**
* Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, North Carolina 27599; Department of
Biology, Genetics and Medical Chemistry, University of Torino, 10126 Torino, Italy; § Belozersky Institute of Physico-Chemical
Biology, Moscow State University, 117311 Moscow, Russia;
Max-Planck-Institute for Biochemistry, Martinsried D-82152,
Germany; ¶ Biogen Inc., Cambridge, Massachusetts 02142; ** Department of Psychology, University of Rome, Italy; and
Institute of Biology, University of Palermo, 90133 Palermo, Italy
Expression of muscle-specific 1D integrin
with an alternatively spliced cytoplasmic domain in
CHO and GD25,
1 integrin-minus cells leads to their
phenotypic conversion.
1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial
activity, retarded spreading, reduced migration, and
significantly enhanced contractility compared with
their
1A-expressing counterparts. The transfected
1D is targeted to focal adhesions and efficiently displaces the endogenous
1A and
v
3 integrins from
the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata
and leads to elevated stability of focal adhesions in
1D
transfectants. Whereas a significant part of cellular
1A
integrin is extractable in digitonin, the majority of the
transfected
1D is digitonin-insoluble and is strongly
associated with the detergent-insoluble cytoskeleton. Increased interaction of
1D integrin with the actin cytoskeleton is consistent with and might be mediated by
its enhanced binding to talin. In contrast,
1A interacts
more strongly with
-actinin, than
1D. Inside-out
driven activation of the
1D ectodomain increases ligand binding and fibronectin matrix assembly by
1D
transfectants. Phenotypic effects of
1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular
ligands. They parallel the transitions that muscle cells
undergo during differentiation. Modulation of
1 integrin adhesive function by alternative splicing serves as a
physiological mechanism reinforcing the cytoskeleton-
matrix link in muscle cells. This reflects the major role
for
1D integrin in muscle, where extremely stable association is required for contraction.
INTEGRINS are a large family of transmembrane heterodimeric receptors that play a key role in cell adhesion
to extracellular matrix (Hynes, 1992 Integrin functions within the cell can be regulated at different levels. These include cell type-specific biosynthesis
of certain integrin heterodimers, maturation and processing of the receptors, as well as their transport to the cell
surface (Hynes, 1992 So far, four cytoplasmic domain variants of the Upon transfection into nonmuscle cells, Antibodies and Reagents
The following antibodies against Blocking anti-mouse Human plasma Fn was from GIBCO BRL. Recombinant, 12-kD cell-binding Fn fragment corresponded to the tenth Arg-Gly-Asp-containing (cell-binding), type III Fn repeat. Cytochalasin D was from Sigma Chemical Co.. Digitonin was purchased from Sigma Chemical Co. and purified
by dissolving in water, filtering, and lyophilization before use. 35S-Translabel and methionine- and cysteine-free medium were from ICN Biomedicals Inc. (Costa Mesa, CA). Na125I and [32P]orthophosphate were from
Dupont-NEN (Boston, MA).
Expression Constructs, Transfection, and Cell Culture
Human full-length cDNAs encoding Morphological Analysis and Spreading
For analysis of cell phenotype, CHO transfectants were plated and cultured for 1 d on Fn-coated dishes. Phase-contrast photographs of live
Measurements of the Ligand-Binding Affinity
The binding of the 125I-labeled Fn(III)10 fragment to Flow Cytometry
Cell surface expression of the transfected human Fn Matrix Assembly Assays
To quantitate Fn incorporation into deoxycholate-insoluble matrix, confluent Migration Assays
Migratory properties of For time lapse videomicroscopy, Analysis of the Association of Localization of the Transfected To localize the transfected and the endogenous To study whether the solubility of integrins in digitonin correlates with
their cytoskeletal association, 35S-labeled To assess the association of the transfected and the endogenous Analysis of the Association of To compare the association of Interaction of Talin and Talin was purified from human platelets as described earlier (Collier and
Wang, 1982 Full-length cytoplasmic domain peptides of Measurements of Cellular Contractility and Myosin
Light Chain Phosphorylation
Silicone rubber substrata for assessing cellular contractility were made as
described previously (Harris et al., 1980 Possible changes in myosin light chain phosphorylation were examined
as described (Chrzanowska-Wodnicka and Burridge, 1996 The levels of surface expression of the transfected human
Table I.
Expression Levels and Activation States of
Transfected Human
To determine possible effects of
Table II.
Cell Shape Parameters of ). Integrin receptors serve a dual purpose, linking extracellular matrix to
the actin cytoskeleton and providing bidirectional transmission of signals between the extracellular matrix and the
cytoplasm (Schwartz et al., 1995
; Yamada and Miyamoto, 1995
; Burridge and Chrzanowska-Wodnicka, 1996
). At
least two major actin-binding proteins, talin and
-actinin,
are thought to interact directly with the cytoplasmic domain of several
subunits, providing a link to the actin cytoskeleton (Horwitz et al., 1986
; Otey et al., 1990
; Hemler
et al., 1994
). Among integrins,
1 is typically the most
abundant and ubiquitously expressed subunit associated with a number of
subunits to form distinct heterodimers.
These interact with a variety of extracellular matrix and cell
adhesion molecules (Hynes, 1992
). The entire structure of
the
1 integrin cytoplasmic domain is critical for integrin-
cytoskeleton interaction (Hayashi et al., 1990
; LaFlamme
et al., 1992
; Reszka et al., 1992
; Ylanne et al., 1993
; Lewis
and Schwartz, 1995
).
). Another level of control of integrin
function is through regulation of the ligand-binding affinity of integrins on the cell surface. This type of regulation
involves conformational changes within integrins. The conformational state of the extracellular domains (activation)
of integrins is regulated via their cytoplasmic tails and is
referred to as inside-out signaling (Ginsberg et al., 1992
;
O'Toole et al., 1994
; Schwartz et al., 1995
). Thus, deletions
or mutations of certain residues in the cytoplasmic domains of
and
subunits can either increase or inhibit the ligand-binding activity of integrin receptors (Takada et al., 1992
; O'Toole et al., 1994
, 1995
). The activation state of integrins can also be controlled by some lipid metabolites
(Hermanowski-Vosatka et al., 1992
; Smyth et al., 1993
) and
small GTP-binding proteins (Zhang et al., 1996
; Hughes
et al., 1997
). Finally, functional properties of integrin receptors can be modulated by alternative splicing involving
their cytoplasmic tails.
1 integrin subunit have been described. Besides the major
1A
isoform, characteristic for all known cell types except red
blood cells and terminally differentiated striated muscles,
two minor cytoplasmic domain isoforms of
1 integrin,
1B and
1C, have been characterized (Altruda et al.,
1990
; Languino and Ruoslahti, 1992
). Although their functions remain uncertain, it has been speculated that
1B
can serve as a negative regulator of cell adhesion during development, whereas
1C can strongly inhibit cell growth
(Balzac et al., 1993
, 1994
; Meredith et al., 1995
). The alternatively spliced sequences of
1B and
1C have no homology to the major
1A isoform and are unable to localize
to cell-matrix adhesion sites apparently because of impaired interaction with the actin cytoskeleton (Balzac et al.,
1993
; Meredith et al., 1995
). Interestingly,
1B and
1C
variants have been found only in humans, whereas the fourth
1 isoform,
1D, is highly conserved at least throughout vertebrate evolution, suggesting an important role for
this muscle-specific variant (van der Flier et al., 1995
; Zhidkova et al., 1995
; Baudoin et al., 1996
; Belkin et al., 1996
).
1 integrin is localized at junctional structures of striated muscles (Bozyczko et al., 1989
). Expression of the
1
integrin subunit as well as the ligand occupation of
1-containing heterodimers is essential for myodifferentiation
and the formation of sarcomeric cytoarchitecture (Menko
and Boettiger, 1987
; Volk et al., 1990
). Integrin-mediated
cytoskeleton-matrix linkage has to be distinct in muscle cells
because of high tensile forces transmitted across the membrane and enhanced stability of muscle adhesive structures. This implies a modified function for
1 integrin in
muscles. This function is now attributed primarily to
1D
cytoplasmic domain variant, which is a major
1 isoform
that completely displaces
1A integrin in differentiated
striated muscles (Belkin et al., 1996
). Its cytoplasmic domain is highly homologous to that of
1A, including conservation of both NPXY motifs involved in the regulation
of ligand-binding affinity (Tamkun et al., 1986
; Argraves et al., 1987
; Zhidkova et al., 1995
; van der Flier et al.,
1995
).
1D accumulates at all major cell-matrix adhesion
sites both in skeletal muscle fibers and cardiomyocytes.
7
1D is a predominant integrin in adult skeletal and
heart muscle tissues (Belkin et al., 1996
). However, other
subunits, including
5 and
6A, can pair with
1D in developing heart muscle (Brancaccio et al., 1997
). The data
obtained so far lead to the suggestion that
1D integrin plays a crucial role in linking the subsarcolemmal cytoskeleton to the surrounding extracellular matrix in muscle tissues (Belkin et al., 1996
; Fassler et al., 1996
).
1D is targeted
to focal adhesions, proving that the muscle-specific isoform of
1 integrin is able to interact with the nonmuscle
cytoskeleton as well (Belkin et al., 1996
). To get an insight
in the functional properties of this integrin, we have expressed human
1D and
1A cytoplasmic domain isoforms in CHO cells and the mouse GD25 cell line. GD25
cells lack endogenous
1 integrin as a consequence of gene
inactivation (Wennerberg et al., 1996
). Here we report that
the expression of
1D integrin in nonmuscle cells leads to a conversion of cellular phenotype. The observed alterations in cell morphology, inhibition of spreading and motility, as well as an increase in the ligand-binding affinity,
fibronectin matrix assembly and contractility, are caused
by an enhanced association of
1D integrin with both the
actin cytoskeleton and extracellular matrix ligands.
1D-mediated enhancement of actin-membrane attachment is,
at least in part, due to a higher affinity interaction of this
integrin with the focal adhesion protein, talin. The altered structure of the
1D cytoplasmic domain causes a conformational change of its ectodomain via inside-out signaling
mechanisms, leading to activation of ligand binding. Reinforcement of the cytoskeleton-matrix association by
1D
reflects a key role for this integrin as a cytoskeleton-matrix
linker, strengthening adhesive structures in muscle tissues.
Materials and Methods
1 integrin were used in this study: TS2/
16 mAb to human
1 subunit, which activates ligand binding by
1-containing heterodimers was a gift from Dr. M. Hemler (Dana-Farber Cancer
Institute, Boston, MA) (Hemler et al., 1984
; Arroyo et al., 1992
); function-blocking P4C10 mAb against human
1 integrin (Carter et al., 1990
) was
from GIBCO BRL (Gaithersburg, MD); 102DF5 mAb against human
1
integrin (Ylanne and Virtanen, 1989
); 12G10 mAb, which reacts with activated (high affinity conformation for ligand binding) human
1 (Mould et
al., 1995
); 9EG7 mAb reacting with ligand-, Mg2+-, Mn2+-induced, Ca2+-
inhibited epitope on human
1 integrin subunit (Bazzoni et al., 1995
);
A1A5 mAb against human
1 integrin (Hemler et al., 1984
), conjugated
with fluorescein and rabbit polyclonal antibody against human
1 integrin
(Belkin et al., 1990
). 7E2 mAb against hamster
1 integrin and inhibitory
PB1 mAb against intact hamster
5
1 heterodimer were generous gifts
from Dr. R. Juliano (University of North Carolina, Chapel Hill, NC)
(Brown and Juliano, 1985
, 1988
). Isoform-specific antibodies against
1A
and
1D integrins were described earlier (Belkin et al., 1996
).
v H9.2B8 mAb (Moulder et al., 1991
) was obtained from PharMingen (San Diego, CA). Rabbit polyclonal antibodies against
v,
3, and
5 cytoplasmic domains were described earlier (Defilippi et al., 1992
; Balzac et al., 1994
). mAb 8d4 against talin was obtained
from Sigma Chemical Co.(St. Louis, MO) and mAb 1682 against
-actinin
was from Chemicon International, Inc. (Temecula, CA). Rabbit polyclonal
antibody against platelet myosin II, cross-reacting with nonmuscle myosin, was a gift from Dr. R. Adelstein (National Institutes of Health, Bethesda, MD). Rabbit polyclonal antibody against human plasma fibronectin (Fn) was provided by Dr. L.B. Chen (Dana-Farber Cancer Institute).
1A integrin or
1D integrin in
SV40-based expression vector pECE (Ellis et al., 1986
), were transfected
into CHO cells or
1-minus GD25 cell line (Wennerberg et al., 1996
), and
transfectants were selected as described (Belkin et al., 1996
). More than
95% of cells in each population expressed human
1 integrin; the expression levels of the transfected
1A and
1D integrins were very similar and
comparable to the level of the endogenous hamster
1 integrin subunit in
CHO cells and close to the levels of the endogenous
v and
3 integrins in
GD25 transfectants. CHO transfectants were cultured in Ham's F12 medium with 10% FBS, and GD25 transfectants were cultured in DME plus 10% FBS.
1A-CHO and
1D-CHO cells were taken on an inverted microscope. The outlines of randomly chosen cells not in contact with other cells were
analyzed by the computer Tracer V1.0 software (Dunn and Brown, 1986
).
The spread area, cell perimeter, and two morphometric parameters of cell
shape, cell dispersion and elongation, were calculated as characteristics of
cell spreading and polarization. For analysis of the time course of spreading,
1A- and
1D-transfected CHO and GD25 cells were plated in serum-free medium on Fn, laminin, vitronectin, or on immobilized mAb
TS2/16 to human
1 integrin (Balzac et al., 1994
; Belkin et al., 1996
). After
specific periods of time, cells were fixed with formaldehyde, stained with
Coomassie brilliant blue (Balzac et al., 1994
), and then photographed.
1A-CHO,
1D-CHO,
1A-GD25, and
1D-GD25 cells in suspension was quantified as
described (O'Toole et al., 1990
; Wu et al., 1995
). Since CHO cells express
the endogenous
5
1, and
1-minus GD25 cells express
v
3 as a major
Fn-binding integrin (Wennerberg et al., 1996
), inhibitory mAbs PB1
against hamster
5
1 or H9.2B8 against mouse
v were used for CHO
and GD25 cells, respectively. In some experiments, blocking P4C10 mAb
against human
1 integrin was used in combination with either PB1 mAb
(for CHO cells) or H9.2B8 mAb (for GD25 cells). Cells (0.2 ml of 5 × 106
cells/ml) in Tyrode's buffer were incubated with specified concentrations of 125I-labeled Fn(III)10 fragment (sp act 0.12 mCi/nM) for 30 min at 37°C
either alone or in the presence of 10 µg/ml of purified TS2/16 mAb, which
activates human
1 integrins. Coincubation with an excess of unlabeled
Fn(III)10 fragment (0.5 mg/ml) was used to determine and subtract the
nonspecific background binding. 50-µl aliquots were layered on 0.3 ml of
20% sucrose in Tyrode's buffer and centrifuged for 3 min at 12,000 rpm.
Radioactivity associated with the cell pellet was determined in a gamma
counter.
1A or
1D integrins in
CHO and GD25 transfectants was assessed with 102DF5 and TS2/16 mAbs,
whose binding to the
1 subunit is conformation independent. Their expression levels were compared to those of the endogenous hamster
1 integrin (examined with 7E2 mAb). 12G10 mAb reacting with activated human
1 integrin subunit (Mould et al., 1995
) and conformation-specific
9EG7 anti-human
1 mAb (Bazzoni et al., 1995
) were used either in the
absence of Mn2+ ions or in the presence of 1 mM Mn2+. Fluorescein-
labeled, affinity-purified donkey anti-mouse IgG (Chemicon International,
Inc.) was used as secondary antibody.
1A-CHO,
1D-CHO, as well as
1A-GD25 and
1D-GD25 cells did not
assemble Fn matrix well when confluent cell monolayers were cultured in
growth medium containing 1% FBS for 2 d. To boost the formation of Fn
matrix, exogenous human plasma Fn was added at 200 nM concentration
for 2 d to the confluent cell monolayers grown on glass coverslips. Inhibitory mAbs PB1 against hamster
5
1 and H9.2B8 against mouse
v were
used to block Fn matrix assembly by the endogenous Fn-binding integrins
in CHO and GD25 cells, respectively. Activating TS2/16 and inhibitory
P4C10 mAbs were used for the transfected human
1A and
1D integrins. After 2 d, cell monolayers were fixed and stained with anti-Fn antibody. Stained cells were observed using a Zeiss epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY) and representative fields were photographed using equal exposure lengths on Kodak T-Max 400 film (Eastman Kodak, Rochester, NY).
1A-CHO,
1D-CHO,
1A-GD25, and
1D-GD25 cultures were
incubated for 2 d with 100, 200, or 300 nM of 125I-labeled Fn (sp act 0.08 mCi/nM) in growth medium containing 1% FBS and blocking and activating mAbs as specified above. Deoxycholate-insoluble fraction was obtained from cell monolayers as described (McKeown-Longo and Mosher,
1985
; Wu et al., 1993
, 1995
).125I-labeled Fn incorporated into the deoxycholate-insoluble extracellular matrix was analyzed by reducing SDS-PAGE (6% running gel) and autoradiography. Iodinated Fn bands were
cut out and counted in a gamma counter.
1A-CHO,
1D-CHO,
1A-GD25, and
1D-GD25 cells were examined by a wound closure assay and time lapse videomicroscopy. For the wound closure assay, confluent cell monolayers grown on Fn-coated coverslips were wounded by dragging a sterile 1-mm
pipette tip across the monolayer to create cell-free fields (Romer et al.,
1994
). 2 d later, glass coverslips were fixed with formaldehyde, stained
with Coomassie blue, and then photographed.
1A- and
1D-transfected CHO and
GD25 cells were plated on plastic dishes coated with 10 µg/ml of human
plasma Fn. Five to six cells were scanned per field in eight different fields,
every 20 min for 4 h. The displacement of the cell center as a function of
time was calculated for each cell using nonoverlapping time intervals. To
block the endogenous Fn receptors, PB1 mAb was used for CHO transfectants and H9.2B8 mAb for GD25 transfectants. TS2/16 was used as the
activating mAb and P4C10 as the blocking mAb for the transfected human
1A and
1D integrins.
1A and
1D Integrins
with
Subunits
1D-CHO cells as well as
1A- and
1D-GD25 transfectants were lysed
in buffer containing 1% Triton X-100 in 50 mM TrisCl, 150 mM NaCl, pH
7.5, and protease inhibitors. Each lysate was clarified by centrifugation, divided into four equal parts, and the transfected human
1 integrins were
immunoprecipitated using TS2/16 mAb, whereas
3,
5, and
v subunits
were immunoprecipitated with antibodies against cytoplasmic domains of
these integrins. The resulting immunoprecipitates were run on 10% gel
and blots were probed with the isoform-specific antibodies against
1A or
1D integrins.
1A and
1D Integrins
and the Endogenous
1A and
v Subunits and Analysis
of their Association with the Actin Cytoskeleton
1 integrins, as well as the
endogenous
v integrins in the transfectants, cells cultured on Fn-coated
coverslips were fixed with formaldehyde and permeabilized with 0.5%
Triton X-100 in PBS. CHO transfectants were costained with fluorescein-labeled A1A5 mAb to human
1 and rhodamine-labeled 7E2 mAb to
hamster
1 integrin. GD25 cells were double stained with mouse fluorescein-labeled A1A5 mAb and rabbit anti-
v antibody followed by
rhodamine-labeled donkey anti-rabbit antibody (Chemicon International
Inc.). Stained cells were observed using epifluorescence with a Zeiss Axiophot microscope and photographed using Kodak T-Max 400 film.
1A-CHO and
1D-CHO cells,
either untreated or treated for 1 h with 10 µM of cytochalasin D, were
fractionated into soluble and cytoskeleton-associated fractions by sequential extraction at 4°C with 0.1% digitonin in 50 mM Pipes, 1 mM MgCl2,
1 mM EGTA, 1 mM EDTA, pH 6.9, and then with radioimmunoprecipitation assay (RIPA) buffer (50 mM TrisCl, 150 mM NaCl, 1% Triton X-100,
0.5% Na-deoxycholate, and 0.1% SDS, pH 7.5). Both buffers contained 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 0.5 mM PMSF as protease inhibitors. The transfected
1A and
1D integrins were immunoprecipitated from
digitonin- and RIPA-soluble fractions using TS2/16 mAb. 35S-labeled
1
immunoprecipitates were run on 10% gels and analyzed by autoradiography.
1 integrin subunits and the endogenous
v integrins with the actin cytoskeleton, 35S-labeled CHO and GD25 transfectants were sequentially extracted with digitonin and RIPA buffers as described above. Immunoprecipitation of the transfected human
1A and
1D integrins from both cellular fractions was performed with TS2/16 mAb. 7E2 mAb antibody was used
for the endogenous hamster
1A integrin. Rabbit anti-
v antibody was
used to immunoprecipitate the endogenous
v
3/
v
5 integrins from
GD25 transfectants. 35S-labeled
1 and
v immunoprecipitates were analyzed by SDS-PAGE on 10% gels and subsequent autoradiography.
1A and
1D Integrins
with Talin and
-Actinin by Coimmunoprecipitation
1A and
1D integrins with talin and
-actinin, 5 × 106 transfected cells were incubated in suspension with 10 µg of either purified TS2/16 mAb, 12G10 mAb, or 7E2 mAb at 4°C for 30 min on a rotator. In the case of 12G10 mAb, cells were either preincubated for 5 min with 1 mM Mn2+ or used in the absence of Mn2+. Cells
were centrifuged (1,000 rpm, 3 min) and the pellets were extracted for 3 min on ice with buffer containing 0.5% digitonin in 50 mM Pipes, 1 mM
MgCl2, 1 mM EGTA, 1 mM EDTA, pH 6.9, with 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 0.5 mM PMSF. Under these conditions ~80-90% of
cellular
1 integrins was extracted. Cell extracts were centrifuged (12,000 rpm, 30 min, 4°C) and the resulting supernatants incubated at 4°C for 45 min with donkey anti-mouse IgG immobilized on protein A-Sepharose
beads. Immunoprecipitates were washed with the same buffer, boiled in
SDS sample buffer, and then run on 10% gels. Proteins were transferred
onto Immobilon membranes (Millipore Corp., Bedford, MA) and blotted
with either rabbit polyclonal antibody to human
1 integrin, 8d4 mAb
against talin, or 1682 mAb against
-actinin. To verify the equal amount of
1A and
1D isoforms in the immunoprecipitates, the blots were stripped and reprobed with the isoform-specific antibodies against
1A
and
1D (Belkin et al., 1996
).
-Actinin with
1A and
1D
Cytoplasmic Domain Peptides
).
-Actinin purification from chicken gizzards was performed
as described (Otey et al., 1990
). Talin and
-actinin were iodinated using
125I and Iodobeads (Pierce Chemical Co., Rockford, IL). The proteins were labeled to a specific activity of 1.2 × 106 cpm/µg for talin and 7.5 × 106 cpm/µg for
-actinin.
1A and
1D integrins (Belkin et al., 1996
) were iodinated using Iodogen method. Both peptides
were initially tested for their binding to the microtiter wells in the range of
1-150 µM concentrations in buffer containing 50 mM TrisCl, 150 mM
NaCl, pH 7.5. Since they bound similarly to the wells, saturating 50 µM
concentration of
1A and
1D peptides was used in subsequent experiments to immobilize them on plastic 96-well microtiter plates for 1 h at
37°C. After blocking with 2% BSA in 50 mM TrisCl, 150 mM NaCl, wells
with the bound peptides were incubated with 1 nM of 125I-talin or 125I-
-actinin and 1 nM to 1 µM concentrations of unlabeled talin or
-actinin
in the same buffer with 0.1% BSA for 4 h at 37°C. After the incubations,
wells were washed three times with the same buffer and bound radioactivity was measured in a gamma counter. Nonspecific background was determined and subtracted for talin and
-actinin binding to BSA-coated wells.
; Danowski, 1989
). The UV glow
discharge polymerization was used in combination with gold-palladium
coating (Chrzanowska-Wodnicka and Burridge, 1996
). 5- and 12-s polymerization was used for CHO and GD25 transfectants, respectively. Cells
were plated on the cross-linked rubber substrata in growth medium with
10% FBS and photographed on the next day.
). Briefly, subconfluent
1A-CHO and
1D-CHO cells cultured on Fn-coated, 35-mm
dishes, were labeled for 4 h with 20 µCi/ml of 35S-Translabel and 100 µCi/ml
of [32P]orthophosphate in phosphate-free medium. Cells were washed
with PBS and equal amounts of material, as judged by 35S-incorporated
radioactivity, were taken for immunoprecipitation with antimyosin antibody, followed by protein A-Sepharose beads. Immunoprecipitates were
washed, boiled in SDS sample buffer, and then run on 15% polyacrylamide gel. Phosphorylated myosin light chain bands were visualized by autoradiography using three sheets of aluminum foil to block traces of 35S radiation.
Results
1D Integrin Alters Cell Morphology and
Inhibits Spreading
1A and
1D, measured with mAbs 102DF5 and TS2/16,
were very close to each other in both CHO and GD25
transfectants (Table I). They were also similar to the levels
of the endogenous
1A in CHO transfectants and
v integrins in GD25 cells (see Fig. 9, I and J). No difference in
association of the transfected
1A and
1D with endogenous
subunits was found in the two types of transfectants
(Fig. 7).
1A and
1D Integrins in CHO and
GD25 Transfectants
Fig. 9.
1D interacts more strongly than
1A with the actin cytoskeleton and displaces the endogenous
1A and
v integrins from
focal adhesions. (A-D) Localization of the transfected
1A and
1D and the endogenous
1A integrins in CHO transfectants.
1A-CHO (A and C) and
1D-CHO (B and D) cells were double stained for human
1A (A) and hamster
1A (C) integrins, or for human
1D (B) and hamster
1A (D) integrins. (E-H) Localization of the transfected
1A and
1D and the endogenous
v integrins in GD25
transfectants.
1A-GD25 (E and G) and
1D-GD25 (F and H) cells were double stained for human
1A (E) and mouse
v (G) integrins, or for human
1D (F) and mouse
v (H) integrins. Note colocalization of the transfected
1A with the endogenous
1A and
v
integrins, whereas transfected
1D integrin displaces the endogenous
1A from focal adhesions in CHO transfectants and the endogenous
v integrins from focal adhesions in GD25 transfectants. (I and J) Association of the transfected
1A and
1D and the endogenous
1A and
v integrins with the actin cytoskeleton in CHO and GD25 transfectants. 35S-Labeled integrins were immunoprecipitated
from digitonin-soluble (S) and digitonin-insoluble (I) fractions of
1A- and
1D-transfected CHO (I) and GD25 (J) cells. (T), transfected human
1A or
1D integrin; (E), endogenous hamster
1A (I) or mouse
v
3/
v
5 (J) integrins. Long arrows point to the mature
1 integrin subunit, short arrows mark the precursor of the
1 subunit. The large arrowhead marks the
v subunit and the small arrowhead marks the associated
3 and
5 subunits, not resolved under conditions of this electrophoresis. Unlike the transfected
1A,
1D is present predominantly in the digitonin-insoluble (cytoskeletal) fraction, whereas the endogenous hamster
1A in
1D-CHO
and mouse
v
3/
v
5 integrins in
1D-GD25 cells are mostly digitonin-soluble. Bars, 50 µm.
[View Larger Versions of these Images (48 + 56 + 112K GIF file)]
Fig. 7.
Association of 1A
and
1D integrins with
subunits in CHO and GD25
transfectants. Immunoprecipitates containing the
transfected human
1 integrin subunit (1),
3 subunit
(2),
5 subunit (3), or
v subunit (4) from
1D-CHO (A
and B),
1A-GD25 (C and
D), and
1D-GD25 (E and
F) cells were run on 10% gel and subjected to immunoblotting
with the isoform-specific antibodies against
1A (A, C, and E)
and
1D (B, D, and F) integrins.
[View Larger Version of this Image (52K GIF file)]
1A and
1D integrin
expression on cell morphology, CHO transfectants were
grown on Fn for 1 d (Fig. 1, A and B).
1D-CHO cells appear more rounded with fewer cytoplasmic extensions at
their periphery than their
1A-transfected counterparts.
Statistical analysis of the cell shape was performed for randomly chosen
1A-CHO and
1D-CHO cells (Dunn and
Brown, 1986
). This demonstrated that spread areas were
similar for
1A and
1D transfectants, but the average cell
perimeter was ~20% lower for
1D-CHO cells (Table II).
Two cell shape parameters, dispersion and elongation, representing measures of cell multipolarity and bipolarity, respectively, were significantly lower for
1D transfectants.
These data indicated that pseudopodial activity and cell
polarization were reduced in CHO cells expressing
1D integrin.
Fig. 1.
Altered morphology and inhibited spreading of CHO
and GD25 cells expressing 1D integrin.
1A-CHO (A) and
1D-CHO (B) cells were plated on Fn and cultured for 1 d.
1A-CHO (C, E, and G) and
1D-CHO (D, F, and H) cells were
plated in serum-free medium on Fn for 30 min (C and D), or 1 h
(E and F); or on TS2/16 mAb against human
1 integrin for 2 h
(G and H).
1A-GD25 (I and K) and
1D-GD25 (J and L) cells
were plated in serum-free medium on Fn (I and J) or vitronectin
(K and L) for 1 h. Cells were fixed with formaldehyde and
stained with Coomassie blue. Bar, 50 µm.
[View Larger Version of this Image (155K GIF file)]
1A-CHO and
1D-CHO
Cells
To analyze the time course of spreading, 1A and
1D
transfectants were plated on purified Fn (Fig. 1, C-F).
Spreading of CHO cells expressing
1D integrin on Fn
was significantly delayed compared with
1A transfectants.
When both
1A and
1D transfectants were plated on
TS2/16 mAb specific for human
1 integrin,
1D-CHO were less spread than
1A-CHO cells (Fig. 1, G and H).
Notably,
1D transfectants also spread more slowly on
laminin, vitronectin, and 7E2 mAb against hamster
1 integrin (data not shown), indicating that inhibition of spreading was not limited to a certain type of substrate. Likewise,
spreading of
1D-GD25 cells on both Fn and vitronectin
was significantly delayed compared with the spreading of
1A-GD25 cells (Fig. 1, I-L). These observations suggested that retardation of spreading was a general phenomenon of
1D expression.
Constitutive Activation of the 1D Integrin
Ectodomain Increases Ligand Binding
To characterize the affinity states of 1A and
1D integrins,
the binding of the Fn(III)10 fragment to
1A- and
1D-transfected CHO and GD25 cells in suspension was examined. In the presence of function-blocking mAbs PB1 and
H9.2B8, which inhibited endogenous Fn-binding hamster
5
1 and mouse
v
3 integrins, respectively,
1D transfectants exhibited a significant increase in binding the soluble ligand compared with
1A-expressing cells (Fig. 2, A
and B). TS2/16 mAb, which activates human
1 integrin,
markedly increased Fn(III)10 fragment binding to both
1A-CHO and
1A-GD25 cells, whereas the ligand binding by
1D transfectants in the presence of TS2/16 mAb
remained unaffected (Fig. 2, A and B). Blocking P4C10
mAb against human
1 integrin completely abolished the
binding of Fn(III)10 fragment to all types of transfectants (Fig. 2, A and B), therefore proving its specificity.
To further assess the difference in the conformation of
the ectodomains of the transfected 1A and
1D, a flow
cytometry analysis was used with mAb 12G10. This antibody recognizes preferentially a Mn2+- and ligand-induced
conformation of the human
1 integrin subunit (Mould
et al., 1995
; Mould, 1996
). The amount of 12G10 mAb
bound to the cell surface was significantly higher for
1D-expressing CHO and GD25 cells, than for
1A-expressing
cells. Moreover, the binding of 12G10 mAb was almost unchanged for
1D-CHO and
1D-GD25 cells in the presence of Mn2+, whereas the 12G10 mAb binding to both
types of
1A transfectants was dramatically increased by
Mn2+ (Table I). Another mAb, 9EG7, whose binding to
human
1 integrin is stimulated by ligands and Mn2+, but
inhibited by Ca2+ (Bazzoni et al., 1995
), also reacted more
strongly with
1D-GD25 compared with
1A-GD25 cells
(Table I). Together, these results showed that in
1D-
expressing cells in suspension the majority (~77-88%) of
1D integrins were constitutively activated on the cell surface, whereas only ~27-44% of the transfected
1A integrins were present in the activated state.
1D Enhances Fn Matrix Assembly
Fn biosynthesis and secretion levels in CHO and GD25
cells were not altered by 1A or
1D expression and appeared to be identical for each pair of transfectants (data
not shown). Fn matrix assembly by
1A- and
1D-transfected cells was analyzed with the exogenous Fn by both
immunofluorescence and measurements of 125I-Fn incorporation into deoxycholate-insoluble matrix (McKeown-Longo and Mosher, 1985
; Wu et al., 1993
, 1995
). Using different concentrations of the exogenous Fn, we consistently
observed an enhanced Fn matrix assembly by
1D transfectants compared to their
1A-expressing counterparts
(Fig. 3 A). In our subsequent experiments we used 200 nM
of exogenous Fn for matrix assembly studies with the
transfectants. There was no difference in Fn matrix assembly between
1A and
1D transfectants in the presence of
blocking P4C10 mAb against human
1 integrin, showing
that the observed effects can be ascribed specifically to the
expressed
1D (Fig. 3 B).
In the presence of blocking anti-hamster 5
1 integrin
mAb PB1 (for CHO cells) or inhibitory anti-mouse
v integrin mAb H9.2B8 (for GD25 cells),
1D transfectants
assembled more abundant meshwork of Fn fibrils, than
1A-expressing counterparts (Fig. 4, A, B, E, F, and I, a, b,
e, and f). Activating mAb TS2/16 significantly increased
Fn matrix assembly by
1A-CHO and
1A-GD25 cells,
but did not change the levels of assembly for
1D-transfected CHO and GD25 cells (Fig. 4, C, D, G, H, and I, c, d,
g, and h). Quantitation of 125I-Fn incorporated into the extracellular matrix showed a five- to sixfold increase in Fn
assembly by
1D compared with
1A integrin in CHO
and GD25 cells (Fig. 4, I and J). Interestingly, whereas mAb TS2/16 caused two- to threefold increase in Fn matrix assembly by
1A integrin for both types of transfectants, these levels appeared still much lower than those exhibited by
1D integrin (Fig. 4, I and J).
1D Integrin Inhibits Cell Migration
Initially, migratory properties of 1A- and
1D-transfected CHO and GD25 cells were analyzed using a monolayer wounding assay. In 2-d wound closure experiments,
1D transfectants exhibited significantly slower migration
rates than
1A-expressing cells (Fig. 5, A-D). When migratory behavior of
1A and
1D transfectants was analyzed on Fn substrate by time lapse videomicroscopy,
1D-expressing cells migrated three- to fourfold slower than
1A-transfected cells. Again, blocking P4C10 mAb against
human
1 integrin completely abolished the effects of
1D
on cell migration (Fig. 5 E).
Migration of 1A- and
1D-expressing cells was also examined on Fn by time lapse videomicroscopy in the presence
of blocking mAbs PB1 for CHO and H9.2B8 for GD25
transfectants (Fig. 6). In both cases when the endogenous
Fn-binding integrins were inhibited, the mean cell speed of
1D transfectants appeared to be drastically reduced compared to that of
1A-expressing cells. Activating TS2/16 mAb significantly decreased migration mediated by
1A
integrin but did not alter the migratory behavior of
1D-expressing cells. The migration rates of
1A-transfected
CHO and GD25 cells treated with TS2/16 mAb still exceeded
substantially the migration rates of
1D-transfectants.
Interaction of 1A and
1D Integrins with
Subunits
in CHO and GD25 Transfectants
Many of the observed properties of 1D including enhanced ligand binding, elevated Fn matrix assembly, and
decreased cell motility could be explained by different
mode of association of
1A and
1D integrins with
subunits in the transfected cells. Therefore, we examined
subunit association for
1A and
1D in CHO and GD25
transfectants. Among various
1-associated
subunits,
3,
5, and very small amount of
v were detected in both
types of transfectants by immunoprecipitation (Fig. 7). Immunoblotting of the corresponding immunoprecipitates
from
1D-CHO (Fig. 7, A and B),
1A-GD25 (Fig. 7, C
and D), and
1D-GD25 (Fig. 7, E and F) cells showed that
equal amounts of
1D and
1A integrins were associated
with
3 and
5 subunits in CHO and GD25 transfectants.
Cytoskeletal Association of 1A and
1D Integrins
Correlates with Their Insolubility in Digitonin
To determine whether there is a difference in cytoskeletal
association between 1A and
1D integrins, we designed
a method of sequential extraction using digitonin and
RIPA buffers for 35S-labeled cell cultures, followed by integrin immunoprecipitation from both cellular fractions. To
test whether integrin insolubility in digitonin is determined by the mode of integrin-cytoskeleton association,
we compared
1 integrin immunoprecipitates from digitonin and RIPA fractions of untreated and cytochalasin D-treated
1A- and
1D-transfected CHO cells (Fig. 8). Treatment
of cultured cells with cytochalasin D shifted almost all
1A and the majority of
1D to the digitonin-soluble fraction. These experiments demonstrated that insolubility of
1 integrins in digitonin depends on their association with
the actin cytoskeleton.
1D Integrin Displaces the Endogenous
1A and
v
Subunits from Focal Adhesions and Associates Strongly
with the Digitonin-insoluble Cytoskeleton
Since 1D and
1A integrins have structurally different
cytoplasmic domains, and the two types of transfectants
displayed dissimilar phenotypes, we next attempted to
compare cytoskeletal interactions of the
1A and
1D isoforms. To localize the transfected and the endogenous
1
integrins in CHO transfectants, cells grown on Fn were
double stained with anti-human
1 and anti-hamster
1
mAbs. In
1A-CHO cells, both the transfected and the endogenous
1A subunits colocalized at focal adhesions
(Fig. 9, A and C).
1D integrin was prominently localized
at focal adhesions of
1D-CHO cells. Surprisingly, no endogenous
1A integrin was detected in focal adhesions of
1D transfectants grown on Fn or other extracellular matrix proteins (Fig. 9, B and D; and data not shown). Similarly, both the transfected
1A and
1D integrins were
targeted to focal adhesions of GD25 transfectants on Fn
(Fig. 9, E and F). The endogenous
v subunit of Fn-binding
v
3 integrin in
1A-GD25 cells was at least partially
colocalized with
1A at sites of cell-matrix contact (
v
does not pair with
1 integrins in GD25 cells; Wennerberg et al., 1996
). In contrast,
1D displaced the endogenous
v
integrins from focal adhesions (Fig. 9, G and H). Therefore, the displacement of
1A and
v integrins from focal
adhesions by expressed
1D was a general phenomenon,
suggesting a considerably stronger association of
1D integrin with the actin cytoskeleton.
To define biochemically the modes of 1A and
1D interaction with the cytoskeleton, both the transfected
1A
and
1D, as well as the endogenous
1A and
v integrins
were immunoprecipitated from soluble and cytoskeleton-associated fractions of 35S-labeled transfectants grown on
Fn (Fig. 9, I and J). In
1A-CHO cells, the transfected
1A was equally distributed between the soluble and the
cytoskeletal fractions, wheras the majority of the endogenous hamster
1A subunit was associated with the cytoskeleton. In contrast,
1D was found exclusively in the cytoskeletal fraction, whereas almost all the endogenous
1A integrin was present in the soluble fraction of
1D-CHO cells, displaced from the cytoskeleton (Fig. 9 I). Cytoskeletal associations of the transfected
1A and
1D
integrins in GD25 transfectants were similar to those observed in CHO transfectants. Again, much of the transfected
1A integrin was digitonin soluble. However, the
majority of
1D integrin was digitonin insoluble, whereas
most of the endogenous
v
3/
v
5 integrins were displaced from their association with the cytoskeleton by the
transfected
1D (Fig. 9 J).
Differential Interaction of 1A and
1D Integrins with
Talin and
-Actinin
At least two cytoskeletal proteins, talin and -actinin, are
known to interact in vitro with the cytoplasmic domain of
1A integrin (Horwitz et al., 1986
; Otey et al., 1990
). To
identify cytoskeletal proteins, associated preferentially
with the
1D integrin subunit in vivo, antibody clustering
of the transfected
1A and
1D integrins on the surface of
CHO and GD25 transfectants was used in combination
with subsequent immunoprecipitation and analysis of the
immunoprecipitates (Miyamoto et al., 1995
). Several cytoskeletal proteins, including actin, talin,
-actinin, and vinculin coprecipitated with the transfected
1 integrins
(data not shown). When
1A and
1D immunoprecipitates, obtained with activating TS2/16 anti-human
1 mAb,
were compared by immunoblotting with mAb 8d4 against
talin, a significantly stronger talin band was detected in association with
1D integrin in both CHO and GD25 transfectants (Fig. 10, A, a, a
, b, and b
; and D, a, a
, b, and b
).
This preferential association of talin with
1D compared with
1A integrin did not depend on the nature of anti-
1
mAb used for clustering. 12G10 mAb, which recognizes a
Mn2+- and ligand-induced conformation of human
1 integrin, precipitated more talin from
1D-CHO than from
1A-CHO cells both in the absence or in the presence of 1 mM Mn2+ (Fig. 10, A, c, c
, d, and d
). In control immunoprecipitations with the 7E2 mAb against the endogenous
hamster
1 integrin, equal amounts of talin were detected
in association with the endogenous
1A in CHO transfectants (Fig. 10, A, e, and e
). Interestingly, when anti-human
1 integrin immunoprecipitates from both types of transfectants were probed for
-actinin, more
-actinin was detected in association with
1A than with
1D integrin
(Fig. 10, A, f, and f
; and D, c, and c
). To ensure equal
amounts of these isoforms in the immunoprecipitates, all
the samples used in these experiments were also examined
with the isoform-specific antibodies against
1A (Fig. 10,
B and E) and
1D (Fig. 10, C and F) integrins (Belkin et
al., 1996
). Besides the immunoprecipitates from
1A-CHO and
1D-CHO cells with the conformation-specific mAb 12G10 in the absence of Mn2+ (Fig. 10, B, c; and C,
c
), all other samples contained equal amounts of
1 integrins (Fig. 10, B, C, E, and F).
To compare further the interactions of 1A and
1D integrins with talin and
-actinin, in vitro solid phase binding
assays were performed with 125I-talin, 125I-
-actinin, and
immobilized full-length synthetic peptides, corresponding to the
1A and
1D cytoplasmic domains (Fig. 11). First,
we tested the binding of 125I-labeled
1A and
1D cytoplasmic domain peptides to the microtiter wells (Fig. 11 A).
The amounts of the
1A and
1D peptides adsorbed to
the wells were almost indistinguishable within the wide
range of concentrations examined.
Then, using the solid phase binding assay we more fully
characterized the interactions of 125I-talin and 125I--actinin with the
1A and
1D cytoplasmic domain peptides
(Fig. 11, B and C). We found that the binding of 125I-talin
to the
1D peptide was severalfold higher than the binding to the
1A peptide. The apparent dissociation constants of 4.2 × 10
8 and 5.9 × 10
9 M were calculated from
the competition binding curves for talin interactions with
the
1A and
1D cytoplasmic peptides, respectively (Fig.
11 B). Conversely, 125I-
-actinin bound more strongly to
the
1A than to the
1D cytoplasmic domain peptide (Fig.
11 C). In this case the dissociation constants were 1.2 × 10
8 M for
-actinin-
1A binding and 8.8 × 10
8 M for
-actinin-
1D binding. Together, the coimmunoprecipitation analyses and in vitro binding data demonstrated that
talin interacts more strongly with the cytoplasmic domain
of
1D than
1A integrin, whereas
-actinin interacts
preferentially with the
1A integrin isoform.
1D Integrin Increases Cellular Contractility
The enhanced association of 1D integrin with the actin
cytoskeleton prompted us to examine whether the
1D integrin-cytoskeleton interaction increases contractility. When
1A and
1D transfectants were plated on flexible silicone
rubber substrata for 1 d,
1D-transfected cells generated
prominent wrinkles within the substrata. However,
1A
transfectants were essentially unable to wrinkle these substrata (Fig. 12, A-D). Interestingly, the enhancement of
contractility was not accompanied by substantially elevated phosphorylation of myosin regulatory light chains in
1D-CHO cells (Fig. 12 E). Therefore, the observed increase in cellular contractility of
1D transfectants appears to be mostly because of enhancement of actin-membrane attachment, and did not depend on significant changes of myosin ATPase activity.
In this work we have analyzed muscle 1D integrin, comparing its properties with those of the common
1A isoform. We found that the unique cytoplasmic sequence of
1D endows this molecule with the distinctive functional
properties.
1D integrin displays an increased affinity for
both the extracellular matrix ligands and the actin cytoskeleton. Expression of
1D causes a marked phenotypic
conversion of both CHO and
1-deficient GD25 cells. The
1D phenotype includes altered morphology, retarded
spreading, enhanced ligand binding, and extracellular matrix assembly, as well as reduced migration and significantly increased contractility.
Together, the increased integrin-ligand and integrin-
cytoskeleton associations mediated by the 1D isoform,
cause a significant reinforcement of the entire cytoskeleton-matrix link. Unlike two other
1 integrin variants
1B
and
1C, which are distributed uniformly at the cell surface and are unable to interact with the actin cytoskeleton,
1D is readily targeted to sites of cell-matrix adhesion
upon expression in nonmuscle cells. Furthermore, significantly stronger association of
1D with the actin cytoskeleton can be mediated by its enhanced interaction with
talin and might change the organization of focal adhesions
in
1D transfectants compared with these structures in
1A-expressing cells. However, by immunofluorescence with a large panel of antibodies against cytoskeletal and
focal adhesion proteins, we did not observe prominent differences in the organization of stress fibers and focal adhesions between
1D and
1A transfectants. The intensities
of immunostaining for talin, vinculin,
-actinin, paxillin,
focal adhesion kinase, and phosphotyrosine at focal adhesions were similar in both types of transfectants (data not
shown). Apparently, the differences in the affinities between
1A/
1D and talin/
-actinin might be insufficient
to detect preferential accumulation of these cytoskeletal proteins at focal adhesions of
1A- and
1D-transfected
cells by immunofluorescence. Nevertheless,
1D transfectants appeared to be more contractile without changes in
myosin light chain phosphorylation and displayed increased resistance of focal adhesions against disassembly
by contractility inhibitors BDM and H7 (data not shown). These observations pointed to the increased stability of focal adhesions in cells expressing
1D integrin that occurs
primarily because of enhancement of actin-membrane attachment.
Simultaneously, displacement of the endogenous 1A
subunit from sites of cell-matrix adhesion by
1D generates a unique "dominant-positive phenotype" of the transfected nonmuscle cells. In this situation, the
1 integrin-mediated, cytoskeleton-matrix link is built exclusively by
the
1D integrin and lacks the
1A isoform. Displacement
of the major
1A integrin isoform from cell-matrix adhesion sites appears to be a general phenomenon of
1D expression, caused by its enhanced association with the actin
cytoskeleton. This displacement is observed on various extracellular matrix substrata in a number of nonmuscle cells
(Belkin, A.M., unpublished data). Similarly, in GD25 transfectants the major endogenous
v
3 integrin appeared to
be displaced from the sites of cell-matrix contact. The altered structure of the
1D integrin cytoplasmic domain also
leads to a conformational change in its ectodomain that increases the ligand-binding affinity of
1D-containing heterodimers. The constitutive activation of
1D on the cell
surface, combined with its stronger interaction with the actin cytoskeleton, enhance Fn matrix assembly by
1D transfectants (Wu et al., 1995
).
Recently, Kucik et al. (1996) demonstrated that nonadhesive inactive state of LFA-1 integrin in lymphocytes is
maintained by the cytoskeleton. Both PMA and cytochalasin D were shown to stimulate lymphocyte adhesion, which
was accompanied by activation of their major
2 integrin
and its release of cytoskeletal constraints. This points to
the opposite mechanisms of cytoskeletal control of integrin activation between
2 and
1 integrin subfamilies and
might reflect profoundly dissimilar physiology of nonadherent lymphocytes and adhesion-dependent cells, including striated muscles.
Previously, modulation of the ligand-binding affinity of
integrin subunits was demonstrated using either activating antibodies (Arroyo et al., 1992
; Faull et al., 1993
) or
point mutations in the
1 and
3 subunit cytoplasmic domains (Ginsberg et al., 1992
; Takada et al., 1992
; O'Toole
et al., 1994
, 1995
; Schwartz et al., 1995
). Here we present
the results showing an existence of physiological mechanism reinforcing the cytoskeleton-matrix link in muscle
cells based on modulation of integrin adhesive function. This
mechanism involves a novel type of inside-out integrin signaling where activation of the extracellular domain of the
integrin
subunit is controlled by alternative splicing of its
cytodomain. Since the expression of
1D and
1A isoforms is developmentally regulated in muscle (Belkin et al.,
1996
), differentiating muscle cells can control the overall
strength of cytoskeleton-matrix attachment via alternative splicing of the
1 integrin subunit.
The organization of focal adhesions in 1D-transfected
nonmuscle cells appears to be very similar to that found in
differentiated muscle cells. In both situations,
1D is the
only
1 isoform localized at cell-matrix adhesive structures (Belkin et al., 1996
). Therefore, the enhanced cytoskeleton-matrix association and stabilization of focal adhesions, mediated by
1D in the transfectants, might reflect the major role for this integrin in muscle. Since
talin, a major structural component of focal adhesions, accumulates at muscle adhesions (Belkin et al., 1986
; Tidball
et al., 1986
), it can also serve as a key cytoskeletal element
linking
1D integrin to actin filaments in muscle. In contrast, we found that
-actinin, a focal adhesion component
that interacts with
1A in vitro (Otey et al., 1990
), binds
1D less strongly than
1A. This correlates with the absence of
-actinin at myotendinous junctions, the major sites of force transmission in muscle (Tidball, 1987
). Additionally, certain muscle-specific cytoskeletal proteins, including dystrophin, may contribute to linking
1D integrin
to the subsarcolemmal cytoskeleton.
Many existing models of focal adhesion assembly imply
that their formation proceeds from outside the cell inward,
starting from integrin clustering by their ligands on the cell
surface (Yamada and Miyamoto, 1995; Craig and Johnson,
1996
). However, rho-stimulated contractility has been shown
to drive the formation of integrin-containing focal adhesion complexes from inside the cell (Hotchin and Hall,
1995
; Burridge and Chrzanowska-Wodnicka, 1996
; Chrzanowska-Wodnicka and Burridge, 1996
). Also, recent data on muscle integrins in Drosophila melanogaster point to
certain ligand-independent intracellular mechanisms directing localization of
ps integrins (analogous to
1 integrins in vertebrates) to sites of their function in embryonic
muscles (Martin-Bermudo and Brown, 1996
). The altered
structure of the
1D cytoplasmic domain and the enhanced interaction of this integrin with the cytoskeleton might determine its ligand-independent targeting to muscle adhesive structures.
Increased Fn matrix assembly by CHO cells expressing
1D can be driven by a combination of higher ligand-binding affinity of this integrin, reinforced actin-membrane association and enhanced contractility of
1D transfectants.
Recently, it was shown that the appearance of new Fn matrix assembly sites on the cell surface is stimulated by lysophosphatidic acid, an agent that promotes contractility
(Jalink and Moolenaar, 1992
; Zhang et al., 1994
; Chrzanowska-Wodnicka and Burridge, 1996
). Wu and coworkers demonstrated that both integrin activation on the cell
surface and integrin-cytoskeleton interactions (postoccupancy events) are essential for the assembly of a Fn matrix
(Wu et al., 1995
). Our data are in agreement with these
findings. The increased Fn matrix assembly mediated by
1D correlates well with the larger proportion of activated
integrins on the cell surface, the more stable integrin-
cytoskeleton linkage and enhanced contractility in these
transfectants. Interestingly, the observed enhancement of
Fn matrix assembly by
1D appeared to be greater than
the increase in ligand-binding affinity for
1D transfectants. Even though an activation of
1A integrin with TS2/
16 mAb significantly increased Fn matrix assembly by
1A transfectants, this still did not convert them entirely
to the
1D phenotype. These differences might reflect the
enhanced integrin-cytoskeleton association and contractility in
1D transfectants.
Finally, the increased ligand-binding, contractility, and
Fn matrix deposition contribute to the reduced cell migration of 1D transfectants. In accordance with recent findings of Palecek et al. (1997)
, activation of the
1A ectodomain reduced the migration of the transfectants at substrate
concentrations and integrin expression levels used in our
experiments. Notably, integrin activation in the case of
1A transfectants did not decrease their migration rates to
the levels characteristic for
1D-expressing cells, again suggesting an important role for integrin-cytoskeleton interactions in the generation of the
1D phenotype. Together, our data demonstrate that the whole set of alterations displayed by cells expressing
1D, is determined by
the distinctive structure of the
1D cytoplasmic domain.
The alternatively spliced sequence of this
1 integrin isoform both enhances its association with the actin cytoskeleton and increases receptor-ligand interaction due to constitutive activation of the
1D ectodomain. Both these
factors contribute to increased Fn matrix assembly and decreased migration of
1D transfectants.
The changes of nonmuscle cells triggered by expression
of the 1D isoform are analogous to the transitions that
muscle cells undergo during differentiation, accompanied
by a gradual increase in the expression of this integrin
(Belkin et al., 1996
). Thus, growing myotubes possess large,
extremely stable adhesions, their spreading is greatly inhibited and even early immature myotubes cease to migrate. Although Fn matrix assembly is strongly decreased in muscle cells, upregulation of synthesis and enhanced
deposition of certain laminin isoforms is typical for myodifferentiation both in culture and in vivo. During myodifferentiation, a dramatic increase in cellular contractility
has to be counterbalanced by the reinforcement of the cytoskeleton-matrix link. All these phenotypic transitions
characteristic for differentiating muscle cells generate a requirement for the novel integrin, strengthening the association between the actin cytoskeleton and the surrounding
extracellular matrix. This requirement determines the distinctive properties of
1D and defines a critical role for
this
1 integrin cytoplasmic domain in the organization
and function of adhesive structures in muscle tissues.
Received for publication 16 May 1997 and in revised form 3 July 1997.
Address all correspondence to A.M. Belkin, Department of Biochemistry, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: (301) 738-0725. Fax: (301) 738-0794. E-mail: Belkina{at}usa.redcross.orgWe are grateful to Dr. L. Arnold (University of North Carolina at Chapel Hill) for his advice and help with fluocytometry.
This work was supported by National Institutes of Health grants R29 CA72961 to A.M. Belkin, and GM29860 and HL45100 to K. Burridge, and grants from the Italian Association for Research on Cancer, Biomed and Telethon to G. Tarone.
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