1 Institute of Molecular Biology of the Austrian Academy of Sciences, A-5020,
Austria
2 Department of Physiology, University of Massachusetts Medical School,
Worcester, Massachusetts 01605, USA
3 Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr.
108, Dresden, D-01307, Germany
* Author for correspondence (e-mail: jvsmall{at}imb.oeaw.ac.at )
Accepted 21 March 2002
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Summary |
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Key words: Microtubules, Actin cytoskeleton, Adhesion, Tension, Mechanosensor
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Introduction |
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Cell locomotion not only involves the mutual and dynamic reorganisation of
the actin cytoskeleton and its associated adhesion sites but also mechanisms
that confer polarity on these structural changes. Only then can traction
forces in the actin cytoskeleton be converted into net movement (e.g.
Beningo et al., 2001). Earlier
findings attributed this polarising function to microtubules
(Vasiliev and Gelfand, 1976
),
and more recent studies have revealed how they may achieve this role
(Kaverina et al., 1998
;
Kaverina et al., 1999
;
Kaverina et al., 2000
). It has
been shown in fibroblasts that microtubules specifically target substrate
adhesion sites and that these targeting events are followed by the turnover of
adhesion sites or their dislocation from the substrate
(Kaverina et al., 1998
;
Kaverina et al., 1999
). Given
the dependence of adhesion site maintenance on contractility, it was
speculated that microtubules destabilise adhesions by delivering signals that
antagonise the contractility pathway. This contention was supported by the
demonstration that dissociation of adhesion sites at the cell edge could be
mimicked by the local application of inhibitors of actomyosin contractility
(Kaverina et al., 2000
).
The question addressed in the present study is what is the mechanism by which microtubules are guided to adhesion sites. We formerly showed that the local application of contractility inhibitors to a cell edge produced a rapid and local depolymerisation of microtubules towards the cell centre. In the present work, we have used alternative approaches to modulate radial stress at the cell periphery. By this means, we demonstrate that microtubules specifically invade regions where stress is locally increased. These findings highlight a stress-dependent feedback mechanism that presumably plays an important role in the selection of adhesion sites for targeted modulation via microtubules.
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Materials and Methods |
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Goldfish fin fibroblasts (line CAR, ATCC) were maintained in basal Eagle
medium with Hanks' BSS and non-essential amino acids and with 15% FBS at
25°C. They were transfected transiently as described previously
(Kaverina et al., 1999). The
stable clone Tub3 was produced by selection with 1 mg/ml G418.
Primary keratocytes from scales of black molly fish or Alpine trout were
prepared and maintained as described previously
(Anderson and Cross, 2000).
The following vectors, kindly provided by J. Wehland and co-workers
(Braunschweig, Germany), were used for expression of EGFP-fused proteins: (1)
mouse beta 3 tubulin in a pEGFP-C2 vector; (2) human zyxin in a pEGFP-N1
vector (Rottner et al., 2001);
and (3) human VASP in a pEGFP-C2 vector (Rottner et al., 1999).
Mouse h1 calponin in pEGFP-C1
(Danninger and Gimona, 2000)
was kindly provided by M. Gimona (Salzburg, Austria). CLIP-170 GFP
(Hoogenraad et al., 2000
) was
a generous gift of A. Akhmanova (Rotterdam).
The B16F1 cell line stably expressing EGFP-beta-actin
(Ballestrem et al., 1998) was
kindly provided by C. Ballestrem (Geneva).
Microinjection and cell manipulation
Injections were performed with sterile Femtotips (Eppendorf, Hamburg) held
in a Leitz Micromanipulator with a pressure supply from an Eppendorf
Microinjector 5242. Cells were injected with a continuous outflow mode from
the needle under a constant pressure of between 20 and 40 hPa. For local
application of drugs, performed with the same system, a constant pressure of
50-100 hPa was used.
Cell manipulations were performed using the same micromanipulator with flamed, curved microinjection needles.
Tetramethyl rhodamine (5-TAMRA; Molecular Probes, USA) conjugated vinculin from turkey gizzard was kindly provided by K. Rottner and M. Gimona. Cy3-conjugated tubulin was kindly provided by J. Peloquin and G. Borisy (Chicago, USA).
For local application through a microneedle, drugs were dissolved in microinjection buffer (2 mM Tris-Acetate pH 7.0, 50 mM KCl). The inhibitor of myosin light chain kinase, ML-7 (Alexis Corporation, Switzerland) was used at a concentration of 300 µM, the actomyosin inhibitor 2,3-butanedione 2-monoxime (BDM) at a concentration 250 mM and H7 (Sigma) at a concentration of 1 mM.
Polyacrylamide substrates
Flexible substrates composed of 5% acrylamide and 0.08% Bisacrylamide were
prepared as previously described (Wang and
Pelham, 1998; Beningo et al.,
2001
). 100 µmL poly-D-lysine in PBS was covalently coated to
the substrates overnight at 4°C following a previously published procedure
(Wang and Pelham, 1998
). After
rinsing with PBS, the substrate was coated with 25 µg/mL Laminin (dialyzed
in PBS 3 hours on ice) for 1 hour at room temperature just prior to
plating.
Video microscopy and image analysis
Cells were injected and observed in an open chamber at room temperature for
CAR cells and keratocytes and in a heated chamber (Warner Instruments,
Reading, UK) at 37°C for B16 cells on an inverted microscope (Axiovert
135TV; Zeiss, Austria) equipped for epifluorescence and phase contrast
microscopy. Injections were performed at an objective magnification of
40x (NA 1.3 Plan Neofluar), and video microscopy was performed with a
100x/NA 1.4 Plan-Apochromat with or without 1.6 optovar intermediate
magnification. Tungsten lamps (100 W) were used for both transmitted and
epi-illumination. Data were acquired with a back-illuminated, cooled CCD
camera from Princeton Research Instruments driven by IPLabs software (both
from Visitron Systems, Germany) and stored as 16-bit digital images. The
microscope was additionally equipped with shutters (Optilas GmbH, Germany) to
allow separate recordings of video sequences in phase contrast and
fluorescence channels and with a filter wheel for two fluorescent channels.
Times between frames were 10 to 25 seconds.
For quantitative analysis of microtubule penetration in lamella, a line was drawn perpendicular to the direction of tension or, for controls, to the direction of protrusion, such that between 5-15 microtubules crossed the line at time `0'. In consecutive video frames the line was fixed at a constant distance from the cell front. Depending on the cell measured, the distance ranged from 4 to 10 µm. The number of microtubules crossing the line was then counted after 1 minute for flexible substrate experiments and after two minutes for cell manipulations.
Fluorescence recovery after photobleaching
Fluorescence recovery after photobleaching (FRAP) was performed in a LSM 5
Pascal confocal microscope (Ziess) using cells expressing GFP-tubulin. A line
was bleached across lamella regions and the time-lapse video of the
microtubule pattern recorded. ML-7 was applied locally as described above.
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Results |
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Lamella regions of B16F1 melanoma cells situated behind rapidly protruding
lamellipodia characteristically show few microtubules that extend close to the
cell front (see also Ballestrem et al.,
2000). However, when stress was applied via a microneedle to the
cell body of B16 cells that had been transfected with GFP-tubulin,
microtubules were seen to extend towards the base of the protruding
lamellipodium (Fig. 1B).
Measurements of 20 cells for a period of 2 minutes showed that there was an
approximately three-fold increase in the number of microtubules that
penetrated a given region of a lamella following the application of stress
(Fig. 2A).
|
In GFP-CLIP-170-transfected cells, the number of fluorescent microtubule plus ends in peripheral lamella regions increased dramatically upon stress application (Fig. 1C, Fig. 2C). Since CLIP-170 binds only to polymerising microtubule tips, these findings show that the stress-induced invasion of microtubules is caused by the stimulation of microtubule polymerisation and not by the transport of pre-existing microtubules towards the cell front by other means. By doubly transfecting cells with GFP-tubulin and GFP-zyxin, we could further show that the microtubules that polymerised to the cell front targeted the adhesion sites that were independently amplified at the cell periphery by the increased tension (Fig. 3).
|
It is notable that the same regions behind rapidly protruding lamellipodia
in B16 cells that are depleted of microtubules also lack well defined bundles
of actin filaments (Ballestrem et al.,
1998). Instead, there commonly exists a loose network of actin
filaments that extends from the base of the lamellipodium into the perinuclear
region, where bundles become more evident
(Rottner et al., 1999b
;
Small et al., 1999b
). In view
of the observed growth of both adhesion sites and microtubules in response to
applied stress, it was important to establish the accompanying changes in the
actin cytoskeleton under the same conditions. For this purpose, we used cells
transfected either with GFP-actin (Fig.
4A) or GFP-calponin. The actin-binding protein calponin is a
particularly useful probe as it binds preferentially to bundles of actin
filaments and not to actin meshworks in lamellipodia or to looser actin
filament arrays in cultured cells (Gimona
and Mital, 1998
). As shown in
Fig. 4B, mechanical stress
induced a rapid and dramatic appearance of actin bundles extending from the
cell centre to the periphery, consistent with the parallel amplification of
peripheral focal adhesions that occurs under the same conditions (Figs
1 and
3). The stress exerted in
lamellae by cell body displacement was estimated on the basis of manipulations
of cells spread on flexible substrates (see below) to be of the order of
5x105 dynes/cm2. This result revealed the
continuity of actin filaments in the cytoplasmic network with peripheral
anchorage sites as well as the competence of these filaments to form bundles
under stress.
|
Stress application to melanoma cells via a flexible substrate
A second method of applying mechanical stress to adherent cells is to use
flexible substrates for growth and to distort the substrate locally with a
micropipette (Lo et al.,
2000). In this case, the distortion can be monitored by the
incorporation of marker beads into the substrate. Accordingly, B16 cells
transfected with GFP-tubulin were plated onto flexible polyacrylamide
substrates, and the substrate was stretched to induce localised stress at the
cell periphery (Fig. 5). The
stress exerted on the cell was estimated to be of the order of 106
dynes/cm2 on the basis of the displacement of the substrate and
previous simulations (Beningo et al.,
2001
). As with cell body manipulation, the application of radial
stress caused a dramatic induction of microtubule growth, so that after 1
minute of tension application there was an average 2.5-fold increase in the
number of microtubules in anterior lamella zones
(Fig. 2B).
|
Using GFP-CLIP-170-transfected cells we could show that this effect was again caused by the enhancement of microtubule polymerisation, as indicated by a dramatic increase in the number of fluorescent microtubule plus ends in stressed regions (Fig. 2C).
Recovery from inhibition of contractility in fibroblasts
An increase in stress in the actin cytoskeleton is also stimulated during
the recovery of cells from an inhibition of actomyosin contractility. As shown
previously, the inhibition of myosin-dependent contractility in fibroblasts
causes the dissolution of focal adhesions and the relaxation of substrate
traction (Chrzanowska-Wodnicka and
Burridge, 1996). When applied locally to a cell edge, myosin
inhibitors not only induce the diminution and release of substrate adhesions
but also the rapid shrinkage of microtubules
(Kaverina et al., 1999
). In
this study, we monitored the short-term behaviour of microtubules and stress
fibres in fish fibroblasts during transient, local treatment with one of three
inhibitors (BDM, ML-7 or H-7). Within 2-3 minutes of inhibitor treatment,
microtubules withdrew from the cell edge
(Fig. 6C). Using the
fluorescence recovery after photobleaching (FRAP) technique in combination
with inhibitor application, we could confirm that the withdrawal of
microtubules was caused by depolymerisation and not by the passive retraction
of the microtubule network (Fig.
6C). Following removal of the inhibitor and the recovery of
tension in the actin cytoskeleton, microtubules repolymerised and targeted the
peripheral adhesion sites (Fig.
6A). Essentially the same result was obtained with all three
inhibitors.
|
The result of using the same experimental protocol with cells transfected with GFP-calponin is presented in Fig. 6B. Typically, the withdrawal of the cell edge was accompanied by the partial dispersion of small peripheral actin bundles, labelled with GFP-calponin. The restoration of contractility following recovery from the inhibitor was marked by bundle growth (Fig. 6B), which is indicative of an increase in the stress level above that before inhibitor treatment. Experiments with GFP-actin-transfected cells gave essentially the same result (data not shown).
Cell body restraint in epidermal keratocytes
In the rapidly migrating fish keratocyte, microtubules are concentrated
around the cell body and are not required for polarised locomotion
(Euteneuer and Schliwa, 1984).
These cells also lack typical focal adhesions and stress fibres but exhibit
small punctate adhesion complexes beneath lamellipodia
(Anderson and Cross, 2000
).
Restraint of the keratocyte cell body with a microneedle caused a dramatic
enhancement in the size of adhesion sites in the lamellipodium
(Fig. 7A), which is indicative
of an increase in stress in the actin network. By microinjecting fluorescent
tubulin into keratocytes we were able to monitor changes in microtubule
dynamics in response to the same mechanical manipulations. As shown in
Fig. 7B, restraint of the cell
body leads to the invasion of microtubules into the lamellipodium, which
extended in the opposite direction from the applied stress.
|
![]() |
Discussion |
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Local tension increases were also induced at the periphery of
Aplysia growth cones by the application and restraint of beads coated
with attachment molecule ligands, and, in line with our findings, microtubules
grew towards the restrained beads (Suter et al., 1998a). Taken together with
an earlier demonstration (Kaverina et al.,
1999) (this study) that localised relaxation of tension causes the
depolymerisation of microtubules away from the cell periphery, our results
highlight the existence of a tension-sensing mechanism for microtubule growth.
The phenomenon we describe should not be confused with the stabilisation of
microtubules at adhesion foci (Kaverina et
al., 1998
) or with the long-term microtubule stabilisation that
depends on RhoA and its effector mDia
(Cook et al., 1998
;
Palazzo et al., 2001
).
Interestingly, the application of tension to chromosomes was recently shown to
amplify the number of kinetochore microtubules, again linking tension with
microtubule growth or stability (King and
Nicklas, 2000
). Two primary processes must be invoked to explain
the effects we have described: a tension-linked stimulation of microtubule
polymerisation and the guidance of microtubules to peripheral sites.
Various possibilities exist for establishing linkages between actin and
microtubule networks (reviewed in Gavin, 1996;
Goode et al., 2000;
Waterman-Storer et al., 2000
)
that may be relevant for the guidance of microtubules to substrate
adhesions.
One possibility is suggested by the demonstration that kinesin and
unconventional myosins can cooperate in the transport of vesicles along
microtubules and actin filaments (Rodionov
et al., 1998). However, the involvement of heterodimeric
myosin-kinesin motor complexes (Huang et
al., 1999
; Beningo et al.,
2000
) in the guidance of microtubules along actin filaments to
adhesion sites (Goode et al.,
2000
) is unlikely since a block in kinesin activity has no effect
on the ability of microtubules to target substrate adhesions
(Krylyshkina et al.,
2002
).
As elaborated in detail by Goode et al., the interaction of microtubules
with actin filaments represents a common feature of morphogenetic events in a
variety of cell types (Goode et al.,
2000). Interestingly, actin cables are needed for maintaining
yeast spindle orientation (Palmer et al.,
1992
; Theesfeld et al.,
1999
), and this process requires myosin V
(Yin et al., 2000
), which can
bind to the microtubule-associated protein Kar 9. Thus, the sliding of a
myosin along actin filaments may serve to direct microtubules toward the
cortex (see also Suter et al., 1998b). Myosin V was also found by
Waterman-Storer et al. to colocalise with dynamic, co-linear arrays of actin
filaments and microtubules observed in Xenopus extracts
(Waterman-Storer et al.,
2000
). A possible mode of linkage for an unconventional myosin to
microtubules has also been suggested by the interaction of a class VI myosin
with a CLIP-170 homologue in Drosophila
(Lantz and Miller, 1998
), but
so far an association of myosin VI with microtubules has not been observed
(Lantz and Miller, 1998
;
Buss et al., 1998
). In this
connection, CLIP-170 (Perez et al.,
1999
) and other proteins at the tips of growing microtubules
(reviewed in Schroer, 2001
;
Tirnauer and Bierer, 2000
;
Schuyler and Pellman, 2001
)
including APC (Mimori-Kiyosue et al.,
2000a
), EB1 (Mimori-Kiyosue et
al., 2000b
), members of the dynactin complex
(Vaughan et al., 1999
) and the
CLASP family (Akhamanova et al.,
2001
) are strategically located where microtubule dynamics as well
as the guidance of microtubule growth are most probably controlled.
With regard to the link between microtubule guidance and stress, the
results with B16 cells transfected with the abundant smooth muscle isoform of
calponin (h1) are particularly interesting. As shown by Gimona and Mital,
calponin binds primarily to stress fibre bundles and not to actin meshworks or
loose actin filament arrays (Gimona and
Mital, 1998). The dramatic appearance of calponin-positive bundles
in the lamella regions of B16 cells after tension application suggests that
tension induces conformational changes in actin filaments, or proteins
associated with them, that facilitate calponin binding. It has already been
shown that actin-binding proteins can induce changes in the twist of the long
pitch helices of actin filaments (Bamberg et al., 1999;
Galkin et al., 2001
): by the
same token the application of torsion to a `relaxed' filament should induce
structural changes allowing interaction with other binding partners. We
suggest that conformational changes in actin induced by stretch play a primary
role in signalling for microtubule polymerisation and guidance. One plausible
scenario includes the linkage of an unconventional myosin to a component of
the microtubule tip complex, whereby this myosin is only competent to bind to
actin filaments that are under tension. This interaction could induce further
conformational changes in the microtubule tip complex itself that promote
microtubule polymerisation. Whether or not such a scenario pertains to
microtubules and whether other regulatory factors are involved that bind to
microtubules (Best et al.,
1996
; Ren et al.,
1998
; Glaven et al.,
1999
) or that influence microtubule assembly
(Carazo-Salas et al., 1999
) in
response to stress-induced signal transduction at adhesion sites remains to be
elucidated. Whatever the mechanism, we appear to have revealed a tension-based
feedback loop that plays a role in the promotion of substrate adhesion
disassembly via microtubules.
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Acknowledgments |
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Footnotes |
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