Expression of Chicken Vinculin Complements
the Adhesion-defective Phenotype of a Mutant Mouse F9
Embryonal Carcinoma Cell
Michael Samuels,* Robert M. Ezzell,* Timothy J. Cardozo,* David R. Critchley,§ Jean-Luc Coil,* and
Eileen D. Adamson*
* La Jolla Cancer Research Foundation, La JoUa, California 92037; * Surgery Research Laboratory, Department of Anatomy and
Cellular Biology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129; and
§ Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, United Kingdom
Abstract. A mutant cell line, derived from the mouse
embryonal carcinoma cell line F9, is defective in cell-
cell adhesion (compaction) and in cell-substrate adhe-
sion. We have previously shown that neither uvomoru-
lin (E-cadherin) nor integrins are responsible for the
mutant phenotype (Calogero, A., M. Samuels, T. Dar-
land, S. A. Edwards, R. Kemler, and E. D. Adamson.
1991. Dev. Biol. 146:499-508). Several cytoskeleton
proteins were assayed and only vinculin was found to
be absent in mutant (5.51) cells. A chicken vinculin
expression vector was transfected into the 5.51 cells
together with a neomycin-resistance vector. Clones
that were adherent to the substrate were selected in
medium containing G418. Two clones, 5.51Vin3 and
Vin4, were analyzed by Nomarski differential inter-
ference contrast and laser confocal microscopy as well
as by biochemical and molecular biological techniques.
Both clones adhered well to substrates and both exhib-
ited F-actin stress fibers with vinculin localized at
stress fiber tips in focal contacts. This was in marked
contrast to 5.51 parental cells, which had no stress
fibers and no vinculin. The mutant and complemented
F9 cell lines will be useful models for examining the
complex interactions between cytoskeletal and cell ad-
ARLIER studies have established that the cytoplasmic
domains of two transmembrane glycoproteins, uvo-
morulin (Nagafuchi and Takeichi, 1988) and/31 inte-
grins (Hayashl et al., 1990; Marcantonio et al., 1990; Reszka
et al., 1992) are essential for transmembrane linkage to the
cytoskeleton and for cell adhesion. The former glycoprotein
links cells together in tight aggregates and epithelial sheets,
the latter attaches cells to the extraceUular matrix. Neither
activity can be maintained without a set of interlinked cyto-
plasmic accessory proteins that are actively disassembled
and reassembled onto the F-actin cytoskeleton. Uvomoru-
lin, catenins or, B, 7 (Kemler and Ozawa, 1989) and plako-
globin (Cowin et al., 1986) are components unique to the
zonulae adherens junctions between cells. Others such as
a-actinin, tenuin, vinculin, zyxin, are common to both zon-
ulae adherens and cell-substrate plaques. Yet other proteins
are unique to cell-substrate locations; certain integrins,
fimbrin, paxillin, talin, and tensin for example (Geiger and
We have previously described an F9 mutant cell line that
is defective in both cell-cell and cell-matrix adhesion (Grover
Address correspondence to Dr. Adamson at La Jolla Cancer Research Foun-
dation, 10901 North Torrey Pines Road, La Jolla, CA 92037.
M. Sarnuels and Dr. Ezzell contributed equally to this work.
et al., 1987). The F9 att-5.51 line (called 5.51 hereafter) was
produced by ethylmethanesulfonate mutagenesis, a process
that can lead to a single base change in the genome (Ingle
and Drinkwater, 1989) or to a wide range of mutational types
(Wood and Moses, 1989). Unlike wild-type F9 cells, 5.51
cells cannot differentiate into an epithelial layer after treat-
ment of cell aggregates with retinoic acid (5 nM RA), prob-
ably because they cannot aggregate into tight cell masses. In-
stead, they grow in suspension even in tissue culture dishes
as floating, loose and uncompacted aggregates. RA treat-
ment of 5.51 cells results in only parietal-type endoderm
differentiation; epithelial layers of polarized visceral endo-
derm cells are not seen (Grover et al., 1987).
We have attempted to define the mechanism underlying the
inability of the mutant cell line to undergo compaction by
studying the expression of uvomorulin in 5.51 cells.
Uvomorulin was expressed at 30-50% wild-type F9 levels
in mutant 5.51 cells while the mRNA was barely detectable
because it was highly unstable (Adamson et al., 1990).
When uvomorulin was overexpressed in 5.51 cells, the cells
aggregated better but did not form an epithelial layer of
visceral endoderm cells and did not adhere to a substrate
(Calogero et al., 1991). In short, the replacement of uvo-
morulin did not complement the cell-cell mutant phenotype.
We examine here the expression of several cytoskeletal
© The Rockefeller University Press, 0021-9525/93/05/909/13 $2.00
The Journal of Cell Biology, Volume 121, Number 4, May 1993 909-921 909
proteins in 5.51 cells and find that, whereas some minor
differences can be found in the expression of several proteins
in mutant cells, vinculin is completely missing. We present
evidence that this is the fundamental cause of the aberrant
cell-cell and cell-matrix interactions displayed by 5.51 cells.
Vinculin (Mr 117 kD) localizes at focal contacts (Geiger,
1979; Burridge and Feramisco, 1980; Geiger et al., 1980)
and intercellular adherens junctions (Geiger et al., 1981;
Geiger and Ginsberg, 1991). Therefore, any cell that makes
contact with other cells or with substratum, expresses vincu-
lin. By inference, this includes all anchorage-dependent cul-
tured cells (Otto, 1990). Vinculin is evolutionarily highly
conserved among nematodes (Barstead and Waterston,
1989), birds (Price et al., 1989), and man (Weller et al.,
1990). The structural and functional domains of vinculin
have been summarized recently (Westmeyer et al., 1990;
Critchley et al., 1991). There is a single gene, but a differ-
ence in splicing gives rise to the 150-kD metavinculin vari-
ant in muscle cells (Koteliansky et al., 1992). We describe
here the results of the transfection of an expression vector
coding for chicken vinculin into the mouse 5.51 cell line and
the isolation of several clones that express different levels of
vinculin protein. The conclusions are: (a) that the mutant
phenotype is largely corrected, because both compaction
and substrate adhesion are significantly improved; (b) actin
stress fibers lacking in the mutant line are restored in the
transfected lines; (c) cell surface topography and motility are
restored in clones that express moderate amounts of exoge-
nous vinculin protein but not in those that express very high
levels. Our results suggest that subtle and complex regula-
tory interactions with other components in the cytoskeletal
network are necessary for normal function.
Materials and Methods
Wild-type F9 BIM cells were cultured as described earlier (Grover et al.,
1983). The mutant F9 cell line was cultured in suspension in the same
medium (Grover et ai., 1987). Transfected cell lines are designated as
5.51UM1 and 5.51Vin for clones expressing uvomorulin or vinculin, respec-
tively. PI9 embryonal carcinoma cells are cultured similarly to F9 cells but
in noncoated dishes (McBurney et al., 1982).
Antibodies Used in Immunoblotting
Rabbit antibodies to uvomorulin, ~fodrin, and ankyrin were kindly
provided by R. Kemler (Max Planck Institut, Freiburg, Germany), E.
Repnsky (Roswell Park Cancer Institute, Buffalo, NY) (Black et al., 1988),
A. Dutton (University of California, San Diego, CA) and W. J. Nelson
(Stanford University, Stanford, CA) (Nelson and Veshnock, 1986), respec-
tively. Rabbit anti-taiin antisera were from K. Burridge (University of North
Carolina, Chapel Hill, NC) and M. C. Beckerle (University of Utah, Salt
Lake City, UT). Anti-o~-actinin was from A. Dutton and S. J. Singer
(University of California, San Diego, CA). Three anti-vinculin antibodies
were used: for immunoprecipitation, a polyclonai rabbit antibody to por-
cine smooth muscle vinculin from A. Dutton; for immunoblotting, mouse
mAb V4505 (anti-chicken) and V9131 (anti-human) vinculin from Sigma
Chemical Co. (St. Louis, MO). Anti-filamin has been described previously
(Ezzell et al., 1988).
Immunofluorescence and Confocal Microscopy
F9 cells were cultured for 24 h (48 h for 5.51 mutant and 5.51Vin-clones
3 and 4) on polylysine-coated coverslips. Coverslips with attached cells
were fixed for 8 rain in 4 % paraformaldehyde in PBS and permeabilized
for 2 rain in 0.2% Triton X-100 in PBS. After rinsing, the coverslips were
incubated for I h in a blocking solution containin$ 3 % BSA (Sigma Chemi-
cal Co.) and 1% normal donkey serum (Jackson Immunoresearch Labs,
Inc., West Grove, PA). grdd-type F9 ceils were stained with mouse anti-
human smooth muscle vinculin (diluted 1:50, Chemicon International, Inc.,
Temecula, CA, catalog number MABI624), and 5.51Vin-clones 3 and 4
were stained with mouse anti-chicken gizT~rd smooth muscle vinculin
(diluted 1:10, Sigma Chemical Co., catalog number VIN-I1-5) for I h. Both
antibodies were diluted in blocking solution. The coverslips were washed
(four changes, 15 rain each), and incubated for 1 h in fluorescein (DTAF)-
conjugated F(ab')2 fragment of donkey anti-mouse IgG (diluted 1:200 in
blocking solution, Jackson Immunoresearch Labs). The coverslips were
washed overnight and then stained with rhodamino-phalloidin (diluted
1:100, Molecular Probes, Inc., Eugene, OR) to visualize F-actin. After
washing for 15 min, the coverslips were mounted in a drop of 1 mg/ml
p-phenylenediamine (Sigma Chemical Co.) to reduce photobleaching in
90% glycerol/PBS, pH 8.5. All specimens were examined in a confocai mi-
croscope (BioRad MRC 600 [BioRad Microsciences, Cambridge, MA]) at-
tached to a Zeiss Axiovert 35 with a I00x Plan-Neofluar objective. Confo-
cal images were collected and digitally stored using photon-counting Biorad
71me-lapse Iruleo Microscopy
For time-lapse video microscopy, F9 cells were cultured on 25 mm circular
coverslips coated with gelatin (for wild-type and 5.51 V'm clones 3 and 4)
or polylysine (for 5.51 cells). The cells were maintained in a Leiden cover-
slip chamber (Medical Systems Corporation, Greenvale, NY) and examined
in a Zeiss Axiovert 10 inverted microscope using a 100x Plan-Neoflnar ob-
jective and Nomarski differential interference contrast (DIC) I optics. A
Zeiss environmental microscope chamber was used to maintain the cell cul-
tures at 37°C and 5% CO2. Video images obtained with a Hamamatsu
C2400 Newvicon camera were recorded onto a Sony LVR 5000 laserdisk
recorder at 15-s intervals.
(a) For probing Northern blots, a 3-kb human vinculin cDNA, HV6, was
used 0Veller et al., 1990) after labeling by the random oligonucleotide
method (Feinberg and Vogelstein, 1984). (b) An expression vector encod-
ing full-length chicken cDNA was constructed from cVinl and cVin5 (Ben-
dori et al., 1989). The promoter was the Simian Vh-'us-40 late promoter de-
rived from pSVL. (c) A plasmid containing the 1.2-kb EcoRl fragment of
the mouse vinculin cDNA (Ben-Ze'ev et al., 1990) in Bluescript KS
(Stratagene Corp., San Diego, CA) was linearized with BamH1 and cRNA
of 220 bp corresponding to nts 1435-1649 of vinculin (Price ct ai., 1989)
was synthesized from the T3 promoter according to the method of Melton
et al. (1987). This was used in ribonuclease protection assays together with
a probe that detects ribosomal protein mRNA, L32, used as a control for
equal loading of RNAs, as described by Darland et al. (1991). The protected
fragment of mouse vinculin RNA was 205 bp.
Mutant F9 cells grow as loose aggregates in suspension. In contrast, we
might expect to find adherent cells if vinculin expression rescued the inabil-
ity to adhere. Dishes (10 cm) containing 1-2 × 106 suspended 5.51 ceils
were treated with 18 t~g plasmid DNA as a mixture of 15/~g pSVL-cVIN
(expressing chicken vinculin cDNA) and 3/tg pSV2neo (for G418 resis-
tance). The method described by Graham et ai. (1974) was followed and
colonies resistant to 0.4 mg/ml G418 were selected in tissue culture dishes
that were coated with gelatin. Clones that could adhere to the substratum
were selected as well as less-adherent ceil clones. Two clones, 5.51Vin3 and
4, are described in the most detail.
RNA Extraction and Analysis
RNA was extracted from cells as described by Chirgwin et al. (1974).
Northern analysis was performed on formnidehyde-denaturing gels (Mania-
tis et al., 1982) with 20 ~g of total RNA, after transfer to Nytran mem-
branes (Schleicher and Schuell, Inc., Keene, Nil). RNase protection assays
were as described above with 50 ~,g total RNA.
1. Abbreviations used in this paper: DIC, differential interference contrast.
The Journal of Cell Biology, Volume 121, 1993 910
Figure I. Morphology of wild-type F9 (,4) and 5.51 (B) cells examined with Nomarski differential interference contrast optics. The wild-type
cell is well-spread and has filopodia (arrows) and lameUipodia (arrowheads). In contrast. 5.51 cells are rounded and have only filopodia
(arrows). 5.51 cells also grow as loose strings or clusters of non-adherent cells. Bar, 10 #m for A, and 6 #m for B.
Radiolabeling and Immunoprecipitation
Radiolabeling and immunoprecipitation were performed as in Darland et
al. (1991). In brief, cells were labeled for 1 or 2 h in methionine-free me-
dium containing 200 t~Ci/ml [3SS]methionine and cysteine (ICN Im-
munoBiolngicals, Lisle, IL, TranSlabel >1,000 Ci/mrnol). Cells were lysed
in RIPA buffer (1% deoxycholate, 1% Nonidet 40, 0.1% SDS, 25 mM Tris
pH 7.5, 0.15 M NaC1, with protease inhibitors: 1 mM PMSE 2/~g/ml
leupeptin and 2 ~g/mi aprotinin) and aliquots containing equal amounts of
radioactive proteins were subjected to immunoprecipitation. Fixed Staphy-
lococcus aureus (Boehringer-Mannheim Corp., Indianapolis, IN) precipi-
tated complexes were analyzed by SDS-PAGE on 7 or 5% polyacrylamide
gels. The radioactive proteins were detected by ttuorography.
Known numbers of cells (105 to 106 cells per lane) were lysed and heated
in sample buffer (Laemmli, 1970). SDS-PAGE analysis was followed by
transfer to Immobilon membranes (Miilipore Corporation, Bedford, MA).
Rainbow Markers (Amersham Corporation, Arlington Heights, IL) were
used to indicate the migration of proteins. Incubation of membranes in
1:200 to 1:500 dilutions of specific antibodies followed by peroxidase-
labeled second antibodies at 1:500 was done according to the manufacturer's
instructions (NOVEX, Encinitas, CA). Peroxidase was detected by staining
using hydrogen peroxide and diaminobenzidine. Immunoblots in Fig. 9
were digitized by scanning with a Hewlett Packard ScanJet Plus attached
to an Apple Macintosh IIci computer. A software program called Adobe
Photoshop 2.0 (Adobe Systems, Mountain View, CA) was used to increase
the contrast of the image. The contrast-enhanced digitized images were then
photographed onto 35 mm Kodak TMAX 100 black and white negative film
with a Lasergraphics LFR Plus film recorder.
Mutant F9 (5.51) Cells Have Altered Morphology and
While wild-type F9 cells adhere and spread on gelatin-
coated plastic, 5.51 cells float in the medium as loose ag-
gregates (Grover et ai., 1987). Wild-type F9 cells become
flattened as they spread but remain in colonies on the sub-
stratum. Time-lapse video and laser confocal microscopy
were used to examine the motile behavior and distribution
of actin filaments (F-actin) and vinculin in wild-type and
5.51 cells. Wild-type F9 cells adhere to surfaces by first
sending out long fingerlike filopodia. This is soon followed
by protrusions of lamellipodia (Fig. 1 A). 5.51 cells have
filopodia but no lamellipodia (Fig. I B).
In wild-type ceils, F-actin is organized into stress fibers
that terminate at focal contacts containing vinculin (Fig. 2,
C and D). 5.51 cells do not have stress fibers and do not stain
with vinculin antibodies (Fig. 2, E and F). Instead, F-actin
is concentrated under the plasma membrane and in filopo-
dia. Wild-type F9 cells observed 15 min after seeding have
already begun to attach to a gelatin-coated substrate and
while still rounded in shape are nevertheless able to organize
vinculin into short nascent focal adhesion points (Fig. 2 A)
even though actin stress fibers are not apparent (Fig. 2 B).
These findings suggest that vinculin is required for attach-
ment and spreading as well as for the formation of stress
Vinculin Levels in F9 Cells: Biochemical and
Molecular Biological Evidence
Because vinculin staining is not evident in the mutant cells
shown in Fig. 2 E, we extracted equal numbers of wild-type
F9 and 5.51 cells to analyze their vinculin content by immu-
noblotting. The results show that wild-type IX) and P19 EC
cells contain very similar steady-state levels of vinculin (Fig.
3, lanes 1, 2, and 5) whereas 5.51 and its uvomorulin-
expressing clone 1 (Calogero et al., 1991) have no visible
Because vinculin could be unstable in 5.51 cells, it might
be detectable with greater sensitivity if measured by meta-
bolic labeling of newly synthesized protein. Equal amounts
of radioactively-labeled lysate were analyzed with a poly-
clonal anti-vinculin to compare immunoprecipitates from
wild-type and mutant cells (Fig. 4). Only wild-type F9 cells
gave the expected ll7-kD band. There was no such labeled
polypeptide in 5.51 cells or with nonimmune serum. Fig. 4
A also shows the electrophoretic migration of uvomorulin in
a similar aliquot of F9 lysate. In this case the F9 cells had
been treated with tunicamycin and both glycosylated and un-
derglycosylated versions of uvomorulin are seen as a doublet
Samuels et al. Vinculin Expression in Mutant F9 Cells
Figure 2. Colocalization of vinculin (A, C, E) and F-actin (B, D, F) in wild-type F9 (A-D) and 5.51 (E and F). Cells were strained with
a mouse monoclonal antibody to human vinculin, and then stained with rhodamine-phalloidin (see Materials and Methods). Confocal im-
ages are of the ventral surface in contact with the substrate (for wild-type cells) and of the midsection of the cell (for 5.51). Wild-type
F9 cells were observed 15 min (A and B) and 2 h (C and D) after seeding. After 2 h, F9 cells are fully spread and stress fibers are seen
to terminate in vinculin-containing focal contacts (arrows). Even after 15 rain, vinculin patches colocalize with F-actin at the cell periphery
and in extending filopodia (arrows). E and F show a 5.51 cell fixed and stained 2 h after addition to the substrate. In this nonadberent
cell type F-actin is concentrated at the cell periphery and in filopodia (arrows). Aggregates of F-actin inside 5.51 cells were frequently
observed. Vinculin staining with antibodies to human (E) or chicken (not shown) vinculin was not detected in 5.51 cells. The round shape
and F-actin organization in 5.51 cells resemble the adhering wild-type F9 cell (compare B with F). Bar, 5 #m for A, B, E, and F,, and
10 #m for C and D.
The Journal of Cell Biology. Volume 121, 1993 912
Figure 3. Immunoblotting to detect vinculin in EC ceils. Equal
numbers of ceils were extracted as described in the Materials and
Methods section. Vinculin was assayed by a two-layer method em-
ploying rabbit polyclonal anti-vinculin and peroxidase labeled sec-
ond antibodies. The stained bands in the left-hand panel indicate
that equal levels of vinculin were detected in monolayer (mono) and
in aggregated (aggs) F9 EC cells and PI9 cells but none were ob-
served in mutant 5.51 or its uvomorulin transfected clone, 5.51
UM1 (denoted C1). PI was nonlmmune serum negative control.
at about 120 kD. Vinculin moves at a slightly different migra-
tion rate between the uvomorulin doublet. To maximize the
ability to detect vinculin in 5.51 cells, threefold more radio-
labeled 5.51 ceils compared to F9 wild-type were analyzed
in Fig. 4 B. Even here, vinculin was not detected in 5.51 ly-
Figure 5. Ribonuclease protection analysis of the relative levels of
vinculin transcripts in F9 cells stimulated with 20% serum and cy-
cloheximide (10/xg/ml) for 1 h; 5.51 similarly stimulated; F9 cells
after RA-stimulated differentiation for 4 d; unstimulated F9 mono-
layers, and 3T3 ceils, respectively. L32 is the signal given by ri-
bosomal protein transcripts and is used to control for RNA loading
on the gel. The relative levels of vinculin mRNA in each sample
are shown below. M, marker lane.
Figure 4. Immunoprecipitation of methionine-labeled F9 and 5.51
mutant cells. (A) Equal amounts of radioactivity were compared as
described in the Materials and Methods section. To demonstrate the
presence of uvomorulin and to distinguish between UM and
vinculin, lane 1 shows uvomorulin protein synthesized in F9 cells
treated with tunieamycin to inhibit the glycosylation of this protein.
The triplet of proteins represents UM precursor (125 kD), mature
UM (120 kD), and the under-glycosylated form. The catenins (Cat)
coprecipitate with UM as indicated (Ozawa et al., 1989). Vinculin
also migrates at ,'~120 kD and is seen only in lane 2, an extract of
F9 ceils. There is no detectable vinculin in 5.51 cells (lane 4). (B)
Here the amount of radioactive 5.51 lysate was threefold that of F9
cells but no immunoprecipitated vinculin could be seen. Lanes
marked PI are controls with a nonimmune rabbit serum. The mi-
gration position of marker proteins is indicated in kD.
sates although a number of other nonspecific proteins were
seen in anti-vinculin as well as nonimmune lanes. We con-
clude that vinculin protein is absent from the mutant F9
The expression of vinculin mRNA was detected by North-
ern blotting as a transcript of 6-7 kb in RNA extracts of F9
and 3T3 mouse fibroblasts, but no signal above background
levels could be seen in 5.51 cells (data not shown). To detect
and quantify vinculin mRNA levels with greater sensitivity,
a ribonuclease protection assay was performed using a
mouse cRNA that protects 205 bp of endogenous vinculin
mRNA. Fig. 5 shows that the levels of vinculin mRNA in F9
cells are lower than in fibroblasts. Note that stimulating ceils
with 20% serum in the presence of cycloheximide (Ben-
Ze'ev et al., 1990) for 60 min, or retinoic acid for several
days, results in changes in the levels of vinculin mRNA. Un-
like Northern blotting, a low level (approximately one-sixth
of that found in F9) of vinculin mRNA was detected in mu-
tant 5.51 cells using the ribonuclease protection assay. These
levels were estimated by reference to the level of L32
ribosomal protein mRNA which was assumed to be in-
Transfection of Vinculin into 5.51 Cells Restores
Adhesion, Morphology and Actin Organization
Co-transfection of an expression vector encoding full-length
chicken vinculin, together with one that conveys neomycin
Samuels et al. Vinculin Expression in Mutant F9 Cells
l~gure 6. Phase contrast mi-
crographs compare the gen-
eral appearance of a popula-
tion of F9 wild-type (A) with
transfeeted cells, 5.51Vin4 (B)
and 5.51Vin3 (C) grown as
monolayers. 5.51Vin3 cells
attach and spread compara-
tively poorly 2 d after seeding
(C), but in suspension culture
aggregate and compact well
(D). Bar in A, 50/am and ap-
plies to all panels.
resistance, allows the selection of stable cell lines carrying
both genes. Selection in G418 gave a mixture of phenotypes,
but we selected cells that either aggregated better or adhered
better to the gelatin-coated substrate. The two properties ap-
peared in parallel, thus supporting the idea that vinculin may
be the major defect in the mutant cells. Two main clones,
5.51Vin3 and 5.51Vin4, were selected and subcloned. Clone
3 was less adhesive than clone 4 in both rate of adhesion and
flattened appearance in a monolayer (Fig. 6 C) but formed
moderately tight aggregates in suspension cultures (Fig. 6
Figure 7. Morphology of 5.51Vin3 (.4) and 5.51Vin4 (B) cells examined with Nomarski difference interference contrast optics. Both clones
are attached to the substrate and have filopodia (arrows) and larnellipodia (arrowheads). ~n3 is not as well spread as Vin4 and wild-type
cells (see Fig. 1). Bar, 10/zm for A and B.
The Journal of Cell Biology, Volume 121, 1993 914
Figure 8. Colocalization of vinculin (A, C) and F-actin (B, D) in 5.51Vin3 (A, B) and 5.51Vin4 (C, D) cells. Cells were stained with
a mouse mAb to chicken vinculin, and then stained with rhodamine-phailoidin. Confocai images are of the ventral plasma membrane
in contact with the substrate. Both clone 3 and 4 cells have aetin stress fibers that terminate in chicken vinculin-containing focal contacts
(arrows). The abundance of stress fibers and vinculin staining in the 5.51Vin3 cell correlates with the increased expression of chicken
vinculin (see Results). Bar, 10/~m for A-D.
D). Clone 4 monolayers adhered well to plastic and spread
sufficiently to resemble wild-type F9 cells (Fig. 6, compare
A with B).
5.51Vin clones 3 and 4 cells were observed with Nomarski
Die optics in order to observe spreading and membrane mo-
bility (Fig. 7). 5.51Vin3 cells did not spread as well as
5.51Vin4 and wild-type cells (see Fig. 1 A), but contrasted
clearly with the rounded shape and absence of lameUipodia
in the 5.51 parental line (compare Fig. 1 B with Fig. 7).
We demonstrated next that the transfected cells not only
expressed vinculin but also that they were able to place vin-
culin at the ends of actin stress fibers which were now re-
stored. Fig. 8 shows that our two transfected 5.51 clones
stained for chicken vinculin in a manner closely resembling
that seen in wild-type F9 cells (see Fig. 2 C and D). Clone
Vin3 had more actin stress fibers than clone 5.51Vin4, and
more vinculin associated with the ends of the stress fibers
(compare Fig. 8, B with D). The vinculin in 5.51Vin4 local-
ized in focal contacts and clustered in small patches dis-
tributed over much of the cell surface. As shown in Fig. 8,
a mouse mAb to chicken vinculin stained both 5.51Vin 3 and
4 cells. The anti-human vinculin antibody used to stain the
wild-type mouse F9 cells (see Fig. 2) did not stain these cells
(data not shown) proving that the vinculin associated with fo-
cal contacts in the transfected 5.51 cells is chicken vinculin
and not endogenous mouse vinculin. These results demon-
strate that chicken vinculin is being expressed in the trans-
fected clones and suggest that this in turn is eifecting the as-
sembly of F-actin and the restoration of a normal F9
Biochemical Evidence for the Expression of Chicken
Vinculin in Transfected 5.51 Clones
Immunoblotting with the polyclonal antibody to vinculin re-
vealed that the two vinculin-transfected clones do indeed ex-
press higher levels of vinculin than even wild-type F9 cells
(Fig. 9, left). We used a 10-fold range of cell lysates to com-
pare the levels. At this point, we also tested a mixture of
mouse mAb to human and chicken vinculin in order to try
to increase the sensitivity of detection in cells that contained
both antigens. It is clear that the differential recognition of
Samuels et al. Vinculin Expression in Mutant F9 Ceils
Figure 9. Immunoblotting to compare the levels of expression of vinculin in transfected clones. Lysates of wild-type (a), 5.51 (b), 5.51UMI
(c), 5.51Vin3 (d), 5.51Vin4 (e) cells were analyzed as described in the Materials and Methods section. Vineulin was detected on blots
with the polyclonal antibody used in immunopreeipitation studies (left) and with a mixture of mouse mAb to human and chicken vinculin
(r/ght). The second layer peroxidase-labeled antibodies were detected with diaminobenzidine and hydrogen peroxide. The number of cells
assayed is shown above each lane: Ix ffi lO s, 5x ffi 5 × lO s, 10x = 106 cells.
antigens by antibodies in different techniques can give rise
to different signal strength (Fig. 9, righO. We may be un-
derestimating the expression of vinculin in wild-type cells
(compare lanes a in Fig. 9), but even with this increased sen-
sitivity, there is no vinculin protein in either the 5.51 cells
or the uvomorulin-transfected clones (Fig. 9, b and c). We
have not assayed the floating transfected cells for vinculin
Because the rabbit polyclonal antibody recognizes both
mouse and chicken vinculin albeit with different sensitivi-
ties, we used it to examine and compare vinculin biosynthe-
sis in metabolically-labeled ceils. Fig. I0 (upper left) con-
firmed that vinculin synthesis was not detectable in 5.51 cells
in contrast to F9 cells. 5.5 IVin3 synthesized chicken vinculin
at extremely high rates while in 5.51Vin4 cells the rate was
similar to F9 wild-type cells. Because all the vinculin-
transfected clones gave more compact aggregates in suspen-
sion cultures, we were interested in seeing if uvomorulin
synthesis might have been increased by the expression of ex-
tra vinculin. We routinely observed unchanged levels of
uvomorulin synthesis in vinculin-transfected and parental
5.51 cells: 30-50% of the level in wild-type F9 ceils (Fig.
10, upper right; see also Adamson et al., 1990). We have
never observed the induction of vinculin synthesis by the ex-
pression of exogenous uvomorulin in 5.51 ceils.
These and other clones were assayed for their steady-state
levels of mouse vinculin mRNA by ribonuclease protection
assay. Using the mouse cRNA probe (this probe could not
detect chicken transcripts), we could detect little difference
in the levels of transcripts between 5.51 and any of the trans-
fected 5.51 clones (data not shown). Therefore, the expres-
sion of an exogenous chicken vinculin gene did not affect the
low levels of endogenous mouse vineulin mRNA in 5.51
Other Cytoskeletal Protein Levels in F9 Cells
Because vinculin is known to interact with talin and with
~-actinin, these and several other cytoskeletal proteins were
analyzed in immunoprecipitation assays and in immunoblot-
ring. We made the assumption that the solubility of cytoskel-
etal proteins of each cell type remained the same and hence
that our extraction procedure was equally effective in F9 and
mutant cells. Little difference was observed in the biosyn-
thetic rates (Fig. 10, bottom) of the cytoskeletal proteins fo-
drin (cx-spectrin), ankyrin (not shown) and filamin in 5.51,
transfected 5.51 or wild-type cells. The rates of synthesis of
filamin and fodrin were somewhat variable, but the differ-
ences between F9 mutant and transfected cell lines were not
The steady-state levels of cytoskeletal proteins were also
compared by immunoblotting. Fig. 11 shows that ~-actinin
was quite similar in all clones while filamin and fodrin
steady-state levels were somewhat variable. Similar results
were obtained for talin and ankyrin (not shown). However,
there seemed to be a much lower level of filamin in 5.51 cells,
while transfected clones derived from 5.51 all expressed
higher levels of filarnin. We think that it is unlikely that the
excess expression of either uvomorulin or vinculin could
have caused this increase and so again we conclude that
clonal differences account for this result. In summary, there
are no large consistent differences in the expression of
cytoskeletal proteins among the five cell lines examined.
The Mutant Phenotype Is Complemented by the
Expression of Vinculin
The defect in adhesion and actin organization in 5.51 cells
The Journal of Cell Biology, Volume 121, 1993 916
Figure 10. Biosynthesis ofvin-
culin in trandect~ clones. Met-
abolically-labeled cells were
examined by immunoprecipi-
tation using the rabbit poly-
clonal antibody that recognizes
both mouse and chicken vin-
culin. Analyses of the expres-
sion of vinculin (upper le~)
were made before the immu-
noprecipitation of uvomorulin
from the same lysates (upper
right). Fodrin (lower left) and
filamin (lower right) antibod-
ies were applied sequentially
to an identical set of samples
a through e, described in Fig.
9. All samples except c con-
tained equal amounts of total
radioactive protein; c contained
half the amount. The position
of the markers is shown as
kD on the left. PI is nonim-
mime serum. See Materials
and Methods for details of
scanning and imaging.
is corrected by the expression of the transfected chicken vin-
culin gene. This is evidenced by the nearly normal rate and
extent of substrate adhesion in two clones expressing chicken
vinculin, especially in 5.51Vin4. In addition, cell-cell adhe-
sion was improved. The introduction of moderate levels of
vinculin into 5.51 cells (as in 5.51Vin4) allowed the mutant
cells to adhere to each other and to the dish surface. We con-
clude that the major defect in the mutant cells is the loss of
vinculin protein expression. A similar approach has been
used by Barstead and Waterson (1989) to show that vinculin
is essential for muscle development and function. They
identified a population of nematodes that are paralyzed, fail
to organize normal muscle structure, and have reduced levels
of vinculin. This lethal mutation was corrected by injection
of a cloned wild-type copy of the vinculin gene. On the other
hand, microinjection of a mAb to chicken vinculin disrupted
stress fibers and led to loss of focal contacts in chicken
fibroblasts (Westmeyer et al., 1990). These observations are
consistent with the hypothesis that vinculin is one of a num-
ber of interacting proteins which link F-actin to the cytoplas-
mic face of transmembrane glyeoproteins involved in cell
Samuels et al. Vinculin Expression in Mutant F9 Cells
Figure 11. Steady-state levels of other cytoskeletal proteins in wild-type, mutant and rescued ceils. (Left) Immunoblotting of c~-actinin in
the five samples (a-e) described in Fig. 9. (Middle) A similar analysis of filamin in an equal number of cells (5 × 105) in identical lysates;
(right) analysis of the steady-state levels of fodrin.
Vinculin Expressed in Mutant Cells Is Correctly
Localized and Restores the Actin Organization
We argue that the induced expression of vinculin in 5.51
Vin3 and 5.51Vin4 is causal in restoring the ability of the mu-
tant cell line to adhere to substrates and to form aedn-
containing stress fibers (Fig. 8). Most of the over-expressed
vinculin in 5.51Vin clones is located at the peripheral ends
of actin stress fibers although some vinculin is also seen dis-
persed throughout the cytoplasm and this is not normally
seen in wild-type F9 cells (Fig. 2). As observed by others
(Pfeiffer and Vogl, 1991; Horvath et al., 1992), vinculin ap-
pears to be present in ceils in a pattern similar to actin but
at the peripheral ends of actin stress fibers. The presence of
stress fibers is clearly correlated with the shape of the cell
and to its ability to adhere to substrates via vinculin-
containing cell matrix junctions.
The Complementation of Mutant Cells Is Imperfect
At least two explanations might account for the slightly in-
complete complementation of the mutant phenotype by
chicken vinculin to that exhibited by F9 parental cells. One
is that chicken vinculin is sufficiently different to be unable
to replace the mouse-type protein. This is unlikely, however,
because there is 95 % similarity between chick and human
vinculin (Weller et al., 1990). This question can be studied
when a mouse vinculin vector becomes available. The sec-
ond possibility is that exactly the right amount of vinculin
is needed. This hypothesis is supported by our observation
that 5.51Vin3 which expresses very high levels of vinculin is
less able to adhere than 5.51Vin4 which expresses moder-
ately high levels (Fig. 9). The presence of excess vinculin as
in 5.51Vin3 cells could inhibit cell spreading by over-
stabilization of stress fibers and by affecting the disassembly
and reorganization of actin filaments. However, we have not
yet detected a transfected 5.51 clone that adheres and spreads
perfectly on the substratum so we are unable to test the hy-
pothesis satisfactorily. The work of Fernandez et al. (1992a
and b) strongly suggests that the level of vinculin expressed
in a cell can alter cell behavior, because the over-expression
of vinculin in BALB/c3T3 cells by as little as 20% drastically
reduces their motility (Fernandez et al., 1992a). Further, the
restoration of vinculin levels in several transformed cell lines
that express little or no vinculin results in an increase in sub-
strate adhesiveness and in suppressed tumorigenicity (Fer-
nandez et al., 1992b).
Although we could detect no consistent differences in the
levels of several cytoskeletal proteins in 5.5 l-derived clones,
only filamin might be expressed at lower levels in 5.51 cells
compared to F9 cells and we have previously documented
that uvomorulin is present at 30-50% of wild-type F9 cells
(Adamson et al., 1990). It is therefore possible that there are
differences in the incorporation of these proteins into the
cytoskeleton and that the mutant phenotype is exacerbated
by lower levels of filamin and uvomornlin. Current studies
also suggest that the transfected cells are not able to complete
the process of epithelium formation after the addition of
retinoic acid to induce differentiation. The cells are not resis-
tant to retinoic acid, however, because they do differentiate
into parietal endoderm, a nonepithelial cell type. It should
be noted that the ability of the mutant cells to proliferate is
completely normal even in the absence of vinculin.
We believe that the major defect in 5.51 cells is in the vin-
culin gene or in a vinculin regulatory gene. We have never
been able to select an F9 nonadhesive clone without muta-
genesis. Therefore, this defect is not merely a characteristic
of a clonal variation. Restriction enzyme analysis suggest
that the structure of the vinculin gene in 5.51 cells is not de-
tectably different from that in parental F9 cells and therefore,
either a relatively subtle mutation has occurred or a regula-
tory gene is aberrant in the mutant cells.
The Journal of Cell Biology, Volume 121, 1993 918
The Role of Vinculin in Cell-Substrate Adhesion
The work of DePasquale and Izzard (1987, 1991) shows that
talin is first located at the tips of the focal contact precursors
and then accumulates in the adhesion plaque after the focal
contact forms and before vinculin is seen there. The
filopodia-like protrusions in 5.51 cells may be focal contact
precursors because, based on the above findings, vinculin is
not required for their assembly. In 5.51 cells, talin may be
at the tips of the filopodia but focal contacts and actin con-
nections cannot be made because vinculin is absent.
The difficulty of proper assembly of the cytoskeleton is
reinforced by our growing knowledge of the level of com-
plexity of interactions between the cytoskeletal proteins.
Adhesion of cells to a substrate occurs through a chain of in-
teractions from/~-integrins to ct-actinin and actin (Burridge
et al., 1988; Geiger and Ginsberg, 1991; Critchley et al.,
1991; Turner and Burridge, 1991; Hynes, 1992). However,
although vinculin binds avidly to talin (Jones et al., 1989),
vinculin lacking the talin binding site retains the ability to
associate with focal contacts (Bendori et al., 1989). This in-
teraction could occur through a binding site on vinculin, pos-
sibly the paxillin binding site, which is located toward the
COOH-terminus of the molecule (Turner et al., 1990). Vin-
culin can also self-associate (Molony and Burridge, 1985).
Furthermore, vinculin binds to t~-actinin and through the lat-
ter protein to actin. Interestingly, a,-actinin appears to bind
directly to B1 integrin (Otey et al., 1990) even though it ap-
pears to be located some distance from the membrane (Chen
and Singer, 1982).
We have shown here that vinculin is necessary for cell at-
tachment, however, the spreading of cells after attachment
is more complex and may not be a function of vinculin (al-
though see Lehtonen et al., 1983). BALB/c-3T3cells re-
plated from suspension cultures attach and spread exten-
sively even in serum-free cultures when only basal levels of
vinculin are present and vinculin is not detectable in the focal
contacts. When serum is present however, vinculin expres-
sion is induced and large vinculin-positive plaques form
(Ben-Ze'ev et al., 1990). Ben-Ze'ev et al. (1990) suggest that
there may be two kinds of focal contacts, with vinculin pres-
ent in only one type. It is possible that our complemented
clones, 5.51Vin3 and 4, are unable to organize the nonvincu-
lin focal adhesions. This may explain the incomplete appear-
ance of adhesion. Vinculin expression is transcriptionally in-
duced by serum growth factors and increases in vinculin
synthesis requires both initial attachment to the substratum
and serum factors. An autoregulatory mechanism linking
the mode of vinculin organization to the growth factor-
mediated control of vinculin gene expression has been sug-
gested (Ben-Ze'ev et al., 1990). Such a control mechanism
has been demonstrated for tubulin regulation (Ben-Ze'ev et
al., 1979; Cleveland et al., 1981) and may operate for other
cytoskeletal elements. Nevertheless, serum induction of
cells in suspension culture is not effective in up-regulating
vinculin expression; cell attachment to the substrate is
necessary for induction. The mechanism of this requirement
remains obscure. Our observations suggest that cell attach-
ment is a catalyst for further adhesion and spreading. Al-
though 5.51Vin clones attach at different speeds after tryp-
sinization (perhaps depending on their level of expression of
vinculin), all clones eventually appear similar in morphol-
ogy, adhesion and strength of attachment.
The Role of Vinculin in Cell-Cell Compaction and
Wild-type F9 cells express uvomorulin at high levels (Pey-
rieras et al., 1985). Immunoprecipitation of uvomorulin
coprecipitates ct,/~, and 3' catenins (Vestweber and Kernler,
1984; Ozawa et al., 1989). Interestingly, ~catenin has
~30% similarity to vinculin (Nagafuchi et al., 1991; Her-
renknecht et all., 1991). The cytoplasmic portion of uvo-
morulin is necessary to associate with c~-catenin (Nagafuchi
and Takeichi, 1989; Ozawa et al., 1990). The hypothesis is
that uvomorulin forms a molecular complex with the actin-
based cytoskeleton via the catenins and other proteins at the
cytoplasmic domain. This association is essential for uvo-
morulin to act as a cell-cell adhesion receptor. The adherens
junctions have tightly-associated actin filaments (Geiger et
al., 1980; Tsukita et al., 1989; Tsukita et al., 1992) and al-
though coprecipitation ofvineulin does not occur in the pres-
ence of antibodies to actin, its distribution is markedly simi-
lar to actin patterns (Pfeiffer and Vogl, 1991; Horvath et al.,
1992). It is possible that these associations are the trigger for
the homotypic association between uvomorulin on adjacent
cells that leads to cell-cell compaction and the formation of
It has been observed that the shape of a cell, the nature of
the substratum and the degree of contact with adjacent cells
(Shirinsky et al., 1991) directly regulate the synthesis of vin-
culin in cultured fibroblasts (Ungar et al., 1986). In addition,
vinculin is an immediate early gene, responding rapidly to
the addition of serum growth factors by up-regulating its
transcription (Ben-Ze'ev et al., 1990; Bellas et al., 1991).
Fibronectin (Dean et al., 1990), ~l-integrin, ot-tropomyosin
and -y and fl-actin (Ryseck et al., 1989) are also early re-
sponse genes. It is clear, therefore, that cell interaction, cell
shape, molecular interactions at the cytoplasmic domains of
adherens junctions and early signal transduction are all
cross-regulated and correlated with long-term signals and
cell behavior. The mutant F9 cell line will be an important
model for studies of the molecular interactions between the
components involved in adhesion and the consequences of
perturbations made to single components.
We are grateful for the many donated antibody samples indicated in
Materials and Methods. We thank Dr. Avri Ben Ze'ev for generously
providing the 1.2-kb mouse vinculin cDNA. S. Delgado and M. Hashim
assisted in the preparation of the manuscript. We thank R. Oshima and D.
Mercola for advice and comments.
This work was supported by United States Public Health Service grants
CA 54233 (E. D. Adamson) and IRG-173-C from the American Cancer
Association, funds from the Massachusetts General Hospital Center for the
Study of Inflammatory Bowel Disease (DK 43351) from the NIH (R. Ez-
zell) and The Medical Research Council (UK) (D. R. Critchley). The As-
sociation pour la Recherche sur le Cancer, Villejuif, France supported J-L.
Received for publication 30 July 1992 and in revised form 18 February
Adamson, E. D., H, Baribault, and R. Kemler. 1990. Altered uvomorulin ex-
pression in a non-compacting mutant cell line of F9 embryonal carcinoma
cells. Dev. BioL 138:338-347.
Barstead, R. J., and R. J. Waterston. 1989. The basal component of the nema-
tode dense-body is vinculin. J. Biol. Chem. 264:10177-10185.
Bellas, R. E., R. Bendori, and S. R. Farmer. 1991. Epidermal growth factor
activation of vinculin and/~ 1 integrin gene transcription in quiescent Swiss
Samuels et al. Vinculin Expression in Mutant F9 Cells
3T3 cells: regulation through a protein kinase C-independent pathway. J.
Biol Chem. 266:12008-12014.
Ben-Ze'ev, A., S. R. Farmer, and S. Penman. 1979. Mechanisms of regulating
tubulin synthesis in cultured mammalian cells. Cell. 17:315-319.
Ben-Ze'ev, A., R. Reiss, R. Bendori, B. Gorodecki. 1990. Transient induction
of vinculin gene expression in 3T3 fibroblasts stimulated by serum growth
factors. Cell Regul. 1:621-636.
Bendori, R., D. Salomon, and B. Geiger. 1989. Identification of two distinct
functional domains on vinculin involved in its association with local contacts.
J. Cell Biol. 18:2383-2393.
Black, J. D.. S. T. Koury, R. B. Bankert, and E. A. Repasky. 1988. Heteroge-
neity in lymphocyte spectrin distribution: ultrastructural identification of a
new spectrin-ricb cytoplasmic structure. J. Cell. Biol. 106:97-109.
Burridge, K., K. Fath, T. Kelly, G. Nuckolls, and C. Turner. 1988. Focal adhe-
sions: transmembrane junctions between the extracellular matrix and the
cytoskeleton. Annu. Rev. Cell Biol. 4:487-525.
Burridge, K., and J. Feramisco. 1980. Microinjection and localization of a
130 K protein in living fibroblasts: a relationship to actin and fibronectin.
Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D.
Adamson. 1991. Over-expression of uvomorulin in a compaction-negative
F9 mutant cell line. Dev. Biol. 146:499-508.
Chen, W. T., and S. J. Singer. 1982. Immunoelectron microscopic studies of
the sites of cell substratum and cell-cell contacts in cultured fibroblasts. J.
Cell Biol. 95:205-222.
Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. 5. Rutter. 1974.
Isolation of biologically active ribonucleic acid from sources enriched in
ribonuclease. Biochemistry. 18:5294-5299.
Cleveland, D. W., M. A. Lopata, P. Sherline, and M. W. Kirschner. 1981.
Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell.
Cowin, P., H. P. Kapprell, W. W. Franke, J. Tamkon, andR. O. Hynes. 1986.
Plakoglobin: a protein common to different kinds of intercellular junctions.
Critchley, D. R., A. Gilmore, L. Hemmings, P. Jackson, A, McGregor, V.
Ohanian, B. Patel, G. Waites, and C. Wood. 1991. Cytoskeleton proteins
in adherens-type cell-matrix junctions. Biochem Soc. Trans. 19:1028-1033.
Darland, T., M. Samuels, S. A. Edwards, V. P. Sukhatme, and E. D. Adam-
son. 1991. Regulation of egr-I (zpf-6) and c-fos expression in differentiating
emhryonal carcinoma cells. Oncogene. 6:1367-1376.
Dean, D. C., J. J. McQuillan, and S. Weintraub. 1990. Serum stimulation of
fibrunectin gene expression appears to result from rapid serum-induced bind-
ing of nuclear proteins to a cAMP response element. J. Biol. Chem.
DePasquale, J. A., and C. S. Izzard. 1987. Evidence for an actin-containing
cytoplasmic precursor of the focal contact and the timing of incorporation
of vinculin at the focal contact. J. Cell. Biol. 105:2803-2809.
DePasquale, J. A., and C. S. Izzard. 1991. Accumulation of talin in nodes at
the edge of the lamellipodium and separate incorporation into adhesion
plaques at focal contacts in fibroblasts. J. Cell. Biol. 113:1351-1359.
Ezzell, R. M., D. M. Kenney, S. Egan, T. P. Stossel, andJ. H. Hartwig. 1988.
Localization of the domain of actin-binding protein that binds to membrane
glycoprotein Ib and actinin human platelets. J. Biol. Chem. 263:13303-
Feinberg, A. P., and B. Vogelstein. 1984. A technique for radiolabelling DNA
restriction endonuclease fragments to high specific activity. Anal. Biochem.
Fernandez, J. L. R., B. Geiger, D. Salomon, and A. Ben-Ze'ev. 1992a. Over-
expression of vinculin suppresses cell motility in Balb/c 3T3 cells. Cell
Motil. Cytoskeleton. 22:127-134.
Fernandez, J. L. R., B. Geiger, D. Salomon, I. Sabanay, M. Zoller, and A.
Ben-Ze'ev, 1992b. Suppression of tumorigenicity in transformed ceils after
transfection with vinculin cDNA. J. Cell, Biol. 119:427--438.
Geiger, B. 1979. A 130K protein from chicken gizzard: its localization at the
termini of microfilament bundles in cultured chicken cells. Cell. 18:193-
Geiger, B., and D. Ginsberg. 1991. The cytoplasmic domain of adherens-type
junctions. Cell Motil. Cytoskeleton. 20:1-6.
Geiger, B., A. H. Dutton, K. T. Tokuyasu, and S. 5. Singer. 1982. Immuno-
electron microscope studies of membrane-microfilament interactions: distri-
bution of a-actinin, tropomyosin and vinculin in intestinal epithelial cells.
J. Cell Biol. 91:614-628.
Geiger, B., K. T. Tokuyasu, A. H. Dutton, and S. J. Singer. 1980. Vinculin,
an intracellular protein localized at specialized sites where microfilament
bundles terminate at cell membranes. Proc. Natl. Acad. Sci. USA. 77:
Graham, F. L., P. J. Abrahams, C. Muldner, H. L. Meijneker. S. Warnaar,
F. A. de Vries, W. Fiers, and A. J. van der Eb. 1974. Studies of in vitro
transformation by DNA and DNA fragments of human adenovirus and sim-
ian virus 40. Cold Spring Harbor Syrup. Quant. Biol. 39:637-650.
Grover, A., R. G. Oshima, and E. D. Adamson. 1983. Epithelial layer forma-
tion in differentiating aggregates of F9 embryonal carcinoma ceils. J. Cell
Grover, A., M. J. Rosenstraus, B. Sterman, M. E. Snook, and E. D. Adamson.
1987. An adhesion-defective variant of F9 embryonal carcinoma cells fails
to differentiate into visceral endoderm. Dev. Biol. 119:1-11.
Hayashi, Y., B. Haimovitch, A. Reszika, D. Boettiger, and A. Horwitz. 1990.
Expression and function of chicken integrin 81 subunit and its cytoplasmic
domain mutants in mouse NIH3T3 cells. J. Cell Biol. 110:175-184.
Herrenknecht, K., M. Ozawa, C. Eckerskorn, F. Lottspeich, M. Lenter, and
R. Kemler. 1991. The uromorulin-anchorage protein a-catenin is a vinculin
homologue. Proc. Natl. Acad. Sci. USA. 88:9156-9160.
Horvath, A. R., G. M. Asijee, and L. Muszbek. 1992, Cytoskeletal assembly
and vinculin-cytoskeleton interaction in different phases of the activation of
bovine platelets. Cell Motil. Cytoskeleton. 21:123-13 I.
Hynes, R. O. 1992. Integrins: versatility, modulation and signaling in cell adhe-
sion. Cell. 69:11-25.
Ingle, C. A., and N. R. Drinkwater. 1989. Mutational specificities of l'-
acetoxysafrole, N-benzoyloxy-N-methyl-4-aminoazobenzene, and ethyl
methanesulfonate in human cells. Murat. Res. 220:133-142.
Jones, P., P. Jackson, G. J. Price, B. Patel, V. Ohanion, A. L. Lear, and D. R.
Critchley. 1989. Identification of a talin binding site in the cytoskeletal pro-
tein vinculin. J. Cell Biol. 109:2917-2927.
Kemler, R., and M. Ozawa. 1989. Uvomorulin-catenin complex: cytoplasmic
anchorage of a Ca++-dependent cell adhesion molecule. Bioessays. I l:
Koteliansky, V. E., E. P. Ogrysko, N. I. Zhidkova, P. A. Weller, D. R. Critch-
Icy, K. Vancompernolle, J. Vandekerckhove, P. Strasser, M. Way, M. Gi-
mona, and J. V. Small. 1992. An additional exon in the human vinculin gene
specifically encodes meta-vinculin-specific difference peptide: cross-species
comparison reveals variable and conserved motifs in the meta-vinculin in-
sert. Eur. J. Biochem. 204:767-772.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature (Lond.). 227:680-685.
Lehtonen, E., V.-P. Lehto, R. A. Badley, and I. Virtanen. 1983. Formation
of vinculin plaques precedes other cytoskeletal changes during retinoic acid-
induced teratocarcinoma cell differentiation. Exp. Cell Res. 144:191-197.
Maniatis, T., E. F. Fritsch, and J. Sambrook, editors. 1982. Molecular Clon-
ing: A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring
Marcantonio, E. E., J. L. Guan, J. E. Trevithich, and R. O. Hynes. 1990. Map-
ping of the functional determinants of the integrin 81 cytoplasmic domain
by site-directed mutagenesis. Cell Regul. 1:597-604.
McBurney, M. W., E. M. V. Jones-Villeneuve, M. K. S. Edwards and P. J.
Anderson, 1982. Control of muscle and neuronal differentiation in a cultured
embryonal carcinoma cell line. Nature (Lond.). 299:165-167.
Melton, J. A., P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn, and M. R.
Green. 1984. Efficient in vitro synthesis of biologically active RNA and
RNA hybridization probes from plasmids containing a bacteriophage Sp6
promoter. Nucleic Acids Res. 12:7035-7056.
Molony, L., and K. Burridge. 1985, Molecular shape and self-association of
vinculin and metavinculin. J. Cell Biochem. 29:31-36.
Nagafuchi, A., and M. TakeichL 1988. Cell binding function of E-cadherin is
regulated by the cytoplasmic domain. EMBO (Eur. Mol. Biol. Organ.) J.
Nagafuchi, A., M. Takeichi, and S. Tsukita. 1991. The 102 kd cadherin-
associated protein: similarity to vinculin and post-transcriptional regulation
of expression. Cell. 65:849-857.
Nagufuchi, A., and M. Takeichi. 1989. Transmembrane control of cadherin-
mediated cell adhesion: a 94kDa protein functionally associated with a
specific region of the cytoplasmic domain of E-cadherin. Cell Regul.
Nelson, W. J., and P. J. Veschnock. 1986. Dynamics of membrane-skeleton
(fodrin) organization during development of polarity in Madin-Darby canine
kidney epithelial cells. J. Cell Biol. 103:1751-1765.
Otey, L. A., F. M. Pavalko, and K. Burridge. 1990. An interaction between
c¢-actinin and the 81 integrin subunit in vitro. J. Cell Biol. 111:721-729.
Otto, J. J. 1990. Vinculin. Cell Motil. Cytoskeleton 16:1-6.
Ozawa, M., H. Baribault, and R. Kemler. 1989. The cytoplasmic domain of
the cell adhesion molecule uvomorulin associates with three independent
proteins structurally related in different species. EMBO (Fur. Mol. Biol. Or-
gan.) J. 8:1711-1717.
Ozawa, M., M. Ringwold, and R. Kemler. 1990. Uvomorulin-catenin complex
formation is regulated by a specific domain in the cytoplasmic region of the
cell adhesion molecule. Proc. Natl. Acad. Sci. USA. 87:4246--4250.
Peyrieras, N., D. Louvard, and F. Jacob. 1985. Characterization of antigens
recognized by monoclonal and polyclonal antibodies directed against
uvomorulin. Proc. Natl. Acad. Sci. USA. 82:8067-8071.
Pfeiffer, D. C., and A. W. Vogl. 1991. Evidence that vinculin is co-distributed
with actin bundles in ectoplasmic "junctional ~ specializations of mammalian
Sertoli cells. Anat. Rec. 231:89-100.
Price, G. J., P. Jones, M. D. Davison, B. Patel, R. Bendori, B. Geiger, and
D. R. Critchley. 1989. Primary sequence and domain structure of chicken
vinculin. Biochem. J. 259:453~,61~
Reszka, A. A., Y. Hayashi, and A. F. Horwitz. 1992. Identification of amino
acid sequences on the integrin 81 cytoplasmic domain implicated in
cytoskeletal association. J. Cell. Biol. 17:1321-1330.
Ryseck, R.-P., H. MacDonald-Bravo, M. Zerial, and R. Bravo. 1989. Coor-
dinate synthesis of fibronectin, fibronectin receptor, tropomyosin and actin
genes in serum-stimulated fibroblasts. Exp. Cell Res. 180:537-545.
The Journal of Cell Biology. Volume 121, 1993 920
Shirinsky, V. P., K. G. Birukov, V. E. Koteliansky, M. A. Glukhova, E. Span- Download full-text
didis, J. D. Rogers, J. H. Campbell, and G. R. Campbell. 1991. Density-
related expression ofcaldesmon and vinculin in cultured rabbit aortic smooth
muscle cells. Exp. Cell Res. 194:186-189.
Tsukita, S., S. Tsukita, A. Nagafuchi, and S. Yonemura. 1992. Molecular link-
age between cadherins and actin filaments in cell-cell adherens junctions.
Curr. Opin. Cell Biol. 4:834-839.
Tsukita, S., M, Itoh, and S. Tsukita. 1989. A new 400 kD protein from isolated
adherens junctions: Its localization at the undercoat of adherens junctions and
at microfilament bundles such as stress fibers and circumferential bundles.
J. Cell Biol, 109:2905-2915.
Turner, C.E., and K. Burridge. 1991. Transmembrane molecular assemblies
in cell-extracellular matrix interactions. Curt. Opin. Cell Biol. 3:849-853.
Turner, C. E., J, R. Glenney, Jr., and K. Burridge. 1990. Paxillin: a new
vinculin-binding protein present in focal adhesions. J. Cell Biol. 111:
Ungar, F., B. C-eiger, and A. Ben-Ze'ev. 1986. Cell contact- and shape-
dependent regulation of vinculin synthesis in cultured fibroblasts. Nature
Vestweber, D., and R. Kemler. 1984. Some structural and functional aspects
of the cell adhesion molecule uvomorulin. Cell Differ. 15:269-273.
Weller, P. A., E. P. Ogryzko, E. B. Corben, N. I. Zhidkova, B. Patel, G. J.
Price, N. K. Spurt, V. E. Koteliansky, and D. R. Critchley. 1990. Complete
sequence of human vinculin and assignment of the gene to chromosome 10.
Proc. Natl. Acad. Sci. USA. 87:5667-5671.
Westmeyer, A., K. Ruhoau, A. Wegner, and B. M. Jocicusch. 1990. Antibody
mapping of functiooal domains in vineulin. EMBO (Eur. Mol. Biol. Organ.)
Wood, C. M., and R. E. Moses. 1989. Ethyl methane sulfonate- and
bloomycin-generated deletion mutations at HPRT locus in xeroderma pig-
mentosum complementation group D fibroblasts. 15:345-357.
Samuels et al. Vinculin Expression in Mutant b "v ) Cells