Department of Molecular Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel
* Author for correspondence (e-mail: benny.geiger{at}weizmann.ac.il)
Accepted 19 November 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell-matrix adhesion, Focal adhesions, Tyrosine phosphorylation, Vinculin, Paxillin, Focal adhesion kinase, FAK
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The assembly and maintenance of cell-matrix adhesions is regulated by
signaling via small GTPases of the Rho family
(Clark et al., 1998;
Rottner et al., 1999
). It was
shown that the activation of Rac-1 induces focal complex formation
(Nobes and Hall, 1995
;
Rottner et al., 1999
), whereas
the introduction of constitutively active Rho-A leads to the formation of
large FAs and stress fibers (Ridley et
al., 1992
). Rho acts by activating several immediate targets,
including Rho-associated kinase (ROCK) and mDia that synergistically affect
cell-matrix adhesions and the associated microfilament network
(Watanabe et al., 1999
;
Takaishi et al., 2000
;
Tominaga et al., 2000
;
Riveline et al., 2001
). Active
ROCK inhibits myosin light chain phosphatase
(Kimura et al., 1996
), thus
causing the stimulation of actomyosin contractility. In turn, application of
local forces by actomyosin contractility stimulates FA and stress fiber
formation (Chrzanowska-Wodnicka and
Burridge, 1996
; Leopoldt et
al., 2001
), whereas inhibitors of actomyosin contractility induce
disruption of these structures (Volberg et
al., 1994
; Helfman et al.,
1999
; Zamir et al.,
1999
; Balaban et al.,
2001
). Growth of FAs and focal complexes can also be induced by
the application of external local force
(Riveline et al., 2001
;
Kaverina et al., 2002
).
Another key regulatory mechanism affecting FA formation and stability is
tyrosine phosphorylation. FAK (Calalb et
al., 1995) and members of the pp60Src family
(Brown and Cooper, 1996
) are
involved in the phosphorylation of several FA proteins, including paxillin
(Turner, 2000
),
p130Cas (Vuori and Ruoslahti,
1995
), tensin (Bockholt and
Burridge, 1993
) and FAK itself
(Schaller, 2001
). This
phosphorylation provides docking sites for additional molecules that contain
phosphotyrosine (PY)-binding domains such as Src-homology 2 (SH2) (for
reviews, see Zamir and Geiger,
2001
; Pawson et al.,
2001
). Serum starvation, as well as treatment with actomyosin
relaxants or tyrosine kinase inhibitors, reduce PY levels and block the
development of FAs (Ridley and Hall,
1994
; Chrzanowska-Wodnicka and
Burridge, 1994
; Barry and
Critchley, 1994
; Bershadsky et
al., 1996
). Furthermore, strong activity of PY phosphatases can
disrupt FAs (Schneider et al.,
1998
), whereas their inhibition can stimulate FA assembly, at
least transiently (Retta et al.,
1996
; Ayalon and Geiger,
1997
). Interestingly, overexpression of deregulated
pp60v-Src results in high levels of tyrosine phosphorylation and
disrupts FAs (Kellie et al.,
1986
), and Src (/) cells form FAs that fail to turn
over into fibrillar adhesions (Volberg et
al., 2001
). Taken together, these data suggest that tyrosine
phosphorylation is involved in the formation and turnover of matrix
adhesions.
FA growth can also be triggered by microtubule depolymerization in a Rho-
and tyrosine phosphorylation-dependent manner
(Bershadsky et al., 1996;
Enomoto et al., 1996; Zhang et al.,
1997
; Liu et al.,
1998
; Pletjushkina et al.,
1998
). Although the exact mechanism is still unclear, microtubule
disruption appears to promote FA growth by increasing overall actomyosin
contractility (Bershadsky et al.,
1996
). Interestingly, microtubules also seem to influence the
formation and turnover of cell-matrix adhesions locally
(Kaverina et al., 1999
) and
the effect of microtubule disruption on FAs and cell motility can be abolished
by local application of contractility inhibitors (Kaverina et al., 2000).
Together, these results suggest that microtubules suppress FA growth by local
inhibition of actomyosin contractility. Nocodazole-mediated stimulation of FA
growth might therefore mimic a natural release of FA from this negative
regulation.
In this study, we investigated the involvement of tyrosine phosphorylation in nocodazole-induced FA assembly. In addition to analysis of fixed specimens using quantitative immunofluorescence microscopy, we introduced a new technique that made it possible to monitor PY dynamics in live cells. For this purpose, we employed a YFP derivative of the PY-binding SH2 domain of pp60c-Src (YFP-dSH2). Using this approach, we demonstrated that the increase in tyrosine phosphorylation occurs only after an apparent recruitment of several FA proteins takes place and does not directly correlate with FA growth. Thus, microtubule-disruption-induced tension stimulates the binding of new molecules such as vinculin, paxillin and FAK to FAs independently of a local increase in tyrosine phosphorylation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunochemical reagents
The rabbit anti-PY antiserum was kindly provided by Israel Pecht and Arie
Licht (The Weizmann Institute, Rehovot). Anti-vinculin (-Hvin)
monoclonal antibody was from Sigma. Anti-FAK and anti-paxillin monoclonal
antibodies were from Transduction Laboratories (Lexington, KY). All secondary
antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).
Generation of YFP-SH2 constructs
The pp60Src SH2 domain (corresponding to amino acids 142-251)
(Martinez et al., 1987) was
amplified by PCR, adding 5' HindIII and 3' XbaI
sites to the sequence. This DNA was then inserted in-frame into the
corresponding sites in the EYFP-C3 vector modified from EGFP-C3 vector
(Clontech, Palo Alto, CA). To generate the YFP-dSH2 construct, the SH2 domain
was amplified again, but this time with a HindIII site attached to
both ends of the fragment. This piece of DNA was then ligated to the
HindIII site of YFP-SH2, and clones containing the insert in the
correct orientation were selected. To create the mutated SH2 domain, a primer
containing the desired changes (R183A) was used to generate the part of the
SH2 upstream of the mutation. The resulting PCR fragment was then used as a
5' primer in a second PCR as previously described
(Landt et al., 1990
). The PCR
fragment of the mutated SH2 domain was then cloned into EYFP-C3 analogous to
the wild-type construct to generate mutated YFP-SH2.
Digital microscopy
Immunofluorescence microscopy of fixed samples was carried out with a Zeiss
Axiovert 100 microscope equipped with a x100/1.4 NA plan-Neofluar
objective (Zeiss, Oberkochen, Germany). Images were acquired with a
DeltaVision system (Applied Precision Inc., Issaqua, WA) as previously
described (Zamir et al.,
1999), except that the pixel size was 0.066 µm. Image
acquisition and processing were performed with Resolve3D and Priism programs
as described (Zamir et al.,
1999
).
For dynamic studies, cells were cultured in coverslip-bottom dishes (MatTek corporation, Ashland, MA). 12 hours after transfection, the cells were serum starved for another 12 hours. The carbonate-buffered DMEM was then exchanged for DMEM containing 25 mM HEPES (Bio Lab, Jerusalem, Israel) and nocodazole was added 30 minutes thereafter. Live-cell imaging was carried out with a back-illuminated frame transfer grade 1 Quantics CCD camera equipped with an EEV 57-10 G1 Chip (Photometrics, Tucson, AZ), generating 12-bit digital data. The microscope contained a filter set for Cyan GFP (No. F31-044, AHF Analysentechnik, Tübingen, Germany) and a nonselective FITC filter set (Zeiss, Oberkochen, Germany), which was used to record YFP. The objective and stage were heated to maintain the cells at a stable temperature of 37°C throughout the experiment. Images were taken every minute for 30 minutes after addition of nocodazole and processed in the same way as in the case of fixed samples. For the measurements of YFP-dSH2 fluorescence, we first determined the range of fluorescence intensity, which corresponds to YFP-dSH2 expression level exerting no apparent effects on PY staining and appearance of FAs. All further measurements were conducted on cells expressing YFP-dSH2 in that range.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
By 10 minutes of incubation with nocodazole, the adhesion sites became elongated, reaching several µm in length. The labeling intensity for all three tested proteins and of PY also increased (Fig. 4). The internal distribution of PY and the three proteins within the newly formed FA was often non-uniform; commonly, vinculin was enriched at the pole of FAs, pointing to the cell center (Fig. 1, arrowheads). A similar shift was also occasionally observed with paxillin. Upon longer incubation (up to 30 minutes), the local intensities of vinculin and paxillin, as well as the size of FAs, continued to increase, and then declined. By contrast, FAK and PY intensity reached a plateau at 10 minutes and sharply declined at 1 hour after addition of the drug. Taken together, these data indicate that the tension-induced binding of FA components and FA growth precede the stimulation of local tyrosine phosphorylation, and that different FA components may display different recruitment kinetics.
The dynamics of tyrosine phosphorylation following nocodazole
treatment: use of YFP-dSH2 as a PY reporter in live cells
In view of the rapid local changes in tyrosine phosphorylation observed
upon nocodazole stimulation, it was desirable to examine, in real-time, the
dynamic changes of PY levels in FAs of live cells. Towards this end, we
constructed novel, intrinsically fluorescent, `PY reporters', namely
YFP-fusion proteins containing one or two copies of the PY-binding domain
(SH2) of pp60Src (Fig.
5A,B). Both molecules localized in cultured cells to FAs, although
theYFP-SH2 fusion protein (C) produced a higher cytoplasmic background than
YFP-dSH2 (D). By contrast, a SH2 domain mutated at Argßb5 (R183A), which
has a 200-fold reduced affinity to PY
(Bradshaw et al., 1999), did
not show any FA localization at all. The FA localization of YFP-SH2 was not
retained after fixation and permeabilization of the cells (E), whereas
FA-associated YFP-dSH2 was also found in FA of fixed cells (F). We therefore
used the YFP-dSH2 construct for live-cell experiments.
|
In order to test whether YFP-dSH2 can be used as a reliable quantitative reporter for local PY levels, we transfected SV80 cells with YFP-dSH2, fixed the cells 24 hours later and stained them with an anti-PY antibody. The images showing the YFP-dSH2 distribution (Fig. 6A,B) and the corresponding anti-PY staining (C,D) were then subjected to ratio imaging and quantitative analysis. The images in Fig. 6 (E,F) represent, in the spectrum scale, the ratio between the intensities of YFP fluorescence and the fluorescence of the fluorochrome (Cy3) conjugated with the secondary antibody used for visualizing PY. This ratio was highly uniform inside the same cell, and throughout individual FAs (E,F).
|
The relationship between the PY antibody and YFP-dSH2 labeling was studied further by plotting, against each other, the intensities of the two labels measured for individual FAs. As can be seen in Fig. 6 (G,H), the two labels show a linear correlation over a wide range of expression levels. These experiments indicated that expression of particularly high levels of YFP-dSH2 leads to a marked elevation (up to fivefold, compared with nontransfected cells) in PY levels (G,H), as well as to a moderate increase in the average size of FAs (quantitative data not shown). By contrast, moderate levels of expression of YFP-dSH2 (see, for example, cell 5 in Fig. 6) have no detectable effect on the intensity of PY labeling as compared with nontransfected cells. Moreover, the high correlation between YFP-dSH2 and anti-PY labeling indicated that the recruitment of the former into phosphorylated adhesion sites is very rapid. Apart from its effect on endogenous PY, we tested the influence of YFP-dSH2 expression on the levels of vinculin, paxillin, FAK and tensin in FAs. These studies indicated that expression of YFP-dSH2 at levels that do not disturb the PY distribution has no effect on the level and organization of these proteins either (data not shown). On the basis of these data, we have regarded YFP-dSH2, when expressed at appropriate levels (e.g. cell 5, but not cells 1 or 4, in Fig. 6), as an essentially nonperturbing quantitative PY reporter. YFP-dSH2 localization to FAs was also observed in several other cell lines, including fibroblasts REF 52, NIH 3T3 and MEF, as well as HeLa and MDCK epithelial cells where cell-cell junctions were also labeled (data not shown).
In order to compare the recruitment of vinculin and the increase in tyrosine phosphorylation in the same cells in real-time, SV80 cells were co-transfected with CFP-vinculin and YFP-dSH2, then serum starved for 12 hours and subjected to nocodazole treatment (see Movie 1, http://jcs.biologists.org/supplemental). Before nocodazole treatment, the residual adhesion sites in the starved cells contain very little vinculin (Fig. 7). Within 2 minutes after application of nocodazole, vinculin starts to accumulate at the cell margins, inside and around peripheral focal complexes and small FAs. After 6 minutes, vinculin becomes confined to FAs, which continue to grow until 10 minutes. By contrast, the early changes in PY levels are much less dramatic and uniform. After 2 minutes, the majority of FAs maintain their initial level of tyrosine phosphorylation, and sometimes show a decrease in tyrosine phosphorylation or even vanish (Fig. 7), whereas a few show a mild increase in PY. By 6 minutes, most adhesions show an increase in YFP-dSH2 levels, which continues until 10 minutes. FA growth according to both labels is almost exclusively oriented towards the cell center. As was already observed with fixed cells (Fig. 1), vinculin is shifted towards the cell center relative to PY.
|
|
The two-color movies were very effective for comparing, in real-time, the
distribution of different components, but were limited to a relatively small
number of double exposures (usually 10) owing to possible photodamage. To
monitor tyrosine phosphorylation dynamics over a longer period and to compare
the changes in PY intensity with the growth of FAs, we examined, by time-lapse
fluorescence microscopy, cells transfected with YFP-dSH2 alone. The cells were
serum starved for 12 hours, treated with nocodazole and examined
microscopically for up to 30 minutes. Fig.
9 shows images derived from such a movie (see also Movie 2,
http://jcs.biologists.org/supplemental).
Each image is the ratio of two different time points (fluorescence ratio of
images, `FRIT image'), highlighting the increase in the area and intensity of
PY-positive matrix adhesions induced by nocodazole. The color scale was
selected so that structures that remain unchanged between the two time points
appear yellow, whereas new structures are depicted in red and those that
disappeared are depicted in blue. As shown, some adhesions were formed de novo
following addition of nocodazole (Fig.
9, arrow), whereas others disappeared (arrowheads). As was already
observed in the vinculin/dSH2 movie, expansion of the tyrosine-phosphorylated
area of FA is primarily oriented towards the cell center. Quantitative
analysis of FA phosphorylation in selected adhesions (marked on the 6'/2'
frame of Fig. 9) shows that all
but one of the tested adhesions grow substantially in area (1.5- to 3-fold)
during the 30 minutes of stimulation, and the recorded growth and PY dynamics
are in good agreement with the data obtained for fixed cells and in the
two-color movie. The increase of PY levels in individual adhesions started
only 3-5 minutes after nocodazole application and reached maximal values
between 6 and 18 minutes after nocodazole stimulation (average time
10
minutes). It is noteworthy that remarkable differences were observed between
the kinetics of tyrosine phosphorylation of individual (sometimes even nearby)
FAs within the same cell. For example FA I and III reached maximal intensity
of YFP-dSH2 at 6 minutes (Fig.
9), whereas FA II and IV reached maximal intensity at 18 minutes.
Moreover, the extent of local increase in YFP-dSH2 intensity did not correlate
with the rate of growth of the adhesion site. For example, the dynamics of
local tyrosine phosphorylation were similar for FA I and III (peak at 6
minutes and then decline), yet FA I grew during the whole period, whereas FA
III hardly changed.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FA assembly is a multistage process that involves the transformation of
small, dot-like focal complexes into large FAs (see
Geiger et al., 2001). Previous
studies have shown that application of mechanical force to focal complexes,
either by increasing cytoskeletal contractility or by external perturbation,
stimulates this transformation
(Chrzanowska-Wodnicka and Burridge,
1996
; Bershadsky et al.,
1996
; Leopoldt and Rozengurt, 2001;
Riveline et al., 2001
) and
that this process is centrally coordinated by Rho-family GTPases
(Ridley and Hall, 1992
;
Rottner et al., 1999
). It was
further proposed that matrix adhesions contain a putative `mechanosensor'
whose activation by local tension leads to a cascade of molecular events,
including the recruitment of FA proteins, growth of adhesion sites and
stimulation of integrin-mediated signaling (reviewed by
Geiger and Bershadsky, 2001
;
Geiger and Bershadsky, 2002
;
Riveline et al., 2001
).
Determining how these three processes are interrelated and elucidating the
role of tyrosine phosphorylation were the main objectives of the present
work.
We have considered here two alternative mechanisms for force-induced assembly of FAs. One possibility is that mechanical perturbation directly activates local tyrosine phosphorylation which, in turn, creates new docking sites that bind SH2-containing proteins, leading to the recruitment of different plaque proteins and, consequently, to FA consolidation and growth. Alternatively, the activation of the mechanosensor could directly stimulate the binding of new FA proteins, independently of PY signaling.
The present study supports the latter view. We show here that
nocodazole-induced changes in FA composition and size are not synchronous.
Thus, FA growth is apparent essentially immediately after stimulation, and is
accompanied by an increase in the local densities of its constituents,
vinculin, paxillin and FAK. By contrast, PY levels are not significantly
altered during the first 2-3 minutes after stimulation, and start to increase
only 3-5 minutes after addition of the drug. Thus, the increase in tyrosine
phosphorylation appears to be a consequence of nocodazole-induced protein
recruitment rather than its cause. The notion that protein recruitment is the
primary response to mechanical perturbation to integrin-mediated adhesions is
in line with a recent report of Sawada and Sheetz
(Sawada and Sheetz, 2002), who
showed that the recruitment of FA molecules such as FAK and paxillin can be a
direct consequence of tension-induced changes in the actin cytoskeleton and/or
the integrin-associated adhesion plaque in membrane-permeabilized cell
models.
Furthermore, the recruitment kinetics of different components after
stimulation was not the same, leading to time-dependent changes in FA
composition. As shown here, the initial increase in local intensities of all
three proteins occurred essentially simultaneously, yet FAK levels reached a
plateau at 10 minutes after addition of nocodazole, whereas vinculin and
paxillin levels usually reach maximal values at about 30 minutes. It is
interesting to note that PY levels also reached a plateau at 10 minutes
(Fig. 4), supporting the view
that FAK is indeed a major regulator of tyrosine phosphorylation in FAs.
The immunofluorescence data discussed above provided strong, yet indirect, evidence for sequential molecular changes in FAs, induced by nocodazole, starting with the recruitment of FA plaque proteins and followed by tyrosine phosphorylation. To substantiate these findings directly at a single cell, or even single FA, level we had to develop an in vivo approach for visualizing tyrosine phosphorylation at a high spatial and temporal resolution. The YFP-dSH2 construct described here for the first time appears to serve as an accurate, quantitative PY reporter for live cells. This fusion protein readily localizes to FAs, its local intensities correlate linearly with those of anti-PY immunostaining and, at moderate expression levels, the protein has no apparent effect on the organization, PY levels and molecular composition of FAs.
As shown here, we have noticed that overexpression of YFP-dSH2 induces an
increase in PY levels, most probably due to the protection of
tyrosine-phosphorylated sites from phosphatases. Previous studies demonstrated
that inhibition of PY phosphatases by chemical inhibitors can induce a rapid
increase in the levels of FA-associated PY
(Retta et al., 1996;
Ayalon and Geiger, 1997
). To
avoid such effects, we have carefully defined a suitable range of expression
levels of YFP-dSH2, which enables visualization of adhesion sites without
altering their properties.
Another issue that should be considered here is the specificity of
YFP-dSH2. Although SH2 domains, in general, may be selective for certain
phosphorylated targets (Sawyer,
1998), the pp60Src-SH2 domain was found to bind to a broad
spectrum of tyrosine-phosphorylated proteins, even when competing with other
SH2 domains (Nollau and Mayer,
2001
). Moreover, the distribution of YFP-dSH2 was essentially
identical to that of several antibodies to PY, suggesting that the YFP-dSH2
molecule is a spatially and temporally faithful, and broad-specificity,
reporter of local tyrosine phosphorylation in cell-matrix adhesions.
As discussed above, the YFP-dSH2 construct was designed and used primarily for directly timing the molecular events induced by nocodazole. For this purpose, we prepared two-color time-lapse movies in which YFP-dSH2- and CFP-vinculin-containing cells were exposed to the drug. Frame-by-frame analysis of the relative changes induced in YFP and CFP fluorescence revealed a strong early increase in vinculin levels, accompanied by FA growth, which takes place in almost every adhesion in the tested cells (Figs 7, 8). By contrast, the changes in local YFP-dSH2 levels during the same period were limited and non-uniform, confirming the notion that vinculin recruitment precedes the local increase in tyrosine phosphorylation by several minutes.
Another intriguing observation that emerged from the live-cell movies depicting the dynamics of tyrosine phosphorylation following nocodazole treatment is the lack of synchrony in the changes occurring in different adhesions of the same cell following stimulation. Thus, whereas in most adhesions the tyrosine-phosphorylated area was induced to grow following nocodazole treatment, the onset and rate of its expansion in individual adhesions were highly variable. This suggests that, even when the stimulation of FA assembly is `global', changes in individual adhesions may be locally regulated or `fine-tuned', owing to variations in local tension or other factors. These results also suggest that changes in local PY level and FA extension are not tightly linked and display complex, nonlinear relationships.
What is the sequence of molecular events leading to FA development
following application of nocodazole? We propose
(Fig. 10) that disruption of
microtubules is accompanied by cellular contractility, which increases the
mechanical tension at focal complexes or small FAs (Bershadsky, 1996). This
mechanical stimulation activates, in turn, the recruitment of structural FA
components, such as vinculin, paxillin and FAK, into focal complexes, leading
to the growth of the adhesive structure. These responses are rather rapid, and
are apparent within less than 2-3 minutes after addition of the drug. The
accumulation of FAK and its pp60src-induced activation result in an
increase in local PY levels, which is typically delayed by 1-3 minutes after
the protein recruitment. The increase in local tyrosine phosphorylation might,
in turn, be involved in the downregulation of FA growth and the promotion of
their turnover. This is in agreement with recent studies showing that FAs of
pp60src-null (Volberg et al.,
2001) and FAK-null cells (L. H. Romer, T. Volberg and B.G.,
unpublished) fail to transform into fibrillar adhesions, and that excessive
phosphorylation (e.g. by the deregulated pp60v-src) leads to
destruction of FAs (Kellie et al.,
1986
). Apparently, the first protein whose accumulation in growing
FAs is arrested shortly after local phosphorylation increases is FAK itself,
raising the possibility that its phosphorylation might downregulate its
association with FAs. This notion is supported by recent studies (B. Z. Katz,
L. H. Romer, S. Miyamoto, T. Volberg et al.., unpublished), showing that
tyrosine phosphorylation of a C-terminal site on FAK interferes with its
association with FAs.
|
The detailed molecular mechanisms involved in the nucleation and growth of
FAs, as well as the precise role of tyrosine phosphorylation in FA development
and maturation, are still poorly defined and remain to be elucidated in the
future. Interestingly, several recent approaches might be instrumental in such
studies. Among these are various fluorescent fusion proteins that can be
expressed in cells, localize to FAs, and serve for dynamic analyses
(Zamir et al., 2000;
Laukaitis et al., 2001
;
Rottner et al., 2001
).
Particularly intriguing is the possibility of tracing tyrosine phosphorylation
in live cells, using fluorescent derivatives of an SH2 domain, as described
here. In addition, new approaches were recently developed for the direct
measurement of mechanical forces in adhesion sites of live cells
(Balaban et al., 2001
;
Riveline et al., 2001
;
Beningo et al., 2001
). A
combination of such approaches might provide us with deeper understanding of
the molecular events underlying the formation and turnover of
cell-matrix-adhesions, and shed new light on the role of tyrosine
phosphorylation in these events.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ayalon, O. and Geiger, B. (1997). Cyclic
changes in the organization of cell adhesions and the associated cytoskeleton,
induced by stimulation of tyrosine phosphorylation in bovine aortic
endothelial cells. J. Cell Sci.
110,547
-556.
Balaban, N. Q., Schwarz, U. S., Riveline, D., Goichberg, P., Tzur, G., Sabanay, I., Mahalu, D., Safran, S., Bershadsky, A., Addadi, L. and Geiger, B. (2001). Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3,466 -472.[CrossRef][Medline]
Barry, S. T. and Critchley, D. R. (1994). The
RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated
with increased tyrosine phosphorylation and the recruitment of both pp125FAK
and protein kinase C-delta to focal adhesions. J. Cell
Sci. 107,2033
-2045.
Beningo, K. A., Dembo, M., Kaverina, I., Small, J. V. and Wang,
Y. L. (2001). Nascent focal adhesions are responsible for the
generation of strong propulsive forces in migrating fibroblasts. J.
Cell Biol. 153,881
-888.
Bershadsky, A., Chausovsky, A., Becker, E., Lyubimova, A. and Geiger, B. (1996). Involvement of microtubules in the control of adhesion-dependent signal transduction. Curr. Biol. 6,1279 -1289.[Medline]
Bockholt, S. M. and Burridge, K. (1993). Cell
spreading on extracellular matrix proteins induces tyrosine phosphorylation of
tensin. J. Biol. Chem.
268,14565
-14567.
Bradshaw, J. M., Mitaxov, V. and Waksman, G. (1999). Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase. J. Mol. Biol. 293,971 -985.[CrossRef][Medline]
Brown, M. T. and Cooper, J. A. (1996). Regulation, substrates and functions of src. Biochim. Biophys. Acta. 1287,121 -149.[CrossRef][Medline]
Calalb, M. B., Polte, T. R. and Hanks, S. K. (1995). Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol. Cell. Biol. 15,954 -963.[Abstract]
Chrzanowska-Wodnicka, M. and Burridge, K.
(1994). Tyrosine phosphorylation is involved in reorganization of
the actin cytoskeleton in response to serum or LPA stimulation. J.
Cell Sci. 107,3643
-3654.
Chrzanowska-Wodnicka, M. and Burridge, K. (1996). Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell Biol. 133,1403 -1415.[Abstract]
Clark, E. A., King, W. G., Brugge, J. S., Symons, M. and Hynes,
R. O. (1998). Integrin-mediated signals regulated by members
of the rho family of GTPases. J. Cell Biol.
142,573
-586.
Enomoto, T. (1996). Microtubule disruption induces the formation of actin stress fibers and focal adhesions in cultured cells: possible involvement of the rho signal cascade. Cell Struct. Funct. 21,317 -326.[Medline]
Geiger, B. and Bershadsky, A. (2001). Assembly and mechanosensory function of focal contacts. Curr. Opin. Cell Biol. 13,584 -592.[CrossRef][Medline]
Geiger, B., Bershadsky, A., Pankov, R. and Yamada, K. M. (2001). Transmembrane extracellular matrixcytoskeleton crosstalk. Nat. Rev. Mol. Cell. Biol. 2, 793-805.[CrossRef][Medline]
Geiger, B. and Bershadsky, A. (2002). Exploring the neighborhood. Adhesion-coupled cell mechanosensors. Cell 110,139 -142.[Medline]
Helfman, D. M., Levy, E. T., Berthier, C., Shtutman, M.,
Riveline, D., Grosheva, I., Lachish-Zalait, A., Elbaum, M. and Bershadsky, A.
D. (1999). Caldesmon inhibits nonmuscle cell contractility
and interferes with the formation of focal adhesions. Mol. Biol.
Cell 10,3097
-3112.
Kaverina, I., Krylyshkina, O. and Small, J. V.
(1999). Microtubule targeting of substrate contacts promotes
their relaxation and dissociation. J. Cell Biol.
146,1033
-1043.
Kaverina, I., Krylyshkina, O., Beningo, K., Anderson, K., Wang,
Y. L. and Small, J. V. (2002). Tensile stress stimulates
microtubule outgrowth in living cells. J. Cell Sci.
115,2283
-2291.
Kellie, S., Patel, B., Wigglesworth, N. M., Critchley, D. R. and Wyke, J. A. (1986). The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity. Exp. Cell Res. 165,216 -228.[Medline]
Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamon, B., Feng, J., Nakano, T., Okawa, K. et al. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273,245 -248.[Abstract]
Landt, O., Grunert, H. P. and Hahn, U. (1990). A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96,125 -128.[CrossRef][Medline]
Laukaitis, C. M., Webb, D. J., Donais, K. and Horwitz, A. F.
(2001). Differential dynamics of alpha 5 intregrin, paxillin, and
alpha-actinin during formation and disassembly of adhesions in migrating
cells. J. Cell Biol.
153,1427
-1440.
Leopoldt, D., Lee, H. F. and Rozengurt, E. (2001). Calyculin A induces focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin in Swiss 3T3 cells. J. Cell Physiol. 188,106 -119.[CrossRef][Medline]
Liu, B. P., Chrzanowska-Wodnicka, M. and Burridge, K. (1998). Microtubule depolymerization induces stress fibers, focal adhesions, and DNA synthesis via the GTP-binding protein Rho. Cell Adhes. Commun. 5,249 -255.[Medline]
Martinez, R., Mathey-Prevot, B., Bernards, A. and Baltimore, D. (1987). Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. Science 237,411 -415.[Medline]
Nobes, C. D. and Hall, A. (1995). Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81,53 -62.[Medline]
Nollau, P. and Mayer, B. J. (2001). Profiling
the global tyrosine phosphorylation state by Src homology 2 domain binding.
Proc. Natl. Acad. Sci. USA
98,13531
-13536.
Pankov, R., Cukierman, E., Katz, B.-Z., Matsumoto, K., Lin, D.
C., Lin, S., Hahn, C. and Yamada, K. M. (2000). Integrin
dynamics and matrix assembly; tensin-dependent translocation of alpha(5) beta
(1) integrins promotes early fibronectin fibrillogenesis. J. Cell
Biol. 148,1075
-1090.
Pawson, T., Gish, G. D. and Nasha, P. (2001). SH2 domains, interaction modules and cellular wiring. Trends Cell Biol. 11,504 -511.[CrossRef][Medline]
Pletjushkina, O. J., Belkin, A. M., Ivanova, O. J., Oliver, T., Vasiliev, J. M. and Jacobson, K. (1998). Maturation of cell-substratum focal adhesions induced by depolymerization of microtubules is mediated by increased cortical tension. Cell Adhes Commun. 5,121 -135.[Medline]
Retta, S. F., Barry, S. T., Critchley, D. R., Defilippi, P., Silengo, L. and Tarone, G. (1996). Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity. Exp. Cell Res. 229,307 -317.[CrossRef][Medline]
Ridley, A. J. and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70,389 -399.[Medline]
Ridley, A. J. and Hall, A. (1994). Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J. 13,2600 -2610.[Abstract]
Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A. (1992). The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 70,401 -410.[Medline]
Riveline, D., Zamir, E., Balaban, N. Q., Schwarz, U. S.,
Ishizaki, T., Narumiya, S., Kam, Z., Geiger, B. and Bershadsky, A. D.
(2001). Focal contacts as mechanosensors: externally applied
local mechanical force induces growth of focal contacts by an mDia1-dependent
and ROCK-independent mechanism. J. Cell Biol.
153,1175
-1186.
Rottner, K., Hall, A. and Small, J. V. (1999). Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9,640 -648.[CrossRef][Medline]
Rottner, K., Krause, M., Gimona, M., Small, J. V. and Wehland,
J. (2001). Zyxin is not colocalized with
vasodilator-stimulated phosphoprotein (VASP) at lammelipodial tips and
exhibits different dynamics to vinculin, paxillin and VASP in focal adhesions.
Mol. Biol. Cell 12,3103
-3113.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. New York: Cold Spring Harbour Laboratory Press.
Sastry, S. K. and Burridge, K. (2000). Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics. Exp. Cell Res. 261,25 -36.[CrossRef][Medline]
Sawada, Y. and Sheetz, M. P. (2002). Force
transduction by Triton cytoskeletons. J. Cell Biol.
156,609
-615.
Sawyer, T. K. (1998). Src homology-2 domains: structure, mechanisms, and drug discovery. Biopolymers. 47,243 -261.[CrossRef][Medline]
Schaller, M. D. (2001). Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim. Biophys. Acta. 1540, 1-21.[Medline]
Schneider, G. B., Gilmore, A. P., Lohse, D. L., Romer, L. H. and Burridge, K. (1998). Microinjection of protein tyrosine phosphatases into fibroblasts disrupts focal adhesions and stress fibers. Cell Adhes. Commun. 5,207 -219.[Medline]
Takaishi, K., Mino, A., Ikeda, W., Nakano, K. and Takai, Y. (2000). Mechanisms of activation and action of mDia1 in the formation of parallel stress fibers in MDCK cells. Biochem. Biophys. Res. Commun. 274,68 -72.[CrossRef][Medline]
Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S. A. and Alberts, A. S. (2000). GTPase and Src tyrosine kinase signaling. Mol. Cell 5, 13-25.[Medline]
Turner, C. E. (2000). Paxillin and focal adhesion signalling. Nat. Cell Biol. 2,E231 -E236.[CrossRef][Medline]
Volberg, T., Geiger, B., Citi, S. and Bershadsky, A. D. (1994). Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system. Cell. Motil. Cytoskeleton. 29,321 -338.[Medline]
Volberg, T., Romer, L., Zamir, E. and Geiger, B.
(2001). pp60(c-src) and related tyrosine kinases: a role in the
assembly and reorganization of matrix adhesions. J. Cell
Sci. 114,2279
-2289.
Vuori, K. and Ruoslahti, E. (1995). Tyrosine
phosphorylation of p130Cas and cortactin accompanies
integrin-mediated cell adhesion to extracellular matrix. J. Biol.
Chem. 270,22259
-22262.
Watanabe, N., Kato, T., Fujita, A., Ishizaki, T. and Narumiya, S. (1999). Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat. Cell Biol. 1, 136-143.[CrossRef][Medline]
Zamir, E. and Geiger, B. (2001). Molecular
complexity and dynamics of cell-matrix adhesions. J. Cell
Sci. 114,3583
-3590.
Zamir, E., Katz, B.-Z., Aota, S., Yamada, K. M., Geiger, B. and
Kam, Z. (1999). Molecular diversity of cell-matrix adhesions.
J. Cell Sci. 112,1655
-1669.
Zamir, E., Katz, M., Posen, Y., Erez, N., Yamada, K. M., Katz, B. Z., Lin, S., Lin, D. C., Bershadsky, A., Kam, Z. and Geiger, B. (2000). Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts. Nat. Cell Biol. 2, 191-196.[CrossRef][Medline]
Zhang, Q., Magnusson, M. K. and Mosher, D. F. (1997). Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction. Mol. Biol. Cell 8,1415 -1425.[Abstract]
Related articles in JCS: