1 Department of Pediatrics and Pharmacology, University of Wisconsin, 1300
University Avenue, University of Wisconsin Medical School, Madison, WI 53706,
USA
2 Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg A-5020,
Austria
3 Department of Cell and Molecular Physiology, University of North Carolina,
Chapel Hill, NC 27599, USA
* Author for correspondence (e-mail: huttenlocher{at}facstaff.wisc.edu)
Accepted 12 June 2002
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Summary |
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Key words: Migration, Calpain, Cytoskeleton, Focal adhesion, -actinin
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Introduction |
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Our recent studies support a role for the calcium-dependent protease
calpain in cell detachment (Huttenlocher
et al., 1997). Calpain is a cysteine protease with two
characterized isoforms, µ- and m-calpain
(Croall and De Martino, 1991
).
Both contain an 80 kDa catalytic subunit and a 30 kDa regulatory subunit.
Calpain has many substrates in vitro including the focal adhesion proteins
integrin (Du et al., 1995
),
FAK (Cooray et al., 1996
),
ezrin (Potter et al., 1998
)
and talin (Beckerle et al.,
1987
). The relevance of these calpain substrates to calpain
function in vivo has yet to be determined. However, the critical importance of
this protease to normal embryonic development has recently been demonstrated
by studies in which targeted disruption of the 30 kDa calpain subunit, CAPN4,
is embryonic lethal at day 10-11 and causes defects in vascular development
(Arthur et al., 2000
). We
previously reported that cell permeable calpain inhibitors reduce cell
migration rates and cell detachment during migration
(Huttenlocher et al., 1997
;
Palecek et al., 1998
).
Further, we showed that calpain inhibition stabilizes peripheral adhesive
complexes that contain vinculin and integrin. Interestingly, disruption of
other proteins implicated in regulating cell migration, including FAK
(Ilic et al., 1995
;
Sieg et al., 1999
) and Src
(Klinghoffer et al., 1999
),
also promote the formation of strong focal adhesions at the cell periphery.
Together, these findings suggest that calpain and calpain substrates, such as
FAK and Src, may be acting by related mechanisms to regulate cell
migration.
Recent evidence supports a central role for microtubules in the specific
targeting of adhesive complex sites and their disassembly during cell
migration (Kaverina et al.,
1998). Kaverina et al. proposed a model whereby microtubules
specifically target and deliver `relaxing' signals to focal contact sites to
promote their dissociation (Kaverina et
al., 1999
; Kaverina et al.,
2000
). However, the mechanisms through which microtubules mediate
the disassembly of adhesions have not been defined. Signaling molecules that
are probably involved include the Rho family of GTPases, since microtubule
disruption leads to Rho activation and the formation of focal adhesions and
stress fibers (Danowski et al., 1989;
Bershadsky et al., 1996
;
Enomoto, 1996
). Recent
evidence supports a role for the microtubule-dependent regulation of
contractility with localized inhibition of contractility as a mechanism to
promote adhesion complex disassembly
(Kaverina et al., 1999
).
In this study we sought to determine whether calpain modulates the dynamics
and composition of adhesive contacts. We found that inhibiting calpain through
overexpression of the endogenous inhibitor of calpain, calpastatin and
pharmacological inhibitors results in an inhibition of adhesive complex
disassembly with a stabilization of GFP-vinculin and GFP-zyxin at the cell
periphery, which implicates calpain as a regulator of adhesive complex
dynamics in adherent cells. We also found that calpain inhibition disrupts
-actinin localization to focal contacts and that localization of
-actinin is important for the subsequent disassembly or translocation
of zyxin-containing contact sites.
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Materials and Methods |
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Antibodies and reagents
Fibronectin was purified from human plasma by affinity chromatography as
described (Ruoslahti et al.,
1982). Fibronectin for the CAR fibroblast experiments was
purchased from Boehringer Mannheim. Anti-vinculin antibody was purchased from
Sigma (St Louis, MO) and used at a 1:500 dilution. Rhodamine-phalloidin was
obtained from Molecular Probes (Eugene, OR) and used at a dilution of 1:1000.
Anti-tubulin antibody was obtained from J. Wehland (GBF, Braunschweig).
Nocodozole was purchased from Sigma and was used at a concentration of 2.4
µg/ml. Calpain inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (ALLN),
was obtained from Calbiochem (San Diego, CA) and used at a concentration of 50
µg/ml and stored as a 10 mg/ml stock in ethanol at -20°C. PD150606 was
used at a concentration of 1 µM and stored as a stock of 100 mM in DMSO at
-20°C.
Cells and transfections
Goldfish fin fibroblasts (CAR) were obtained from American Type Culture
Collection (ATCC) and maintained in basal Eagle medium supplemented with Hanks
BSS, non-essential amino acids, and 15% fetal calf serum at 25°C. (Sigma)
Chinese hamster ovary (CHOK1) cells were obtained from ATCC and maintained in
Dulbecco's modified Eagles medium (Cellgro®, Mediatech, Herden, VA)
supplemented with non-essential amino acids, 10% fetal bovine serum, 100 U/ml
penicillin, and 100 µg/ml streptomycin (Sigma) at 37°C with 10%
CO2. CAR fibroblasts were transfected as described
(Kaverina et al., 1999).
Briefly, a monolayer of cells was cotransfected with 1 µg EGFP-zyxin and 2
µg EGF-tubulin using 15 µl of superfect reagent as described (Qiagen,
Valencia CA) (Kaverina et al.,
1999
). Cells were used 24 hours after transfection. CHO cells were
transfected with 4 µg of DNA and 14 µl of lipofectamine as described by
the manufacturer (Gibco BRL Life Technologies, Grand Island, NY). Cells were
used between 36 and 48 hours after the start of the transfection. For
co-transfections, the GFP-tagged cDNA was transfected at a 1:6 ratio with
either control vector or calpastatin cDNA. We found that over 95% of the cells
that expressed the GFP construct also expressed calpastatin by direct staining
for calpastatin in transfected cells. A similar protocol was used for dual
imaging experiments.
Time-lapse videomicroscopy
For live fluorescent imaging, CHO cells were plated in CCM1 on a coverslip
mounted on a 35-mm plate. The coverslip was coated with 5 µg/ml fibronectin
for 1 hour and blocked with 2% BSA for 30 minutes. Prior to videotaping, media
was replaced with HAMS media supplemented with 4 mM L-glutamine, 1 mM
non-essential amino acids, 10% fetal bovine serum, 100 U/ml penicillin, and
100 µg/ml streptomycin (Sigma). Cells were placed directly on a heated
stage and supplemented with CO2 to maintain a pH of 7.0-7.5.
Fluorescent images were captured every 3 minutes for 1.5-2 hours using a
heated 60x/1.4NA objective on a Nikon inverted microscope attached to an
ORCA II cooled CCD camera (Hamamatsu, Japan). The objective was heated to
35°C by a Bioptechs objective controller (Bioptechs, Butler, PA). An
electronic shutter (Ludl, Hawthorn, NY) controlled the illumination from a 100
watt mercury bulb. Cooled CCD control and image processing was done using ISEE
imaging software on an SGI computer from Inovision Corporation (Raleigh, NC).
The experimental results show representative cells from a minimum of five
different experiments. For all studies, similar results were found using
co-transfection with calpastatin, GFP-calpastatin or treating cells with the
cell-permeable calpain inhibitor, ALLN.
Immunofluorescence microscopy
Glass coverslips were acid washed, coated with fibronectin and blocked with
2% BSA using a previously described protocol
(Huttenlocher et al., 1998)
with minor modifications. Cells were allowed to adhere for 12 hours at
37°C and 10% CO2 after which they were fixed and stained as
described (Huttenlocher et al.,
1996
).
Supplemental processing
Supplemental processing of videotapes from SGI® format to
QuickTime® 4.0 format was done using Adobe Premier 5.0®. Captured
images were processed using Adobe PhotoShop® 5.0. Focal adhesion turnover
was quantified using previously described methods
(Kaverina et al., 2000). In
brief, vinculin containing focal complexes were tracked for a 30 minute
interval and the number of contacts that disappeared over this interval were
counted and presented as a percentage of the total initial contact sites.
Co-localization was determined for each condition in at least 14 cells using
Isee analytical software. Co-localization was defined as the number of red
pixels above background that overlapped with the green pixels above background
divided by the total number of red pixels. Background fluorescence for each
channel was determined in the interior of the cell and used to eliminate noise
or un-localized signal.
Online supplemental material
The online version of this article contains movies that accompany several
of the figures
(http://jcs.biologists.org/supplemental).
Movie 1: cells co-transfected with GFP-vinculin and control vector. Movie 2:
cells co-transfected with GFP-vinculin and calpastatin. Movie 3: cells
co-transfected with GFP-zyxin and control vector. Movie 4: cells
co-transfected with GFP-zyxin and calpastatin. Movie 5: cells co-transfected
with hrEGFP and RFP-zyxin. Movie 6: cells co-transfected with
hrEGFP-calpastatin and RFP-zyxin. Movie 7: cells co transfected with
GFP-actinin and control vector. Movie 8: cells co transfected
with GFP
-actinin and calpastatin. Movies 9 and 10: control cells
co-transfected with RFP-zyxin and GFP
-actinin. Movie 11: calpain
inhibitor (ALLN)-treated cells co-transfected with RFP-zyxin and
GFP
-actinin. Movie 12: RFP-zyxin in cells cotransfected with
full length
-actinin. Movie 13: RFP-zyxin in cells co-transfected with
the head domain of
-actinin. Movie 14: RFP-zyxin in cells
co-transfected with rod domain of
-actinin.
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Results |
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|
To determine whether calpain modulates focal complex turnover, we
characterized the effects of transient expression of calpastatin or treatment
of cells with cell permeable calpain inhibitors on the dynamics and turnover
of vinculin- and zyxin-containing adhesions. To this end, we co-transfected
vinculin or zyxin with either control vector or calpastatin at a 6:1 ratio as
described in Materials and Methods. We found that the control cells formed
small punctate vinculin- or zyxin-containing complexes that were most
prominent at the cell periphery (Fig.
2). These complexes were highly dynamic and generally dispersed
within 30 minutes after formation. In contrast, cells that expressed
calpastatin or were treated with ALLN (Figs
1,
8) had prominent vinculin- or
zyxin-containing complexes at the cell periphery. These complexes frequently
elongated along the cell periphery and were less likely to disassociate than
the adhesive complexes from control cells
(Fig. 2). The difference
between these complexes was clearly demonstrated in enlargements of small
regions of the cell periphery. In control cells, marked contact sites
dispersed and new ones formed in subsequent images
(Fig. 2). In contrast, in cells
that expressed calpastatin or were treated with calpain inhibitors, there were
fewer contact sites and these sites were more stable in the time frame of
observation. To quantify this effect, we analyzed ECM-contact fate in seven
cells that express EGFP-vinculin, in several experiments using a variation of
previously described methods (Kaverina et
al., 1999). In control cells, 66% of 113 contact sites disappeared
after 30 minutes. In contrast, 27% of 57 contact sites dispersed in the cells
that express calpastatin. In general, the contact sites in the calpastatin
cells remained attached to the substratum and did not translocate towards the
cell center. However, we observed a few contact sites at the cell's rear that
were able to detach from the substratum and translocate intact along the edge
of the cell. Similar results were obtained in cells co-transfected with
RFP-zyxin and GFP-calpastatin (Fig.
2). Together, these findings demonstrate that calpain modulates
the disassembly of adhesive complex sites that contain vinculin and zyxin.
|
|
Calpain is required for microtubule-mediated focal complex
disassembly after nocodazole wash-out
Previous reports have demonstrated that treatment of cells with nocodazole
promotes the stabilization of peripheral adhesive complexes that contain focal
adhesion components such as zyxin
(Kaverina et al., 1999). These
studies have demonstrated that microtubules specifically target and promote
the disassembly of adhesive contact sites. Treatment of cells with the
microtubule-disrupting agent nocodazole inhibits microtubule targeting and
focal complex disassembly. After nocodazole wash-out, microtubule targeting of
focal complexes occurs and promotes focal complex turnover and cell spreading.
To determine whether calpain is required for the microtubule-mediated turnover
of adhesive complexes, we treated cells with the calpain inhibitor ALLN and
assayed focal complex fate in goldfish fin fibroblasts (CAR cells). CAR cells
were used for these studies since these cells were previously used to study
the effects of microtubule targeting on focal contact fate
(Kaverina et al., 1999
). We
found that recovery of focal complex turnover after nocodazole wash-out did
not occur normally in the presence of ALLN, suggesting that calpain is
required for microtubule-mediated focal complex disassembly after nocodazole
wash-out. Fig. 3 shows examples
of contact dynamics in spread cells after recovery from nocodazole in the
presence (Fig. 3b) and absence
(Fig. 3a) of ALLN. After
recovery from nocodazole, control cells showed increased cell spreading and
turnover of adhesive complexes that contained EGFP-zyxin (53% of contact sites
disassembled; Fig. 3B). In
contrast, in the presence of ALLN, the turnover of adhesive contact sites was
inhibited (21% of contact sites disassembled). These findings suggest that
calpain is required for microtubule-mediated disassembly of adhesive complex
sites after nocodazole wash-out.
|
To determine whether calpain modulates focal complex disassembly by affecting microtubule function, we studied the effects of the calpain inhibitor ALLN, on microtubule targeting after recovery from nocodazole in the presence and absence of ALLN. Control cells showed targeting of peripheral adhesive complexes stained for paxillin 10 minutes after nocodazole wash-out and focal complex disassembly 1.5 hours after wash-out (Fig. 4). In contrast, in the presence of ALLN, targeting of adhesive complexes occurred normally but 1.5 hours after nocodazole recovery the prominent peripheral paxillin-containing adhesive complexes remained intact despite normal microtubule targeting. These results demonstrate that calpain does not affect microtubule organization or targeting but is required for microtubule-mediated substrate contact site disassociation after nocodazole wash-out.
|
Calpain modulates -actinin localization and dynamics in live
cells
To examine how calpain modulates the organization and disassembly of
adhesive contact sites we examined the effects of calpain inhibition on the
localization of different focal adhesion components. In contrast to the other
components studied (zyxin, FAK, paxillin, integrin, talin and vinculin), the
actin-binding protein -actinin showed reduced localization to adhesive
contact sites after calpain inhibition in CHOK1 cells
(Fig. 5).
-Actinin is an
actin-binding protein that crosslinks actin filaments and integrin-containing
adhesive complexes, and localizes both along actin stress fibers and in focal
adhesions (Otey et al., 1993
).
Using both antibody staining for
-actinin and expression of an
EGFP
-actinin fusion protein, we found that
-actinin
showed reduced localization to adhesive complex sites after calpain inhibition
with ALLN or calpastatin (Fig.
5). After calpain inhibition, we found localization of
-actinin along the cell periphery. In live imaging studies using CHOK1
cells co-transfected with the EGFP
-actinin and control vector or
calpastatin, calpain inhibition inhibited the dynamics and localization of
-actinin in adhesive complex sites
(Fig. 5). In control cells,
EGFP
-actinin was highly dynamic and localized in adhesive
complex sites at the cell center and periphery. In contrast, expression of
calpastatin induced a peripheral distribution of
-actinin and inhibited
-actinin dynamics. Interestingly and in contrast to
-actinin,
zyxin, which normally localizes to both focal complexes and the actin
cytoskeleton, was present predominantly in focal complexes after calpain
inhibition and its co-localization with
-actinin was reduced in CHOK1
cells (Fig. 6). In control
cells,
-actinin showed 80% co-localization with zyxin but after
treatment with cell permeable calpain inhibitors only 31% co-localization was
observed. Immunofluorescence or co-transfection was used to characterize the
zyxin-containing structures. We found that in both vehicle control and
ALLN-treated cells the zyxin complexes contained
5-GFP-integrin
(Laukaitis et al., 2001
)
(Fig. 7a), vinculin
(Fig. 7b), paxillin, FAK and
talin (data not shown). Notably,
5-integrin and vinculin localization
to the peripheral focal complexes was more prominent in cells treated with
ALLN. To determine whether nonspecific disruption of the actin cytoskeleton
results in similar changes in
-actinin and zyxin localization, cells
were treated with the actin-disrupting drug cytochalasin D. We found that, in
contrast to calpain inhibition, cytochalasin D disrupted focal complex
formation with a loss of focal adhesions at both the cell center and
periphery, and did not result in a specific disruption of
-actinin and
zyxin co-localization (data not shown). Together, these findings suggest that
calpain regulates the composition of focal adhesions and the specific
localization of
-actinin into adhesive complex sites.
|
|
|
Effects of calpain inhibition on -actinin and zyxin dynamics
in live cells
To characterize the mechanism by which calpain regulates focal adhesion
disassembly we examined the dynamics of zyxin and -actinin in live
cells using dual imaging studies of cells co-transfected with RFP-zyxin and
GFP
-actinin. This is the first study to describe the temporal
and spatial distribution of zyxin and
-actinin in live cells. As
described above, control cells showed greater than 80% co-localization of
zyxin and
-actinin. We found that localization of
-actinin into
zyxin-containing focal complexes in control cells may be associated with
different contact fates including stabilization of the complex, disassembly
and translocation (Fig. 8).
Interestingly, at areas of cell retraction, common fates included contact site
disassembly and translocation to the cell center. In fact, a temporal and
spatial relationship between
-actinin localization into zyxin contact
sites and their translocation was observed in areas of cell retraction. In
contrast, in areas of protrusion, zyxin and
-actinin were less
associated and we did not commonly observe contact site translocation or
disassembly (Movies 9-11;
http://jcs.biologists.org/supplemental).
These findings suggest that zyxin
-actinin association may
promote contact site translocation and disassembly, thereby facilitating cell
retraction. In the presence of cell permeable calpain inhibitors ALLN (Figs
6,
8) and PD150606 (data not
shown), co-localization of
-actinin and zyxin was reduced. In live
imaging studies, ALLN inhibited the co-localization of zyxin and
-actinin and, under these conditions, we did not observe contact site
translocation or disassembly. Together these findings suggest that
-actinin localization to focal complexes may promote their subsequent
disassembly or translocation, and that this effect may be mediated by
calpain.
Characterization of the role of -actinin during focal adhesion
disassembly
Previous studies suggest that -actinin localization to the focal
complex is important for focal complex disassembly
(Laukaitis et al., 2001
;
Pavalko and Burridge, 1991
).
Pavalko and Burridge have shown that microinjection of purified
-actinin 53 kDa rod domain localizes to the focal complex while the 23
kDa head domain localizes to actin stress fibers
(Pavalko and Burridge, 1991
).
The
-actinin rod and head domains were thought to act as dominant
negatives, competing with full-length
-actinin for binding to the focal
complex or the actin cytoskeleton, respectively. To determine whether
-actinin localization to the focal complex is important for focal
complex disassembly and translocation, GFP fusion proteins to the 23 kDa head
domain and the 53 kDa rod domain were generated and their effects on the
dynamics of RFP-zyxin were determined using live imaging. The 53 kDa
rod-domainGFP fusion protein (
-actinin-rodGFP) localized
to focal adhesions while the 23 kDa head-domainGFP fusion protein
(
-actinin-headGFP) localized predominantly to actin stress
fibers, which is similar to results previously published
(Pavalko and Burridge, 1991
)
(Fig. 9). Interestingly,
disrupting
-actinin localization to focal complexes through the
expression of
-actinin-rodGFP inhibited focal adhesion
disassembly similar to calpain inhibition. When cells were co-transfected with
-actinin-rodGFP and RFP-zyxin, fewer zyxin-containing focal
complexes disassembled (21% of focal complexes disassembled) after 30 minutes
compared with expression of full-length
-actininGFP (44% of
focal adhesions disassembled P<0.003) or
-actinin-headGFP (49% of focal adhesions disassembled
P<0.002) (Fig. 9).
In addition, cells that express
-actinin-rodGFP did not show
translocation of RFP-zyxin to the cell center, supporting a role for
-actinin in complex site translocation (Movies 11-13). These results
demonstrate that the localization of
-actinin to focal complexes is
important for focal complex disassembly and translocation.
|
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Discussion |
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Recent studies have supported the importance of microtubules as regulators
of the turnover and dynamics of focal adhesions in adherent cells. Kaverina et
al. have demonstrated that the specific targeting of microtubules to substrate
contact sites is required for their turnover and cell detachment in migrating
cells (Kaverina et al., 1999).
Treatment of cells with nocodazole, which depolymerizes microtubules, promotes
the stabilization of peripheral adhesions and reduces cell protrusion and
spreading. After nocodazole wash-out and recovery of the microtubular network,
specific targeting of peripheral adhesions promotes their turnover and allows
for cell spreading, Rac activation
(Waterman-Storer et al., 1999
)
and cell polarization (reviewed by
Waterman-Storer and Salmon,
1999
). The mechanism by which microtubules promote adhesive
complex turnover and cell detachment has not been elucidated, although
previous studies have implicated a possible role for members of the Rho family
of GTPases and/or localized regulation of contractility
(Kaverina et al., 1999
). We
found that calpain inhibition had no effect on microtubule targeting of focal
adhesion sites, but that calpain was required for microtubule-mediated
turnover of focal complexes after nocodazole wash-out. Together these findings
suggest that calpain may act downstream of microtubules to mediate focal
complex disassembly.
In contrast to the microtubule cytoskeleton, we and others have found that
calpain inhibition disrupts the actin cytoskeleton and inhibits the formation
of Rho-mediated stress fibers (Kulkarni et
al., 1999; Bialkowska et al.,
2000
). In this paper we found that calpain inhibition specifically
perturbs the co-localization of zyxin and
-actinin at focal contacts.
The importance of zyxin
-actinin binding for cell migration has
been recently demonstrated by a study showing that a peptide inhibitor that
blocks zyxin
-actinin interactions affects zyxin localization and
cell migration (Drees et al.,
1999
). Furthermore, earlier studies demonstrated that disruption
of
-actinin function by microinjection of
-actinin 27 kDa or 53
kDa fragments results in the disassembly of the stress fibers, but the
persistence of focal adhesions at the cell periphery
(Pavalko and Burridge, 1991
),
similar to the phenotype observed after calpain inhibition. These findings
suggest that the association of
-actinin with both actin-containing
stress fibers and focal adhesions may be important for focal complex
disassembly and/or translocation to the cell center. Our live imaging studies
now provide direct evidence to support this possibility. We found a temporal
and spatial relationship between zyxin and
-actinin co-localization,
focal contact site translocation or disassembly and the retraction of a region
of the cell. These findings suggest that zyxin
-actinin
co-localization at regions of cell retraction may mediate contact site
translocation and disassembly. Zyxin
-actinin co-localization was
disrupted after calpain inhibition. Accordingly, there was an inhibition of
contact site translocation, disassembly and cell retraction. In addition, we
found that disrupting
-actinin localization to the focal complex
through expression of the
-actinin rod domain also caused an inhibition
of focal complex disassembly and translocation similar to calpain inhibition.
Together, these findings suggest that the ability of
zyxin
-actinin to co-localize may be important for focal complex
disassembly and translocation to the cell center, and that the
zyxin
-actinin interaction at focal contact sites may be mediated
by calpain.
The critical substrates for calpain's effects on focal complex disassembly
and cell motility have remained elusive. Possibilities are broad since many
focal adhesion components and related signaling proteins have been identified
as calpain substrates in vitro. Likely candidates include FAK and Src, both of
which are important mediators of cell migration and adhesive complex turnover.
Cells deficient in FAK (Ilic et al.,
1995) and Src (Klinghoffer et
al., 1999
) have been reported to show strong focal adhesions that
occur primarily at the cell periphery, consistent with a defect in adhesive
complex turnover. However, we do not find evidence for differential cleavage
or phosphorylation of FAK (Dourdin et al.,
2001
) or Src activation (using Src kinase activity assays) in
calpain-deficient embryonic fibroblasts (data not shown). Furthermore, the
distribution of
-actinin in Src and FAK-deficient cells is not
comparable with the results observed after calpain inhibition or in
calpain-deficient embryonic fibroblasts (data not shown), suggesting that FAK
and Src are not the critical substrates for calpain's effects on focal contact
disassembly. Alternatively, the focal adhesion component talin is an
attractive candidate. Our recent studies demonstrate the differential cleavage
of talin in control and calpain-deficient embryonic fibroblasts
(Dourdin et al., 2001
), which
supports a critical role for talin in the regulation of the actin cytoskeleton
and focal adhesions by calpain. Similarly, inhibition of calpain in CHOK1
cells led to a 50% decrease in talin cleavage while having no detectable
effect on other focal complex proteins such as
-actinin, FAK, vinculin
or paxillin (data not shown). It is possible that calpain cleaves talin and
this modification of the adhesion complex permits the localization of
-actinin into the adhesive contact site. In support of this hypothesis
is the recent observation that the regulated interaction of the
ß2-integrin with
-actinin rather than talin requires a
conformational changes that unmasks a cryptic
-actinin-binding domain
in the integrin cytoplasmic domain
(Sampath et al., 1998
). The
authors proposed a model in which a proteolysis event cleaves talin and
promotes a conformational change in the integrin cytoplasmic domain that
allows for
-actinin binding to integrin. The importance of calpain in
lymphocyte adhesion and spreading, suggests that the proteolysis event
described by Sampath et al. (Sampath et
al., 1998
) may be mediated by calpain
(Stewart et al., 1998
).
In summary, we have shown that calpain regulates adhesive complex
disassembly and that inhibiting calpain with calpastatin, stabilizes
peripheral adhesive complexes and inhibits contact site disassembly and
translocation. Further, we found that calpain is required for
microtubule-mediated focal complex turnover but not targeting after nocodazole
wash-out. Finally, our findings suggest that -actininzyxin
co-localization at focal contacts may be an important component of the
calpain-dependent mechanism that regulates focal complex turnover and
translocation. A challenge for future studies will be to identify the critical
substrate or substrates that mediate calpain's effects on focal adhesion
disassembly and cell migration.
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Acknowledgments |
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Footnotes |
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