Worcester Foundation for Biomedical Research, an Affiliate of the University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.
INTERACTIONS between the plasma membrane and the
actin cytoskeleton are involved in controlling cell
shape, in organizing membrane proteins into domains, and in regulating membrane domain function (52).
Some of these domains contain membrane-spanning proteins, such as ion channels and adhesion molecules, which
are localized because of interactions with cortical meshworks of spectrin-cross-linked actin filaments (7, 27). Other
actin-based membrane skeletons underlie membrane domains responsible for cell-substrate and cell-cell adhesion
(64, 80). For instance, intercellular adhesion at adherens
junctions, which is mediated by Ca2+-sensitive transmembrane proteins called cadherins, depends upon cadherin
attachment to actin filaments through linker proteins, such
as Membrane-actin linkages are also important for the assembly and control of more dynamic structures, e.g.,
pseudopods, filopodia, and microvilli (10, 19, 52, 55). For
example, the actin-binding glycoprotein ponticulin (86) is
required for pseudopod stabilization, efficient chemotaxis,
and normal multicellular development of Dictyostelium
discoideum amebae (42, 74). The Dictyostelium F-actin-
bundling protein, p30a, also modulates the structure and
function of cell surface extensions. This protein is concentrated in filopodia (29), stabilizes actin filaments at sites of
cell-cell interaction (30), and is required for normal
filopodial structure and function (67). The actin-bundling
protein called drebrin may play a similar role in mammalian cells since overexpression in fibroblasts induces the
formation of long cell extensions that are relatively resistant to actin destabilization by cytochalasin D (46, 72). Finally, the ezrin, radixin, and moesin (ERM)1 proteins may
help stabilize both adherens junctions and dynamic cell
surface extensions (79). Simultaneous reduction of the intracellular levels of these structurally related, F-actin-
binding proteins inhibits both cell-cell adhesion and the formation of microvilli, filopodia, and membrane ruffles (77).
One approach to the identification of proteins that bind
actin at the plasma membrane is the use of 125I-labeled
F-actin blot overlays. This technique is particularly useful
for identifying F-actin-binding proteins in membrane skeletons because such proteins, which are often difficult to
solubilize, can be solubilized with SDS before analyzing
for actin binding activity (17, 66). This approach has been
used successfully to monitor the distribution and/or purification of ponticulin (17, 18), p30a (17, 36), drebrin (54),
and the ERM proteins (66). Although it is not clear why
this technique selectively identifies many membrane-associated, as opposed to strictly cytoplasmic, actin-binding
proteins, this has proved to be true so far.
One membrane-associated protein that we have identified using F-actin blot overlays exhibits an apparent Mr of
205 on SDS-polyacrylamide gels. This protein (p205), which
binds to the sides of actin filaments, is especially prominent in crude membranes from bovine neutrophils (66) and
in whole cell extracts and crude membrane fractions from
cervical carcinoma (HeLa) cells (54; our unpublished observations). Because p205 is not immunocrossreactive with
antibodies against myosin II, fodrin, talin, or tensin (66),
and because myosins I and V do not bind F-actin on blot
overlays under our conditions (54), we have speculated that
p205 may be a new actin-binding membrane protein (66).
In this study, we demonstrate that p205 is, in fact, a previously uncharacterized component of both the neutrophil
and MDBK cell membrane skeletons. This protein is a
tightly bound peripheral protein that cofractionates with
Cell Culture
Cells were grown with 10% FBS unless otherwise specified. MDBK cells
were grown in MEM with Earl's balanced salts; SHSY5Y human neuroblastoma cells in RPMI-1640; NIH-3T3 cells and COS-7 monkey kidney
cells in DME; pig kidney epithelial cells (LLC-PK1) in medium 199 with
3% FBS; NRK cells in F-12 nutrient mixture; HeLa-S3 cells in Joklik
MEM with 5% FBS (Irvine Scientific, Santa Ana, CA). The SHSY5Y
neuroblastoma cells were a gift of Dr. A.H. Ross and the HeLa cells were
provided by Dr. T. Pederson, both from the Worcester Foundation for
Biomedical Research (Shrewsbury, MA). The remaining cell lines were
obtained from the American Type Culture Collection (Rockville, MD).
Reagents for tissue culture were purchased from GIBCO Laboratories
(Grand Island, NY).
Neutrophils and Plasma Membranes
Neutrophils were isolated from 8 to 16 liters of fresh bovine blood by differential lysis, followed by fractionation on preformed gradients of isotonic Percoll (Pharmacia Biotechnology, Piscataway, NJ) and were disrupted by nitrogen cavitation (24, 60, 66). Plasma membranes, secretory
vesicles, pooled granules, and cytosol were separated by flotation of the
postnuclear supernatant into a step Percoll gradient (21). Briefly, cavitates
were centrifuged to remove nuclei and mixed with an equal volume of a
1.12 g/ml Percoll solution in relaxation buffer (66). Gradients consisted of
5 ml of 1.12 g/ml Percoll in relaxation buffer, 14 ml of the cavitate/Percoll
mixture, 14 ml of a 1.04 g/ml Percoll suspension in relaxation buffer, and 5 ml
of relaxation buffer. After centrifugation at 65,000 g for 20 min at 4°C, the
plasma membrane fraction was collected from the top of the 1.04 g/ml
Percoll layer. Secretory vesicles were harvested from the interface between the 1.04 g/ml Percoll layer and the layer that initially contained cavitate. Pooled granules were collected from the bottoms of the centrifuge tubes. "Cytosol" was defined as the supernatant obtained after centrifuging the cavitate-containing layer at 141,000 g for 2 h at 4°C. The various
membrane-containing fractions were centrifuged under the same conditions to remove Percoll, and the less-dense organellar pellets were resuspended in relaxation buffer and stored in aliquots at Electron Microscopy
Purity of the plasma membrane fraction was assessed by transmission
electron microscopy. The plasma membrane and secretory vesicle fractions (0.5 ml each) were overlaid onto 2-ml cushions of 64% sucrose in relaxation buffer and centrifuged at 200,000 g for 30 min at 4°C to pellet residual Percoll. Membranes were collected from the tops of the sucrose
cushions, diluted with relaxation buffer, and recentrifuged into a tight pellet at 200,000 g for 15 min at 4°C. The pellets were fixed for 1 h at 0°C in 2.5% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.4, washed three times with cacodylate buffer, and then postfixed with 2% OsO4, 0.1 M sodium cacodylate, pH 7.4. After three more washes with buffer, the pellets
were stained en bloc with 0.5% aqueous uranyl acetate, dehydrated in ethanol/acetone, and then embedded in EMBED 812 - DER 736 (Electron
Microscopy Sciences, Ft. Washington, PA). Sections of ~65 nm were cut
both parallel and perpendicular to the axis of centrifugation and then
stained with 5% aqueous uranyl acetate for 30 min and Reynolds' lead citrate
for 30 s before visualization on an electron microscope (EM 301; Philips
Electron Optics, Inc., Mahwah, NJ) at an accelerating voltage of 60 kV.
Membrane Extractions
Detergent extractions were carried out at 0°C for 60 min with either 1%
Triton X-100, 3% octylglucoside, or 0.1% SDS in 1 mM EGTA, 2.5 mM
MgATP, 0.3 µM aprotinin, 2 µM leupeptin, 3 µM pepstatin, 1 mM PMSF,
25 mM Tris-HCl, pH 7.5, and either 50, 150, or 250 mM NaCl. As a positive control, membranes were extracted at 70°C for 10 min with 1% SDS.
Supernatants and pellets were collected after centrifugation at 200,000 g
for 60 min.
For extraction with salt or alkali, plasma membranes were suspended in
20 mM sodium phosphate, pH 7.5, containing 1 mM EDTA, 1 mM DTT,
and the above-mentioned protease inhibitors. Membranes were extracted
at 0°C for 60 min with either 2.5 mM MgATP, 0.25 M KCl, 1.0 M KCl, or
for 10 min in 0.1 M sodium carbonate or 0.1 M NaOH. Supernatants and
pellets were collected after centrifugation at 200,000 g for 60 min through
a 10% sucrose cushion prepared in the above buffer. Samples were denatured for 10 min at 70°C in Laemmli sample buffer before analysis on
SDS-PAGE (51).
Phalloidin Shift Experiments
Plasma membranes (1 mg/ml), in the presence or absence of 10 µM phalloidin (Boehringer Mannheim GmbH, Mannheim, Germany), were extracted for 1 h at 0°C with Triton X-100 extraction buffer (TEB): 1% Triton X-100, 250 mM NaCl, 2.5 mM MgATP, 2 mM EGTA, 0.3 µM aprotinin,
2 µM leupeptin, 3 µM pepstatin, 1 mM PMSF, 25 mM Tris-HCl, pH 7.4. Samples (0.8 ml) were centrifuged at 200,000 g for 16 h at 4°C into 20-55%
linear sucrose gradients (3.6 ml) over a 64% sucrose cushion (0.5 ml).
Fractions (0.3 ml) were collected from the top of the gradient with a density gradient fractionator (Isco, Lincoln, NE) and analyzed for the presence of cytoskeletal proteins after SDS-PAGE and electrotransfer to nitrocellulose (78). Calibration standards and accepted values (75) for
Svedberg coefficients were: Immunoblots and F-Actin Blot Overlays
Nitrocellulose blots were probed for moesin, ezrin, and p205 using 125I-
labeled F-actin (17, 66). Other cytoskeletal proteins were visualized with
either monoclonal antibodies ( Purification and Microsequencing of p205
Plasma membranes (160 mg) at a concentration of 1 mg/ml were extracted
for 60 min at 0°C with TEB. After centrifugation at 141,000 g for 60 min,
the pellet was solubilized by sonication for 1 min at 0°C (bath sonicator) in
10 ml of 4% SDS, 0.1 mM DTT, 10 mM Tris-HCl, pH 7.5, and heated for
10 min at 70°C. The suspension was clarified by centrifugation at 240,000 g
for 30 min and concentrated to 3 ml in a Centricon-100 microconcentrator
(Amicon, Bedford, MA). High molecular weight polypeptides were resolved by electrophoresis into a ~15-cm-long 5% SDS-polyacrylamide
gel. When a visible myosin standard (Amersham Corp.) had migrated to
~10 cm, the proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore Corp., Bedford, MA) and visualized
by staining with Ponceau S. The band just under myosin, which corresponded to p205, was excised, washed extensively with sterile water, and then digested with either sequencing grade N-1-tosylamide-2-phenylethylchloromethyl ketone-trypsin (Promega Corp., Madison, WI) or Endo-LysC (Promega Corp.). Peptides were purified on a micropore HPLC and
sequenced by Dr. J.D. Leszyk at the Worcester Foundation for Biomedical Research, W.M. Keck Protein Chemistry Facility (Shrewsbury, MA).
Anti-p205 Antibodies
Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and
VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).
Antipeptide antibodies were produced against each peptide (peptides A
and B, respectively) after conjugation to keyhole limpet hemocyanin.
ELISA titers (40) of antibodies directed against peptide A ranged from
108,600 to 300,000; titers of antibodies against peptide B were much lower
(4,300-6,700). Antibodies were affinity purified against the cystinyl aminocaproic acid derivative of the appropriate peptide conjugated to immobilized chicken egg white lysozyme (47). IgG was purified by ammonium
sulfate precipitation and DE52 chromatography (45) and was incubated
overnight at 4°C with the appropriate affinity matrix. After extensive
washes with PBS (150 mM NaCl, 10 mM sodium phosphate buffer, pH
7.5) and with 0.5 M NaCl, 10 mM Tris-HCl, pH 7.6, high affinity antipeptide antibodies were eluted with 3.5 M MgCl2, 50 mM Tris-HCl, pH 7.2. These antibodies were immediately diluted with 1 mg/ml BSA, 10 mM
Tris-HCl, pH 7.6, dialyzed against 150 mM NaCl, 10 mM Tris-HCl, pH
7.6, and concentrated and stored at Immunoprecipitations
For most experiments, p205 was immunoprecipitated with antibodies
against peptide A, but some experiments also used antibodies against peptide B. To show that both peptides originated from p205, neutrophil
plasma membranes (6 mg) were extracted with TEB, solubilized with 1%
SDS (0.6 ml) at 70°C for 10 min and diluted 10-fold with RIPA buffer
(150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM
Tris-HCl, pH 8.0) lacking SDS to generate a final concentration of 0.1%
SDS. The suspension was clarified by centrifugation at 200,000 g for 30 min, preadsorbed for 3 h at 4°C with nonspecific rabbit IgG bound to protein A-agarose beads (Bio-Rad Laboratories, Hercules, CA) (31), and
then centrifuged for 30 s at 100 g. p205 was precipitated from 0.5-ml aliquots of the supernatant by overnight incubation at 4°C with antisera and
protein A-agarose beads. Controls employed either preimmune sera or
immune sera in the presence of the appropriate competing peptide (66.6 µg/ml). Proteins bound to the agarose beads were sedimented through
1 M sucrose in RIPA buffer and solubilized with Laemmli sample buffer.
To demonstrate coimmunoprecipitation of actin with p205, neutrophil
plasma membranes (1.0 mg/ml) were solubilized for 1 h at 0°C with TEB,
preadsorbed, and then clarified, as described above. Clarified suspensions
(0.25 ml) were incubated overnight at 4°C with protein A-agarose beads
containing 150 µg of either affinity-purified antibodies against peptide A
or nonspecific rabbit IgG. The beads were centrifuged for 3 min at 200 g
through 1 M sucrose in TEB and washed three times with the high stringency RIPA buffer before solubilization for analysis by SDS-PAGE.
To demonstrate coprecipitation of p205 with actin, plasma membranes
were treated briefly at 0°C with either fluorescein-phalloidin (Molecular
Probes, Inc., Eugene, OR) or unlabeled phalloidin (Boehringer Mannheim GmbH) at a ratio of 1 µg of phalloidin/mg of membrane protein.
Membranes (1 mg/ml) were extracted for 1 h at 0°C with TEB, preabsorbed, and clarified, as above. Clarified suspensions (0.45 ml) were incubated overnight at 4°C with protein A-agarose beads and 400 µg of either
purified antifluorescein IgG (53) or nonspecific IgG. This amount of antifluorescein IgG is approximately equimolar to the amount of phalloidin
bound to membrane actin. Beads were centrifuged through 1 M sucrose in
TEB and solubilized in Laemmli sample buffer.
Immunofluorescence Microscopy
Confluent or subconfluent MDBK cells grown on coverslips were washed
twice in PBS and fixed for 20 min at room temperature with PBS containing 1% EM grade formaldehyde (Electron Microscopy Sciences). EGTA-treated cells were washed with Dulbecco's PBS (138 mM NaCl, 2.7 mM
KCl, 8.1 mM Na2HPO4, 1.2 mM KH2PO4, pH 7.0) and incubated at 37°C
with 3 mM EGTA in the same buffer for 10, 20, and 30 min before fixation. After three washes with PBS, the fixed cells were permeabilized with
1% Triton X-100 in PBS for 1 min at room temperature, and then washed
three more times. The coverslips were blocked with 10% horse serum,
1% BSA, and 0.02% sodium azide in PBS for ~2 h at room temperature
or overnight at 4°C. Coverslips were then incubated 2-4 h at 37°C with either a monoclonal pan-cadherin antibody (Sigma Chemical Co.) diluted 1:1,000 in blocking solution, and/or affinity-purified antibodies against
p205 peptide A at 150 µg/ml in blocking solution. After three washes in
PBS, coverslips were incubated with a 1:750 dilution of Texas red-labeled,
goat anti-rabbit IgG (Cappel Laboratories, Durham, NC) and/or a 1:1,000
dilution of Oregon green-labeled, goat anti-mouse IgG (Molecular
Probes, Inc.) for 1 h at room temperature. In some experiments, the secondary antibody solution also contained 10 units/ml Bodipy-phallicidin
(Molecular Probes, Inc.). At these antibody concentrations, no significant
background was observed in the absence of primary antibody, and no
bleed through fluorescence was detected in samples labeled singly with either primary antibody. After the final washes, samples were mounted with
Slowfade-Light Antifade Medium (Molecular Probes, Inc.) and observed
using a confocal microscope (MRC 1024; Bio-Rad Laboratories) equipped with LaserSharp Version 2.1A software.
Molecular Cloning
Cloning of the p205 Peptide A Sequence.
Degenerate oligonucleotide primers (5 3 5 Gene-specific PCR.
Nondegenerate oligonucleotide primers (5 Sequence Determination and Comparison.
Full-length and deletion constructs generated from internal AatII and PstI restriction sites were sequenced in both directions by primer walking at the Iowa State University
DNA Sequencing and Synthesis Facility (Ames, IA). The final sequence
encoding supervillin (p205) (these sequence data are available from GenBank/EMBL/DDBJ under accession No. AF025996) represents the consensus sequence from 18 overlapping cDNAs. For each nucleotide, 6-13
independently generated cDNAs were sequenced in both directions.
RNA Isolation and Northern Analysis.
MDBK mRNA was isolated
from log-phase and confluent cultures with the Poly(A)Pure mRNA Isolation Kit. For Northern analyses, 5 µg of polyadenylated (poly(A)+)
RNA and 5 µg of RNA molecular weight markers II (Ambion) were separated per lane on a 0.8% agarose-formaldehyde gel, transferred to Duralon filters (Stratagene, La Jolla, CA) by capillary blotting, and UV cross-linked to the membrane (28). The blots were stained with methylene blue
to visualize the markers and rRNA bands, and then hybridized with a 32P-labeled, random-primed probe (28) produced using the Prime-A-Gene kit
(Promega Corp.), using as a template either a 659-bp HindIII/SacI fragment from the 5 Sequence Analysis.
Predicted secondary structure was determined using
the PeptideStructure subroutine in the Genetics Computer Group, Inc.
(Madison, WI) sequence analysis software package. Percent identity and
homology were assessed using the GAP and PileUp subroutines in this
same package. GAP was used for individual, optimized sequence comparisons, and PileUp for multiple sequence alignments.
p205 Is a Peripheral Plasma Membrane Protein
As detected by blot overlays with 125I-labeled F-actin (17),
moesin, ezrin, and a polypeptide with an Mr of 205,000 (p205) were previously described as major actin-binding proteins
in a bovine neutrophil membrane fraction called the
To explore the nature of the interaction between p205
and the plasma membrane, we extracted purified neutrophil plasma membranes with a series of salt and detergent
solutions (Fig. 2). We found that buffers containing 2.5 mM
MgATP, a reagent that extracts most of the similarly sized
myosin II, had no effect on the membrane association of
p205 (Fig. 2 A, lanes 2S and 2P). Similarly, moderately high salt concentrations (0.25 M) that extract significant
amounts of membrane-bound moesin and ezrin, had no
detectable effect on the extractability of p205 (Fig. 2 A,
lanes 3S and 3P). Even sodium carbonate, a reagent that
extracts many peripherally bound proteins (44), had no effect on the membrane association of p205 (Fig. 2 A, lanes
5S and 5P). On the other hand, p205 was partially extracted at salt concentrations >1 M (Fig. 2 A, lanes 4S and
4P) and was almost completely extracted by 0.1 M NaOH
(Fig. 2 A, lanes 6S and 6P), indicating that p205 is a tightly
bound peripheral protein, and not an integral component,
of the plasma membrane.
In agreement with this assessment, p205 also was resistant to extraction by a number of nonionic detergents (Fig.
2 B). At salt concentrations up to 250 mM, p205 remained
insoluble in the presence of 1% Triton X-100 (Fig. 2 B,
lanes 2-4), 3% octylglucoside (Fig. 2 B, lanes 5S and 5P), or
0.1% SDS (Fig. 2 B, lanes 6S and 6P). The only detergent
that effectively solubilized p205 was 1% SDS (Fig. 2 B, lanes
7S and 7P). Interestingly, a large fraction of the membrane-associated actin was retained in the p205-enriched pellet after extraction with buffers containing 250 mM
NaCl and either 1% Triton X-100 or 3% octylglucoside
(Fig. 2 B, lanes 4 and 5), conditions that extracted essentially all moesin and ezrin. Thus, both p205 and actin appear to be detergent-resistant components of a membrane
skeleton that does not require either moesin or ezrin for at
least some of its integrity.
p205 Is a Previously Uncharacterized Protein
The inextractability of p205 under conditions that solubilized most membrane and membrane skeleton proteins, in
conjunction with the large amounts of plasma membrane
obtainable from bovine neutrophils, suggested that this
protein should be readily purified by differential extraction followed by preparative SDS-PAGE. We thus extracted plasma membranes with a buffer containing 1%
Triton X-100, 0.25 M NaCl, and 1 mM MgATP to identify
proteins that are tightly associated with the neutrophil
plasma membrane skeleton (Fig. 3 A, lane 2 vs. lane 1).
The inclusion of MgATP in this buffer resulted in the removal of most of the myosin II that migrated near p205 on
SDS gels. When run on a long, 5% polyacrylamide gel, there was a clear separation of p205 from residual myosin
and other similarly sized membrane skeleton proteins
(Fig. 3 A, lanes 2-5). Microsequencing of the band corresponding to p205 generated a total of eight peptide sequences (Fig. 3 B). None of these peptide sequences were
significantly similar to any protein sequence in the nonredundant, updated protein databases, indicating that p205 is a previously uncharacterized protein.
To confirm that the peptide sequences in Fig. 3 B were
derived from the F-actin-binding polypeptide called p205,
antibodies against the two longest peptides (pepA, pepB)
were used to specifically immunoprecipitate p205 from
neutrophil membrane extracts (Fig. 4). Although pepA
was much more immunogenic than pepB, antisera against
either peptide, generated in each of four rabbits (Fig. 4,
lanes 1-4, respectively), specifically immunoprecipitated
p205 from SDS- and heat-denatured membrane extracts
(Fig. 4, center, lanes 1-4). Specificity was indicated by the
absence of p205 from immunoprecipitates generated with
preimmune sera (Fig. 4, left) or with immune sera in the
presence of the appropriate peptide antigen (Fig. 4, right).
Similar results were obtained with the corresponding affinity-purified antibodies (data not shown). Thus, both
pepA and pepB are derived from p205.
p205 Associates with Actin Filaments In Vivo
The high speed centrifugations used in the extraction experiments (Fig. 2) were expected to sediment essentially
all membrane vesicles and large protein complexes. To determine whether p205 and endogenous As expected for an F-actin-binding protein, p205 exhibited a reproducible phalloidin-induced increase in S value
in TEB extracts of neutrophil plasma membranes (Fig. 5).
In the presence of phalloidin, p205 sedimented as a component of a ~30 S complex (Fig. 5, A and B) that also contained the bulk of the membrane-associated
More evidence for the association of p205 with actin in
situ was obtained by demonstrating that p205 and F-actin
cosediment in reciprocal immunoprecipitation assays (Fig.
6, A and B). Because antiactin antibodies were less than
optimal in these assays, we developed a procedure in
which high affinity polyclonal antibodies against fluorescein (53, 83) were used to pellet actin filaments stabilized
with fluorescein-labeled phalloidin. Neutrophil plasma membranes were incubated with fluorescein-phalloidin, and
then phalloidin-bound actin filaments were precipitated
from detergent-solubilized extracts with antifluorescein
IgG (see Materials and Methods). Essentially all of the
p205 in the initial extract (Fig. 6 A, lane 1) coprecipitated
with F-actin bound to fluorescein-phalloidin (Fig. 6 A, lane
3), whereas little or no p205 was found in control experiments with nonspecific IgG (Fig. 6 A, lane 2) or with unlabeled phalloidin (Fig. 6 A, lane 4).
In converse experiments, we found that actin copelleted
with p205 immunoprecipitated with affinity-purified antibodies against pepA (Fig. 6 B, lane 2). Less than 5% as
much actin coprecipitated with equivalent amounts of nonspecific IgG under these conditions (Fig. 6 B, lane 1). Thus,
both the phalloidin shift experiments and the coimmunoprecipitation assays suggest that the direct association between p205 and rabbit p205 Is Present in Many Cell Types
Based on immunoblot analyses with affinity-purified antibodies against p205, this protein is present in a number of
cell types (Fig. 7). Both human cervical carcinoma cells
(Fig. 7, A and B, lane 1) and bovine kidney epithelial cells
(Fig. 7 A, lane 2; and B, lane 4) contained at least as much
p205 as bovine neutrophils (Fig. 7 A, lane 3). The anti-pepA antibody was highly specific for p205 in these tissue
culture cells since they lacked a ~90-kD immunocrossreactive protein (Fig. 7 A, arrowhead) that is present in
whole neutrophil extracts and pooled granules, but absent
from purified neutrophil plasma membranes (data not
shown). Other transformed and nontransformed cell lines
also contained a 205-kD band recognized by both affinity-purified, anti-pepA antibodies (Fig. 7 B, top) and 125I-labeled
F-actin (Fig. 7 B, bottom). For instance, p205 was present in neuroblastoma SHSY5Y cells (Fig. 7 B, lane 2), in 3T3
fibroblasts (Fig. 7 B, lane 3), and in a number of epithelial
cell lines (Fig. 7 B, lanes 4-7). Although the affinity-purified pepA antibodies appeared to be specific for p205 in
these cells, a second polypeptide with a slightly slower mobility was observed in F-actin blot overlays of 3T3 mouse
(Fig. 7 B, lane 3) and normal rat kidney (Fig. 7 B, lane 5)
cells. Thus, while p205 is found in many cell types, it is not
the only protein in this size range that can be visualized by
125I-labeled F-actin on blot overlays.
p205 Localization in Epithelial Cells
As was the case in neutrophils (Fig. 1), p205 was associated with plasma membranes in HeLa (not shown) and
MDBK (Figs. 8-10) cells. First, p205 was enriched ~10-fold,
about the same fold enrichment as a biotin cell surface
marker, in crude plasma membrane fractions (4) from all
three cell types (not shown). Second, immunofluorescence
localization with affinity-purified, anti-pepA antibodies
showed label at the plasma membrane in both low density (Fig. 8) and confluent (Fig. 9) MDBK cells. Low density
cells (Fig. 8, A and C) and subconfluent cells (Fig. 9 A)
also contained appreciable amounts of signal in punctate
dots throughout the cytoplasm and within the nucleus. In
nonjunctional regions of the plasma membrane, p205 staining was definite, but not pronounced, and did not necessarily colocalize with antibody against the cell-cell adhesion protein, E-cadherin (Figs. 8 and 9, hollow arrows). As the MDBK cells became confluent, the ratio between the
plasma membrane and the internal p205 signal increased,
as did the colocalization with E-cadherin at sites of both
initial (Fig. 8, C and D) and established (Fig. 9, A and B)
cell-cell contact (solid white arrows). In confluent cells,
nearly all of the p205 staining was concentrated at lateral
cell borders (Fig. 9 C), where it colocalized almost perfectly at the light level with E-cadherin (Fig. 9 D). Because p205 staining was not observed in apical microvilli, nor
with actin meshworks at basal cell surfaces (not shown),
we infer that the colocalization with cadherin-containing,
adherens-type junctions is specific and may reflect a role
for p205 in the formation or stabilization of these structures.
To explore further the extent of the association between
p205 and cadherin-containing junctional complexes, confluent monolayers of MDBK cells were treated with 3 mM
EGTA, which induces the release of intercellular contacts
and a coordinate internalization of vesicles containing
E-cadherin and bound junctional proteins (49). At early
times after treatment with EGTA, p205 (Fig. 10 A) colocalized with ringlike structures of bundled actin filaments
(Fig. 10 B) and E-cadherin (not shown) during their contraction into the cytoplasm away from the membrane (49,
82). At later times after EGTA-mediated cell-cell dissociation, p205 (Fig. 10 C) and E-cadherin (Fig. 10 D) continued to colocalize in fragmented barlike structures near the
nucleus. As these structures broke down with time (49,
82), p205 became dissociated into cytoplasmic punctae
(Fig. 10 E) that no longer colocalized significantly with the
large juxtanuclear structures containing cadherin (Fig. 10
F). However, p205 and cadherin continued to colocalize at
sites of residual cell-cell contact (Fig. 10, E and F). No significant intranuclear staining for either p205 or cadherin
was observed at any time after addition of EGTA.
These results indicate an extensive association, direct or
indirect, between E-cadherin, p205, and F-actin in adherens-type junctions and suggest that p205 also can reside in
a separate, punctate cytoplasmic compartment, e.g., on
vesicles. These observations further suggest that the nuclear signal observed in low density cells (Fig. 8, A and C)
may represent yet another p205-containing intracellular
compartment. On the other hand, the preponderance of
nuclear staining artifacts in immunofluorescence microscopy (57) remains an important caveat.
Molecular Cloning of p205
Primers with both inosines and degeneracies were designed from each end of the pepA microsequence (Fig. 3 B)
and used in a PCR reaction to obtain the nondegenerate
central portion of the pepA sequence (see Materials and
Methods). Once the central region of the pepA sequence
was known, primers designed from this sequence were
used in a 3 To confirm the approximate message size, Northern
analysis was performed with MDBK poly(A)+ RNA (Fig.
11 B). Both a probe from the 5 Primary Structure of p205
The consensus DNA sequence encoded a protein of 1,792 amino acids (Fig. 12 A) that is absent from current protein
databases. The predicted mol wt of 200,626 (isoelectric
point ~6.44) (8) is in good agreement with that predicted
for p205 by SDS-PAGE (Fig. 1). Relative to an "average"
protein (20), p205 is high in arginine (7.1% vs. 4.7%) and
glutamine (9.2% vs. 6.2%) and low in tyrosine (2.3% vs.
3.5%) and cysteine (1.3% vs. 2.8%), variations that are
consistent with its relative insensitivity to staining by silver
(Fig. 3 A) (62). The deduced amino acid sequence includes
all eight peptides obtained from purified p205 (Figs. 3 B
and 12 A, double underlines). Since antibodies against two
of these peptides immunoprecipitate the F-actin-binding
protein of ~205,000 D from SDS-solubilized neutrophil
plasma membranes (Fig. 4), the sequence in Fig. 12 A undoubtedly encodes p205.
Analysis of the deduced amino acid sequence suggests
that p205 is a bipartite protein with distinctly different
NH2- and COOH-terminal domains (Fig. 12 B). The NH2-terminal half (first ~935 amino acids) contains numerous
charge clusters in the context of a primarily Sequence analysis also suggests a potential mechanism
for the regulation of p205 accumulation in the nucleus.
The putative nuclear localization signals are surrounded
by 43 serines and threonines that are potentially phosphorylatable by protein kinase A (Fig. 12 B, asterisks), protein
kinase C, and/or casein kinase II (not shown). Thus, targeting of p205 to the nucleus could be regulated by Ser/
Thr phosphorylation, a mechanism documented for other
proteins that conditionally localize to the nucleus (37).
The COOH-terminal half of p205 contains 24 potentially phosphorylatable serines and threonines and a consensus
site for tyrosine phosphorylation (Tyr-1157; Fig. 12, A
black box and B, black dot), protein modifications known
to regulate adherens junction structure (3).
The most striking characteristic of the p205 COOH-terminal domain, by far, is its extensive homology (Figs. 12 B
and 13) with the villin/gelsolin family of cytosolic F-actin-
binding proteins (85). Many short stretches of sequence
similarity were identified by the BLASTP search algorithm (2) between sequential segments of p205, starting at
about Asn-979, through virtually the entire lengths of villin
and gelsolin (Fig. 13 A). In individual, optimized comparisons of each of these sequences with that of the COOH
terminus of p205, the overall percent identities were 29%
(villin) and 28% (gelsolin), and the overall similarities
were 48 and 50%, respectively.
The nature of the similarity with villin and gelsolin is
best appreciated when the percent identities are plotted as
a function of position along the length of p205 (Fig. 13 A).
Regions of very high sequence identity are interspersed
with regions exhibiting little or no similarity, usually due
to the presence of additional residues in p205. In particular, this analysis identified three localized regions of
~50% sequence identity between p205 and sites in villin
and/or gelsolin (Fig. 13, A and B). Interestingly, two of
these sites include sequences that have been previously
shown to bind F-actin (85). The first site, amino acids
1,023-1,032, is very similar to a sequence found in the segment-2 region of both gelsolin and villin that, when dimerized, can crosslink actin filaments (23). The second of
these sites is the COOH terminus of p205, which is extremely similar to the COOH-terminal "headpiece" region
of villin, a sequence involved in bundling actin filaments in
vitro (38, 48) and in vivo (32, 35). Interestingly, most of the
conserved residues that are required for structural stability
or F-actin binding of the villin headpiece (26, 56) are also
found in p205 (Fig. 13 B, bottom), suggesting that this region of p205 also may bind F-actin.
The third region of high sequence similarity between
p205, gelsolin, and villin corresponds to COOH-terminal
residues in segment five of the latter two proteins (Fig. 13,
A and B). Intriguingly, proteolytic fragments (12) and bacterially expressed proteins (84) containing segments four
through six of gelsolin contain an otherwise unmapped
Ca2+-dependent site for binding F-actin. It is thus possible
that this third region of high homology to villin and gelsolin also corresponds to a sequence that can bind F-actin. In
any case, this segment-five homology region in p205, as
well as many of the other peaks of sequence identity shown
in Fig. 13 A, apparently represents an important structural
or functional site common to all three of these proteins.
Other proteins exhibiting high structural similarity with
p205 are protovillin, a ~100-kD F-actin capping protein
from Dictyostelium and an open reading frame (ORF) in
the Caenorhabditis elegans genome that is predicted to encode a ~113-kD protein (Fig. 13 B). Optimized alignments
along the length of each protein indicate that protovillin is
27% identical (49% similar) and that the C. elegans ORF
is 25% identical (46% similar) to the p205 COOH terminus. More distant relationships with other members of the
villin/gelsolin superfamily, including adseverin, scinderin,
severin, and fragmin (85), also were observed (not shown).
Thus, at ~200,000 D p205 is the largest member of the villin/gelsolin superfamily. In recognition of this relationship,
we propose the name, "supervillin."
In this paper, we have identified and characterized a novel
plasma membrane-associated, F-actin-binding protein (supervillin/p205), which is present in bovine neutrophils and
in various transformed and nontransformed cell lines. We
have shown previously that this protein fractionates with
crude neutrophil membranes and binds directly and specifically to the sides of We also show here that the structure of supervillin is
novel (Figs. 3, 11, and 12). Not only is this protein currently
unrepresented in the protein databases, but it is unique in
that it contains both a strong homology to cytosolic actin-binding proteins in the villin/gelsolin family (Fig. 13) and
four nuclear localization signals (Fig. 12). The demonstrated tight binding of supervillin to actin filaments (Figs. 1
and 6) is reflected by the prediction from the primary sequence that this protein may contain as many as three
binding sites for F-actin (Fig. 13 B). Based upon the extent
of the sequence similarities and the known properties of the homologous sequences in villin and gelsolin, supervillin may also bundle actin filaments. Interestingly, amino
acids required for filament severing in gelsolin (Lys-150
through Gln-160; see reference 41) and villin (Arg-137; see
reference 23) are not conserved in supervillin (Fig. 11 B),
suggesting that, like protovillin (43), supervillin probably
lacks this activity. At least one of the F-actin-binding sites
in supervillin is insensitive to the presence of free calcium
ions since F-actin binding on blot overlays is similar in the
presence of either 1 mM EGTA or 0.1 mM CaCl2 (data
not shown). However, the definitive determination of the
nature of the interaction(s) between supervillin, actin filaments, and calcium ions awaits the identification of a
source from which biochemically significant amounts of
native supervillin are readily obtainable.
The colocalization of supervillin with E-cadherin at sites
of initial (Fig. 8) and established (Fig. 9) cell-cell contact and its internalization with E-cadherin and actin during
EGTA-mediated cell dissociation (Fig. 10) suggest that supervillin may be involved in the formation and/or stabilization of actin filament bundles at adherens junctions.
Such an activity would be analogous to that documented
for villin in the microvilli of highly organized brush borders. Villin both nucleates microvillar assembly in transfected cells and cross-links actin filaments in the mature
microvillar core (34). A second precedent may be the actin
bundling protein, p30a, which stabilizes filaments against
depolymerization (89) and apparently potentiates their association with intercellular junctional membranes, even
though p30a does not itself bind tightly to the membrane
(30). An even more intriguing paradigm may be Because the COOH terminus of supervillin apparently
constitutes the actin-binding domain of the molecule, an
attractive hypothetical function for the NH2 terminus is the
targeting of supervillin to appropriate intracellular compartment(s). The localization of supervillin at regions of
lateral cell-cell contact (Figs. 8, 9) is quite distinct from the
observed concentration of villin in apical microvilli (11, 33).
Also, no supervillin is observed in association with the actin filament meshwork at the basal surfaces of MDBK
cells. Thus, either the unique supervillin NH2 terminus or
one of the supervillin-specific, "linker" sequences interspersed between the villin/gelsolin homology regions must
contain a sequence responsible for targeting to some component of adherens junctions. The punctate cytoplasmic
distribution observed in nonadherent cells implies that this
target may be membrane associated.
Another intracellular destination for supervillin might
be the nucleus. The nuclear localization predicted from
the presence of NH2-terminal targeting signals is supported by the observation that nuclei of low density cells
label with an antibody against a supervillin peptide (Fig.
8). Although nuclear localization artifacts are common in
fixed cells (57), no significant nuclear staining is observed
in EGTA-treated cells (Fig. 10), suggesting that the localization observed in Fig. 8 is not a consequence of our fixation conditions or a fortuitous cross-reaction with a similar
epitope in a nuclear protein.
A role for supervillin outside the adherens junction is
also suggested by its comparatively high abundance in bovine neutrophils (Figs. 1 and 7 A) (66) and HeLa cells (Fig.
7 B). These cells are not adherent and either lack (neutrophils) or are grossly deficient (HeLa cells) in classical cadherins detectable by antibodies against the highly conserved cadherin cytoplasmic domain (data not shown).
While these cell types might contain a divergent cadherin with an immunologically distinct cytoplasmic domain (76),
it is also possible that supervillin plays different roles in
nonadherent and adherent cells. Such a multiplicity of functions is supported by the changing intracellular localization of supervillin as a function of the growth and adhesive
state of MDBK cells (Figs. 8 and 9).
Assuming that subsequent analyses with additional antibodies and with epitope-tagged supervillin confirm its
multiple intracellular localizations, this protein is an excellent candidate for a signaling molecule that transduces information to the nucleus from the membrane skeleton at
sites of cell-cell adhesion. Precedents include In conclusion, we have shown that supervillin is a novel
F-actin-binding protein that cofractionates with endogenous actin, binds peripherally but tightly to neutrophil
plasma membranes, and conditionally localizes with E-cadherin at sites of intercellular adhesion. Future work will be
directed towards elucidating supervillin function and regulation in adherent vs. nonadherent cells and determining
the role of this protein in the modulation of adhesion, motility, and adhesion-mediated signal transduction.
While this paper was in press, Mandai, K., H. Nakanishi, A. Satoh, H. Obaishi, M. Wada, H. Nishioka, M. Itoh, A. Mizoguchi, T. Aoki, T. Fujimoto, et al. (1997. J. Cell Biol. 139:517-528) described
a 205-kD rat brain cytosolic protein (afadin) that binds to F-actin on blot
overlays and exhibits a distribution in epithelial cells similar to that reported here for supervillin. However, the deduced amino acid sequences
of supervillin and afadin are not significantly similar, except for the existence of multiple predicted nuclear targeting sequences in both proteins.
Thus, afadin is a good candidate for the second ~205-kD F-actin-binding polypeptide that we observe in some cell lines (Fig. 7), but it is not a member
of the villin/gelsolin superfamily, nor is it structurally related to supervillin.
- and
-catenins (1). Similarly, indirect interactions between actin filaments and integrins, mediated through numerous other linker proteins in focal contacts, can regulate
integrin binding to ligands in the extracellular matrix and
thus affect cell-substrate adhesion (13, 87).
-actin and
-actin from neutrophil plasma membranes
under stringent conditions and colocalizes with E-cadherin
at sites of adhesion between MDBK cells. In subconfluent MDBK cells, punctate staining also is observed throughout the cytoplasm and within the nucleus. Using amino
acid sequences derived from SDS-PAGE-purified p205
and PCR-RACE (rapid amplification of cDNA ends), we
obtained a series of overlapping clones that encode the
complete p205 cDNA. The deduced protein sequence includes a COOH terminus that is very similar to the villin/
gelsolin family of actin-binding proteins. The NH2 terminus of p205 is novel and contains several potential nuclear
localization signals. Based on the striking similarity with
villin/gelsolin, p205 is apparently the largest known member of the villin superfamily, and accordingly, we suggest
the name "supervillin." The primary sequence motifs and
cellular localization(s) of supervillin suggest that this protein is a structural component of the plasma membrane
skeleton that may also play a role in cell-cell adhesion
and/or information transfer to other cell compartments.
Materials and Methods
80°C.
-amylase (9 S), bovine thyroglobulin (19 S), and Escherichia coli small ribosomal subunit (30 S). Changes in the distribution of p205 were assessed by quantification of bound 125I-labeled F-actin
on overlays as described below.
-actin) or polyclonal antibodies (
-actin,
fodrin, and myosin II). Dr. J.C. Bulinski (Columbia University College of
Physicians and Surgeons, New York) generously supplied the antibody
against
-actin (63), and the antifodrin (nonerythroid spectrin) antibody
(14) was a gift from Dr. K. Burridge (University of North Carolina,
Chapel Hill, NC). Antibodies against
-actin and nonmuscle myosin II
were obtained from Sigma Chemical Company (St. Louis, MO) and Biomedical Technologies, Inc. (Stoughton, MA), respectively. Polyclonal antibodies were visualized with 0.1 µCi/ml 125I-labeled rProtein ATM (Dupont NEN, Boston, MA). Antibody against
-actin was visualized by
incubating blots either with 0.25 µg/ml 125I-labeled goat anti-mouse IgG
(Amersham Corp., Arlington Heights, IL) or with 5 µg/ml rabbit anti-
mouse IgG (Pierce Chemical Co., Rockford, IL), followed by incubation
with 125I-labeled rProtein ATM. After exposure to film, relative amounts of
labeled protein were quantified with a scanning densitometer (PDI, Huntington Station, NY).
20°C in this buffer containing 50%
glycerol.
-CCIGTIGARYTIGAYGARGA-3
and 5
-CKIGGIGCRTAIGGRTCRAA-3
) corresponding to 20 bp at each end of the p205 peptide A
microsequence were used with Advantage KlenTaq polymerase (CLONTECH, Palo Alto, CA) in a touchdown thermal cycle reaction (OMN-E
cycler; Hybaid Ltd., Long Island, NY) to amplify a 53-bp product from
MDBK cDNA. The cDNA was prepared using the Marathon cDNA Amplification Kit (CLONTECH), and mRNA made with the PolyATtract
mRNA Isolation System IV (Promega Corp.) from total RNA prepared
using Tri Reagent (Molecular Research Center, Cincinnati, OH). Products were cloned into the pGEM-T vector (Promega Corp.), and propagated in JM-109 chemically competent cells (Promega Corp.). Plasmids
were purified by boiling minipreps (5), screened for inserts by digestion
with AatII/PstI, and sequenced (Sequenase Version 2.0, Amersham
Corp.), yielding the nondegenerate central nucleotides of the MDBK peptide A sequence.
-RACE.
A degenerate oligonucleotide primer (5
-AGTTNGATGAGGATTTCGATGTCATTTTYGAYCC-3
) and the CLONTECH
Marathon Adaptor Primer 1 (AP1) were used with KlenTaq enzyme mix
in touchdown PCR program No. 1 (CLONTECH) to generate a 3-kb, 3
-RACE product from the double-stranded cDNA template. Correct clones
were identified by digestion with AatII/PstI; sequencing verified the presence of known codons downstream of the primer.
-RACE.
Primers designed initially from 3
-RACE products and subsequently from 5
-RACE products were used in two sequential rounds of 5
-RACE reactions with the CLONTECH AP1 primer and KlenTaq enzyme mix to generate overlapping clones corresponding to the full-length cDNA encoding p205. Gene-specific primers used in these reactions
were 5
-CTCGCGGCCAGCATCTTCAGGG-3
, 5
-GATCTTCCCTCGCGGCCAGCATCTTCAGGG-3
, 5
-TCAAACGACTTCTCCATCTCCCTGAAGAGC-3
, or 5
-GTCAGGTTCTCCCTGCTCAGCAAATCTTT-3
. Reaction products were cloned into pGEM-T, and colonies were screened using a modification of a standard protocol (71). Briefly, nitrocellulose filters
were placed onto plates containing 100-300 medium-sized colonies (1.0 mm diam) for 30 s, and holes were punched for alignment. Filters were denatured, neutralized twice, washed twice, air dried, stacked individually
between sheets of aluminum foil, autoclaved (3 min to sterilize, 3 min to dry),
and then screened for proper inserts with end-labeled oligonucleotides
corresponding to sequences upstream of the gene-specific primer (5).
-GAGCCAGGTCAACTTCAAATTCAGAAATG-3
and 5
-TATTAAGGTAGAAAGGTGGATTCGCACAGA-3
) and the Expand Long Template
PCR System (Boehringer Mannheim GmbH) were used in a touchdown
PCR reaction with first strand MDBK cDNA. The cDNA was prepared
with the SuperScript Preamplification System for First Strand cDNA Synthesis (GIBCO Laboratories) from mRNA prepared using the Poly(A)Pure
mRNA Isolation Kit (Ambion, Austin, TX). The 5,198-bp product was ligated into pGEM-T and completely sequenced.
end of the p205 cDNA or a 465-bp AatII fragment from
the 3
end of the sequence.
Results
fraction (66). The
fraction contains both large sheets of
plasma membrane and "secretory vesicles," which are intracellular vesicles of similar density and composition that
are believed to represent mobilizable intracellular stores
of plasma membrane proteins involved in cell adhesion
and other activation-associated surface processes (9). Taking advantage of a recently described technique for separating human neutrophil membranes containing latent vs.
surface-exposed alkaline phosphatase (21), we found that
the use of a modified Percoll gradient does indeed separate the
fraction into two membrane populations (Fig.
1). The less dense fraction (density <1.04) is enriched in
large membrane sheets with associated amorphous filamentous structures (Fig. 1 A) and corresponds to the peak of surface-exposed alkaline phosphatase (not shown).
Thus, the less dense fraction appears to be mostly plasma
membrane. The denser fraction (density between 1.04 and
1.06) corresponds to the peak of latent alkaline phosphatase (not shown) and contains predominantly small osmophilic vesicles (Fig. 1 B). These vesicles probably represent secretory vesicles although some mitochondria are
also observed in this fraction. While large amounts of all three major F-actin-binding proteins (p205, ezrin, moesin)
were found in the plasma membrane fraction (Fig. 1 C,
lane 1), the enrichment was greatest for p205 since ezrin
and moesin also were present in the secretory vesicle (Fig.
1 C, lane 2) and cytosolic (Fig. 1 C, lane 3) fractions. As reported previously (66), no F-actin-binding proteins were
observed in the pooled granule fraction. There was at least
20-fold more p205 in the plasma membrane fraction than
in the secretory vesicle fraction, and 10-15-fold more than in cytosol. This large enrichment suggests an intimate association of p205 with the plasma membrane.
Fig. 1.
Neutrophil plasma
membranes are highly enriched in p205. Fractions
highly enriched in plasma
membranes (A) or secretory
vesicles (B) were analyzed for the presence of p205 by F-actin
blot overlay (C). Aliquots (100 µg protein) were fractionated on
a 5% SDS-polyacrylamide gel, transferred to nitrocellulose, and
then probed with 125I-labeled F-actin. Lane 1, plasma membranes; lane 2, secretory vesicles; and lane 3, cytosol. E/M, the
position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3).
[View Larger Versions of these Images (89 + 31K GIF file)]
Fig. 2.
p205 is a peripheral component of the neutrophil
plasma membrane skeleton. (A) Neutrophil plasma membranes
were extracted with either buffer alone (lane 1), or buffer containing a final concentration of 2.5 mM MgATP (lane 2), 0.25 M
KCl (lane 3), 1.0 M KCl (lane 4), 0.1 M sodium carbonate (lane 5),
or 0.1 M NaOH (lane 6). For each extraction condition, high
speed supernatants (S) and pellets (P) from 130-µg membranes
were electrophoresed, blotted, and then probed with 125I-labeled
F-actin and with specific antibodies. (B) Neutrophil plasma
membranes (100 µg per treatment) were extracted with either buffer alone (lane 1), or buffer containing 1% Triton X-100 (lane 2), 1% Triton X-100, 50 mM NaCl (lane 3), 1% Triton X-100, 250 mM NaCl (lane 4), 3% octylglucoside, 250 mM NaCl (lane 5), or 0.1% SDS (lane 6) and processed as above. A positive control
consisted of membranes extracted with 1% SDS at 70°C for 10 min (lane 7). The higher mobility F-actin-binding polypeptide
present in B is consistently observed after detergent treatment;
this band also reacts with an antibody against p205 sequences
(see below), suggesting a close structural relationship with p205.
[View Larger Version of this Image (66K GIF file)]
Fig. 3.
Purification of p205 by SDS-PAGE. (A) Neutrophil
plasma membranes (lane 1) and Triton X-100-insoluble pellets
(lanes 2-5) were separated on a 5% polyacrylamide gel and
stained with silver (lanes 1 and 2), or electrotransfered to nitrocellulose and probed with either 125I-labeled F-actin (lane 3), or
with antibodies against myosin II (lane 4), or nonerythroid spectrin/fodrin (lane 5). Loads represent 100 µg membranes or equivalent amounts of Triton X-100-insoluble pellets. The location of
p205 is indicated (arrowheads). (B) Eight microsequences were
obtained from proteolytic digests of SDS-PAGE-purified p205.
Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences (pepA and pepB). Residues at variance with the deduced amino acid sequence (Fig. 11 A) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.
[View Larger Versions of these Images (63 + 25K GIF file)]
Fig. 11.
Strategy used for the
cloning of sequences representing the full-length supervillin
cDNA (A), and Northern blots
with probes from the 5 and 3
ends of the predicted sequence.
(A) A schematic of the supervillin cDNA showing the 5,376-bp
coding region (gray box), the 5
and 3
untranslated regions, and
the sequences used as probes for
the Northern blots (hatched
bars). 18 overlapping clones encoding the full-length supervillin
cDNA (~6.5 kb) were produced
by PCR. Left arrows, 5
-RACE
products; right arrows, 3
-RACE
products, and no arrows, products obtained with two gene-specific primers. (B) Poly(A)+ RNA
(5 µg) was separated on a 0.8%
formaldehyde-agarose gel, blotted onto Duralon membranes
and probed with the 32P-labeled
probes indicated in A. Both the
659-bp probe 1 (lane 1) and the
465-bp probe 2 (lane 2) recognize a message of ~7.2 kb. The
smaller band of ~1.8 kb observed in lane 2 may represent cross-hybridization with comigrating, residual ribosomal RNA
(not shown).
[View Larger Versions of these Images (9 + 25K GIF file)]
Fig. 4.
F-actin blot overlay shows that p205 is specifically immunoprecipitated from bovine neutrophil plasma membranes by
antibodies against p205 peptides, pepA, and pepB. Proteins were
immunoprecipitated from SDS-solubilized, Triton X-100-insoluble pellets by preimmune (Preimmune) or immune (Immune)
sera from four different rabbits (lanes 1-4) that were immunized
with either peptide A (lanes 1 and 2) or peptide B (lanes 3 and 4).
Antibody specificity is indicated by the absence of p205 from immunoprecipitates generated either with preimmune sera or with
immune sera plus the appropriate competing peptide (Immune + pepA/pepB). S, p205 in the initial RIPA supernatant.
[View Larger Version of this Image (48K GIF file)]
-actin sedimented
together in the same detergent- and salt-resistant protein
complex, we used the phalloidin shift assay originally described by Carraway and colleagues (15). In this approach,
membranes are extracted with a Triton X-100-containing buffer in the presence and absence of 10 µM phalloidin.
Microfilament-associated proteins are those that exhibit
increased S values when sedimented in the presence of stabilized (plus phalloidin) vs. destabilized (no phalloidin) actin filaments. In our version of this assay, we used a buffer
containing relatively high concentrations of Triton X-100
(1%), NaCl (250 mM), and MgATP (2.5 mM) to depolymerize significant amounts of the total actin and to dissociate most membrane skeleton proteins.
-actin (Fig.
5 A) and
-actin (data not shown). Although variable
amounts of a ~13 S moiety were observed in two experiments, most p205 sedimented as a ~26 S complex in the
absence of phalloidin (Fig. 5 B). Surprisingly, whereas
much of the actin was rendered monomeric by the harsh
buffer conditions, significant amounts continued to sediment with high S values in the absence of phalloidin stabilization (Fig. 5 A). In contrast to the behavior of p205 and
actin, little or no fodrin, myosin, ezrin, or moesin exhibited
significant phalloidin-induced shifts in sedimentability under these conditions (Fig. 5 A). Thus, p205 apparently
forms large complexes with endogenous actin under conditions that suggest an extremely tight association, direct or indirect, in the neutrophil membrane skeleton.
Fig. 5.
Treatment of membranes with phalloidin increases the
sedimentability of p205, as well as actin. (A) Neutrophil plasma
membranes treated without () or with (+) phalloidin were solubilized in TEB and fractionated on 20-55% sucrose gradients.
The initial membrane extract (Load) and gradient fractions
(lanes 1-17) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards (9S, 19S, and 30S) are indicated. (B) Phalloidin-
induced shift in the sedimentability of p205. Average distribution
of p205 in gradient fractions, expressed as a percent of the total
F-actin binding at 205 kD on F-actin blot overlays (n = 3). Similar results were observed when blot strips were re-probed with
anti-pepA antibody, indicating that a single 205-kD F-actin-binding polypeptide is present in the 13S, 26S, and 30S complexes.
[View Larger Versions of these Images (71 + 23K GIF file)]
Fig. 6.
p205 cosediments
with endogenous, phalloidin-stabilized actin (A), and actin
coimmunoprecipitates with
p205 (B). (A) Neutrophil plasma membranes (lane 1)
were treated with either fluorescein-phalloidin (lanes 2 and 3) or unlabeled phalloidin (lane 4), solubilized in
TEB, and then incubated
with nonspecific IgG (lane
2) or antifluorescein IgG
(lanes 3 and 4) bound to protein A-agarose. p205 was visualized by staining with affinity-purified pepA IgG
and by F-actin blot overlays (not shown). (B) IgG and actin in
immunoprecipitates generated with either nonspecific rabbit IgG
(lane 1) or affinity-purified, anti-pepA antibody (lane 2) after
three washes with RIPA buffer. The relative amounts of actin
cited in the text were normalized by reference to the amounts of
IgG visualized by labeling with radiolabeled secondary IgG.
[View Larger Versions of these Images (15 + 54K GIF file)]
-actin observed on F-actin blot
overlays (Figs. 3 and 4; [66]) also occurs between p205 and
actin on the neutrophil plasma membrane.
Fig. 7.
p205 is present in many
cell types but is not the only ~205-kD F-actin-binding protein. (A) Nitrocellulose blots of HeLa cells (lane
1), MDBK cells (lane 2), and bovine neutrophils (lane 3) stained
with affinity-purified anti-pepA antibodies. Only the neutrophils contain a cross-reactive protein at ~90
kD (arrowhead). This 90-kD protein cofractionates with pooled neutrophil granules (66), specialized
vesicles involved in host defense,
and is distinct from the ~90-kD cytosolic actin-binding protein in Fig. 1
C, lane 3. (B) Blots of HeLa (lane
1), SHSY5Y neuroblastoma (lane
2), 3T3 (lane 3), MDBK (lane 4),
NRK (lane 5), LLC-PK1 (lane 6), and COS-7 cells (lane 7) were stained in parallel with antibodies to pepA and F-actin. A higher molecular mass protein that binds F-actin, but not anti-pepA, is observed in lanes 3 and 5 (*).
[View Larger Versions of these Images (34 + 30K GIF file)]
Fig. 8.
Distribution of p205
(A and C) and E-cadherin (B
and D) in MDBK cells grown
at low cell density. Cells were
double labeled with affinity-purified, rabbit anti-pepA
IgG and with monoclonal anticadherin antibodies, as
described in Materials and
Methods. Regions of p205
and cadherin colocalization
at the plasma membrane (white arrows), and regions
of anti-pepA staining alone
(hollow arrows) are indicated. Bar, 10 µm.
[View Larger Version of this Image (138K GIF file)]
Fig. 9.
Distribution of p205
(A and C) and E-cadherin
(B and D) in MDBK cells
grown to high cell density.
Cells were double labeled with affinity-purified, rabbit
anti-pepA IgG and with
monoclonal anticadherin antibodies. Regions of p205 and
cadherin colocalization at the
plasma membrane (white
arrows) and regions of anti-pepA staining alone (hollow
arrows) are indicated. Bar,
10 µm.
[View Larger Version of this Image (119K GIF file)]
Fig. 10.
Colocalization of
p205 (A, C, and E) with
F-actin (B) and cadherin (D
and F) in ringlike structures
and cytoplasmic aggregates 10 min after EGTA treatment to disrupt cell adhesions. After 30 min (E and
F), most of the cadherin is
dissociated from p205, which
is diffusely distributed in cytoplasmic puncta. Cells were double labeled with affinity-purified pepA antibodies and
either fluorescein-phalloidin
or anticadherin antibodies.
Bar, 10 µm.
[View Larger Version of this Image (117K GIF file)]
-RACE reaction with oligo-dT primed cDNA
to obtain four 3-kb clones corresponding to the 3
end of
the p205 cDNA (Fig. 11 A, right arrows). Nondegenerate
primers designed from these sequences were used in a 5
-RACE reaction to generate five clones containing 5
sequences. A second round of 5
-RACE with new primers
was used to ensure that the 5
end of the p205 cDNA had
been identified. A total of six clones were obtained that all
begin at the same nucleotide (Fig. 11 A, left arrows). As a check for cDNA production artifacts, two gene-specific
primers were used to generate 5,198-bp clones that contained most of the p205 coding sequence (Fig. 11 A, no arrows). Products of this size were obtained from two different commercial cDNA libraries (data not shown).
end of the sequence (Fig.
11 A, probe 1) and a probe from the 3
half of the sequence (Fig. 11 A, probe 2) recognized a ~7.2-kb message
in MDBK cells (Fig. 11 B, lanes 1 and 2). This message size
is consistent with the ~6.5-kb cDNA obtained by PCR,
given the presence of a poly(A)+-tail of unknown size. A
third probe near the 5
end of the sequence showed a single ~7.2-kb band on Northern blots (data not shown). The
source of the ~1.8-kb band visualized with probe 2 (Fig.
11 B, lane 2) is not known, but this band comigrates with
residual 18 S rRNA.
Fig. 12.
Predicted sequence and domain structure of bovine
p205 (supervillin). (A) The amino acid sequence starting with the
first methionine of the translated ORF includes all eight peptides
obtained from purified p205 (double underline). The NH2-terminal half contains four putative nuclear targeting signals (gray boxes)
the longest of which resembles the nucleoplasmin targeting signal
(single underline). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site (black). Amino
acid positions are indicated by numbers in the left margin. These
sequence data are available from GenBank/EMBL/DDBJ under
accession number AF025996. (B) Schematic representation of the
domain structure showing the NH2-terminal region with putative
nuclear targeting regions (gray boxes) juxtaposed with potential
protein kinase A phosphorylation sites (asterisks). The COOH-terminal domain (cross-hatched) shows extensive similarity to villin and gelsolin, with three regions of especially high homology
that correspond to potential F-actin-binding sites (black boxes).
The potential tyrosine phosphorylation site is indicated (·).
[View Larger Versions of these Images (89 + 15K GIF file)]
-helical secondary structure. One 17-residue motif and three short
clusters of positively charged amino acids (Fig. 12 A, gray boxes) fit the consensus sequences for, respectively, nucleoplasmin- and SV40-like nuclear targeting signals (16, 68).
Because the nucleoplasmin targeting signal is found in
56% of all nuclear proteins but only ~4% of non-nuclear
proteins (25) and because nuclear localization signals are
additive (37), the PSORT protein localization prediction
program (61) indicates a 96.4% probability that p205 partitions into the nucleus. Hence, an analysis of the p205 primary sequence supports the immunocytological observation of anti-pepA signal within the nuclei of subconfluent
cells (Fig. 8, A and C).
Fig. 13.
Detailed comparison of the COOH terminus of supervillin (p205) with
the full-length protein sequences of mouse gelsolin,
mouse villin, chicken villin,
Dictyostelium protovillin, and
a predicted C. elegans protein (These sequence data
are available under GenBank/EMBL/DDBJ accession numbers P13020,
M98454, P02640, P36418, and
U88311, respectively). (A)
Mouse villin, mouse gelsolin, and the COOH terminus of
supervillin were aligned with
PileUp, and the percentage
of identical residues in every
consecutive 30-amino acid
segment of supervillin were
plotted vs. the number of the
last residue in the segment.
The locations of the gelsolin and villin homology segments
(S1-S6) and the villin headpiece domain (HP) are
drawn to scale. (B) The regions of highest identity between supervillin and the
other proteins in this family
include portions of segments
2 and 5, which are present in
both gelsolin and villin, and
the villin headpiece domain,
which is absent from gelsolin.
Both segment 2 and the villin
headpiece contain known actin-binding motifs, indicated by asterisks above the sequence (23, 38, 48). Villin headpiece amino acids implicated in binding to F-actin and in stabilization of headpiece structure (26, 56) are designated by and
, respectively. Identical residues (black boxes) and conservative replacements (gray boxes), defined as matches scoring
0.6 on the Dayhoff matrix (22), are highlighted.
[View Larger Versions of these Images (28 + 48K GIF file)]
Discussion
-actin filaments in blot overlay assays (66). We show here that supervillin is a tightly bound
peripheral protein (Fig. 2) that can associate with the plasma
membranes of both neutrophils (Fig. 1) and MDBK cells (Figs. 8-10). We further show that supervillin can be isolated from neutrophil plasma membranes as part of a high
molecular weight complex with endogenous actin (Fig. 5)
and that the interaction with actin persists after immunoprecipitation and high stringency washing (Fig. 6). Although
many tissue culture cell lines contain both supervillin and
another F-actin-binding protein of similar size (Fig. 7 B),
all of our results to date suggest that supervillin is the only
205-kD F-actin-binding protein in bovine neutrophils. The
presence of supervillin in numerous cell lines (Fig. 7) suggests that this protein is one of a small, but growing group
of membrane skeleton proteins known to bind actin at the
peripheries of many cells (73, 88).
-actinin,
an actin-bundling protein that also appears to facilitate the
attachment of stress fibers to integrins at focal contacts (64). We hypothesize that supervillin plays an analogous
role in actin filament bundling and/or attachment to the
membrane at adherens junctions.
-catenin and the Drosophila melanogaster armadillo protein, both
of which bind cadherin, potentiate cell-cell adhesion, and
function in the Wnt-1/Wingless signal transduction pathway (39, 65). When present at high levels, both
-catenin
(6, 58) and armadillo protein (81) can functionally interact
with the LEF-1/Tcf family of transcription factors, an interaction that provides one explanation for the apparent
involvement of
-catenin in tumor progression (50, 59, 69).
Thus, supervillin may be one of a small group of candidate
proteins, which also includes the focal contact proteins zyxin and cCRP (70), that could function as relatively direct signaling molecules between the nucleus and the actin
cytoskeleton at sites of cell adhesion.
Note Added in Proof.
Received for publication 6 June 1997 and in revised form 21 August 1997.
R.K. Pope was supported by an American Cancer Society postdoctoral fellowship (No. PF-4297), and J.D. Wulfkuhle by a National Institutes of Health (NIH) postdoctoral training fellowship (No. 5T32HD07312-12). This research was supported by NIH grant GM33048 and also benefited from grants to the Worcester Foundation for Biomedical Research from the J. Aron Charitable Foundation, and from the Stork Foundation.The authors would like to thank C.P. Strassel for production of MDBK cell cDNA and for expert technical assistance. We also thank Dr. J. Leszyk for protein digestion and peptide microsequencing and Dr. J. Aghajanian for electron microscopy of neutrophil membranes and assistance with confocal microscopy. We are also grateful for the assistance of L. Ohrn and M. Martineau for preparation of media/reagents and assistance with laboratory supplies. We also thank Dr. H.G. Hills, Dr. G. Polking, S. Nelson, and W.C. Chu of the Iowa State DNA Sequencing and Synthesis Facility for primer production and automated DNA sequencing.
ERM, ezrin, radixin, and moesin; ORF, open reading frame; RACE, rapid amplification of cDNA ends; TEB, Triton X-100 extraction buffer.
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