, right panels, lane 2). By contrast, no detectable SV-(1-174) was observed in pellets with F-actin. SV-(1-342) was only partially sedimented (Fig. 2A), consistent with the presence of a single F-actin-binding site. Thus, the supervillin N terminus contains at least three sites capable of binding to actin filaments.

F-actin blot overlays of lysates from COS7 cells expressing fusion proteins containing EGFP and supervillin sequences supported the assignment of an F-actin-binding site in each of these three supervillin sequences (Fig. 2B). EGFP fusion proteins on nitrocellulose blots after SDS-PAGE and electrotransfer were identified with antibodies against EGFP (Fig. 2B, left panel) and probed with 125I-labeled F-actin (Fig. 2B, right panel). Although no radioactivity was observed in association with EGFP (Fig. 2B, lanes 1) or with EGFP-SV-(1-174) (Fig. 2B, lanes 2), EGFP-SV-(171-342), EGFP-SV-(343-571), and EGFP-SV-(570-830) each bound directly to 125I-labeled F-actin (Fig. 2B, lanes 3-5). By contrast, no binding was observed to SV-(423-571) tagged with EGFP at the C terminus (data not shown), suggesting that amino acids 342-422 are required for F-actin binding to this supervillin fragment in the blot overlay assay.

By taking advantage of the proteolysis or incomplete translation of bacterially expressed chimeric proteins containing N-terminal GST and C-terminal supervillin sequences, we used partially purified GST proteins to map further the approximate locations of the F-actin-binding sites within each supervillin fragment (Fig. 2C). Supernatants and pellets after sedimentation of each of these proteins with actin filaments were analyzed by immunoblotting with antibodies against GST. The sizes of the GST-tagged polypeptides that did, and did not, bind to F-actin were estimated to approximate the location of the actin-binding site(s) within each of the GST-tagged supervillin fragments. Polypeptides with both N-terminal GST and a C-terminal actin-binding site co-pelleted with F-actin (Fig. 2C, lanes P); shorter N-terminally tagged GST fusion proteins that had lost the F-actin-binding site from their C termini were enriched in the F-actin supernatants (Fig. 2C, lanes S).

As expected, full-length GST-SV-(171-342), GST-SV-(343-571), and GST-SV-(570-830) co-sedimented with F-actin (Fig. 2C, top panel, lanes 6, 8, and 10), but GST (Fig. 2C, top panel, lane 2) and GST-SV-(1-174) (Fig. 2C, top panel, lane 4) did not. F-actin pellets were enriched in full-length GST-SV-(171-342) relative to a truncated protein that lacked ~7 kDa of C-terminal sequence (Fig. 2C, top panel, lane 6), suggesting that the missing sequences are important for actin binding activity. Similarly, full-length GST-SV-(570-830) (Fig. 2C, top panel, lane 10), but not the next smaller related GST-tagged polypeptides (Fig. 2C, top panel, lane 9), sedimented with actin filaments, indicating that the C-terminal ~7 kDa of sequence in this domain is essential for binding to F-actin. By contrast, essentially all truncated GST fusion proteins containing SV-(343-571) sequences were pelleted (Fig. 2C, top panel, lane 8), consistent with the presence of an F-actin-binding site near the N terminus of this fragment. Thus, the supervillin N terminus apparently contains F-actin-binding sites in the vicinity of amino acids ~280-342, ~344-422, and ~700-830.

Supervillin-induced Changes in Actin Filament Organization—To determine whether supervillin N-terminal F-actin-binding sequences can directly induce changes in actin filament organization, we measured the low shear viscosity of solutions containing actin filaments and supervillin proteins predicted to contain one (SV-(1-342)) or two (SV-(171-571) and SV-(343-830)) binding sites for F-actin (Fig. 3). The low shear viscosity of a solution containing F-actin and associated proteins is very sensitive to the geometry and extent of the resulting structures, permitting sensitive detection of changes in actin organization (63). Solutions containing F-actin polymerized in the presence of SV-(1-342) exhibited approximately the same viscosities as a solution containing F-actin alone (Fig. 3, open circles), indicating that final filament lengths and filament organization were essentially unaffected by these sequences. By contrast, inclusion of SV-(171-571) induced large increases in solution viscosity (Fig. 3, open triangles), a profile reminiscent of observations made for F-actin cross-linking proteins (63). SV-(343-830) induced an ~3-fold increase in viscosity at ~100 µg/ml but resulted in viscosities that were even less than that of actin alone at higher concentrations of SV-(343-830) (Fig. 3, closed squares). This pattern of viscosity changes was similar to that reported for the F-actin bundling protein, {alpha}-actinin (64), raising the possibility that SV-(343-830) also might induce actin filament bundling.



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  		FIG. 3.
Viscosities of solutions containing F-actin and supervillin sequences. Viscosity, in centipoise (cp), as a function of the concentration of the supervillin fragments SV-(1-342), SV-(171-571), and SV-(343-830). G-actin (0.5 mg/ml) was added to purified supervillin fragments and polymerized for 1 h at 28 °C before a low shear assay for viscosity. Inclusion of SV-(171-571) ({triangleup}) and SV-(343-830) ({blacksquare}), but not SV-(1-342) ({circ}), resulted in increased viscosities of mixtures containing polymerized actin.

 
Solutions of F-actin polymerized in the presence of supervillin sequences also were examined for gross changes in organization by negative-stain electron microscopy (Fig. 4). Filament organization was not obviously affected by the presence of SV-(1-342) (Fig. 4, B versus A) or SV-(171-571) (Fig. 4C). However, actin filaments were noticeably bundled in solutions containing SV-(343-830) (Fig. 4D). Thus, the actin-binding sites present in SV-(343-830) are sufficient for the formation of actin filament bundles.



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  		FIG. 4.
Effects of supervillin sequences on actin filament organization. Electron micrographs of negatively stained actin filaments (150 µg/ml) polymerized in the absence (A) and in the presence of purified supervillin fragments (40 µg/ml), SV-(1-342) (B), SV-(171-571) (C), or SV-(343-830) (D). The mixture also contained 100 mM KCl, 40 mM PIPES, pH 6.9, 2 mM MgCl2, 1 mM ATP, 1 mM DTT, 0.004% NaN3, 10 µM CaCl2, 0.1 mM EGTA. Large bundles of actin filaments are observed in the presence of SV-(343-830). Bar, 200 nm.

 
To determine the effects of the supervillin N-terminal actin-binding sites on actin filament organization in vivo, we overexpressed each of the sequences under study here as an EGFP fusion protein in COS7 cells (Fig. 5). Consistent with expectations based on viscosity assays and electron microscopic observations, EGFP-tagged SV-(1-342) was more closely associated with microfilaments (Fig. 5d) than was EGFP alone (Fig. 5a) but did not obviously affect the organization of phalloidin-stained basal microfilament bundles (Fig. 5, e versus b, arrows). In agreement with the prediction that they should each contain two F-actin-binding sites, EGFP-SV-(171-571) and EGFP-SV-(343-830) co-localized strongly with actin filaments. EGFP-SV-(171-571) associated with F-actin in short cell surface extensions (Fig. 5, g-i) and increased the prevalence of these structures (Fig. 5h, arrowhead), at the apparent expense of basal microfilaments. EGFP-SV-(343-830) (Fig. 5, j-l) localized in similar surface extensions and increased the amount and extent of microfilament bundles around the cell peripheries (Fig. 5k, arrowheads).



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  		FIG. 5.
Localizations of EGFP-tagged supervillin sequences with phalloidin-stained actin filaments in transfected COS7 cells. COS7 cells expressing EGFP (a-c) or EGFP-tagged supervillin sequences (d-x), as indicated, were fixed, imaged with fluorescein optics (EGFP), and counter-stained with rhodamine-labeled phalloidin to visualize filamentous actin (F-actin). Composite images (Composite) show the superimposition of the EGFP (green) and phalloidin (red) signals; areas of overlap appear yellow. All these EGFP-tagged supervillin sequences show enhanced co-localization with F-actin, as compared with EGFP alone, although some of these sequences are associated with peripheral membrane structures (arrowheads) rather than with basal surface F-actin bundles (arrows). Bar, 10 µm.

 
Surprises arose from the localizations of the shortest subdomains of the supervillin N terminus. Based on the absence of binding to F-actin in vitro (Fig. 2), EGFP-SV-(1-174) was expected to lack an association for microfilaments. However, this supervillin subdomain was consistently found along microfilaments and/or at microfilament-associated punctae (Fig. 5, m-o). EGFP-SV-(171-342), EGFP-SV-(343-571), and EGFP-SV-(570-830) were predicted to associate with actin filaments but to have marginal, if any, effects on cytoskeletal organization. This prediction was largely true for EGFP-SV-(171-342) (Fig. 5, p-r) and EGFP-SV-(570-830) (Fig. 5, v-x), and EGFPSV-(343-571) was associated with peripheral F-actin structures (Fig. 5, s-u, arrowheads). However, moderate overexpression of EGFP-SV-(343-571) (Fig. 5, s-u) decreased the percentage of transfected cells with basal microfilament bundles to ~35% from the 92-95% observed for cells expressing EGFP alone or with most of the other supervillin fragments (Fig. 5, arrows). EGFP-SV-(171-342) also may have induced a modest decrease in the numbers of basal microfilaments, but at least 82% of the COS7 cells transfected with EGFP-SV-(171-342) contained at least some of these structures. Taken together, these results suggested the presence of cytoskeleton-associated binding sites in the supervillin N terminus in addition to the three F-actin-binding sites already described. The most striking of these results was the strong correlation between basal microfilament bundles and structures containing SV-(1-174), a sequence that lacks detectable F-actin binding activity.

Filamin and Myosin II Binding to SV-(1-174)—To identify potential binding partners for SV-(1-174) that might explain the localization of this sequence with microfilaments in vivo (Fig. 5, m-o), we fractionated a low salt extract of chicken gizzard smooth muscle (57) by affinity chromatography on columns containing GST-SV-(1-174) or GST alone (Fig. 6A). Polypeptides with molecular masses of ~250 and ~210 kDa were prominent in fractions specifically eluted from GST-SV-(1-174) columns (Fig. 6A, lane 4) but did not bind to columns containing GST alone (Fig. 6A, lane 2).



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  		FIG. 6.
Identification of filamin and smooth muscle myosin II as potential binding partners of SV-(1-174). A, Coomassie Blue-stained SDS-polyacrylamide gel showing that two major high molecular mass polypeptides from a chicken gizzard extract bind to GST-SV-(1-174) but not to GST alone. Bacterially expressed recombinant GST (lane 1) or GST-SV-(1-174) (lane 2) was bound to glutathione-SepharoseTM beads. Beads were incubated with extracted gizzard proteins, washed, and eluted with glutathione (lanes 3-5). Eluates from GST (lane 3), GST-SV-(1-174) (lane 4), and glutathione beads without a bound GST protein (lane 5) were analyzed by SDS-PAGE. Polypeptides with apparent molecular masses of ~250 and ~210 kDa were selectively recovered in association with SV-(1-174) sequences (lane 4). B, MALDI-TOF and tandem mass spectrometry (MS/MS) identified the ~250 and ~210-kDa proteins as filamin (NCBI Protein Database accession number BAB63943 [GenBank] ) and chicken smooth muscle myosin II heavy chain (NCBI Nucleotide Database accession number X06546 [GenBank] ), respectively. Assignments of SwissProt/PIR/GenBankTM accession numbers (Accession Number) were based on the high ratios of matching fragments to total fragments (MALDI-TOF) and the highly significant MOWSE Scores (76). Identifications were confirmed with peptide sequences obtained by MS/MS via post-source decay (PSD). C, the presence of filamin (Filamin) and myosin II heavy chain (Myosin) was further confirmed by immunoblot analyses of the ~250- and ~210-kDa polypeptides in the chicken gizzard extract (lane 1) and in eluates from glutathione beads containing bound GST-SV-(1-174) (lane 2) or GST (lane 3). Immunoblotting with anti-actin antibodies (Actin) indicated that this protein was not detectable in pellets with glutathione-SepharoseTM beads containing either GST-SV-(1-174) or GST alone.

 
The ~250- and ~210-kDa polypeptides that bound specifically to SV-(1-174) sequences were identified as filamin and smooth muscle myosin II heavy chain, respectively (Fig. 6, B and C). Database searches with peptide masses obtained by MALDI-TOF mass spectrometry from tryptic digests of these two polypeptides matched well with masses predicted for Gallus gallus filamin (NCBI Protein Database accession number BAB63943 [GenBank] ) and smooth muscle myosin II heavy chain (MYHB, NCBI Nucleotide Database accession number X06546 [GenBank] ). Details of these matches are provided in the Supplemental Material Table II. Molecular weight search scores were highly significant (Fig. 6B), and tandem mass spectrometry (MS/MS) via MALDI (PSD) of selected peptides confirmed the presence of filamin and smooth muscle myosin II sequences (Fig. 6B, Supplemental Material Table II).

The presence of filamin and myosin II in fractions eluted specifically from GST-SV-(1-174) was further confirmed by immunoblot analyses of the ~250- and ~210-kDa polypeptides (Fig. 6C). As described originally (57), filamin and myosin II were present in the low salt gizzard extract (Fig. 6C, lane 1). Filamin and myosin II also bound to the GST-SV-(1-174) column (Fig. 6C, lane 2) but not to columns containing GST alone (Fig. 6C, lane 3). Although detectable in the gizzard extract, no actin could be detected in the fractions eluting with glutathione from either GST-SV-(1-174) or GST columns (Fig. 6C). Thus, although this assay could not differentiate between direct and indirect binding of filamin and myosin II to GST-SV-(1-174), any indirect associations were not caused by mutual binding to F-actin.

Direct Binding to Smooth Muscle Myosin II—A single polypeptide of ~210-kDa in gizzard extracts and in glutathione-eluted fractions from GST-SV-(1-174) was recognized by 35S-labeled SV-(1-174) in a blot overlay assay (Fig. 7A). Given the presence of significant amounts of the similarly sized myosin heavy chain in these fractions (Fig. 6), we used purified gizzard muscle myosin in a "pulldown" assay with glutathione-SepharoseTM beads and either GST or GST-SV-(1-174) (Fig. 7B). Smooth muscle myosin co-sedimented with beads containing bound GST-SV-(1-174) (Fig. 7B, lanes 2, 4, 6, and 8) but not to beads with GST only (Fig. 7B, lanes 1, 3, 5, and 7). The interaction appeared to be of relatively high avidity because myosin binding was retained after washing with increased levels of salt (Fig. 7B, lanes 2 and 4) or detergent (Fig. 7B, lanes 6 and 8). However, increased salt did lower the amount of bound myosin heavy chain (Fig. 7B, lanes 2 and 4), raising a question about whether binding required that the myosin be filamentous.



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  		FIG. 7.
SV-(1-174) binds directly and tightly to smooth muscle myosin II. A, blot overlay with in vitro translated, 35S-labeled SV-(1-174) of polypeptides in a chicken gizzard extract (lane 1) and in specifically bound eluates from GST (lane 2) and GST-SV-(1-174) (lane 3) glutathione-SepharoseTM beads. 35S-Labeled SV-(1-174) binds directly to an ~210-kDa polypeptide (lanes 1 and 3), presumably myosin II heavy chain, that is present in both the extract and in the eluate from GST-SV-(1-174) beads. B, purified smooth muscle myosin II binds to SV-(1-174) sequences under a range of solution conditions. Coomassie Blue-stained SDS-polyacrylamide gel showing the binding of myosin purified from turkey gizzard to GST-SV-(1-174) (lanes 2, 4, 6, and 8), but not to GST (lanes 1, 3, 5, and 7), after co-sedimentation with glutathione-SepharoseTM beads. Myosin and GST fusion protein beads were incubated and washed at 4 °C in the presence of increasing salt (lanes 2 and 4) and in the presence of 1% Nonidet P-40 (NP-40)(lane 6) or 1% Nonidet P-40 + 0.05% SDS (lane 8), and then eluted with glutathione. Representative reaction mixtures before centrifugation (lanes 9 and 10) and post-centrifugation supernatants (lanes 11 and 12) also are shown. Molecular mass markers (M) and their masses in kDa are shown on the right. C, binding of double-headed smooth muscle myosin II monomers to affinity columns containing bound GST or GST-supervillin proteins (shown in Fig. 1B). Samples (15 µl) of the void volumes (V, lanes 1), washes with binding buffer (W, lanes 2), washes with the indicated concentrations of NaCl in binding buffer (lanes 3-6), and eluates with 5 mM glutathione (E, lanes 7) were analyzed on Coomassie Blue-stained 6-20% SDS-polyacrylamide gels. Significant amounts of myosin were retained only on columns containing SV-(1-174) or SV-(1-342) (lanes 5-7), suggesting that only one myosin-binding site is present in the supervillin N terminus. D, Coomassie Blue-stained 6-20% (left panel) and 13% (right panel) SDS-polyacrylamide gels showing co-sedimentation of SV-(1-174) (left panel, lanes 1-6) and SV-(1-342) (right panel, lanes 7-12) with filaments of turkey smooth muscle myosin II (Sm). No sedimentation of either supervillin fragment was observed in samples without myosin (-), nor was co-sedimentation observed with filaments composed of rabbit skeletal muscle myosin II (Sk). Supernatants (S) and resuspended pellets (P) after sedimentation of samples containing SV-(1-174) and smooth muscle myosin (lanes 1 and 2), SV-(1-174) alone (lanes 3 and 4), smooth muscle myosin alone (lanes 5 and 6), SV-(1-342) and smooth muscle myosin (lanes 7 and 8), SV-(1-342) and skeletal muscle myosin (lanes 9 and 10), or SV-(1-342) alone (lanes 11 and 12). Arrows denote the migration positions of the myosin heavy chains (MHC) and light chains (LCs); arrowheads show the positions of SV-(1-174) and SV-(1-342). E, little or no double-headed skeletal muscle HMM binds to either GST-SV-(1-174) or GST-SV-(1-342) on affinity columns. Incubations and washes correspond to those used with monomeric smooth muscle myosin II in C.

 
To determine whether double-headed smooth muscle myosin II monomers could bind to supervillin sequences in GST pulldown assays, turkey gizzard myosin in a buffer containing 1 mM MgATP was incubated with glutathione-SepharoseTM beads containing GST or GST chimeric proteins with supervillin sequences (Fig. 7C). Monomeric smooth muscle myosin II bound to columns containing GST-SV-(1-174) or GST-SV-(1-342), even after extensive washing with high salt solutions (Fig. 7C, lanes 7). By contrast, no significant association of myosin to GST-SV-(171-342), GST-SV-(343-571), or GST-SV-(570-830) was observed. Thus, supervillin residues 1-174 are sufficient for binding to monomeric smooth muscle myosin II.

Sedimentation assays with smooth and skeletal muscle myosins were used to determine whether SV-(1-174) and SV-(1-342) bind to filamentous myosin II (Fig. 7D). Both of these supervillin fragments co-sedimented with filaments composed of smooth muscle myosin (Fig. 7D, lanes 2 and 8), but neither SV-(1-174) (not shown) nor SV-(1-342) (Fig. 7D, lanes 10) bound to filaments composed of skeletal muscle myosin II. These results indicated that SV-(1-174) could bind to smooth muscle myosin II filaments, as well as to monomers.

To test whether double-headed monomeric skeletal muscle myosin II might be capable of binding to supervillin sequences, we performed GST pulldown assays with skeletal muscle HMM (Fig. 7E). Under conditions identical to those that supported binding of smooth muscle myosin monomers (Fig. 7C), we found that <=0.001 mol of skeletal muscle HMM bound per mol of either GST-SV-(1-174) or GST-SV-(1-342) (Fig. 7E, lanes 5-7). The lack of significant binding of either filamentous (Fig. 7D)or monomeric (Fig. 7E) skeletal muscle myosin II to supervillin N-terminal sequences suggests that the binding observed for filamentous (Fig. 7B) and monomeric (Fig. 7C) smooth muscle myosin II may represent an isoform-specific interaction.

To determine which region of smooth muscle myosin II interacts with supervillin sequences, we examined the association of SV-(1-174) with myosin fragments (Figs. 8 and 9). Most of these fragments (HMM, HMM{Delta}1, HMM{Delta}2, S1, and S2) were purified after expression in insect cells, as described previously (47). Two fragments (rod, LMM) were purified after proteolytic digestion of turkey gizzard myosin (45, 46). Fig. 8A shows a diagram of these myosin fragments and the residues at which their sequences were truncated. Blot overlays with 35S-labeled SV-(1-174) showed that HMM, HMM{Delta}1, and HMM{Delta}2 bound directly to SV-(1-174) (Fig. 8B, bottom panel, lanes 1, 2, 4, and 5). Binding to HMM{Delta}1 (lane 4) and, especially, HMM{Delta}2 (lane 5) was perhaps reduced slightly, as compared with HMM (lanes 1 and 2), but some variability in binding was observed relative to the amounts of loaded proteins (Fig. 8B, top panel). By contrast, no binding was observed to myosin S1 that had been denatured and processed identically with HMM (Fig. 8B, bottom panel versus top panel, lanes 1). Also, very little binding was observed to the rod domain (Fig. 8B, bottom panel, lane 3), compared with the amount of protein loaded (Fig. 8B, top panel, lane 3). Similarly, only slight binding of SV-(1-342) to the rod was observed in sedimentation assays, and no interaction at all with LMM could be detected after sedimentation (data not shown). Thus, sequences throughout much of the S2 domain of smooth muscle myosin II heavy chain may participate in binding to supervillin. N-terminal sequences within the myosin S2 domain, including sequences at the S1-S2 junction that are cleaved by papain during generation of myosin rod, appear to be especially important for this interaction.



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  		FIG. 8.
Myosin S2 domain sequences are required for binding to SV-(1-174). A, schematic representations of the purified myosin and myosin fragments used in this study: full-length smooth muscle myosin heavy chain (MHC, NCBI Protein Database accession number P10587 [GenBank] ), heavy meromyosin (HMM), truncated heavy meromyosins (HMM{Delta}1, HMM{Delta}2), myosin subfragment-1 (S1), myosin subfragment-2 (S2), rod generated by digestion with papain (Rod), and chymotryptic light meromyosin (LMM). Boundary amino acid residues for each truncation are indicated. All proteins, except rod and LMM, were purified from insect cells overexpressing chicken gizzard smooth muscle myosin heavy chain sequences. B, Coomassie Blue-stained SDS gel (top panel) and blot overlay with in vitro translated, 35S-labeled SV-(1-174) (bottom panel) of HMM and S1 (lane 1), HMM (lane 2), rod (lane 3), HMM{Delta}1 (lane 4), and HMM{Delta}2 (lane 5). Only proteins containing the S2 region of the myosin II heavy chain bind well to SV-(1-174) on blot overlays.

 



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  		FIG. 9.
Myosin S2 is sufficient for SV-(1-174) binding. A, purified myosin S2 domain (bottom panels), but not myosin S1 (top panels), binds tightly to GST-SV-(1-174) (*) on glutathione-SepharoseTM affinity columns (left panels, lanes 5-7). Neither myosin fragment binds to a GST affinity column (right panels, lanes 12-14). Conditions for binding and elution were the same as those used for Fig. 7C. These Coomassie Blue-stained gels were loaded with samples (15 µl) of the column void volumes (V, lanes 1 and 8), washes with binding buffer (W, lanes 2 and 9), washes with the indicated concentrations of NaCl in binding buffer (lanes 3-6 and 10-13), and glutathione eluates from the GST-SV-(1-174) (lanes 7) and GST (lanes 14) columns. B, human nonmuscle myosin IIA sequences (MYH-9, NCBI Protein Database accession number P35579 [GenBank] ) interact with supervillin amino acids 11-174 (SV-(11-174)) in a yeast two-hybrid assay. Yeast transformed with pHybLex/Zeo SV-(11-174) and a plasmid encoding one of three separately identified target sequences (lanes 1-3) or control plasmid pYesTrp2 (lane 4) were patched onto inductive, selective media (galactose/raffinose, Zeocin (0.3 mg/ml), -Ura, -Trp). Colonies were transferred to a nitrocellulose membrane and assayed for {beta}-galactosidase activity according to the manufacturer's protocols (Invitrogen). C, schematic representation of the locations of the target sequences (lines 1-3) identified by yeast two-hybrid screening with respect to the domain structure of human nonmuscle myosin IIA. All target sequences include most of the S2 domain. The smallest interacting sequence, amino acids 874-1095, corresponds to residues 887-1108 in chicken smooth muscle myosin MYHB (NCBI Protein Database accession number P10587 [GenBank] ).

 
To determine whether the myosin S2 domain is sufficient for binding to SV-(1-174), we tested the binding of purified recombinant smooth muscle myosin S1 and S2 domains to GST fusion proteins in a glutathione-Sepharose pulldown assay (Fig. 9). Myosin S1 interacted only slightly with GST-SV-(1-174) (Fig. 9A, top panel, lanes 1-7), with most of the loaded S1 washing off with salt concentrations similar to those that were sufficient to remove S1 from columns with GST (Fig. 9A, top panels, lanes 2-5 versus lanes 9-11). By contrast, nearly stoichiometric amounts of myosin S2 fragment bound to GST-SV-(1-174). In two experiments, ~55 and ~76 µg of myosin S2 bound to columns containing bound GST-SV-(1-174) (100 µg). The bound S2 resisted salt washes and eluted only with 5 mM glutathione (Fig. 9A, bottom panel, lane 7, asterisk). On a molar basis, this represents ~0.94 ± 0.13 mol of myosin S2 bound per mol of SV-(1-174), or roughly one S2 dimer bound per dimer of GST-SV-(1-174). Because essentially no myosin S2 was bound to columns containing GST alone (Fig. 9A, bottom panel, lane 14), the interaction was specific, as well as stoichiometric, and of relatively high avidity.

Myosin S2 was independently identified as a key interaction site for the supervillin N terminus in an untargeted yeast two-hybrid screen of a HeLa cDNA library that was probed with a bait vector encoding supervillin amino acids (11-174) (Fig. 9, B and C). Out of ~108 colonies screened, 10 specifically interacting clones encoding human nonmuscle myosin IIA (MYH-9, NCBI Protein Database accession number P35579 [GenBank] ) were obtained. These colonies supported growth on inductive, selective media and induced expression of {beta}-galactosidase activity (Fig. 9B). Seven of these clones encoded amino acids 874-1095 in human MYH-9 (Fig. 9, B and C, 1), two encoded residues 874-1100 (Fig. 9, B and C, 2), and one encoded amino acids 846-1100 (Fig. 9, B and C, 3). The shortest sequence identified in this assay corresponds to residues 887-1108 in chicken gizzard smooth muscle myosin MYHB (Fig. 8A), based on a sequence alignment of these two proteins (see below). Thus, the untargeted yeast two-hybrid screen also identified sequences within myosin S2 as important interaction sites for the supervillin N terminus.

Co-localization of Nonmuscle Myosin II with Supervillin Sequences in Vivo—Consistent with previous reports (65, 66), nonmuscle myosin II was localized primarily along stress fibers at the basal surfaces of COS7 cells (Fig. 10, b, e, h, k, and n). The supervillin distribution partially overlapped that of myosin II, especially along stress fibers, but much more supervillin than myosin was observed within surface membrane extensions (Fig. 10, d-f). The localization of myosin II was not disrupted by expression of moderately high levels of EGFP (Fig. 10, a-c), moderately high levels of EGFP-tagged full-length supervillin (Fig. 10, d-f), or low levels of EGFP-SV-(1-174) (Fig. 10, g-i). By contrast, moderately high levels of EGFP-tagged SV-(1-174) induced a re-distribution of the nonmuscle myosin II staining into dot-like structures in 32.7 ± 8.9% (S.E., n = 3, p < 0.01) of the transfected cells (Fig. 10, j-l). In these cells, many fewer basal microfilaments stained for myosin II, and of those that did, staining was much less uniform along their lengths (Fig. 10k). Normal distributions of myosin II were observed in cells expressing moderately high levels of SV-(1-342) (Fig. 10, m-o), suggesting that the overexpression phenotype of SV-(1-174) is a dominant-negative effect caused by increased levels of the supervillin myosin-binding site divorced from the other cytoskeleton-associated sequences in this protein. These results show that EGFP-SV-(1-174) can induce a redistribution of myosin filaments in vivo and support the in vitro demonstrations of interactions between myosin II and the supervillin N terminus.



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  		FIG. 10.
Co-localization of nonmuscle myosin II and supervillin sequences. COS7 cells expressing EGFP (a-c) or EGFP-tagged supervillin sequences (d-o), as indicated (EGFP), were fixed and counterstained with anti-nonmuscle myosin IIB and Texas Red-labeled goat anti-rabbit IgG (Myosin II) and imaged. Composite images (Composite) show the superimposition of the EGFP (green) and myosin (red) signals; areas of overlap appear yellow or orange. EGFP-tagged full-length supervillin (d-f), EGFP-tagged SV-(1-174) (g-l), and EGFP-tagged SV-(1-342) (m-o) all co-localized with myosin filament arrays (single arrows). Myosin filament organization was disrupted by moderate levels of overexpressed EGFP-SV-(1-174) (j-l, double arrows) but not by other EGFP-tagged supervillin constructs. Bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have identified four sequences within the supervillin N terminus that mediate associations with the actin-based cytoskeleton. Three of these supervillin sequences mediate direct binding to actin filaments, and a fourth binds directly to myosin II (Fig. 11). Binding of all four supervillin sequences has been demonstrated with purified proteins (Figs. 2, 7, and 9) and by co-localization and reorganization of target cytoskeletal structures in vivo (Figs. 5 and 10).



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  		FIG. 11.
Binding sites identified in this study. A, sequence conservation within the myosin S2 domain supports the hypothesis that much of this region may participate in binding to SV-(1-174). Residues that are identical between the heavy chains of chicken smooth muscle myosin II (Chick SmM) and human nonmuscle myosin IIA (Human Non), but are not conserved with amino acids in rabbit skeletal muscle myosin II (Rabbit SkM), are highlighted in yellow; amino acids conserved among all three myosin heavy chains are shaded with red. NCBI Protein Database accession numbers for these sequences are P10587 [GenBank] (chicken MYHB), NP_002464 [GenBank] (human MYH9), and AAG435721 (rabbit MyHC-EO/IIL). B, highly conserved regions within the supervillin N terminus (amino acids 1-830)) correspond to the approximate locations of mapped binding sites for myosin (Myosin) and F-actin (F-actin). Amino acids encoded by bovine (NP_776615 [GenBank] ) and human (NP_003165 [GenBank] ) supervillin cDNAs were aligned with cognate sequences encoded by murine archvillin cDNA (NP_694793 [GenBank] ) and by related rat genomic (NW_043108) and expressed sequence tag (AI715344 [GenBank] ) sequences. Percent identities among all four amino acid sequences were determined for 10-residue windows and plotted as a function of distance along the bovine supervillin amino acid sequence. Regions of nearly perfect sequence identity are observed within SV-(1-174), the location of the binding site for myosin. Other regions of high sequence conservation are found within amino acids 291-342, 345-450, and 748-805, sequences that correspond to mapped binding sites for F-actin. C, schematic model for supervillin interactions with myosin II (green) and actin filaments (red). Supervillin amino acids 11-174 (M, magenta) bind smooth and nonmuscle myosin II S2 domains, and three F-actin-binding sites (blue), located within supervillin amino acids 291-342 (A1), 345-450 (A2), and 748-805 (A3), respectively, exhibit the potential to mediate actin filament organization. Taken together, these interactions suggest that supervillin may contribute to the organization of actin and myosin filaments at the cytoplasmic surface of the plasma membrane.

 
Minimal sequences required for the binding to myosin II are supervillin amino acids 11-174 and sequences within myosin subdomain S2. Binding of SV-(1-174) to S2 in avian smooth muscle myosin II is approximately stoichiometric and of sufficiently high avidity to resist extensive washing (Figs. 6, 7, 8, 9A). Interactions between SV-(11-174) and S2 sequences from human nonmuscle myosin IIA were also identified in an untargeted yeast two-hybrid assay (Fig. 9, B and C). Furthermore, overexpression of SV-(1-174) induced reorganization of filamentous nonmuscle myosin II in vivo (Fig. 10k). Because supervillin N-terminal sequences did not bind significantly to rabbit skeletal muscle myosin II (Fig. 7, D and E), binding may be selective for nonmuscle and smooth muscle myosins. In agreement with this idea, the region of myosin S2 that binds SV-(1-174) contains even more amino acids that are identical between smooth muscle and nonmuscle myosins but divergent in skeletal muscle myosin (Fig. 11A, yellow shading) than residues that are conserved in all three types of myosin II (Fig. 11A, red shading).

The minimal myosin S2 sequence required for binding to supervillin may lie between smooth muscle myosin Lys-887 and Asn-1030 (Fig. 11A). The smallest myosin sequence identified as a strong positive in the yeast two-hybrid screen corresponds to chick smooth muscle myosin residues 887-1109 (Fig. 11A). The prominent binding of 35S-SV-(1-174) to HMM{Delta}2 in blot overlays (Fig. 8B, lane 5) indicates that sequences N-terminal to Asn-1030 are sufficient for binding, albeit at a possibly reduced affinity. The greatly diminished binding to rod in blot overlays (Fig. 8B, lane 3) supports the importance of residues at or near the S1-S2 junction (amino acids 849-946 (67)), a region of smooth muscle myosin II that has been suggested to play an important role in the regulation of myosin activity and assembly, both directly (46, 68, 69) and through binding of regulatory proteins (70-74).

Supervillin sequences capable of binding to actin filaments include a site near the C terminus of SV-(171-342), the N terminus of SV-(343-571), and the C terminus of SV-(570-830) (Fig. 2C). Each of these three F-actin-binding sites appears to bind along the sides of actin filaments. First, direct binding to 125I-labeled F-actin, nucleated and capped by plasma gelsolin and stabilized by phalloidin, has been demonstrated for each of the three supervillin fragments (Fig. 2B). This assay has been shown previously to be selective for proteins that bind to the sides, rather than to the ends of actin filaments (61). Binding of 125I-labeled F-actin to full-length supervillin is completely competed by excess amounts of myosin S1 in the absence, but not in the presence, of MgATP but is not affected by barbed-end filament capping proteins (29). Multiple lateral associations with actin filaments also are indicated by the increased affinities for sedimented F-actin (Fig. 2A) and the actin filament bundling and cross-linking activities (Figs. 3 and 4) that we have observed for supervillin fragments containing at least two F-actin-binding sites. Thus, the in vivo F-actin binding and bundling activities of the supervillin N terminus (Fig. 5) (31) can be fully explained by the three F-actin-binding sites identified here.

The regions of the supervillin N terminus that contain the actin- and myosin-binding sites have been especially well conserved during evolution. Alignment of bovine, human, murine, and rat supervillin sequences identify six regions with especially high proportions of amino acids that are identical in all four supervillins (Fig. 11B). These highly conserved regions correspond to amino acids 1-60, 93-136, 291-319, 383-444, 695-723, and ~746-809 in the bovine supervillin sequence. Of these, one or both of the first two conserved regions may be involved in binding to myosin S2 (Fig. 11B, Myosin), although residues 1-10 are not required for this interaction (Fig. 9B). The third, fourth, and sixth conserved regions fall within sequences that contain binding sites for F-actin (Fig. 11B, F-actin). We have estimated these binding sites to lie within residues 291-342, 343-450, and 748-830, based on the molecular masses of C-terminally truncated supervillin fragments that do and do not co-sediment with F-actin (Fig. 2C). These correlations are consistent with the presence of highly conserved binding sites for myosin S2 and F-actin in the supervillin N terminus.

Other functional binding domains also may be present within the supervillin N terminus. For instance, filamin binds directly or indirectly to SV-(1-174) at an unknown site (Fig. 6A). In addition, the loss of basal microfilament bundles induced by overexpression of SV-(343-571) (Fig. 5, s-u) is unlikely to be caused by a single actin-binding site. Although we cannot exclude the possibility that an additional binding site for F-actin is present in SV-(343-571), if this fragment does contain more than one binding site for F-actin, they each must be of relatively low avidity. Similar amounts of co-sedimentation with F-actin were observed for SV-(343-571) as for SV-(171-342) and SV-(570-830) (Fig. 2A), each of which contains a single C-terminal actin-binding site (Fig. 2C). Finally, the existence of an N-terminal membrane attachment site is suggested by the membrane association of SV-(1-830) in vivo (31) and by the continued binding of supervillin to cholesterol-rich membrane domains stripped of detectable actin by extraction with sodium carbonate (25). Interestingly, significant amounts of nonmuscle myosin II co-associate with supervillin and intrinsic membrane proteins under these conditions, suggesting the possibility that supervillin may mediate an actin-independent linkage of myosin II to the membrane.

Supervillin also may mediate the interaction between myosin II and actin filaments (Fig. 11C). Such a function is well accepted for other proteins known to bind to both myosin and actin filaments. These proteins include myosin light chain kinase, calponin, and caldesmon (70, 71, 73-75). Although calponin and caldesmon have been investigated primarily in smooth muscle, nonmuscle isoforms for all three of these proteins exist. Thus, one hypothesis for the myosin II redistribution induced by overexpressed SV-(1-174) (Fig. 10, j-l) is a dominant-negative effect caused by competition with other myosin S2-binding proteins. If true, then the normal appearing distribution of myosin II in the presence of overexpressed SV-(1-342) (Fig. 10, m-o), a fragment that contains an actin-binding site as well as the myosin-binding site, implies that supervillin may organize actin and myosin filament interactions in ways similar to those proposed for caldesmon and calponin. The tight association of supervillin with liquid-ordered membrane domains (25) and its localization at actin-rich membrane punctae (25, 31) further suggest that supervillin is well suited to promote the recruitment to, and/or to regulate the interaction between, actin and myosin filaments at these regions of the membrane. As the only protein known so far to be capable of linking liquid-ordered membrane domains to both F-actin and myosin II, supervillin may be an important "adapter" protein for the organization of membrane skeleton attachments at these dynamic regions of the plasma membrane.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Research Grants GM33048 (to E. J. L.), GM55834, and AR41653 (to M. I.) and by the Muscular Dystrophy Association (to E. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Table II. Back

Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: Dept. of Cell Biology, Biotech 4, Ste. 306, 377 Plantation St., Worcester, MA 01605. Tel.: 508-856-8661; Fax: 508-856-8774; E-mail: Elizabeth.Luna{at}umassmed.edu.

1 The abbreviations used are: DRMs, detergent-resistant membranes; DRM-H, DRMs with buoyant densities of ~1.15-1.18 g/ml; DRM-L, DRMs with buoyant densities of ~1.09-1.13 g/ml; {alpha}-H340, affinity-purified antibody directed against amino acids 1-340 of human supervillin; GST, glutathione S-transferase; HMM, heavy meromyosin; LMM, light meromyosin; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; S1, myosin subfragment-1; S2, myosin subfragment-2; SV, supervillin; EGFP, enhanced green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; MS/MS, tandem mass spectrometry; PSD, post-source decay. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Louise Ohrn for solution preparation and Donna Castellanos and Ernestina Bernal for expert glassware washing. We also are indebted to Dr. Gregory Hendricks for assistance with the electron microscopy and to Dr. John Leszyk of the Proteomic Mass Spectrometry Laboratory (University of Massachusetts Medical School, Shrewsbury, MA) for mass spectroscopic analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anderson, R. G., and Jacobson, K. (2002) Science 296, 1821-1825[Abstract/Free Full Text]
  2. Pike, L. J. (2003) J. Lipid Res. 44, 655-667[Abstract/Free Full Text]
  3. Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 31-39[CrossRef][Medline] [Order article via Infotrieve]
  4. Braun, J., Fujiwara, K., Pollard, T. D., and Unanue, E. R. (1978) J. Cell Biol. 79, 409-418[Abstract/Free Full Text]
  5. Gomez-Mouton, C., Abad, J. L., Mira, E., Lacalle, R. A., Gallardo, E., Jimenez-Baranda, S., Illa, I., Bernad, A., Manes, S., and Martinez, A. C. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9642-9647[Abstract/Free Full Text]
  6. Manes, S., Mira, E., Gomez-Mouton, C., Lacalle, R. A., Keller, P., Labrador, J. P., and Martinez, A. C. (1999) EMBO J. 18, 6211-6220[CrossRef][Medline] [Order article via Infotrieve]
  7. Pierini, L. M., and Maxfield, F. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9471-9473[Free Full Text]
  8. Pierini, L. M., Eddy, R. J., Fuortes, M., Seveau, S., Casulo, C., and Maxfield, F. R. (2003) J. Biol. Chem. 278, 10831-10841[Abstract/Free Full Text]
  9. Bourguignon, L. Y., and Bourguignon, G. J. (1984) Int. Rev. Cytol. 87, 195-224[Medline] [Order article via Infotrieve]
  10. Khrebtukova, I. A., Kwiatkowska, K., Gudkova, D. A., Sorokin, A. B., and Pinaev, G. P. (1991) Exp. Cell Res. 194, 48-55[CrossRef][Medline] [Order article via Infotrieve]
  11. Bourguignon, L. Y., Jy, W., Majercik, M. H., and Bourguignon, G. J. (1988) J. Cell. Biochem. 37, 131-150[CrossRef][Medline] [Order article via Infotrieve]
  12. Rizzo, M. A., Kraft, C. A., Watkins, S. C., Levitan, E. S., and Romero, G. (2001) J. Biol. Chem. 276, 34928-34933[Abstract/Free Full Text]
  13. Holowka, D., Sheets, E. D., and Baird, B. (2000) J. Cell Sci. 113, 1009-1019[Abstract]
  14. Aguado-Velasco, C., and Bretscher, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9684-9686[Abstract/Free Full Text]
  15. Pasternak, C., Spudich, J. A., and Elson, E. L. (1989) Nature 341, 549-551[CrossRef][Medline] [Order article via Infotrieve]
  16. Arhets, P., Olivo, J.-C., Gounon, P., Sansonetti, P., and Guillén, N. (1998) Mol. Biol. Cell. 8, 1537-1547
  17. Burns, C. G., Larochelle, D. A., Erickson, H., Reedy, M., and De Lozanne, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 29, 8244-8248
  18. Fukui, Y., De Lozanne, A., and Spudich, J. A. (1990) J. Cell Biol. 110, 367-378[Abstract/Free Full Text]
  19. Dahl, S. C., Geib, R. W., Fox, M. T., Edidin, M., and Branton, D. (1994) J. Cell Biol. 125, 1057-1065[Abstract/Free Full Text]
  20. Kwiatkowska, K., and Sobota, A. (1999) Biochem. Biophys. Res. Commun. 259, 287-293[CrossRef][Medline] [Order article via Infotrieve]
  21. Pradhan, D., and Morrow, J. (2002) Immunity 17, 303-315[CrossRef][Medline] [Order article via Infotrieve]
  22. Pralle, A., Keller, P., Florin, E. L., Simons, K., and Horber, J. K. (2000) J. Cell Biol. 148, 997-1008[Abstract/Free Full Text]
  23. Varma, M., and Leavitt, J. (1988) Mutat. Res. 199, 437-447[Medline] [Order article via Infotrieve]
  24. Galbiati, F., Razani, B., and Lisanti, M. P. (2001) Cell 106, 403-411[CrossRef][Medline] [Order article via Infotrieve]
  25. 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[Abstract/Free Full Text]
  26. von Haller, P. D., Donohoe, S., Goodlett, D. R., Aebersold, R., and Watts, J. D. (2001) Proteomics 1, 1010-1021[CrossRef][Medline] [Order article via Infotrieve]
  27. Kim, M., Jiang, L. H., Wilson, H. L., North, R. A., and Surprenant, A. (2001) EMBO J. 20, 6347-6358[CrossRef][Medline] [Order article via Infotrieve]
  28. Oh, S. W., Pope, R. K., Smith, K. P., Crowley, J. L., Nebl, T., Lawrence, J. B., and Luna, E. J. (2003) J. Cell Sci. 116, 2261-2275[Abstract/Free Full Text]
  29. Pestonjamasp, K., Amieva, M. R., Strassel, C. P., Nauseef, W. M., Furthmayr, H., and Luna, E. J. (1995) Mol. Biol. Cell 6, 247-259[Abstract]
  30. Pestonjamasp, K. N., Pope, R. K., Wulfkuhle, J. D., and Luna, E. J. (1997) J. Cell Biol. 139, 1255-1269[Abstract/Free Full Text]
  31. Wulfkuhle, J. D., Donina, I. E., Stark, N. H., Pope, R. K., Pestonjamasp, K. N., Niswonger, M. L., and Luna, E. J. (1999) J. Cell Sci. 112, 2125-2136[Abstract]
  32. Janmey, P. A., Stossel, T. P., and Allen, P. G. (1998) Chem. Biol. 5, R81-R85[CrossRef][Medline] [Order article via Infotrieve]
  33. Kwiatkowski, D. J. (1999) Curr. Opin. Cell Biol. 11, 103-108[CrossRef][Medline] [Order article via Infotrieve]
  34. Way, M., Gooch, J., Pope, B., and Weeds, A. G. (1989) J. Cell Biol. 109, 593-605[Abstract/Free Full Text]
  35. Pope, R. K., Pestonjamasp, K. N., Smith, K. P., Wulfkuhle, J. D., Strassel, C. P., Lawrence, J. B., and Luna, E. J. (1998) Genomics 52, 342-351[CrossRef][Medline] [Order article via Infotrieve]
  36. Ting, H. J., Yeh, S., Nishimura, K., and Chang, C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 661-666[Abstract/Free Full Text]
  37. Swaffield, J. C., and Johnston, S. A. (1996) in Current Protocols in Molecular Biology (Susubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 16.0.5-16.4.11, John Wiley and Sons, New York
  38. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871[Abstract/Free Full Text]
  39. MacLean-Fletcher, S., and Pollard, T. D. (1980) Biochem. Biophys. Res. Commun. 96, 18-27[CrossRef][Medline] [Order article via Infotrieve]
  40. Chia, C. P., Hitt, A. L., and Luna, E. J. (1991) Cell Motil. Cytoskeleton 18, 164-179[CrossRef][Medline] [Order article via Infotrieve]
  41. Luna, E. J. (1998) Methods Enzymol. 298, 32-42[Medline] [Order article via Infotrieve]
  42. Starr, R., and Offer, G. (1971) FEBS Lett. 15, 40-44[CrossRef][Medline] [Order article via Infotrieve]
  43. Weeds, A. G., and Pope, B. (1977) J. Mol. Biol. 111, 129-157[CrossRef][Medline] [Order article via Infotrieve]
  44. Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 13146-13153[Abstract/Free Full Text]
  45. Seidel, J. C. (1980) J. Biol. Chem. 255, 4355-4361[Free Full Text]
  46. Ikebe, M., and Hartshorne, D. J. (1985) Biochemistry 24, 2380-2387[CrossRef][Medline] [Order article via Infotrieve]
  47. Sata, M., and Ikebe, M. (1996) J. Clin. Invest. 98, 2866-2873[Medline] [Order article via Infotrieve]
  48. Takizawa, N., Schmidt, D. J., Mabuchi, K., Villa-Moruzzi, E., Tuft, R. A., and Ikebe, M. (2003) Am. J. Physiol. 284, C250-C262
  49. Spector, D. L., Goldman, R. D., and Leinwand, L. A. (1998) Cells: A Laboratory Manual, pp. 86.2-86.3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  50. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  51. Mackay, D. J. G., Esch, F., Furthmayr, H., and Hall, A. (1997) J. Cell Biol. 138, 927-938[Abstract/Free Full Text]
  52. Cavaillès, V., Dauvois, S., Danielian, P. S., and Parker, M. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10009-10013[Abstract/Free Full Text]
  53. Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Electrophoresis 20, 601-605[CrossRef][Medline] [Order article via Infotrieve]
  54. Fowler, V., and Taylor, D. L. (1980) J. Cell Biol. 85, 361-376[Abstract/Free Full Text]
  55. Fowler, V. M., Luna, E. J., Hargreaves, W. R., Taylor, D. L., and Branton, D. (1981) J. Cell Biol. 88, 388-395[Abstract/Free Full Text]
  56. Griffith, L. M., and Pollard, T. D. (1978) J. Cell Biol. 78, 958-965[Abstract/Free Full Text]
  57. Feramisco, J. R., and Burridge, K. (1980) J. Biol. Chem. 255, 1194-1199[Abstract/Free Full Text]
  58. Hartwig, J. H., Chambers, K. A., and Stossel, T. P. (1989) J. Cell Biol. 108, 467-479[Abstract/Free Full Text]
  59. Gietz, R. D., and Woods, R. A. (2002) Methods Enzymol. 350, 87-96[CrossRef][Medline] [Order article via Infotrieve]
  60. Hoffman, C. S., and Winston, F. (1987) Gene (Amst.) 57, 267-272[CrossRef][Medline] [Order article via Infotrieve]
  61. Luna, E. J., Pestonjamasp, K. N., Cheney, R. E., Strassel, C. P., Lu, T. H., Chia, C. P., Hitt, A. L., Fechheimer, M., Furthmayr, H., and Mooseker, M. S. (1997) Soc. Gen. Physiol. Ser. 52, 3-18[Medline] [Order article via Infotrieve]
  62. Cuff, J. A., and Barton, G. J. (2000) Proteins 40, 502-511[CrossRef][Medline] [Order article via Infotrieve]
  63. Pollard, T. D., and Cooper, J. A. (1982) Methods Enzymol. 85, 211-233[Medline] [Order article via Infotrieve]
  64. Wachsstock, D. H., Schwartz, W. H., and Pollard, T. D. (1993) Biophys. J. 5, 205-214
  65. Kolega, J. (1998) J. Cell Sci. 111, 2085-2095[Abstract]
  66. Saitoh, T., Takemura, S., Ueda, K., Hosoya, H., Nagayama, M., Haga, H., Kawabata, K., Yamagishi, A., and Takahashi, M. (2001) FEBS Lett. 509, 365-369[CrossRef][Medline] [Order article via Infotrieve]
  67. Ikebe, M., Yamada, M., Mabuchi, K., Kambara, T., and Ikebe, R. (1998) Biochemistry 37, 13285-13290[CrossRef][Medline] [Order article via Infotrieve]
  68. Ikebe, M., and Hartshorne, D. J. (1984) J. Biol. Chem. 259, 11639-11642[Abstract/Free Full Text]
  69. Konishi, K., Kojima, S., Katoh, T., Yazawa, M., Kato, K., Fujiwara, K., and Onishi, H. (2001) J. Biochem. (Tokyo) 129, 365-372[Abstract/Free Full Text]
  70. Ikebe, M., and Reardon, S. (1988) J. Biol. Chem. 263, 3055-3058[Abstract/Free Full Text]
  71. Kohama, K., Ye, L.-H., Hayakawa, K., and Okagaki, T. (1996) Trends Pharmacol. Sci. 17, 284-287[CrossRef][Medline] [Order article via Infotrieve]
  72. Kunst, G., Kress, K. R., Gruen, M., Uttenweiler, D., Gautel, M., and Fink, R. H. (2000) Circ. Res. 86, 51-58[Abstract/Free Full Text]
  73. Morgan, K. G., and Gangopadhyay, S. S. (2001) J. Appl. Physiol. 91, 953-962[Abstract/Free Full Text]
  74. Wang, C.-L. A. (2001) Cell Biochem. Biophys. 35, 275-288[CrossRef][Medline] [Order article via Infotrieve]
  75. Gusev, N. B. (2001) Biochemistry (Moscow) 66, 1112-1121[CrossRef][Medline] [Order article via Infotrieve]
  76. Pappin, D. J. (1997) Methods Mol. Biol. 64, 165-173[Medline] [Order article via Infotrieve]

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 All ASBMB Journals   Molecular and Cellular Proteomics 
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
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