1 Department of Cell and Molecular Biology, Northwestern University Medical
School, Chicago, IL 60611, USA
2 Laboratory of Cell Motility, A. N. Belozersky Institute, Moscow State
University, Moscow, Russia
* Author for correspondence (e-mail: y-komarova{at}northwestern.edu)
Accepted 20 June 2002
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Summary |
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In the steady state, nascent microtubules grew persistently from the centrosome towards the cell margin. Frequently, they arrived at the cell margin without undergoing any transition to the shortening phase. In contrast to the growth of microtubules, shortening of the plus ends from the periphery was non-persistent; that is, rescue was frequent and the extent of shortening showed a distribution of lengths reflecting a stochastic process. The combination of persistent growth and a cell boundary led to a difference in apparent microtubule behavior in the cell interior compared with that near the cell margin. Whereas microtubules in the cell interior showed asymmetric transition frequencies, their behavior near the cell margin showed frequent fluctuations between phases of shortening and growth. Complete microtubule turnover was accomplished by the relatively rare episodes of shortening back to the centrosome. Release from the centrosome with subsequent minus end shortening also occurred but was a minor mechanism for microtubule turnover compared with the plus end pathway.
We propose a life cycle for a microtubule which consists of rapid growth from the centrosome to the cell margin followed by an indefinite period of fluctuations of phases of shortening and growth. We suggest that persistent growth and asymmetric transition frequencies serve the biological function of providing a mechanism by which microtubules may rapidly accommodate to the changing shape and advancing edge of motile cells.
Key words: Microtubules, Dynamics, Mammalian cell culture line
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Introduction |
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In previous studies, the dynamics and turnover of MTs in cultured cell
lines have been analyzed primarily using fluorescently labeled tubulin. The
picture that has emerged from these studies is that MTs in vivo undergo
frequent transitions between phases of growth and shortening
(Sammak et al., 1987;
Cassimeris et al., 1988
;
Shelden and Wadsworth, 1993
;
Waterman-Storer and Salmon,
1997
; Yvon and Wadsworth,
1997
).
However, several considerations suggest that this picture is incomplete.
One basic point is that almost all observations of individual MT dynamics in
vivo have been made near the cell margin. This is because the cell is thinner
and the MT density is lower near the edge of the cell, thus permitting better
visualization of individual MTs. Consequently, MT dynamics in the cell
interior have remained essentially unexplored and the formal possibility
exists that they are different from that at the cell margin. A second point is
that MT dynamics in vivo show complexity not seen in vitro. In vitro, MT
dynamics are characterized by transitions between well defined phases of
growth and shortening (Mitchison and
Kirschner, 1984a; Mitchison
and Kirschner, 1984b
; Horio
and Hotani, 1986
; Walker et
al., 1988
). In contrast, MTs in many cell types do not show well
defined phases. In addition, MTs in vivo frequently are quiescent, neither
growing nor shortening, a behavior termed pause. Growth and shortening are
highly variable in terms of velocity, duration and extent but, in general,
tend to be brief. For example, the mean growth length has been reported as 1.3
µm in PtK1 cells and 3.2 µm in CHO cells
(Shelden and Wadsworth,
1993
).
The difficulty in clearly defining phases of growth and shortening in vivo
led us (Vorobjev et al., 1999;
Vorobjev et al., 1997
) to
introduce an alternative description of MT dynamics. MT dynamics were
considered as a 1-dimensional random walk of their plus ends along the cell
radius and their overall properties were characterized by two parameters
a diffusion coefficient and a drift coefficient. The diffusion
coefficient is a measure of the amplitude (squared) of growth and shortening
excursions per unit time, while the drift parameter represents the imbalance
of growth and shortening excursions over time. This framework provided an
analysis of dynamics not dependent upon detailed assumptions of growth or
shortening behavior. The diffusion and drift parameters permitted prediction
of the steady state length distribution of MTs and the time for turnover of
the MT population. However, turnover times predicted from reported dynamic
instability parameters were substantially greater than experimental
determinations (Vorobjev et al.,
1997
). A similar conclusion was drawn from Monte Carlo analysis
using the dynamic instability model
(Gliksman et al., 1993
).
Proceeding from the assumption that MT plus ends undergo stochastic excursions
such as the random walk observed in the lamellar region of PtK1
cells (Vorobjev et al., 1997
)
or in fish melanophores (Vorobjev et al.,
1999
), the time for a nascent MT plus end starting from the
centrosome to grow to the cell margin (or to shorten from the cell margin back
to the centrosome) will be a few hours for a typical cultured cell of radius
25 µm. Such a long time seems incompatible with the requirements for rapid
cytoskeletal remodeling in cell motility behavior. This disparity between
theoretical and experimental analyses of MT turnover is a third point
suggesting the incompleteness of our understanding of MT dynamics in vivo.
Thus, we considered that the dynamics of MTs in the cell interior may somehow
be different from that near the cell margin. Supporting this view, a few
studies have reported the capacity of MTs in vivo to show behavior other than
rapid fluctuations between shortening and growth. MTs have been observed to
persistently grow into the lamellipodia of newt lung cells
(Waterman-Storer and Salmon,
1997
) and to continuously elongate into newly formed protrusions
of HGF-stimulated PtK1 cells
(Wadsworth, 1999
).
These considerations taken together motivated us to reinvestigate the life cycle of MTs by procedures designed to evaluate their behavior in the cell interior and, in particular, in the vicinity of the centrosome. By employing a combination of novel approaches, we found that MT behavior in the cell interior indeed differed from that near the cell margin. Remarkably, nascent MTs freshly nucleated at the centrosome grew persistently until they approached the cell margin. Only then did they display the frequent fluctuations between growth and shortening that are considered to be the hallmark of dynamic instability. We suggest a revised view of MT dynamics in vivo in which the `default' condition of a nascent MT is persistent growth. In this view, `dynamic instability' at the cell margin results from the behavior of the MT system operating under the constraint of a `boundary condition'.
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Materials and Methods |
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Path photobleaching
Photobleaching was performed on a Zeiss IM-35 inverted microscope using a 3
W argon ion laser as described elsewhere
(Keating et al., 1997), except
that the cells were incubated at 37°C. The laser beam was shaped into an
approximately 20x3 µm bar using a cylindrical lens and a Neofluar
100x, 1.3 NA objective. The zone was placed to photobleach a path across
a cell with the centrosome at its center.
Preparation of cytoplasts
Cytoplasts were prepared by a modification of a described method
(Karsenti et al., 1984).
Briefly, 2 days after plating onto coverslips cells were treated with
nocodazole (1 µg/ml) and cytochalasin D (1.5 µg/ml) for 90 minutes.
Coverslips were then placed `cells-down' into centrifuge tubes containing
culture medium with drugs and were centrifuged at 10,000 g for
25 minutes to enucleate the cells. Enucleation resulted in about equal numbers
of cytoplasts containing or lacking the centrosome. Coverslips were washed
with fresh medium to remove drugs and incubated for 2 hours for complete
recovery of MTs in the cytoplasts. For observation of MTs, cells were
microinjected with Cy3-tagged tubulin before enucleation. For simultaneous
observation of MTs and CLIP-170 (see next section), cells were first
transfected with and allowed to express GFP-CLIP-170, GFP-CLIP-170-positive
cells were microinjected with Cy3-tubulin and then cytoplasts were
prepared.
Transfection of cells by microinjection
Cells were transfected by glass capillary microinjection of DNA into the
nuclei following a previously described procedure
(Perez et al., 1999) with
slight modification and were observed after 10-20 hours. Briefly,
pCB6-myc-GFP-CLIP-170 was used at a needle concentration of 20 µg/ml, which
gave sufficiently low expression levels of recombinant protein without
addition of cycloheximide. For visualization of the centrosome in some
experiments, HsCen-2-GFP DNA (human centrin) was mixed with
pCB6-myc-GFP-CLIP-170 DNA that had the same promoter. The final needle
concentration of each vector was the same, -20 µg/ml.
Subtraction analysis
The position of active ends of MTs and the length of growth or shortening
episodes were analyzed by subtraction of sequential images
(InIn+1) in time-lapse series as described
previously (Vorobjev et al.,
1999). The resultant difference images identified the extent of
growth or shortening as black or white domains, respectively, at the ends of
individual MTs. The threshold (minimal length) for determination of shortening
or growth in subtraction analysis was set to 0.45 µm that is, 5
pixels in the digital image. Lateral displacements of MTs were distinguished
from growth and shortening episodes because they gave a parallel arrangement
of long black and white segments, whereas growth or shortening gave single
short segments that were either black or white but not both.
Analysis of microtubule dynamics
The following parameters of MT dynamics were determined: instantaneous
rates of growth and shortening; drift and diffusion coefficients; and mean
velocity of growth. The lengths of individual MTs were measured from the
centrosome (0,0 position of a centrosome) and life histories of MTs were
plotted as length (µm) vs time (seconds). Instantaneous velocities were
measured as displacement of the plus end divided by the time between
successive images (3-5 seconds) in a time-lapse series. The minimum
displacement that was measurable was two pixels in the digital image,
corresponding to 0.18 µm in the cell. Histograms of instantaneous
velocities were generated for the MT population and the mean values and s.d.
were calculated. The histogram of MT growth rate included all displacements
(growth episodes, rare pauses or shortening episodes) that occurred during MT
growth from the centrosome to the cell margin, defined here as a zone 3 µm
from the cell boundary. MT dynamics were treated as a 1D random walk
characterized by diffusion and drift coefficients. Data handling was performed
using SigmaPlot software (Jandel Scientific, San Rafael, CA) as described
elsewhere (Vorobjev et al.,
1999). The drift coefficient, vd, is a measure of the
imbalance of growth and shortening, whereas the diffusion coefficient D is a
measure of the absolute magnitude (squared) of the growth and shortening
excursions at MT ends per unit time. For calculation of these coefficients for
MT plus ends, we used direct imaging of MTs as well as subtraction analysis of
those images. Originally, life histories of MTs over a 45-60 second interval
were used to construct plots of displacement and variance vs time
(Vorobjev et al., 1999
). Here
we simplified the data collection and used instantaneous displacements to
evaluate the coefficients. Positions of MT plus ends were determined in
sequential images and a histogram of displacements was generated. The mean
displacement calculated from this histogram represents the drift coefficient
and the variance represents an estimate of the diffusion coefficient:
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Mean velocity of MT growth was determined using direct observation of Cy3-labeled MTs and the CLIP-170 approach. For the direct observation approach, MTs were monitored until they reached 90% of cell radius. The velocity of apparent CLIP-170 movement was determined by tracking the head of the individual CLIP-170-positive structure from the centrosome until it disappeared within the cytoplasm or near the plasma membrane.
The extent of persistent growth was expressed both as an absolute distance and as a percentage of the local cell radius. For the latter calculation, the local cell radius was determined as the straight line distance from the centrosome to the cell margin in the direction of MT plus end or CLIP displacement.
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Results |
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MT behavior analyzed after path photobleaching
Cy3-tubulin was microinjected into cells and allowed to incorporate into
MTs. The MTs in CHO cells are arranged in a predominantly radial pattern with
the centrosome at its focus. An argon ion laser and an optical track with a
cylindrical lens were used to photobleach a path across the cell with the
centrosome in its center. MTs that were nucleated at the centrosome after the
photobleaching and which grew in the direction of the path could then be
visualized continuously beginning within seconds of their birth
(Fig. 1A).
|
The nascent MTs displayed remarkable behavior not previously reported in
animal cells. They showed negligible, if any, dynamic instability. Typically,
their leading (presumably plus) ends grew persistently until they were at the
cell periphery (Fig. 1B). As an
operational definition, we consider the cell periphery to be a zone of
15% of the cell radius (3 µm). On average, 68% of the MTs reached the
cell margin without transition to a shortening phase and continuous MT growth
phases sometimes exceeded 20 µm. The growth rate derived from the frequency
histogram of instantaneous rates (Fig.
1C) was 17.8±13.8 µm/minute (n=33 MTs; 10
cells; total time of observation=1228 seconds). The histogram confirmed that
episodes of shortening (8.4%) or pause (0.6%) comprised a minor fraction of
nascent MT behavior. At the same time that nascent MTs were growing
persistently in the interior, life history plots of MTs at the periphery of
the cell showed that they displayed characteristic dynamic instability, both
before and after photobleaching (data not shown). Therefore, the experimental
protocol did not detectably affect MT behavior. Thus, observations after path
photobleaching suggest that nascent MTs in the cell interior have different
behavior than those at the periphery. However, a limitation of this approach
was that only those MTs that were oriented along the path (usually 2-4 per
cell) could be followed, limiting the size of the data set. To obtain a more
comprehensive picture we examined cytoplasts where multiple MTs could be
tracked starting close to the centrosome.
MT behavior in centrosome-containing cytoplasts
Enucleation of cells results in cytoplasts that are smaller and flatter
than whole cells. The cytoplasts generally contained a reduced number of MTs
(50%) compared with those in intact cells, suggesting that some
nucleating material was lost or damaged during enucleation. Nevertheless,
centrosome-containing cytoplasts retained a strong radial array of MTs with
the centrosome at its center. The combination of thinness and reduced number
of MTs allowed the centrosome to be clearly visible and MTs growing from it to
be readily traceable (Fig. 2A).
Thus, it was possible to identify an individual MT shortly after it was
nucleated and to monitor its elongation in cytoplasts without the need for
path photobleaching. MTs grew with rare pauses or shortening episodes
(Fig. 2B) at a rate of
15.8±5.9 µm/minute (50 MTs; 8 cytoplasts), which was similar to that
in whole cells after path photobleaching. Thus, the property of persistent MT
growth was retained after enucleation.
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As a measure of MT persistence, we estimated the frequency of transition from growth to shortening. Times were recorded for the growth of nascent MTs from the moment they could be detected near the centrosome until they had a `catastrophe' (shortening episode >0.5 µm) or arrived at the cell periphery, whichever came first. For MTs that arrived at the cell periphery without a detectable shortening episode, we logged their times but did not log a catastrophe. Since MTs in whole cells and centrosome-containing cytoplasts behaved similarly, we combined data from both protocols. For 52 MTs analyzed, only 12 transitions to shortening were logged in 2337 seconds of observation time for a catastrophe frequency of 0.005 second-1.
As MTs approached the cell margin, their behavior changed. Continued observation of MTs for at least 3 minutes, starting from the centrosome (Fig. 2B) or selecting them near the plasma membrane (Fig. 2C) showed that the majority of plus ends displayed typical dynamic instability near the cell margin. MT behavior within the 3 µm zone from the cell margin was characterized by an apparent catastrophe frequency of 0.08 second-1 (207 events for 2710 seconds, 61 MTs analyzed in 17 cells and cytoplasts), that is, 16-fold higher than that in the cell interior. Thus, as in the path photobleaching experiments, dynamic instability behavior was observed only at the cell periphery.
Selective visualization of growing MT ends with CLIP-170
The apparent difference in MT behavior in the cell interior as opposed to
the cell periphery represents a significant departure from our understanding
of MT dynamics. Therefore, it seemed advisable to seek confirmation of these
conclusions by an independent approach. Ideally, one would want to visualize
MT growing ends without the background contributed by the rest of the MTs.
Such selective visualization seemed possible through the use of CLIP-170 as a
marker for MT elongation (Perez et al.,
1999). Although Perez et al. showed that CLIP-170 co-localized
with the ends of some growing MTs and was not targeted to stationary or
shortening MTs, they were not able to determine whether CLIP-170 was targeted
to all growing MTs or only to a certain subset. Thus, we first needed to
evaluate this point. To do so, we compared MT growth with GFP-CLIP-170
behavior using a double labeling protocol for cytoplasts. Intact cells were
transfected by microinjection of GFP-tagged CLIP-170 DNA into the nuclei. To
avoid potential problems with overexpression, we selected cells at 10 hours
after transfection expressing low levels of fluorescent GFP-CLIP-170. These
GFP-positive cells were then injected with Cy3-labeled tubulin and incubated
for 60-120 minutes prior to cytoplast preparation. Colocalization of
GFP-CLIP-170 (red) and ends of MTs (green) in a centrosome-containing
cytoplast is shown in Fig. 3A.
Displacement of the CLIP-170 label in successive frames was identical to the
growth of MT plus ends (Fig.
3B). CLIP-170 labeling disappeared within 5 seconds (time
resolution of our double channel time-lapse series, 2.5 seconds per channel)
when a MT paused or underwent a transition from growth to shortening
(Fig. 3C). Several typical
CLIP-170 and MT histories are plotted in
Fig. 3D. Invariably
(n>100), CLIP-170 tracks were coincident with MT tip displacement,
thus indicating that CLIP-170 was targeted to all growing ends and validating
their use as a tool for selectively visualizing MT growth.
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Persistent MT growth confirmed by long CLIP-170 tracks
A prediction of persistent growth of MTs starting from the centrosome is
that CLIP-170 will be associated with a MT tip almost continuously until the
MT plus end approaches the cell margin. Therefore, we used the movement of
CLIP-170 labeled zones as a tool to evaluate MT growth in the cell interior.
For visualization of the centrosome in some experiments, GFP-Hscen2 DNA (human
centrin expressing construct) was mixed with GFP-CLIP-170 DNA and they were
used together for transfection.
We evaluated MT growth both in intact cells and in cytoplasts containing a
centrosome. As predicted, GFP-CLIP-170-labeled zones displayed long excursions
over time (continuous duration frequently >1 minute) through the cell
interior (Fig. 4A). Track
diagrams were built from successive positions of CLIP-170-labeled zones on
individual MTs. The tracks showed that CLIP-170 zones moved radially outward
from the centrosome. The mean unbroken length of GFP-CLIP-170 tracks was
17.3±4.8 µm (n=78) in whole cells
(Fig. 4B) and 15.8±5.8
µm/minute (n=40) in cytoplasts containing a centrosome. The mean
velocity of CLIP-170 zones was 16.5±6.0 µm/minute (n=110)
for whole cells and 18.3±4.8 µm/minute (n=39) for the
cytoplasts. The velocity of CLIP-170 movement was similar to that determined
for MT growth by direct observation, both in whole cells and cytoplasts. We
may conclude from these measurements both that CLIP-170 movement is a good
proxy for MT growth and that cytoplasts are a good proxy for intact cells.
Normalizing the length distribution of CLIP tracks against the distance
between the centrosome and the cell margin
(Fig. 4C) shows that the
majority of MTs nucleated at the centrosome reached 85% (84±16%)
of the cell radius without catastrophes or long pauses. This result signifies
that most MTs persistently grow from their time of birth at the centrosome
until their plus end nears the cell margin.
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Because new CLIP-170 labeled zones continuously appeared near the centrosome, it was possible to use their appearance to estimate the frequency of MT formation at the centrosome, defined operationally as a circle of 2 µm radius drawn about the focus of the MT array. With this criterion, we estimated the rate of nucleation of MTs by the centrosome in CHO cells to be 5.6±2.3 per minute (178 CLIP-170 zones; 7 cells; 1997 seconds).
Comparison of persistent growth and treadmilling
The only previously reported instance of persistent plus end growth in
mammalian cells is the MT treadmilling observed in vivo in the absence of a
centrosome (Rodionov and Borisy,
1997; Rodionov et al.,
1999
). In our previous study
(Rodionov et al., 1999
) we
suggested that treadmilling in cytoplasts lacking a centrosome resulted from
exposed MT minus ends whose depolymerization caused an elevated tubulin pool
that suppressed transitions from growth to shortening phase at the MT plus
end. Contrary to that suggestion, this study has uncovered persistent growth
of MT plus ends in the absence of substantial numbers of free minus ends.
Therefore, we reevaluated the question of whether the presence of the
centrosome had any significant effect on the rate of plus end growth. With our
improved procedures, we compared cytoplasts containing and lacking the
centrosome.
In centrosome-free cytoplasts, MTs spontaneously appeared in the cytoplasm, then translocated by means of treadmilling and eventually arrived at a cell margin (Fig. 5A). After reaching the cell margin, plus ends often became paused and MTs disassembled because of minus end depolymerization. The rate of growth of the plus end during treadmilling was measured in the same way as for MTs growing from the centrosome in intact cells or centrosome-containing cytoplasts. From time-lapse observations of Cy3-labeled MTs, the rate of plus end growth during treadmilling was 20.3±8.3 µm/minute (n=66; 11 cytoplasts). In steady-state treadmilling, growth of the plus ends must be balanced by minus end shortening. Consistent with this expectation, the rate of minus end shortening in the cytoplasts lacking a centrosome was 18.2±5.8 µm/minute (n=66; 11 cytoplasts).
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Our measurement by direct observation of mean plus end growth rate includes pauses and shortening episodes. To compare the real growth potential of the plus ends these episodes have to be excluded. The CLIP-170 approach provides a natural realization of this algorithm since CLIP disappears from the plus ends when they stop. The tracks of CLIP-170 in cytoplasts lacking a centrosome (Fig. 5B) gave the MT plus end growth rate as 22.0±10.1 µm/minute (n=75; 7 cytoplasts), while in centrosome-containing cytoplasts, the measured rate was 18.3±4.8 µm/minute (n=39; 5 cytoplasts), a difference of 20%. Thus, by both measures, MT growth was slightly faster in cytoplasts lacking a centrosome, consistent with an elevated tubulin pool. However, since persistent growth is a regular feature of MTs in intact cells and centrosome-containing cytoplasts, the creation of free minus ends is clearly not a prerequisite for this phenomenon.
Treadmilling MTs permitted an additional test of the localization of CLIP-170 exclusively at growing ends. Since one end of a treadmilling MT is shortening while the other is growing, CLIP-170 is expected to be present at the leading (plus) end but absent from the trailing (minus) end. This prediction was confirmed by direct dual label observation (Fig. 5C). Thus, CLIP-170 can be used as a polarity marker for the growing MT plus end.
Shortening of MTs
Polymer balance of treadmilling MTs comes at the level of individual MTs
with shortening from the minus end being equal, on average, to growth at the
plus end. However, in intact cells or centrosome-containing cytoplasts, minus
ends are tethered at the centrosome. Since release from the centrosome and
minus end shortening is infrequent
(Keating and Borisy, 1999;
Waterman-Storer and Salmon,
1997
; Vorobjev et al.,
1999
), most of the balance must come from the dynamics of the plus
end. Therefore, the persistent growth of MT plus ends in the cell interior
must be balanced primarily by shortening of other MT plus ends.
The steady state requirement of balance of growth and shortening holds true for every position in the cell, including the centrosome. Since nascent MTs are born at the centrosome at the rate of 5.6±2.3 per minute, we predicted that an equivalent rate of MT shortening back to the centrosome should be observed. To evaluate this issue, we randomly selected MTs having their plus ends close to the margin and monitored their fate (Fig. 6A). In contrast to growth, shortening was not persistent. Shortening occurred at a velocity of 28.8±14.1 µm/minute (n=474; 10 cells) (Fig. 6B,C). Frequency histograms of the distances shortened showed an approximately exponential decay (Fig. 6D) with the mean distance being 2.9±3.4 µm. These properties are consistent with a first-order process of transition back to the growing state (rescue). The rescue frequency was determined to be 0.12 second-1 (91 transitions, 100 MTs, 735 seconds of observation) by tracking MTs shortening back from the margin until they began to grow. Thus, rescue as opposed to catastrophe was frequent leading to small shortening excursions being common and long ones rare. Nevertheless, shortening back to the centrosome could be directly observed, albeit at low frequency. Since MTs that shortened long distances were generally lost as their plus ends neared the congested area around the centrosome, we scored, as a more reliable estimate, the number of MTs that shortened by two-thirds of the cell radius. Out of all episodes of shortening, we obtained 6.4±2.7 per minute that shortened more than two-thirds of the distance to the centrosome.
|
Besides shortening from the plus end, infrequent releases of MTs from the
centrosome also occurred. Released MTs had a short lifespan and rapidly
depolymerized from the minus end (Fig.
6E). In cytoplasts, the frequency of releases was 1.0±0.5
MT per centrosome per minute (58 releases, 11 cytoplasts). Thus, plus end
death (6.4/minute) + minus end release (1.0/minute) was approximately equal to
plus end birth (5.6 MTs/minute). This rough equality confirms that the MT
system in CHO cells is indeed in steady state and it indicates that balance at
the centrosome in CHO cells comes primarily (85%) from the plus end
pathway with the minus end pathway making a minor (
15%) but significant
contribution.
Length distribution of MTs
The persistent growth of nascent MTs is an apparently paradoxical result
because it is not clear how this behavior can be reconciled with the dynamic
instability behavior observed near the cell margin. To address this question,
we employed the conceptual framework of diffusion plus drift and the procedure
of sequential subtraction analysis to obtain the necessary data.
Sequential subtraction images (Fig. 7A) permit identification of growing and shortening MT ends as black or white line segments, respectively, even in regions of high MT density, which otherwise preclude direct observation of individual MTs. MTs that do not undergo a displacement (i.e. are stable or paused) between successive frames become canceled out and do not appear in the subtraction image.
|
The velocities of growing and shortening were determined from the length of
the line segments in the differential images divided by the time between
acquisitions of the images. Frequency histograms of the velocities were
bimodal with growth events slower than shortening but more frequent. An
example histogram is shown in Fig.
7B for the region between 0.6-0.9 of the cell radius. For the same
region, the fraction of growing MTs, fg, was 0.70 and their
velocity, vg, was 17.0±5.9 µm/minute, n=372,
while the fraction of shortening MTs, fs, was 0.30 and their
velocity, vs, was 29.7±13.4 µm/minute, n=156.
The drift coefficient, vd, reflects the imbalance of growth over
shortening and is calculated either as
or as the mean of the velocity histogram. Based on this histogram analysis,
calculated values of the drift coefficient were
5 µm/minute in the
cell interior, declining somewhat towards the cell margin
(Fig. 7C), indicating that
throughout the cell interior, growth was strongly favored over shortening.
Close to the cell margin (<3 µm) the calculated drift coefficient was
negative (-2.7±0.4 µm/minute; n=235 growth events; 197
shortening events), reflecting the increase in proportion of shortening events
because of the influence of the cell boundary. It should be noted that since
paused MTs do not appear in the subtraction images, they do not contribute to
the calculation of drift. As a check on the result, we also evaluated
displacement histograms obtained from direct fluorescence imaging in the cell
interior. Such histograms were also bimodal although now a population of
paused MTs was detected. In the cell interior, paused MTs were infrequent
(15%) while near the cell margin they were abundant (70%). In the cell
interior, velocities and drift coefficients (data not shown) were similar to
those obtained from subtraction analysis. However, near the cell margin, the
larger fraction of pauses resulted in a proportionate decrease in the absolute
value of the drift coefficient.
The diffusion coefficient represents fluctuations around the behavior
predicted from drift and is calculated as the variance of the velocity
frequency histogram. Calculation gave the apparent diffusion coefficient as
13.1±1.3 µm2/minute for the inner, 15.1±1.1
µm2/minute for the middle and 13.1±1.3
µm2/minute for the outer thirds of the cell, respectively. The
ratio of coefficients, diffusion/drift was 3 µm and did not change
significantly along the cell radius. As shown previously
(Vorobjev et al., 1999
),
positive drift predicts a non-random distribution of MT ends.
To determine the distribution of MT ends along the cell radius, the whole cell was divided into five radial zones. Counts of black or white line segments showed that MT ends, both growing and shortening, were rare near the centrosome and increased exponentially towards the cell margin (Fig. 7D). Thus, in CHO cells, the length distribution of dynamic MT ends is an ascending one and most MTs are long. From an exponential curve fit to the data in Fig. 7D, the ratio of the coefficients was independently estimated as 5.8 µm, which is within a factor of 2 of the measured value. Thus, the diffusion plus drift framework is sufficient to account for the observed distribution of MT ends.
Persistent growth is a characteristic feature of cultured cells
Since persistent growth in CHO cells seemed a remarkable property, it was
important to test its generality. Consequently, we examined the MT life cycle
in NRK cells and cytoplasts obtained from them. For these cells, we observed
persistent growth of MTs towards the plasma membrane at a velocity of
17.4±5.7 µm/minute (data not shown). Sequential subtraction analysis
of NRK cytoplasts gave a bimodal distribution similar to that for CHO cells
(data not shown) with a drift coefficient in the cell interior of
4.0±1.1 µm/minute (n=488, 5 cytoplasts). Additional support
for this conclusion was obtained by calculation of the drift coefficient from
data available in the literature. Using the expression presented earlier,
,
we calculate drift coefficients of 4.4 µm/minute for the lamellae of NRK
fibroblasts (Mikhailov and Gundersen,
1998
), 3.4 µm/minute for the periphery of CHO cells
(Shelden and Wadsworth, 1993
),
and 3.8 µm/minute for MTs growing parallel to the cell edge in newt lung
cells (Waterman-Storer and Salmon,
1997
). Thus, an apparent imbalance of growth over shortening is a
characteristic feature of many cultured cells. We conclude that persistent
growth and positive drift are widespread features of MT dynamics.
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Discussion |
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Persistent growth of nascent microtubules
The frequency of transition from growth to shortening of a MT in the cell
interior was determined to be very low, 0.005 second-1, which
equates to a half-time for the growing phase of 140 seconds. Assuming a radius
of 20 µm for a typical CHO cell and a growth velocity for the MT plus end
of 17 µm/minute, a MT would require only 70 seconds to grow the
distance of the cell radius, which means that a nascent MT typically arrives
at the cell margin before it has had a chance to shorten. Assuming a
first-order process of transition to shortening, we calculate that an average
of 72% of nascent MTs starting at the centrosome would still be in the growth
phase by the time they reached the cell margin. This result is essentially the
same as the experimentally observed value of 68% for cells. Cytoplasts are
smaller than cells and therefore they have a higher percentage of MTs that
arrive at the margin without shortening. Thus, persistent growth of a nascent
MT can be understood as resulting from a combination of high elongation
velocity and low catastrophe frequency. This remarkable feature of the MT life
cycle was not previously recognized. The major explanation for this oversight
is most likely that observations of MT dynamics in vivo have generally been
made where it is technically easier to obtain the data namely, at the
cell periphery, where the cytoplasm is thin and MT density is relatively low,
thus facilitating visualization of individual MTs. An implicit and untested
assumption has been that MT dynamics parameters evaluated at the cell
periphery are valid throughout the cell. Our results demonstrate that, at
least in CHO and NRK cells, this is not the case.
Data consistent with persistent growth can be found in recent papers
focusing on GFP-labeled plus end markers
(Perez et al., 1999;
Mimori-Kiyosue et al., 2000a
).
Although these reports did not emphasize the persistent behavior of MT growth,
they did contain data from which such conclusions could be drawn. In Vero
cells, GFP-CLIP-170 tracks of 10 µm length were common
(Perez et al., 1999
) and a
similar picture emerges from movies of EB-1-GFP tracks in Xenopus A6 cells
(Mimori-Kiyosue et al., 2000a
;
supplementary material). Persistent growth as a property of MTs was also
described in the fission yeast, S. pombe, where the majority of MTs
grew until they reached the cell ends
(Brunner and Nurse, 2000
).
Taking these results together with our observations, we conclude that
persistent growth of MTs in the cell interior is not an exceptional property
of CHO cells. Rather, it may be an evolutionarily selected and widespread
characteristic that has previously escaped notice because it is seen primarily
in the cell interior.
Dynamic instability behavior near the cell margin
As MTs neared the cell margin, their behavior changed to frequent
alternation of brief phases of shortening and growth. Such behavior is how
dynamic instability has come to be characterized in vivo. For example, growth
and shortening lengths in PtK1 cells have been reported as 1.3 and
1.6 µm and in CHO cells as 3.2 and 4.3 µm, respectively
(Shelden and Wadsworth, 1993).
We obtained similar values for CHO cells for MTs near the cell edge. Thus, the
small duration of growth seen for MTs near the cell margin differed from the
persistent growth in the cell interior.
What could be the explanation for this difference in behavior? Are MT
dynamics intrinsically different in the cell interior as opposed to the cell
margin or only apparently so? Closer inspection of MT behavior revealed that
not all parameters depended on position in the cell. Velocities of growth and
shortening (17 and 30 µm/minute, respectively) did not. We obtained the
same values independently of whether the determinations were made at the cell
margin or in the cell interior and they were similar to those obtained
previously at the cell periphery (Shelden
and Wadsworth, 1993). Likewise, the rescue frequency was
essentially constant. In the cell interior, the mean distance of shortening
was small (2.9±3.4 µm) and the rescue frequency was high (0.12
second-1). Shortening of MTs back from the cell margin followed an
exponential decay function, signifying that the probability of transition to
the growing phase did not depend detectably upon the extent of shortening or
position in the cell. The values of shortening length and rescue frequency
were essentially the same as those obtained previously at the cell periphery
(4.3±3.3 µm and 0.13 second-1, respectively)
(Shelden and Wadsworth, 1993
).
Finally, when we evaluated the apparent catastrophe frequency near the cell
margin, we obtained a value (0.08 second-1), similar to that
previously reported (0.06 second-1) (Sheldon and Wadsworth, 1993).
Thus, the major difference in MT behavior is the apparent high frequency of
catastrophe seen at the cell margin versus the low frequency (0.005
second-1) that we find in the cell interior.
We propose that the difference in catastrophe frequency is only apparent and can be accounted for by a `boundary effect' of the cell margin. The boundary prevents growth from continuing, inducing a pause and/or catastrophe. It follows that observations restricted to the cell periphery underestimate intrinsic growth duration and overestimate the catastrophe frequency. A high rescue frequency prevents MTs from shortening all the way back to the centrosome. Instead, shortening MTs are quickly converted to growing ones, which are then driven back to the cell membrane. Thus, the asymmetry of transition frequencies and the boundary effect combine to produce persistent growth in the cell interior but many small fluctuations of MT length near the cell margin.
What is the nature of the boundary? The simplest possibility is that the
membrane itself or the dense actin network that typically underlies it
presents a physical obstacle to growth. Alternatively, a chemical signal
emitted from the cell surface could change the state of the MT. Growing MTs
are marked by the presence of CLIP-170-family proteins (CLIP-170 and CLIP-115
for mammalian cells) (Perez et al.,
1999; Hoogenraad et al.,
2000
), CLASP (Akhmanova et al.,
2001
), EB-1/APC complex
(Mimori-Kiyosue et al., 2000a
;
Mimori-Kiyosue et al., 2000b
)
and dynactin complex (Vaughan et al.,
1999
) at their plus ends. The relationship between the presence of
these proteins and MT dynamics has yet to be established for mammalian cells.
CLIP-170 family proteins could be good candidates for ensuring MT growth in
the cell interior, as the fission yeast ortholog of CLIP-170, tip1p,
stabilizes MT growth by preventing premature catastrophe
(Brunner and Nurse, 2000
).
EB1/APC complex, which promotes MT polymerization in vitro
(Nakamura et al., 2001
), and
increases MT stability in vitro and in vivo
(Zumbrunn et al., 2001
), might
also prevent growing MT tips from depolymerization. If any or all of these
proteins were required to maintain a low catastrophe frequency or a high
rescue frequency and if a factor at the cell surface caused them to dissociate
from the MT plus end, the result would be an induced transition to the
shortening phase.
The steady state
The simplest criterion of steady state is that the number and amount of MT
polymer does not change over time. An apparently paradoxical result is that in
the steady state some MTs persistently grow. Where does the shortening come
from to balance the growth? Fig.
8A provides a qualitative explanation. Neglecting the minor
contribution that the minus end pathway plays in CHO cells, the growth of a
nascent MT is essentially compensated by the complete shortening of another MT
from the plus end. Fluctuations of growth and shortening near the cell margin,
on average, equate to each other because persistent growth up to the cell
boundary restores polymer lost by shortening from the boundary. Thus, cellular
levels of MT polymer remain constant in the presence of persistent growth of
individual MTs.
A deeper paradox is the ascending distribution of MT ends. Why is the distribution not uniform in the cell? The answer is again related to a boundary effect. Persistent growth and non-persistent shortening of MTs is equivalent to stating that the catastrophe frequency is small and the rescue frequency is high. In CHO cells, the catastrophe frequency is so small that nascent MTs typically arrive at the cell margin before having had a chance to shorten. In contrast, MTs typically shorten for only a small distance before being driven back to the cell margin. Since, by definition, a boundary imposes a limit to growth, the asymmetry in transition frequencies will cause MT ends to spend more time and thus to accumulate near the cell margin (Fig. 8B).
A quantitative evaluation of the MT length distribution is provided by the
conceptual framework of treating MT ends as dynamic elements undergoing
1-dimensional diffusion and drift. A diffusion plus drift treatment has been
used previously to evaluate MT dynamics in melanophores
(Vorobjev et al., 1999) and
has been shown theoretically to be a valid approximation of MT dynamic
instability (Maly, 2001
). In
this framework, steady state is attained when fluxes of MT ends due to drift
and diffusion are equal and opposite. This equality may be expressed as
,
where n refers to the number of MT ends at any given plane in the cell and
n/
x is the concentration gradient of ends across the plane.
The diffusion plus drift framework permits calculation of the distribution
of MT plus ends in the steady-state according to the equation
(Vorobjev et al., 1999
). The
ratio of the coefficients D/vd provides an estimate of how steeply
the distribution ascends. For CHO cells, the ratio was
3 µm as
determined kinetically and 5.8 µm as determined from the length
distribution. Taking the value from fitting the length distribution gives a
doubling distance of
. This
signifies that there will be 20 times more MT ends within 5 µm of the cell
margin than within a 5 µm zone around the centrosome.
MT dynamics in vivo versus pure tubulin in vitro
The asymmetry of MT dynamics in vivo stands in contrast to MTs in vitro,
where both growth and shortening tend to be persistent
(Mitchison and Kirschner,
1984b; Horio and Hotani,
1986
; Walker et al.,
1988
). The difference between parameters observed in vitro and in
vivo suggest that there is a regulatory mechanism at the MT plus end. Several
proteins have been suggested to increase the catastrophe frequency including
XKCM1 (Walczak et al., 1996
;
Desai et al., 1999
;
Tournebize et al., 2000
) and
stathmin/Op18 (Belmont and Mitchison,
1996
; Howell et al.,
1999
). Some MT-associated proteins (MAP4, Xenopus XMAP
215 and its human homolog TOGp) can play an opposite role, rescuing MTs by
stabilization and/or assembly-promotion
(Tournebize et al., 2000
;
Spittle et al., 2000
;
Popov et al., 2001
) (for
reviews, see Andersen, 2000
;
Heald, 2000
). In addition, plus
end binding proteins such as CLIP-170
(Perez et al., 1999
) or EB1
(Mimori-Kiyosue et al., 2000b
)
represent potential regulatory factors, although mechanistic studies
demonstrating how they might affect MT dynamics are lacking.
Growth of nascent MTs in CHO cells was not only persistent but rapid. The
velocity we obtained, 15-20 µm/minute, was higher than most values reported
for MT growth in other cells, cell extracts or for purified tubulin (reviewed
by Odde and Buettner, 1995).
However, similar instantaneous values were reported for CHO cells
(Shelden and Wadsworth, 1993
),
GFP-CLIP-170 tracks in Vero cells (Perez
et al., 1999
) and EB-1-GFP tracks in Xenopus A6 cells
(Mimori-Kiyosue et al.,
2000a
). It is not clear what factors are responsible for the
differences in reported rates but our study and the several reports cited
indicate that MT growth in vivo can be astonishingly rapid. The rates in vivo
are almost an order of magnitude higher than those predicted by in vitro
studies of pure tubulin (Walker et al.,
1988
). This consideration suggests that the rate constant for MT
growth in vivo is higher than that determined in vitro or that the cell has a
mechanism to maintain the free tubulin pool substantially above the critical
concentration.
Biological significance of MT life cycle pattern
The life cycle of MTs in CHO and NRK cells displays several characteristic
features including persistent growth in the cell interior, asymmetric
transition frequencies and effects of the cell boundary. What might be the
biological significance of this set of properties? We suggest that the answer
is to provide a mechanism by which the MT array can rapidly `sense' changes of
the cell periphery. Rapid and persistent growth allows nascent MTs to elongate
from the centrosome to the cell boundary in a short time. This would be
advantageous for a locomoting cell in which the cell margin is advancing in
that the MT cytoskeleton could rapidly accommodate to any protrusion or change
in cell shape. Such accommodation could also be important for maintenance of
membrane trafficking within the cell, including transport of vesicles either
to or away from the cell surface. Finally, efficient sensing of the periphery
by MTs may be important for coordinating motile activities around the cell
perimeter to achieve directional motility.
The new insights into MT dynamics obtained from this analysis of the MT life cycle in CHO and NRK cells suggest that similar comprehensive analyses in other cell types will be rewarding. It will be important to determine whether different cell types such as fibroblasts, epithelial or neuronal cells have similar or distinctive MT `lifestyles'. In any event, this study points out the critical necessity of carrying out determinations of MT behavior in the cell interior and in recognizing that the cell is a finite system in which its boundary exerts important effects.
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Acknowledgments |
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References |
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