Department of Anatomy, Johannes Gutenberg-University, Becherweg 13, 55128 Mainz, Germany
* Author for correspondence (e-mail: leube{at}mail.uni-mainz.de)
Accepted 7 August 2002
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Summary |
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Key words: Tyrosine phosphorylation, Keratin, Intermediate filament, Plectin, 14-3-3, Live cell imaging, Green fluorescent protein
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Introduction |
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In terms of molecular diversity, the IF polypeptides of the CK type are
certainly the most complex, comprising more than 50 members, which are
expressed as pairs of type I and type II isoforms in a
differentiation-dependent manner (Moll,
1998; Hesse et al.,
2001
; Coulombe and Omary,
2002
). In contrast to the other major cytoskeletal filaments, IFs
form spontaneously and rapidly in vitro without energy consumption, auxiliary
proteins or other factors (Hofmann,
1998
; Herrmann et al.,
1999
; Herrmann and Aebi,
2000
; Coulombe and Omary,
2002
). In the case of CKs, parallel heterodimers assemble in an
antiparallel fashion into tetramers, which associate longitudinally and
laterally into protofilaments to form the bona fide 8-12 nm IFs
(Herrmann et al., 1999
;
Herrmann and Aebi, 2000
;
Parry and Steinert, 1999
;
Coulombe and Omary, 2002
). In
vivo, soluble tetrameric and larger oligomeric precursors have been identified
which presumably incorporate directly into pre-existing CKFs (e.g.,
Chou et al., 1993
;
Bachant and Klymkowsky, 1996
).
The molecular interactions of IF polypeptides are very strong, thus favouring
the filamentous over the soluble state by far. It takes saturated urea
solutions to completely break up CKFs into their monomeric subunits
(Franke et al., 1983
). In
spite of these extreme biochemical properties, CKs must be in an adjustable
equilibrium between the soluble and filamentous state in vivo to allow
processes that require a less rigid and more pliable cytoskeleton.
Consequently, CKFs can not be viewed simply as rigid and immobile
scaffoldings around which the cell body is arranged. On the contrary, the
arrangement and dynamic properties of the CKF cytoskeleton must be in constant
interplay with cellular requirements. Probably the best examined
physiologically occurring process of major IF rearrangement takes place during
mitosis, when CKFs either disassemble completely into soluble subunits and
granular aggregates or collapse into cage-like thick filament bundles
(Franke et al., 1982;
Lane et al., 1982
;
Jones et al., 1985
;
Kitajima et al., 1985
;
Tölle et al., 1987
;
Windoffer and Leube, 1999
;
Windoffer and Leube, 2001
;
Windoffer et al., 2002
).
Protein modification and altered interactions with IF-associated proteins
(IFAPs) are supposed to be major factors contributing to such CKF network
remodeling. Accordingly, increased levels of phosphorylated CK polypeptides
were detected in mitotic cells (Celis et
al., 1983
; Chou and Omary,
1993
; Liao et al.,
1997
; Omary et al.,
1998
) and also during meiosis
(Klymkowsky et al., 1991
).
Phosphorylation may affect CK organization in a number of different ways: it
leads to a shift in the equilibrium between the soluble and filamentous state
toward the soluble form (Chou and Omary,
1993
); it may directly induce filament disassembly as suggested by
in vitro experiments (Yano et al.,
1991
); and it alters the interaction with IFAPs, as demonstrated
for the binding to the signaling 14-3-3 proteins, which act as solubility
factors for CKs (Liao and Omary,
1996
; Ku et al.,
1998
). Furthermore, phosphorylation of the cytoskeletal
crosslinker plectin may affect CK organisation, as has been shown for
plectinlamin-B and plectin-vimentin interactions
(Foisner et al., 1991
;
Foisner et al., 1996
).
Among the non-filamentous assemblies of CKs, granular aggregates are of
particular relevance for the understanding of disease pathologies. For
example, during chronic liver disease Mallory bodies are formed, which contain
large amounts of hyperphosphorylated CK polypeptides
(Franke et al., 1979;
Cadrin and Martinoli, 1995
;
Stumptner et al., 2000
). In
this case, phoshorylation itself may be the cause of aggregation
(Yuan et al., 1998
), possibly
by preventing ubiquitin-dependent degradation
(Ku and Omary, 2000
;
Coulombe and Omary, 2002
).
Granular aggregates are also a hallmark of various genodermatoses
(Anton-Lamprecht, 1983
;
Coulombe et al., 1991
;
Cadrin and Martinoli, 1995
;
Kobayashi et al., 1999
).
Furthermore, granules are formed in vitro in response to temperature stress or
to drugs that affect the state of cellular phosphorylation
(Schliwa and Euteneuer, 1979
;
Shyy et al., 1989
;
Falconer and Yeung, 1992
;
Kasahara et al., 1993
;
Liao et al., 1995
;
Blankson et al., 1995
;
Toivola et al., 1997
;
Strnad et al., 2001
).
Elucidation of the mechanisms of granule formation may reveal ways to
influence their formation and thereby positively affect disease outcome.
Very little is known about the molecular principles that determine the
dynamic CKF organisation in interphase cells beyond the continuous and
non-selective exchange of subunits throughout the entire CKF network
(Franke et al., 1984;
Miller et al., 1991
;
Miller et al., 1993
). One
would expect that filaments are broken down and reassemble in various cellular
domains to support ongoing cellular functions such as movement of vesicular
carriers and organelles within the cytoplasm and migration of cells or their
rearrangement within a complex tissue. This type of plasticity should fulfil
certain requirements: it must be rapid to respond immediately to dynamic
requirements; it must be reversible to prevent interference with basic CK
functions; and it must be restricted temporospatially to prevent catastrophic
disruption of the IF cytoskeleton and interference with its basic housekeeping
functions. As a first step in the identification of such dynamic principles of
CK organisation, we now show by in vivo fluorescence microscopy that the
tyrosine phosphatase inhibitor orthovanadate (OV) induces alterations in the
CKF network that fulfil the above-mentioned requirements. These observations
give rise to the exciting possibility that transient CKF disruption is
regulated through signaling pathways that involve phosphorylation.
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Materials and Methods |
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To obtain cell lines stably expressing HK18-YFP, we used a modified calcium
phosphate precipitation method (Leube et
al., 1989). Transfected cells were selected using geneticin
sulphate (Invitrogen Life Technologies, Karlsruhe, Germany). Surviving
colonies were picked, transferred into MicrotestM Tissue Culture
Plates (Becton Dickinson Labware, Franklin Lakes, NJ) and amplified for
further analysis.
Cell culture
AK13-1 cells expressing the chimera HK13-EGFP
(Windoffer and Leube, 1999)
and hepatocellular-carcinoma-derived PLC cells stably expressing fusion
protein HK18-YFP were grown in high glucose DMEM (PAA Laboratories,
Cölbe, Germany) at 37°C with 5% CO2. In some instances,
okadaic acid (Sigma, St. Louis, MO) was dissolved in DMSO at 10 µg/ml and
added to cells at final concentrations between 0.1 µg/ml and 1 µg/ml. In
other instances, sodium orthovanadate (OV; Aldrich Chemical Corporation,
Milwaukee, WI) was dissolved in distilled water and stored as a 1 M stock. It
was either used directly or incubated with H2O2 prior to
usage to generate pervanadate (Feng et
al., 1999
). In some cases, especially for timelapse fluorescence
microscopy, phenol-red-free Hanks medium was used (cf.
Strnad et al., 2001
). To
enrich for mitotic cells, cultures were treated for 6 hours with demecolcine
(Sigma) at a final concentration of 20 µM.
Immunofluorescence microscopy
Cells grown on 18 mm glass coverslips to the desired density were fixed
with pre-cooled methanol (5 minutes, -20°C) and acetone (15 seconds,
-20°C), and mounted either directly in elvanol or subjected to indirect
immunofluorescence prior to embedding as described previously
(Windoffer and Leube, 1999;
Strnad et al., 2001
).
Fluorescence was viewed in an epifluorescence microscope (Axiophot, Carl
Zeiss, Jena, Germany) and was recorded with a digital camera (Hamamatsu
4742-95, Hamamatsu, Herrsching, Germany).
Murine monoclonal antibodies were used for detection of plectin (clone
10F6; kindly provided by Gerhard Wiche, Biocenter, Vienna, Austria)
(Foisner et al., 1994) and
-tubulin (Amersham Pharmacia Biotech, Freiburg, Germany). Monoclonal CK
epitope antibodies were generously provided by Bishr Omary and Nam-On Ku
(Stanford University, Palo Alto, CA), reacting with CKs 8 and 18 (L2A1)
(Chou et al., 1993
),
phospho-S73 of CK8 (LJ4) (Liao et al.,
1997
), phospho-S431 of CK8 (5B3)
(Ku and Omary, 1997
) and
phospho-S33 of CK 18 (IB4) (Ku et al.,
1998
). Polyclonal antibodies from rabbits against CK5 were from
Regina Reichelt and Thomas Magin (Department of Biochemistry, Bonn University,
Germany) (Peters et al.,
2001
), against plectin from Harald Herrmann (German Cancer
Research Center, Heidelberg, Germany)
(Schröder et al., 1999
).
For detection of protein 14-3-3, isoform-
-specific and broad-reactive
rabbit antibodies (antibodies sc-1019 and sc-629, respectively) were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). To visualise bound primary
antibodies, Texas-Red-conjugated goat anti-rabbit antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated goat
anti-mouse IgGs (Rockland Laboratories, Gilbertsville, PA) were used.
For actin staining, cells were briefly washed in PBS prior to incubation with Texas-Red-coupled phalloidin (Molecular Probes, Eugene, OR) at room temperature for 30 minutes, which was followed by washing in PBS (10 minutes) and distilled water (2 minutes) before mounting in elvanol.
For statistic analysis, digital images were imported into Image-Pro Plus 4.5 (Media Cybernetics, Silver Spring, CA) to calculate Pearson's correlation coefficients. The resulting data were further analysed for statistically significant differences between groups with two-tailed Wilcoxon-W-test using SPSS software (version 9; SPSS Incorporation, Chicago, IL). The determined Pearson's coefficients were transferred to SigmaPlot 2001 (Jandel Scientific GmbH, Erkrath, Germany) to prepare Whisker-box-blots.
Electron microscopy
Cells were grown on glass slides and washed briefly in PBS prior to a 2
hour fixation in a freshly prepared solution of 2% (w/v) paraformaldehyde and
2.5% (w/v) glutaraldehyde in phosphate buffer (0.05 M
Na2HPO4, 0.05 M NaH2PO4).
Subsequently, cells were washed three times for 5 minutes in phosphate buffer
containing 0.1 M sucrose and were postfixed for 30 minutes with osmium
tetroxide [2% (w/v) in PBS]. After two 5 minute washes in distilled water,
cells were dehydrated in an ascending ethanol series. Embedding was done in
Epon 812 (Serva, Heidelberg, Germany). Ultrathin sections were stained with 8%
(w/v) uranyl acetate for 10 minutes and contrasted with lead citrate for 5
minutes. Sections were viewed in an EM 906 (Zeiss, Oberkochen, Germany).
Immunoelectron microscopy was done as described previously
(Windoffer and Leube, 1999;
Strnad et al., 2001
) using
antibodies against green fluorescent protein that crossreact with enhanced
green fluorescent protein (Molecular Probes, Eugene, OR) in combination with
gold-labelled secondary antibodies and the silver amplification technique.
Time-lapse fluorescence microscopy
To view living cells, Petri dishes with glass bottoms (MatTek Corporation,
Ashland, MA) were used. Images were recorded by epifluorescence microscopy
using inverse optics from Olympus (Hamburg, Germany) and an attached IMAGO
slow scan CCD camera. The microscope was placed in a heated chamber
(37°C). The whole system was controlled by TILLvisION software. Excitation
was adjusted with the monochromator to 496 nm for the detection of EGFP and to
498 nm for EYFP. Image sequences were imported into Image-Pro Plus 4.5 (Media
Cybernetics) and converted into movies (available at
jcs.biologists.org/supplemental).
Photoshop software (Adobe Photoshop 5.0) was used to edit single pictures and
to assemble figures.
Gel electrophoresis and immunoblotting
High salt pellet fractions containing enriched CKs and corresponding
supernatant fractions were prepared in the continued presence of OV (5 mM)
prior to one-dimensional SDS PAGE or two-dimensional gel electrophoresis
employing isoelectric focusing in the first dimension using established
procedures (Achtstaetter et al.,
1986). Separated polypeptides were either stained with Coomassie
Brilliant Blue (Serva, Heidelberg, Germany) or were transferred onto
nitrocellulose membranes for immunoblotting. In addition to the
above-mentioned CK antibodies monoclonal antibodies against phospho-tyrosine
epitopes (P-Tyr-100; New England Biolabs GmbH, Frankfurt, Germany) and CK13
(Ks13.1, Progen Biotechnics, Heidelberg, Germany) were used.
Detection of bound antibodies was accomplished with
horseradish-peroxidase-coupled secondary antibodies (Jackson ImmunoResearch
Laboratories) and an enhanced chemiluminescence system (Amersham Biosciences
Europe, Freiburg, Germany).
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Results |
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In the corresponding electron micrographs, bundled filaments with local thickenings appeared (Fig. 2E). Some of the thickenings contained an amorphous core, probably representing an early state of non-filamentous granular structures (+ in Fig. 2E). Many of the emerging spheroidal granules were in direct continuity with residual filaments (Fig. 2E and inset). Practically all filaments were gone in the final stages and, instead, multiple non-filamentous granules ranging in diameter from less than 200 nm to 500 nm were spread throughout the cytoplasm without apparent linkage (Fig. 2F). Immunoelectron microscopy using antibodies against EGFP confirmed that the dense bundles with local enlargements and the newly formed aggregated material contained HK13-EGFP (Fig. 2G,H).
To examine whether these drastic alterations were reversible, OV was washed out after 10 minutes. Indeed, cells restored their typical outspread morphology within 50 minutes of removal of the drug. Remarkably, all granules were gone by this time, and an extensive CKF network had been re-established (Fig. 1D). This network was finer than the typical interphase web, and the morphological appearance remained practically unchanged for the next 5 hours (data not shown).
To demonstrate that the changes in HK13-EGFP-containing filaments reflected the behaviour of the entire CKF system, the endogenous network was examined by indirect immunofluorescence in wild-type A-431 cells and AK13-1 cells. Direct comparison of CK5 immunofluorescence with HK13-EGFP fluorescence before OV-treatment, 2 and 10 minutes after addition of the drug and 50 minutes after washout showed that both patterns were practically identical (Fig. 1A'-D'). Similar results were also obtained, when antibodies against CKs 8 and 18 were used (data not shown). In most instances, however, staining of granules with CK antibodies was considerably weaker than the corresponding HK13-EGFP fluorescence. In particular, large granules were labelled only on their surface by indirect fluorescence microscopy, indicating that epitopes within granules were either altered or, more likely, not accessible to the antibodies, as it was also evident from immunoelectron microscopy (Fig. 2G,H).
The reaction of all cells in a given experiment was exceptionally uniform,
and the kinetics of granule formation were similar for OV concentrations
between 2 and 10 mM. However, considerably reduced OV sensitivity was often
noted in cells grown in phenol-red-free Hanks medium in comparison to those
maintained in high glucose DMEM [for medium dependency of OV see also
(Huyer et al., 1997)].
Furthermore, OV activation by H2O2 did not affect the
observed pattern of CKF reorganisation, although approximately 10-times lower
concentrations of pervanadate were sufficient to cause comparable alterations.
For the following experiments the non-activated form of OV was used. In some
instances, cell morphology normalised even without removal of the drug,
probably indicating inactivation of OV and/or acquisition of insensitivity to
the drug (see also Huyer et al.,
1997
). Concurrently, granules disappeared to a large extent and
CKFs were restored.
Orthovanadate effects on the cytokeratin filament network show no
apparent correlation to alterations in the microtubule and actin system
Next, we examined whether OV-induced CKF network changes correlate with
alterations of other cytoskeletal components. After 2 and 10 minutes of OV
treatment, the microtubule network was still visible
(Fig. 3B',C'; see
also arrowheads in Fig. 2E).
However, the number of microtubules was slightly reduced, and microtubules
were distributed differently, sometimes in proximity to CK granules
(Fig. 2E, Fig. 3B,B'), which
probably reflected only the cell shape change and not a specific association.
After 2 minutes of OV incubation, an increase in actin stress fibers was noted
(Fig. 3F'). Later on,
actin was further concentrated in the cell cortex
(Fig. 3G'). After washout
of OV, both microtubules and the actin system were largely restored within 50
minutes (Fig.
3D',H'). Taken together, these observations show that
alterations in these systems do not correlate with CKF network remodeling.
|
Orthovanadate-induced changes in plectin distribution reflect altered
cytokeratin filament network organisation and resemble alterations observed
during mitosis
The distribution of the IF-binding protein plectin, which mediates
interactions among all three major cytoskeletal filament networks
(Foisner, 1997;
Wiche, 1998
), was examined
next. Plectin immunofluorescence partially co-distributed with HK13-EGFP
fluorescence in interphase AK13-1 cells when either monoclonal
(Fig. 4A,A') or
polyclonal antibodies were used (data not shown). A significant increase in
colocalisation was apparent after 2 minutes of OV addition
(Fig. 4B,B') and reached
near identity after 10 minutes (Fig.
4C,C'). After removal of OV, newly formed CKFs still showed
a somewhat increased co-distribution with plectin (data not shown). To
quantify the changes in plectin-CK co-distribution, Pearson's correlation
coefficients were determined between HK13-EGFP fluorescence and plectin
immunofluorescence in untreated cells, in cells with completely disrupted CKF
networks (10 minutes of OV treatment) and in partially recovered cells 50
minutes after removal of OV. For each situation, eight typical regions were
chosen on the basis of DNA staining without prior knowledge of the plectin
distribution. The results (Fig.
5) were fully in support of the visual evaluation, revealing a
significant increase in colocalisation between plectin and HK13-EGFP both
after 10 minutes of OV treatment and after an additional 50 minutes recovery
period in comparison to untreated cells with a P value of <0.005.
Furthermore, co-distribution was slightly lower after recovery in comparison
to the time of maximum CKF disruption, although the difference was
statistically insignificant (P=0.08).
|
|
To find out whether the increased colocalisation of plectin and CKs is a
common feature of CKF disruption, we examined two other situations in which
granular aggregates are formed. First, cells were treated with the
serine/threonine-phosphatase inhibitor okadaic acid (see also
Strnad et al., 2001). In this
instance, granules did not significantly co-distribute with plectin at any
time point during CKF disruption (Fig.
4D,D'), which was also evident from Pearson's coefficient
analysis (Fig. 5). The
P value of the analysis for okadaic-acid-treated and untreated
control cells was >0.8, suggesting no significant difference between both
groups, whereas the P value of the Pearson's coefficient analysis for
okadaic-acid-treated cells and cells incubated for 10 minutes with OV was
<0.0002, demonstrating the significant difference between both granule
types. Second, double fluorescence microscopy was performed during mitosis
when granular and rod-like aggregates are formed and additional strong diffuse
fluorescence occurs as a result of increased soluble subunits in the cytoplasm
of AK13-1 cells (Windoffer and Leube,
2001
). Despite the obscuring effect of the diffuse HK13-EGFP
staining, a strong correlation was observed between fluorescent HK13-EGFP
aggregates and plectin immunofluorescence during mitosis
(Fig. 4E,E'). This was
also confirmed by Pearson's coefficient analysis
(Fig. 5), which produced a
P value of <0.0007 for comparison with untreated cells.
Interestingly, the plectin-negative granules formed during okadaic acid
treatment were positive for 14-3-3 proteins
(Fig. 4I,I'), IFAPs that
preferentially associate with soluble and phosphorylated CK subunits
(Liao and Omary, 1996). By
contrast, neither OV-induced granules nor those generated during mitosis
contained significant amounts of 14-3-3 proteins
(Fig. 4G,G',H,H').
Taken together, our results show that aggregates formed during okadaic acid
treatment differ from those formed during OV incubation and mitosis.
Cytokeratin granules are generated directly from cytokeratin
filaments and cytokeratin filament fragments during orthovanadate
treatment
Time-lapse fluorescence microscopy was performed in AK13-1 cells to find
out how CK granules are formed during OV treatment. Using this method, small
granules were seen to rapidly emerge from filaments
(Fig. 6A). Further details of
this process were difficult to image owing to its speed and the simultaneous
rounding of cells, which resulted in focal shifts of fluorescent structures.
For better resolution we therefore established
hepatocellular-carcinoma-derived PLC cells expressing fluorescent CKFs. The
cytoplasm of these cells is much more outspread than it is in A-431 cells, and
the CKF network has a larger mesh size with thicker appearing filaments. A
HK18-YFP chimera was stably introduced into PLC cells. The fluorescent
chimeras were incorporated into a typical CKF network
(Fig. 6B) and co-distributed
with the endogenous CKs (data not shown). Although PLC cells were less
sensitive to OV than A-431 cells, similar alterations were induced
(Fig. 6C). The time until
filament breakdown began was usually longer in PLC than in A-431 cells even
when high concentrations of OV were used. Yet, the breakdown itself was
remarkably fast once it had started and occurred homogeneously throughout the
entire cell. High magnification images of distinct CKF bundles showed that
they fragmented and formed small rodlets and/or granules
(Fig. 6D). Interestingly, CKFs
straightened prior to disruption (arrows
Fig. 6D). The dynamic aspects
of these processes are best appreciated in Movie 1 (available at
jcs.biologists.org/supplemental),
in which precursor-product relationships are unambiguously resolved.
|
Orthovanadate-induced cytokeratin filament network reorganisation is
rapidly reversible
In a few instances, OV treatment resulted only in incomplete disassembly of
the CKF network. In this case, it was possible to trace small CK granules,
which disappeared again after a short time (usually within less than 30
minutes; Fig. 7). Time-lapse
fluorescence imaging suggested that the granules formed as local thickenings
within or next to CKFs and that they were re-integrated directly into the
original filaments afterwards (arrows in
Fig. 7). After re-integration,
the CKF network looked similar to that prior to treatment, although the cells
were still slightly contracted (see Fig.
7, 38 minutes). Movie sequences taken at high frequency provided
further evidence for the reversible nature of OV-induced granule formation by
direct transformation into and from CKFs (Movie 2; available at
jcs.biologists.org/supplemental).
Furthermore, the movies demonstrated that the granules stayed in close
apposition to the filaments during the entire process, suggesting a remaining
connection. Movie 2 also revealed peripheral CKFs that were most probably
anchored to desmosomal contact sites (arrowheads in
Fig. 7; see also arrowheads in
Fig. 6A). Loss of cell adhesion
upon OV treatment resulted in retraction of these filaments, which appeared to
remain in contact with the plasma membrane.
|
To investigate the re-formation of a CKF network from OV-induced granules
in a more controlled fashion, OV was washed out after short incubation periods
prior to time-lapse fluorescence microscopy. In general, the same phenomena
were seen as in cells with spontaneously occurring reversion of CK granule
formation. Even when most of the CKF network was disrupted and the entire
cytoplasm was filled with small granules, fast reformation of an extended CKF
network took place after OV washout (Fig.
8 and corresponding Movies 3,4, available at
jcs.biologists.org/supplemental).
Although no continuous connection between such granules was noticeable, they
still appeared to be anchored either to CKFs that were below the detection
limit and/or to other parts of the cytoskeleton. The CK granules completely
disappeared within 25 minutes of removal of OV, and a very delicate but
extensive and dense network was re-established
(Fig. 8). Reformation of the
network was not restricted to certain cellular subdomains but occurred at
multiple sites throughout the cytoplasm. High magnification revealed further
details of this process (Movie 4). Granules were seen to elongate and to fuse
occasionally. They decreased in size and disappeared gradually whereas thin
filaments were formed, often extending from the vanishing granules. The first
visible filaments were just above background fluorescence, subsequently
gaining intensity and generating a finely woven network. This newly formed
network differed in appearance from the coarser network in most interphase
cells (e.g., Fig. 1A,
Fig. 3A,E,
Fig. 4A,F). Furthermore, the
network was remarkably homogeneous, lacking, most notably, the thick
perinuclear filament bundles and prominent desmosome-anchored filaments.
However, the peripheral part of the newly formed CKF network exhibited the
typical inward-directed movement of CK fluorescence [compare Movie 3 with
movies shown in (Windoffer and Leube,
1999)].
|
The solubility and phosphorylation of the majority of cytokeratin
polypeptides is not significantly altered in orthovanadate-treated cells
To examine if and to what degree CKs were altered by OV, we looked for
biochemical changes in cells with completely disrupted CKF networks, as
assessed by fluorescence microscopy. We found no significant increase in CK
solubility in OV-treated cells in comparison with untreated cells
(Fig. 9A). This was in contrast
to elevated levels of soluble CKs in okadaic-acid-treated and enriched mitotic
cells (Fig. 9A). Next,
two-dimensional gel electrophoresis was performed to search for altered
patterns of CK modification. No major differences between OV-treated and
untreated cells were apparent, although some minor differences were seen, most
notably for CK 5 (Fig. 9B,C).
Furthermore, commercially available phospho-tyrosine antibodies did not reveal
any significant reactivity of cytoskeletal fractions before or after OV
incubation (data not shown). Similarly, antibodies reacting with specific
phosphoserine epitopes of CKs 8 and 18 did not pick up increased
phosphorylation, although some of the sites were clearly elevated after
okadaic acid treatment (Fig.
9D). These observations suggest that the percentage of modified
CKs in OV-treated cells is either below the detection limit of the methods
used so far and/or that other factors are responsible for the observed
effects.
|
![]() |
Discussion |
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Orthovanadate-induced cytokeratin filament remodeling is similar to
that occurring during mitosis but is profoundly different from that mediated
by okadaic acid
The effect of various modulators of phosphorylation on the dynamics and
organization of the CKF cytoskeleton has been the subject of many studies
(Baribault et al., 1989;
Eckert and Yeagle, 1990
;
Cadrin et al., 1992
;
Falconer and Yeung, 1992
;
Ohta et al., 1992
;
Deery, 1993
;
Kasahara et al., 1993
;
Yatsunami et al., 1993
;
Baricault et al., 1994
;
Blankson et al., 1995
;
Toivola et al., 1997
;
Toivola et al., 1998
;
Yuan et al., 1998
;
Feng et al., 1999
; Paramio,
1999; Sanhai et al., 1999
;
Negron and Eckert, 2000
;
Strnad et al., 2001
). One of
the best examined drugs in this context is the serine/threonine phosphatase
inhibitor okadaic acid, which induces complete disruption of the CKF system
and results in formation of granular aggregates
(Kasahara et al., 1993
;
Yatsunami et al., 1993
;
Chou and Omary, 1994
;
Blankson et al., 1995
;
Strnad et al., 2001
). The
effects of okadaic acid, however, differ in several respects from those
induced by OV as reported in this communication. The okadaic-acid-induced
disruption takes several hours to complete, starting only after a lag period
of 1-2 hours in AK13-1 cells, and cells did not recover for several hours
after removal of the drug (Strnad et al.,
2001
) (see also Lee et al.,
1992
) (P.S., R.W. and R.E.L., unpublished). Furthermore, increased
levels of soluble CKs and CK phosphoepitopes were readily identified after
okadaic acid treatment but not after OV. In addition, okadaic-acid-induced
granules were, on the average, more than twice the size of OV granules
[compare Fig. 2E-H with
(Strnad et al., 2001
)]. They
also differ compositionally by the presence of 14-3-3 proteins and the absence
of plectin (this study). Finally, in okadaic-acid-treated cells, CKFs
disappear first in the cell periphery and only later in the perinuclear region
(Strnad et al., 2001
).
Although okadaic acid is also known to induce p38
(Westermarck et al., 1998
;
Chen et al., 2000
) and to
affect p38-mediated phosphorylation of IFs and IFAPs
(Cheng and Lai, 1998
;
Chen et al., 2000
;
Ku et al., 2002
), it must act
differently from OV. We recently proposed that the effect of okadaic acid is
mainly caused by prevention of integration of soluble CK subunits into CKFs,
which occurs preferentially in the cell periphery
(Strnad et al., 2001
). The
observed colocalisation of CK aggregates with 14-3-3 protein, which may keep
CKs `soluble, that is, non-filamentous, further supports this notion
(Liao et al., 1997
). This also
explains the centripetal decrease in filaments in okadaic-acid-treated cells,
where cortical filament reformation
(Windoffer and Leube, 1999
;
Windoffer and Leube, 2001
)
appears to be blocked, thereby resulting in a filament-free peripheral
cytoplasm while perinuclear filament bundles are still intact. However, this
process is rather slow and therefore not sufficient to account for the rapid
CKF disruption observed in OV-treated cells and at the beginning of mitosis
(Windoffer and Leube, 2001
).
The morphological similarities of mitotic CKF breakdown to OV-induced changes
are striking, since small, filament-associated granules were found in both
instances during the rapid disruption process which takes less than 10 minutes
(Windoffer and Leube, 2001
).
Furthermore, we have shown here that mitotic granules contain, similar to
OV-induced granules, plectin and lack 14-3-3 protein. Whether the underlying
molecular mechanisms of granule formation and disassembly are identical in
both situations remains to be determined.
Plectin is important for cytokeratin filament network
reformation
We were particularly intrigued by the observed association of plectin with
OV-induced CK granules. Among the various linker proteins of the plakin type
that connect the cytoskeletal filament systems to each other and to certain
plasma membrane domains (Ruhrberg and
Watt, 1997; Fuchs and Karakesisoglou, 2001), plectin is certainly
the most prominent crosslinker for the IF system (reviewed in
Foisner, 1997
;
Wiche, 1998
). Interestingly,
plectin affects assembly properties of both unassembled and assembled IF
polypeptides, including CKs, in a complex fashion
(Steinböck et al., 2000
).
Furthermore, plectin is subject to dynamic phosphorylation, most notably
during M phase, when increased p34cdc2 kinase-mediated
phosphorylation of plectin is accompanied by its redistribution from a mostly
filament-associated to a diffuse state
(Foisner et al., 1996
). The
observed colocalisation of plectin with OV-induced CK granules was specific,
since other CK-associated proteins, such as the desmosomal proteins
desmoplakin and plakophilin (data not shown) and the signalling molecules of
the 14-3-3 type did not co-distribute. It therefore appears reasonable to
assume that the particular composition of granular CK aggregates determines
their dynamic properties. Hence, given the compositional differences between
okadaic-acid-induced and OV-induced granules we propose that plectin defines,
in a yet unknown manner, CK aggregates that can be rapidly re-integrated into
filaments. A possibility is that plectin maintains anchorage of granules to
the cytoskeleton, since plectin-positive granules were rather immobile in
contrast to those seen during okadaic-acid treatment
(Strnad et al., 2001
). On the
other hand, loss of plectin and association with 14-3-3 proteins abolishes the
capacity of CK aggregates to re-establish a CKF system.
Cytokeratin network formation is determined by two mechanistically
different pathways
We have shown here that the CKF network recuperates after OV-induced
breakdown within a short time. However, two alternative modes of CKF
re-formation were noted. Most frequently, multiple cytoplasmic CK granules
were seen to vanish by integrating into a fine filamentous network. This
multifocal CKF assembly mechanism is highly reminiscent of experiments in
which CKs were introduced into epithelial and non-epithelial cells by
microinjection of epidermal poly-A+-RNA or by transfection with CK
cDNA-constructs, in which multiple sites were shown to be involved in CKF
network formation (Kreis et al.,
1983; Franke et al.,
1984
; Magin et al.,
1990
; Bader et al.,
1991
). The relevance of this mechanism was further strengthened by
the experiments of Miller et al. that demonstrated that microinjected CK
polypeptides first formed granules together with their endogenously expressed
partner polypeptides and integrated subsequently at multiple sites into the IF
cytoskeleton of epithelial cells (Miller
et al., 1991
; Miller et al.,
1993
). Their images are practically indistinguishable from our
photomicrographs of OV-treated cells [compare, for example,
Fig. 2A-C and
Fig. 7 with
Fig. 7 of
(Miller et al., 1993
)].
Furthermore, ultrastructural analyses showed in both instances that the
granules were composed of non-filamentous material and were in direct
continuity with IF bundles. Finally, the speed of integration of granular
material into filaments is comparable.
The other mode of CKF reformation after OV treatment was observed only in a
minority of cells and will be presented in detail elsewhere. In this case,
novel CKFs were exclusively detected in the cell periphery, which was similar
to the recently described CKF network reformation after mitosis in AK13-1
cells (Windoffer and Leube,
2001). This cortex-restricted mode is significantly slower than
multifocal remodeling. Apparently, factors that are needed for successful
cytoplasmic CKF network formation were inactivated in some OV-treated cells
and are lacking after mitosis. Anchorage of CK granules to the cytoskeleton
either by direct linkage to residual CKFs that are below the detection limit
or to other, yet unknown, cytoskeletal components may be necessary for CKF
network formation. In support, cells with multiple cytoplasmic CKF-forming
foci presented granules with low mobility throughout the cytoplasm. By
contrast, extremely mobile granules that were observed in the central
cytoplasm during mitosis (Windoffer and
Leube, 2001
) and sometimes after OV treatment (data not shown)
lost the capacity for multifocal CKF network reformation and were only able to
contribute to cortical CKF formation. We therefore propose that anchorage of
CK granules in the cytoplasm, which may be mediated by plectin, is a
prerequisite for successful integration into a filamentous network. Probably
cortex-dependent and multifocal cytoplasmic CK-reorganization occur
simultaneously but fulfil different functions. The multifocal principle would
be expected to contribute to local and rapid changes of the cytoskeleton and
can be induced by specific requirements in a temporospatially restricted
fashion. Any out of balance situation, such as the elevated presence of CKF
polypeptides in cells that were microinjected with polypeptides or mRNA
(Miller et al., 1991
;
Miller et al., 1993
;
Franke et al., 1984
) or the
OV-induced CKF-disruption, allows the detection of this mechanism that is
otherwise difficult to see in sessile epithelial cell assemblies. The cortical
principle, by contrast, may be a basic housekeeping function of epithelial
cells, sustaining the continuous CKF network replacement. Accordingly, a
continuous inward-directed movement of CK fluorescence has been reported in
living epithelial cells (Windoffer and
Leube, 1999
; Yoon et al.,
2001
). This system would also be invoked in situations of complete
disruption of the CKF network, because its associated structures are able to
initiate de novo filament formation as it occurs after mitosis and after
extended OV treatment (for details, see
Windoffer and Leube,
2001
).
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Acknowledgments |
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References |
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