Author Correction
Paxillin-dependent stimulation of microtubule catastrophes at focal
adhesion sites
Andrey Efimov1, Natalia Schiefermeier2, Ilya Grigoriev3, Ryoma Ohi1, Michael C. Brown4,
Christopher E. Turner4, J. Victor Small5 and Irina Kaverina1,*
1
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA
University of Innsbruck, Innsbruck, Austria
Erasmus University, Rotterdam, The Netherlands
4
SUNY Upstate Medical University, Syracuse, NY, USA
5
IMBA, Institute of Molecular Biotechnology, Vienna, Austria
2
3
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science 121, 405 (2008) doi:10.1242/jcs.03497
There was an error published in J. Cell Sci. 121, 196-204.
The corresponding author regrets that a name was omitted from the author list and wishes to correctly acknowledge Ryoma Ohi for work
presented in this paper.
196
Research Article
Paxillin-dependent stimulation of microtubule
catastrophes at focal adhesion sites
Andrey Efimov1, Natalia Schiefermeier2, Ilya Grigoriev3, Michael C. Brown4, Christopher E. Turner4,
J. Victor Small5 and Irina Kaverina1,*
1
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA
University of Innsbruck, Innsbruck, Austria
3
Erasmus University, Rotterdam, The Netherlands
4
SUNY Upstate Medical University, Syracuse, NY, USA
5
IMBA, Institute of Molecular Biotechnology, Vienna, Austria
2
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 1 November 2007
Journal of Cell Science 121, 196-204 Published by The Company of Biologists 2008
doi:10.1242/jcs.012666
Summary
An organized microtubule array is essential for the polarized
motility of fibroblasts. Dynamic microtubules closely interact
with focal adhesion sites in migrating cells. Here, we examined
the effect of focal adhesions on microtubule dynamics. We
observed that the probability of microtubule catastrophes
(transitions from growth to shrinkage) was seven times higher
at focal adhesions than elsewhere. Analysis of the dependence
between the microtubule growth rate and catastrophe
probability throughout the cytoplasm revealed that a
nonspecific (mechanical or spatial) factor provided a minor
contribution to the catastrophe induction by decreasing
microtubule growth rate at adhesions. Strikingly, at the same
growth rate, the probability of catastrophes was significantly
higher at adhesions than elsewhere, indicative of a site-specific
biochemical trigger. The observed catastrophe induction
Introduction
The dynamics of microtubules in higher eukaryotic cells are
complex, being tightly regulated by a variety of microtubuleassociated proteins. However, the precise mechanisms of this
regulation are only partially understood. It has been well accepted
that microtubules demonstrate random transitions from periods of
growth to shortening and back at their plus-ends, termed ‘dynamic
instability’ (Mitchison and Kirschner, 1984), with most minus-ends
being blocked owing to their association with microtubule
organizing centers (MTOCs). Plus-end dynamics can be described
by five parameters: the rates of growth and shortening, catastrophe
frequency (transitions from growth to shortening), rescue
frequency (transitions from shortening to growth) and time spent
in pauses.
The overall pattern of microtubule dynamics differs between
different cell types (Shelden and Wadsworth, 1993) and depends
on the availability of serum factors (Danowski, 1998), as well as
the phase in the cell cycle (Salmon et al., 1984). Most important,
regional microtubule dynamics can differ within a single polarized
cell. Thus, de-tyrosinated stable microtubules are found
specifically in advancing lamellae of fibroblasts in wound-healing
assays (Nagasaki et al., 1992). Dynamic microtubules in the rear
of motile fibroblasts undergo more catastrophes than those in the
front, whereas certain microtubules in lamellae, so-called ‘pioneer’
microtubules, are characterized by almost no catastrophes
(Wadsworth, 1999; Waterman-Storer and Salmon, 1997).
occurred at adhesion domains containing the scaffolding
protein paxillin that has been shown previously to interact with
tubulin. Furthermore, replacement of full-length paxillin at
adhesion sites by microinjected paxillin LIM2-LIM3 domains
suppressed microtubule catastrophes exclusively at adhesions.
We suggest that paxillin influences microtubule dynamics at
focal adhesions by serving as a scaffold for a putative
catastrophe factor and/or regulating its exposure to
microtubules.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/121/2/196/DC1
Key words: Microtubules, Focal adhesions, Microtubule catastrophe,
Paxillin, Cell motility
Microtubule dynamics in vivo can be specifically regulated by
diverse protein factors (Amos and Schlieper, 2005; Howard and
Hyman, 2007), certain posttranslational tubulin modifications
(Tran et al., 2007) or tensile forces (Kaverina et al., 2002). In the
past few years, more and more data indicate that interactions with
the actin cytoskeleton play a crucial role in the local regulation of
microtubule dynamics (Etienne-Manneville, 2004; Rodriguez et
al., 2003). Indeed, it is the actin system and its regulators that
possess region-specific differences in motile cells. Noteworthy in
this regard, actin anchorage sites – focal adhesions – are able to
precisely influence the dynamics of individual microtubules in the
course of microtubule targeting of focal adhesions (Kaverina et al.,
1998). The mechanism of targeting includes directional growth of
the plus-end to the adhesion site and a short association of the
microtubule tip with the adhesion plaque. Three subsequent
scenarios are possible: first, the microtubule continues to grow,
second it pauses in the adhesion and, third, it undergoes catastrophe
and shrinks (Small and Kaverina, 2003). In this study, we show
that catastrophe events are specifically enriched at focal adhesion
sites. Such precise catastrophe activity has not been described
before at any specific locus of interphase cells.
The nature of microtubule catastrophes can be diverse. They can
be a part of dynamic instability and depend on the availability of
free tubulin. Additionally, in vitro catastrophe frequency can be
enhanced when microtubules polymerize against a stiff obstacle
(Janson et al., 2003). Thus, a possibility exists that the catastrophes
Journal of Cell Science
Microtubule catastrophes at focal adhesions
at adhesion sites are enhanced passively owing to the mechanical
stiffness of adhesions or owing to the spatially restricted
availability of free tubulin at dense adhesion plaques. However, it
is known that catastrophes in cells are often induced by specific
molecular factors, including stathmin or proteins of the kinesin-13
family (Cassimeris, 2002; Wordeman, 2005). Our present data
suggest that a biochemical trigger locally activated at the focal
adhesion plaque is crucial for catastrophe stimulation, whereas the
physical influence of a dense adhesion structure makes a minor
contribution to this process. In order to clarify what makes the
adhesion plaque a preferred site for induction of catastrophe, we
investigated focal adhesion molecules potentially involved in
interacting with microtubules. In particular, we studied the focal
adhesion scaffolding protein paxillin, which previously was found
to bind to tubulin (Herreros et al., 2000).
Paxillin is a 68-kDa scaffold protein that contains many proteinbinding modules and interacts with a variety of structural and
signalling molecules (Brown and Turner, 2004). Paxillin contains
two principle structural domains: five LD domains at the aminoterminus and four LIM domains at the carboxyl terminus. A
number of kinases and phosphatases crucial for adhesion signalling
bind to either LD or LIM domains, making paxillin a central player
in several regulatory pathways (Brown and Turner, 2004). The
paxillin LIM-domain region has been shown to serve as an anchor
to a plasma membrane component and thus assure localization to
focal adhesion sites (Brown et al., 1996), although the protein that
mediates this association has not yet been identified. Of note,
paxillin LIM2-LIM3 domains are also capable of binding to tubulin
(Brown and Turner, 2002; Herreros et al., 2000).
In the present study, we show that the same two LIM domains
of paxillin significantly change microtubule dynamics at adhesion
sites when microinjected into fibroblasts. Substituting full-length
197
paxillin at focal adhesions, LIM2-LIM3 protein reduces the
catastrophe frequency exclusively at these locations, thus
implicating paxillin in the local regulation of microtubule
catastrophes.
Results
The frequency of catastrophes is higher at focal adhesion sites
In order to visualize microtubule dynamics clearly in relation to
focal adhesions, we applied total internal reflectance fluorescence
(TIRF) microscopy to illuminate only the ventral layer of cells
attached to a glass substrate (Axelrod, 1989). Fish fibroblasts were
transfected with mCherry-tubulin or 3xGFP-EMTB to visualize
microtubules and GFP-paxillin or mCherry-paxillin to mark
adhesion sites (Fig. 1A-C). Under TIRF conditions, we assumed
that colocalization of a microtubule with an adhesion site indicates
their close physical interaction. In live-cell time-lapse sequences,
we observed that, when growing microtubules reached an adhesion
site, they frequently underwent catastrophe and shrank. Individual
microtubules often underwent multiple catastrophe events at a
focal adhesion followed by subsequent rescues (Fig. 1A), whereas
only few catastrophes occurred in adhesion-free areas (Fig. 1B).
Analysis of overall adhesion numbers showed that approximately
40% of catastrophes occurred at adhesion sites, 12% at the cell
edge and 48% elsewhere (Fig. 1D). As the overall adhesion area
is considerably smaller than the adhesion-free ventral cell surface,
these numbers indicate that adhesions serve as preferential sites for
catastrophes.
A simple quantification of catastrophe events within a large area
is insufficient for the analysis of the induction of microtubule
catastrophes at local sites. The reason is that, because catastrophe
is an event in which a microtubule switches from growth to
shrinkage, it can occur only to a growing but not to a shortening
plus-end. Thus, only those sites where growing
microtubule ends were present were taken into
consideration. Accordingly, we measured the
length of elongation for microtubules while
growing through adhesions or through an
adhesion-free area. To obtain average persistent
microtubule elongation, we normalized the average
Fig. 1. Microtubule catastrophes at focal adhesions (FA) are
specific events. (A,B) Kymographs of microtubule
dynamics at focal adhesions (A) and in adhesion-free
cytoplasm (B). Upper panels show 3xGFP-EMTB (green),
mCherry-paxillin (red) and microtubule life-history plot
(white line); catastrophes at adhesions (arrows) and a
catastrophe in an adhesion-free area (arrowhead) are
indicated. Lower panels show microtubule images only.
Microtubule shrinkage of 0.5 ␮m or more is considered to
be a catastrophe. (C) Frame from a TIRF video sequence of
fish fibroblasts co-transfected with 3xGFP-EMTB (green)
to visualize microtubules and mCherry-paxillin (red) to
mark focal adhesions. Bar, 10 ␮m. The boxed region is
presented in the kymograph in A. (D) Microtubule
catastrophe distribution in the ventral cell layer. A total of
292 catastrophes were quantified in five 3xGFP-EMTBand mCherry-paxillin-co-transfected cells within 12
minutes. (E) The average microtubule elongation per
catastrophe is reduced by a factor of seven at focal
adhesions (blue, 0.71 ␮m/catastrophe) compared with
elsewhere (red, 4.91 ␮m/catastrophe). A total of 24
microtubules were quantified in five 3xGFP-EMTB- and
mCherry-paxillin-co-transfected cells within 12 minutes.
Error bars are ± s.d.
198
Journal of Cell Science 121 (2)
Journal of Cell Science
microtubule elongation by the average number of catastrophe
events for each group of data. Strikingly, persistent microtubule
elongation without catastrophes was seven times lower at adhesion
sites (Fig. 1E) than elsewhere. These data suggest a specific
mechanism that triggers microtubule catastrophes at adhesion sites.
adhesions, the proportion of slow microtubules increased by up to
50% within the same microtubule population (Fig. 2D). At the
same time, the catastrophe ratio dramatically increased at adhesion
sites. For microtubules approaching adhesions, the probability of
catastrophe increased from less than 5% on the approach route to
~25% at the adhesion (Fig. 2F).
Adhesions trigger catastrophes by combined mechanical and
Combined, these data reveal a correlation between microtubule
biochemical factors
tip speed and catastrophe ratio. The percentage of ‘slow MTs’
A crucial question is whether catastrophes at adhesions are
increases (Fig. 2D, red line) at the same location at inner adhesions
triggered by a specific biochemical mechanism or as a consequence
(zones 0-1) where catastrophes are induced (Fig. 2F, blue line).
of the increased density of actin filaments at the adhesion plaque.
These data suggest that the dense focal adhesion structure
Such nonspecific mechanisms could include mechanical restraint
suppresses microtubule growth and thus increases the probability
or spatial restriction for free tubulin penetration. In order to answer
of catastrophe. This mechanism, however, is not specific for
this question, we analyzed the dynamics of mCherry-EB3-marked
adhesions. We found that the catastrophe ratio of individual
plus-tips of microtubules in the vicinity of GFP-paxillin-containing
microtubules correlated inversely with their plus-tip velocity, both
focal adhesion sites (Fig. 2).
for microtubules growing through adhesions and through adhesionKymographic analysis of the dynamics of microtubule tips
free areas (Fig. 2E), suggesting that the microtubule catastrophe
revealed two scenarios: either EB3 was released from a
rate can be increased by cytoplasmic rigidity or spatially restricted
microtubule at an adhesion site, indicative of a catastrophe (Fig.
concentration of tubulin dimers equally at adhesion plaques and at
2A), or a growing tip proceeded through an adhesion (Fig. 2B). In
other locations. Strikingly, for microtubules with the same growth
the second case, the velocity of microtubule tips was notably
rate, the catastrophe ratio was higher at adhesion sites than
reduced while moving through the adhesion site (Fig. 2B). In
elsewhere (Fig. 2E).
adhesion-free cytoplasm, ~70% of microtubules grew at a rate of
We have further tested whether softening of the adhesion
0.1 ␮m/second or more (‘fast microtubules’) and ~30% of
structure decreases the ability of adhesions to induce catastrophes.
microtubules grew more slowly (‘slow microtubules’). At focal
For this purpose, we plated cells on a soft gelatin gel cushion (Seals
et al., 2005), where strong tensile forces
cannot be developed (Discher et al., 2005).
Adhesions formed under these conditions
were smaller than those on glass substrates
(<2 ␮m versus <4 ␮m in length), but the
catastrophe ratio at such adhesions was
similar (23% on glass and 32% on gelatin
gel). Together, these data indicate that a
specific trigger of biochemical catastrophe
acts at adhesion sites.
In line with this finding, some
microtubules continued to polymerize when
their plus-ends were apparently prevented
from advancing by an obstacle. The
polymerizing plus-ends of such microtubules
remained immobile while the length of the
microtubule increased, resulting in loop
formation (supplementary material Fig. S1).
Thus, mechanical obstacles do not
necessarily cause microtubule catastrophe.
Combined, our results strongly suggest that a
Fig. 2. Microtubule tip dynamics at focal adhesions. (A) Kymograph of a microtubule catastrophe
biochemical mechanism, rather than a
event at a focal adhesion (FA). The microtubule is marked with mCherry-EB3 (red), and the focal
adhesion is marked with GFP-paxillin (green). (B) Kymograph of a microtubule tip (mCherry-EB3,
mechanical mechanism or spatially restricted
red) that does not undergo catastrophe at the adhesion (GFP-paxillin, green). The microtubule tip
concentration of tubulin dimers, drives
changes its growth dynamics at the focal adhesion. (C) An example of assigned area zones for
induction of microtubule catastrophe at focal
microtubule growth paths near focal adhesions (GFP-paxillin, green). Cytoplasmic zones close to the
adhesion sites.
cell center are encoded as zones ‘–2’ and ‘–1’. The zone adjacent to a focal adhesion is encoded as
zone ‘0’. A focal adhesion contains zones ‘1-3’, according to its length. Cytoplasmic zones towards the
cell periphery are encoded as zones ‘1out’ and ‘2out’. Each zone is 1 ␮m in size. (D) The distribution
between fast (growth rate >0.1 ␮m/second) and slow (growth rate <0.1 ␮m/second) microtubules for
each zone. In the cytoplasm (yellow background), ~70% of microtubules grow at a speed of 0.1
␮m/second or faster. At focal adhesions (green background), the percentage of fast microtubules
reduces down to ~50%. (E) Dependence of the microtubule catastrophe ratio (percentage of
approaching microtubules undergoing catastrophe) on the time a microtubule spends in the cytoplasm
zone (zone –1, green), at the adhesion base (zone 0, blue) and in the adhesion (zone 1, red). (F) Total
microtubule catastrophe ratio for each zone. The percentage of approaching microtubules undergoing
catastrophe increases from 2% in the cytoplasm to 25% at focal adhesions. A total of 139 microtubules
were quantified in three 3xGFP-EMTB- and mCherry-paxillin-co-transfected cells within 15 minutes.
Bar, 1 ␮m.
Induction of catastrophe does not depend
on the maturation stage of adhesions
Having determined that a specific
biochemical mechanism at the adhesion sites
enhances microtubule catastrophe activity,
we aimed to identify focal adhesion proteins
potentially involved in the process. As focal
adhesion composition and signalling changes
during their life course, we first investigated
Microtubule catastrophes at focal adhesions
199
paxillin, which is present in the majority of adhesion sites, and
zyxin, which has been shown to incorporate into adhesions at a
later maturation stage (Zaidel-Bar et al., 2003). Simultaneous
transfection of the microtubule marker GFP-EMTB, mCherrypaxillin and Cerulean-zyxin allowed us to compare microtubule
dynamics at early (containing only paxillin) versus late (containing
paxillin and zyxin) adhesions (Fig. 3A,B).
Overall quantification based on time-lapse live-image sequences
revealed that ~90% of adhesion-associated catastrophes occur at
late adhesions, and only ~10% at early adhesions (Fig. 3C).
Nevertheless, taking into account that late adhesions are
approached by a significantly higher number of microtubules, the
final analysis showed that both types of adhesions were equally
effective in catastrophe induction: 90% of approaching
microtubules underwent catastrophe at adhesions (Fig. 3D)
regardless of their maturation stage.
Journal of Cell Science
Paxillin-associated adhesion structures are sufficient for
induction of microtubule catastrophe
Fig. 3. Microtubule catastrophes do not depend on the maturation stage of
focal adhesions. (A,B) A frame from a video sequence of a cell co-transfected
with mCherry-paxillin (A, red) and Cerulean-zyxin (B, cyan). Paxillin marks
both early (hollow arrows) and late (filled arrows) focal adhesions. Zyxin is a
marker for late focal adhesions only (filled arrows). Bar, 5 ␮m.
(C) Microtubule catastrophe ratio at early versus late adhesions.
Approximately 90% of approaching microtubules undergo catastrophe both at
early and late adhesions. A total of 117 catastrophe events in four cells were
quantified.
whether induction of catastrophe depends of the adhesion
maturation stage. To distinguish initial (early) focal adhesions from
mature (late) adhesion sites, we used two adhesion markers:
Fig. 4. Catastrophes can occur in the zyxinfree zone of focal adhesions. (A) A frame
from a video sequence of a cell cotransfected with mCherry-paxillin (red) and
GFP-zyxin (green). Bar 10 ␮m.
(B,C) Enlargement of areas at the leading
edge (B) and trailing edge (C) of the cell
(paxillin, red; zyxin, green). The intensity
profiles of paxillin (red lines) and zyxin
(green lines) along a line (white) of width
one pixel are shown. Paxillin extends more
distantly towards the cell edge. The image is
a representative example from 15 cells.
(D) A frame from a video sequence of a cell
co-transfected with GFP-EMTB (blue) to
visualize microtubules, Cerulean-zyxin
(green) to mark late adhesions and mCherrypaxillin (red) to mark both early and late
adhesions. Bar, 10 ␮m. The boxed area is
enlarged to the right (E,F). (E) Enlarged
frame sequence of microtubules (green) and
paxillin (pink). (F) Enlarged frame sequence
of microtubules (green) and zyxin (red). A
catastrophe occurred at the 20-second timepoint at the distal zyxin-free end of a focal
adhesion. Time, seconds. Arrows show the
direction of microtubule movement.
(G) Enlarged frame sequence of the focal
adhesion from E and F, showing zyxin
(green) and paxillin (red). The arrows point
at the zyxin-free distal end of the focal
adhesion.
Although both zyxin and paxillin are present in late adhesions,
we found that these adhesion proteins are distributed unevenly
within single elongated mature adhesion sites: paxillin extends
more distantly towards the cell edge than zyxin (Fig. 4). Such
uneven distribution was found both at adhesions behind the
leading edge (Fig. 4B) and sliding adhesions at the trailing edge
(Fig. 4C) of the cell. We observed that a considerable number of
microtubules underwent catastrophe at the distal ends of late
adhesions (Fig. 1B). Detailed analysis of these catastrophes
showed that they occurred at paxillin-rich adhesion domains
devoid of zyxin (Fig. 4D-G). These data suggest that paxillinassociated adhesion structures are sufficient for induction of
microtubule catastrophe.
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Journal of Cell Science 121 (2)
Journal of Cell Science
Displacement of paxillin from focal adhesions by microinjection
of LIM domains 2 and 3 decreases the number of catastrophes
specifically at adhesion sites
We further examined the involvement of paxillin in the regulation
of catastrophe by displacement of paxillin from adhesion sites. For
this purpose, individual LIM domains of paxillin, including LIM2,
LIM3 and tandem LIM2-LIM3 domains were produced as GSTfusion proteins (see Materials and Methods) and microinjected into
cells. One hour after injection, cells were fixed and processed for
immunostaining. Single GST-LIM2 (Fig. 5A,B) and GST-LIM3
(data not shown) showed mostly diffuse staining in the cytoplasm,
with only occasional weak focal adhesion localization, whereas
GST-LIM2-LIM3 was localized robustly to focal adhesions (Fig.
5C,D). Moreover, live-cell imaging of GFP-paxillin-expressing
cells showed that injected LIM2-LIM3 domains caused
displacement of GFP-paxillin from adhesion sites within 45
minutes (Fig. 5E,F). Immunostaining of injected cells confirmed
paxillin displacement (data not shown) while another core adhesion
component, vinculin, remained in the adhesions (Fig. 5D).
Notably, although LIM2-LIM3 domains have been shown to
bind to tubulin (Brown and Turner, 2002; Herreros et al., 2000),
no detectable localization to microtubules has been observed for
the injected protein (Fig. 5C). Thus, if the LIM2-LIM3 domains of
paxillin interact with microtubules in vivo, it can only occur in the
vicinity of focal adhesions.
In order to determine whether substitution of paxillin by the
LIM2-LIM3 domain at adhesion sites modulates microtubules at
this location, we microinjected LIM2-LIM3 into fish fibroblasts
co-transfected with GFP–␤-tubulin and dsRed-zyxin. Time-lapse
live-image sequences were recorded before the injection as well as
5 minutes after the injection. We found first that microtubules more
often exhibited persistent growth resulting in an increase of
microtubule length and density at the cell periphery (Fig. 6A,B).
Analysis of microtubule dynamics revealed that, for each cell, the
percentage of catastrophe events in the adhesion sites was 40%
lower than in the same cell before injection (Fig. 6G,H). Typically,
microtubules grew processively through three to four focal
adhesions during a single growth phase in comparison with one to
two adhesions in the same cell before injection (Fig. 6C-F). Along
with a statistically significant decrease of catastrophe frequency at
adhesions, no changes were observed in microtubule catastrophes
in adhesion-free cytoplasm (Fig. 6G,H). This effect appeared to be
specific for LIM2-LIM3 domains as microinjections of the paxillin
LIM1 domain alone and either LIM1-LIM2 or LIM3-LIM4
domains had no influence on microtubule dynamics (data not
shown).
Furthermore, we found that injection of LIM2-LIM3 paxillin
domains in GFP-EB1-expressing cells resulted in an increased
number of growing microtubules but had no effect on the velocity
of microtubule growth. In particular, the number of EB1 comets
indicating polymerizing microtubule plus-tips was increased by
30% 5 minutes after LIM2-LIM3 microinjection (supplementary
material Fig. S2). However, the rate of microtubule growth did not
change significantly (supplementary material Fig. S2), indicating
that, other than decreasing the rate of catastrophe at the adhesion
site, injection of LIM2-LIM3 protein had no detectable effect on
microtubule polymerization.
Discussion
Substrate adhesion turnover in fibroblasts and other cells is
influenced by changes in microtubule dynamics (Small and
Kaverina, 2003; Wittmann and Waterman-Storer, 2001). Our
previous findings suggested that the origin of this interdependence
is a direct cross-talk between microtubules and focal adhesions,
involving their mutual interaction during dynamic targeting events
(Kaverina et al., 2002; Kaverina et al., 1999). However, the
molecular players involved in interactions between microtubules
and focal adhesions remained unclear. Here, we have shown that
microtubule catastrophes are specifically enriched at adhesion
sites. In adhesion-free cytoplasm, a microtubule can grow for 4.9
␮m on average without catastrophes, whereas, at adhesion sites,
catastrophes can occur after only 0.7 ␮m of microtubule extension.
The accumulation of catastrophe events at the focal adhesion
sites is notable for two reasons. First, focal adhesions serve as
platforms where diverse signalling pathways are initiated,
including those controlling cell motility and morphology, as well
as proliferation and differentiation (DeMali et al., 2003; Martin,
2003). Second, microtubule catastrophe leads to a local release of
Fig. 5. LIM2-LIM3 displaces full-length GFP-paxillin from focal adhesions.
Cells microinjected with GST-LIM2 (A,B) and GST-LIM2-LIM3 (C,D)
proteins were stained with antibodies against GST (A,C) and vinculin (B,D).
Only GST-LIM2-LIM3 (C,D) localizes to focal adhesions. Bar 10 ␮m. Cells
expressing GFP-paxillin (E,G) were microinjected with either GST-LIM2LIM3 (F) or GST only (H). By 45 minutes after injection of GST-LIM2-LIM3,
paxillin is displaced from existing focal adhesions (F, arrows) and does not
appear in new adhesion sites. Microinjection of just GST changed neither the
intensity level of paxillin in old adhesions (H, arrows) nor the ability of
paxillin to incorporate into new adhesion sites (H, arrowheads). Bar, 10 ␮m.
Microtubule catastrophes at focal adhesions
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Journal of Cell Science
Fig. 6. GST-LIM2-LIM3 inhibits microtubule catastrophe at focal
adhesions. (A,B) Cell expressing GFP–␤-tubulin (green) and
mRFP-zyxin (red) before (A) and after (B) microinjection of GSTLIM2-LIM3 protein. After injection, the microtubule density
increases as a result of catastrophe suppression at adhesion sites.
Bar 10 ␮m. (C,D) Kymographs of microtubule dynamics at focal
adhesions. A microtubule undergoes catastrophe at an adhesion
before injection (C) but grows through adhesions after injection (D,
arrows). (E,F) Examples of microtubule life-history plots in cells
before (E) and 30 minutes after (F) injecting the GST-LIM2-LIM3
construct. The black line indicates the tracking position of
individual microtubule tips in relation to focal adhesions (FA)
marked with red, green and yellow lines. Note multiple catastrophes
at adhesions in (E), whereas microtubules grow through several
adhesions without catastrophe in (F). (G) Frequency of catastrophes
per square micron before and after microinjection of GST-LIM2LIM3 protein. Upon injection, the frequency of catastrophes
reduces in focal adhesions (upper bracket) but stays the same in the
cytoplasm (lower bracket). Data are shown for 271 catastrophes in
five individual cells recorded both before injection and 30 minutes
after injection. (H) The difference in microtubule catastrophe before
(blue boxes) and after (red boxes) injection of GST-LIM2-LIM3
shown in a ‘box-and-whisker’ plot. The P value was calculated by a
t test (Microsoft Excel). The reduced frequency of catastrophes at
focal adhesions after injection of GST-LIM2-LIM3 was statistically
significant (P<0.001). At the same time, the frequency of
catastrophes in the cytoplasm did not change (P=0.24).
a large group of microtubule-associated regulatory proteins. For
example, microtubule catastrophe and disassembly results in the
release of the microtubule-binding factor Rho GEF-H1 (Krendel et
al., 2002) that mediates cross-talk between microtubules and the
actin cytoskeleton through activation of Rho. Release of this factor
from microtubules drives cell movements during the process of
convergent extension of Xenopus laevis embryos (Kwan and
Kirschner, 2005), making catastrophes crucial regulatory events in
Xenopus development. Additionally, a set of plus-tip-binding
proteins that are concentrated at the growing microtubule plus-ends
is released at catastrophe sites. These proteins include adenomatous
polyposis coli (APC) that is able to bind to members of the Racand Cdc42-regulatory pathways ASEF and IQGAP1 (Kawasaki et
al., 2000; Watanabe et al., 2004). Thus, catastrophes might be
important for modulation of Rac and Cdc42 signalling, controlling
the actin cytoskeleton and cell polarity. Moreover, the release of
APC can induce degradation of ␤-catenin and thus cause silencing
of Wnt signalling (Polakis, 2007), suggesting that microtubule
catastrophes might be involved in the regulation of cell
proliferation and differentiation.
If signalling molecules are released from microtubules
at adhesions, they could be brought into direct association
with molecular factors concentrated at the adhesion
plaques, including, among others, key players of the Src
signalling pathway (Hanks et al., 2003; Parsons, 2003) and
molecules controlling actin polymerization [PAK (Brown
et al., 2002), Arp2/3 (Kaverina et al., 2003)]. The aim of
this study was to understand the mechanisms whereby
adhesions increase the probability of microtubule
catastrophes. We addressed adhesion properties that might
be responsible for enforcement of catastrophe.
Focal adhesions in cultured cells can be distinguished
according to their molecular composition, dynamics and
function (Zamir and Geiger, 2001). Early focal adhesions
(also called focal complexes) are dot-like adhesions that
assemble under the lamellipodium (Nobes and Hall,
1995; Rinnerthaler et al., 1988; Rottner et al., 1999).
Within less than a minute of their formation, early adhesions either
turn over or undergo a force-dependent transformation into late, or
mature, focal adhesions (Zaidel-Bar et al., 2003). Late adhesions
are considerably larger structures that are associated with
actomyosin stress fibers and require contractility for their
maintenance (Bershadsky et al., 2003; Galbraith et al., 2002). The
protein composition of early and late adhesions is similar, although
not identical. Paxillin, the major scaffolding adhesion factor, is one
of the first proteins incorporated into early adhesions and remains
associated during their transformation into mature late adhesions
(Zaidel-Bar et al., 2003), and the majority of adhesion proteins
colocalize with paxillin throughout the cell. By contrast, zyxin has
been shown to be absent consistently from early adhesions and to
be associated only with late adhesions (Rottner et al., 2001; ZaidelBar et al., 2003). Zyxin is also one of the first proteins dissociated
from disassembling adhesions (Rottner et al., 2001). Based on
these facts, we assumed that a paxillin-associated adhesion core
might be considered distinct from zyxin-containing structures, and
we used paxillin only and the zyxin-paxillin combination as
markers for early and late adhesions, respectively.
Journal of Cell Science
202
Journal of Cell Science 121 (2)
Fig. 7. Model of paxillin involvement in the induction
of microtubule catastrophe at adhesion sites. In control
cells: (1) Paxillin (light blue) binds to adhesion sites
(blue) through LIM2-LIM3 domains. Catastrophe
factor (‘Cat F’, red box) binds to paxillin through sites
other than LIM2-LIM3 domains and in this way is
enriched at adhesion sites. (2A) When a microtubule
(green) approaches a focal adhesion, the catastrophe
factor associated with paxillin induces microtubule
catastrophe (red star) and depolymerization (green
arrow). Alternatively (2B), when a microtubule (green)
approaches a focal adhesion, the catastrophe factor
associated with paxillin activates microtubuleassociated catastrophe-inducing factor (‘CIF’, magenta
box) to induce microtubule catastrophe (magenta star)
and depolymerization (green arrow). In LIM2-LIM3injected cells (3), exogenous LIM2-LIM3 (light blue)
binds to the adhesion site and replaces full-length
paxillin. The catastrophe factor cannot bind to the
LIM2-LIM3 mutant protein and is excluded from
adhesion sites. (4) When a microtubule approaches the
focal adhesion, it does not undergo catastrophe and
continues to polymerize (green arrow) because
catastrophe factor is absent or not activated at the
adhesion site.
Early adhesions are associated with poorly developed actin arrays
and their rigidity is low compared with that of stress fiber-bound late
adhesions (Bershadsky et al., 2003; Chen et al., 2004). However,
microtubules undergo catastrophes both at early and late adhesions
with the same efficiency (Fig. 3D). Decreasing the adhesion rigidity
by growing cells on a gelatin cushion also did not decrease their
ability to cause microtubule catastrophes. Additionally, a
microtubule tip hitting an intracellular obstacle often does not result
in a catastrophe. Combined, these observations suggest that
mechanical factors probably play a minor role in promoting
catastrophes. Another option for passive induction of catastrophes
would be the spatially decreased availability of free tubulin at dense
adhesion plaques. However, the density probably varies in the same
way as does rigidity between early and late adhesions (i.e. early
adhesions are less dense) as well as in adhesions under low tension
on a gelatin gel substrate. Thus, our observations indicate that a
mechanical factor does not contribute significantly to induction of
catastrophes at adhesions. The same line of evidence suggests that
spatial restriction of the tubulin concentration at dense adhesions
does not strongly influence induction of catastrophes. We conclude
that the influence of adhesions on microtubule catastrophes is
attributable to a specific biochemical signal.
Catastrophes frequently occur at zyxin-free early adhesions or
at the distal end of late adhesions rich in paxillin but devoid of
zyxin. This finding suggests that the catastrophe factor is coupled
with paxillin-associated, rather than with zyxin-associated,
structures. The association of zyxin with adhesions is promoted by
actomyosin contractility. Zyxin not only localizes to mature stressfiber-connected adhesions but it can bind to stress fibers
themselves under tension (Yoshigi et al., 2005). As catastrophes
tend to occur in zyxin-free loci, the presence of a putative
microtubule catastrophe factor at adhesions is probably neither
actin associated nor tension dependent. Most likely, paxillin itself
or paxillin-associated factors are involved in the induction of
microtubule catastrophes at adhesion sites. Strikingly, a recent
study showed that early adhesions and the distal part of late
adhesions are enriched in phosphorylated paxillin (Zaidel-Bar et
al., 2007). As we describe the same adhesion domains as areas of
preferential induction of catastrophes, one can speculate that
catastrophes could be associated with phosphorylated paxillinbased protein complexes.
It has been shown previously that paxillin binding to tubulin
occurs through the LIM2 and LIM3 paxillin domains and requires
intact LIM zinc-finger structures (Brown et al., 2002). We found that
paxillin LIM2-LIM3 domains microinjected into fibroblasts
localized specifically to adhesion sites, replacing full-length paxillin.
This led to a decreased frequency of microtubule catastrophes at this
location. Based on these data, we suggest a model that acknowledges
the role of paxillin as a scaffolding protein at the adhesion sites (Fig.
7). According to this model, paxillin serves as a docking site for the
catastrophe factor, possibly through phosphotyrosine-containing
domains in the amino terminus (Fig. 7-1). When a microtubule
approaches an adhesion site, a paxillin-associated factor can trigger
it to undergo catastrophe and depolymerization directly (Fig. 7-2A)
or through activation of a microtubule-associated catastropheinducing protein (Fig. 7-2B). Injected LIM2-LIM3 domains replace
full-length paxillin at adhesion sites, thereby potentially displacing
the putative catastrophe factor (Fig. 7-3). As a result, microtubules
do not undergo catastrophe at adhesion sites any more (Fig. 7-4). If,
during adhesion targeting, a microtubule directly binds to paxillin by
means of the paxillin LIM2-LIM3 domain (Brown and Turner,
2002), this might additionally promote catastrophe by bringing the
microtubule and catastrophe factor into close proximity. However,
such an interaction might not be necessary for induction of
catastrophe. Similarly, the putative catastrophe factor might not bind
to paxillin directly but be part of a paxillin-organized adhesion core.
The nature of the catastrophe factor involved in microtubule
depolymerization at the adhesion sites remains to be clarified. One
of the well-known catastrophe-inducing molecules, such as
stathmin (Cassimeris, 2002) or a member of the kinesin-13 family,
Microtubule catastrophes at focal adhesions
for example MCAK (Wordeman, 2005), could be engaged in this
process. However, none of these proteins has been found
concentrated at focal adhesions. Thus, it is more likely that upstream
regulators of these proteins, but not the proteins themselves, are
specifically recruited to the adhesion plaques and play the role of
‘catastrophe factors’ described in our model (Fig. 7-2B). As both
stathmin (Larsson et al., 1997) and kinesin 13 (Andrews et al., 2004;
Ohi et al., 2004) are active in the de-phosphorylated form, an
adhesion-associated phosphatase could serve such a role.
Alternatively, a putative catastrophe factor at adhesions could act
through removal of certain stabilizing factors from the microtubule
tip, thus allowing catastrophe-inducing molecules to complete their
function. In this case, catastrophes might be induced by MCAK that
is found at the tips of polymerizing microtubule in an already
activated form (Moore et al., 2005) and can be delivered to adhesion
sites during the course of microtubule targeting of focal adhesions.
In conclusion, our findings attribute to focal adhesions a decisive
role in local regulation of microtubule catastrophes. Paxillin is
probably a major upstream regulator of catastrophe induction at
focal adhesion sites, although the actual catastrophe factor remains
to be identified in future studies.
Journal of Cell Science
Materials and Methods
Cells
Goldfish fin fibroblasts (line CAR, no. CCL71; American Type Culture Collection)
were maintained in DMEM with nonessential amino acids, 25 mM HEPES and with
10% FBS at 27oC. For experiments, cells were plated onto coverslips or glassbottomed dishes (MatTek) and coated with human serum fibronectin (BD
Biosciences) for at least 48 hours. Fibronectin was coated onto coverslips or glassbottomed dishes (50 ␮g/ml in PBS for 30 minutes at room temperature). Gelatin gel
cushion was prepared essentially as described previously (Seals et al., 2005). In brief,
2.5% gelatin in PBS containing 2.5% sucrose was allowed to polymerize for 30
minutes on glass-bottomed dishes (300 ␮l per dish). Next, the gels were crosslinked
by 0.5% glutaraldehyde for 45 minutes. Free aldehyde groups were blocked by 1
mg/ml NaBH4. Finally, the gels were coated with fibronectin, as described above.
DNA constructs and transfection
mCherry-EB3 and Cerulean-EB3, made by replacing GFP in a pEGFP-EB3 construct
(kind gift of A. Akhmanova, Rotterdam, The Netherlands) with Cerulean (kind gift
of D. W. Piston, Nashville, TN) or mCherry (kind gift of R. Tsien, San Diego, CA),
EGFP-EB1 (kind gift of A. Akhmanova, Rotterdam), pEGFP-tubulin (kind gift of J.
Wehland, Germany), mCherry-tubulin (kind gift of R. Tsien) and 3xGFP-EMTB
[microtubule-binding domain of E-MAP-115 (ensconsin) fused to three copies of
EGFP (Bulinski et al., 1999), a kind gift of J. C. Bulinski, New York, NY] was used
for microtubule plus tips and microtubule visualization. Neither tubulin fusion
proteins (Rusan et al., 2001) nor EMTB (Faire et al., 1999) change microtubule
dynamics and are routinely used for in vivo studies. GFP-paxillin (West et al., 2001),
mCherry-paxillin (kind gift of S. Hanks, Nashville, TN), mCherry-zyxin, made by
replacing GFP in a pEGFP-zyxin construct (kind gift of J. Wehland) with mCherry,
and Cerulean-zyxin, constructed by cloning zyxin from mCherry-Zyxin into a
mCerulean-C1 vector, were used for visualization of adhesion sites. Subconfluent
monolayer cultures on 30 mm Petri dishes were used for transfection. For each dish,
the transfection mixture was prepared as follows: 1.5-2 ␮g total DNA and 12 ␮l of
Superfect lipofection agent (Qiagen) were mixed in 200 ␮l of serum-free medium.
After 30 minutes of incubation at room temperature, a further 1.3 ml of medium
containing 5% serum was added. Cells were incubated in this mixture for 4 hours at
27oC and the medium then replaced by normal medium containing 10% serum. After
24 hours, cells were replated at a desired dilution onto coverslips or glass-bottomed
dishes for microscopy.
Microinjections
Injections were performed with sterile Femtotips (Eppendorf) 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.
Proteins for microinjection
Cy3-tubulin was kindly provided by F. Severin (Max Planck Institute, Dresden,
Germany). It was stored at a concentration of 20 mg/ml in aliquots at –70°C. For
microinjections, Cy3-tubulin aliquots were diluted 1:3 with Tris-acetate injection
buffer (2 mM Tris-acetate, pH 7.0, 50 mM KCl and 0.1 mM DTE) and used on the
203
same day. GST only, GST-LIM1, GST-LIM2, GST-LIM3 and GST-LIM2-LIM3
fusion recombinant proteins were expressed in Escherichia coli and purified as
described previously (Brown et al., 1998). GST, GST-LIM1, GST-LIM2, GST-LIM3
and GST-LIM2-LIM3 were stored at a concentration of 1.6 mg/ml at –70°C.
Video microscopy of transfected cells
TIRF live-cell videos were acquired on a Nikon TE2000E microscope equipped with
a Perfect Focus System and Nikon TIRF2 System for TE2000 using a TIRF 100⫻
1.49 NA oil-immersion lens and back-illuminated EM-CCD camera Cascade 512B
(Photometrics) driven by IPLab software (Scanalytics). A 40 mW argon laser (Melles
Griot) and 10 mW DPSS laser 85YCA010 (Melles Griot) were used for excitation.
A custom-made double-dichroic TIRF mirror and emission filters (Chroma) in a filter
wheel (Ludl) were used. Three-channel movies were made with the TIRF setting as
described above for two channels and wide-field fluorescence using a BrightLineCFP 2432A filter cube (Semrock) for the third channel.
Video microscopy of injected cells
Cells were injected and observed in an open chamber at room temperature on an
inverted microscope (Axiovert 135TV; Zeiss) equipped for epifluorescence and
phase-contrast microscopy. Injections were performed at an objective magnification
of 40⫻ (1.3 NA Plan Neofluar) and video microscopy with a 100⫻ 1.4 NA PlanApochroma objective. Double fluorescence was achieved by a GFP-RFP Pinkel filter
set (Chroma) in a custom-made filter wheel. 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. Paxillin displacement from focal adhesions was observed by live TIRF
imaging of GFP-paxillin-expressing cells, as described above.
Photobleaching
Cells coexpressing mCherry-alpha-tubulin and GFP-paxillin where photobleached
for 10 seconds with a 10 mW DPSS laser 85YCA010 (Melles Griot) by focusing
laser light in the focal plane with a custom-made lens (Nikon) placed in the position
of the filter cube. Two-channel movies were recorded using TIRF microscopy, as
described above, for 2.5 minutes after bleaching.
Immunofluorescence microscopy
For immunostaining, cells were extracted for 1 minute in 0.25% Triton X-100 in
cytoskeleton buffer (10 mM MES, 150 mM NaCL, 5 mM EGTA, 5 mM glucose and
5 mM MgCl2, pH 6.1) and fixed for 20 minutes in 3% paraformaldehyde in
cytoskeleton buffer. Immunostaining was performed using polyclonal goat antibodies
against glutathione S-transferase (GST) and mouse antibodies against vinculin.
Quantitative analysis
Microtubule catastrophe: time-lapse movies of cells expressing 3xGFP-EMTB to
label microtubules and mCherry-paxillin to mark adhesion sites (5 seconds/frame)
were processed by background subtraction and intensity adjustment using ImageJ
software. The coordinates of each catastrophe event were recorded and then checked
to determine whether there was an adhesion site with the same coordinates.
Average microtubule elongation: time-lapse movies of cells expressing mCherrytubulin and GFP-zyxin (5 seconds/frame) were processed by background subtraction
and intensity adjustment using ImageJ software. The trajectories of microtubule plusend movement were followed using a plug-in of ImageJ (‘manual tracking’).
Microtubule elongation in the cytoplasm or at adhesion sites was normalized by the
number of catastrophes in each location for each microtubule. Then the average
elongation distance was calculated for all microtubules. Statistical analysis was
performed in Microsoft Excel.
Tip dynamics at adhesion sites: time-lapse movies of cells expressing mCherryEB3 and GFP-paxillin (5 seconds/frame) were processed for intensity adjustment
using ImageJ. For uniform analysis, only the most common cases when microtubule
tips approached adhesions from their proximal ends were considered. Narrow areas
around adhesion sites were delineated and combined into kymographs. The area was
divided into 1-␮m-long zones (Fig. 2C). For each microtubule tip, the time it spends
in each zone and place of catastrophe was calculated manually. Microtubules that
pass through the zone in 1-2 frames (10 seconds or less) were considered as fast
microtubules and in three frames or more (>15 seconds) as slow microtubules. For
each zone, the number of approaching microtubule tips and disappearing tips was
calculated. For zones –1, 0 and 1, the probability of a microtubule undergoing
catastrophe versus the time the microtubule tip spent in the zone was calculated.
Statistical analysis was performed in Microsoft Excel.
Catastrophes at early and late adhesions: time-lapse movies of cells expressing
3xGFP-EMTB, mCherry-paxillin and Cerulean-zyxin (5 seconds/frame) were used.
Trajectories of microtubule plus-end movement were followed using a plug-in
(‘manual tracking’) of ImageJ software. The coordinates of each catastrophe were
recorded and then checked against the localization of paxillin and zyxin by using
ImageJ software. Statistical analysis was performed in Microsoft Excel.
Analysis of microtubule dynamics after microinjection: calculations of
microtubule growth velocity and catastrophe events were made using MetaMorph
software, and statistical analysis was performed in Microsoft Excel.
204
Journal of Cell Science 121 (2)
We thank Steve Hanks and Ethan Lee for helpful discussion and
advice. This study was funded by Development funds of the
Department of Cell and Developmental Biology, Vanderbilt University
Medical Center and by a pilot project within grant ACS IRG-58-00949 to I.K., by Austrian Science Fund grant #P16743-B09P to J.V.S.
and NIH grant RO1 GM47607 to C.E.T.
Journal of Cell Science
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