Control of Microtubule Nucleation and Stability in
Madin-Darby Canine Kidney Cells: The Occurrence of
Noncentrosomal, Stable Detyrosinated Microtubules
Marie-Hdlbne Br~, T h o m a s E. Kreis, a n d Eric Karsenti
European Molecular Biology Laboratory, D-6900 Heidelberg, Federal Republic of Germany
Centrosomal and noncentrosomal micrombules regrew
in MDCK ceils with similar kinetics after release from
complete disassembly by high concentrations of nocodazole (33 ~tM). During regrowth, centrosomal microtubules became resistant to 1.6 I~M nocodazole before
the noncentrosomal ones, although the latter eventually
predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic
instability model but the presence of factors prevents
them from complete depolymerization. This creates
seeds for reelongation that compete with nucleation off
the centrosome. By using specific antibodies, we have
shown that the abundant subset of nocodazole-resistant
microtubules in MDCK ceils contained detyrosinated
a-tubulin (glu tubulin). On the other hand, the first
microtubnles to regrow after nocodazole removal conrained only tymsinated tubulin. Glu-tubulin became
detectable only after 30 min of micmtubule regrowth.
This strongly supports the hypothesis that a-tubulin
detymsination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount
of glu-tubulin that we found in taxol-treated cells.
EVERAL lines of evidence support the view that microtubules are involved in cytoplasmic organization and
orientation of intracellulartransport. Together with
micmtubule-organlzing centers (MTOCs) 1 they are thought
to determine the axis of polarity in many ceils (8, 24, 32).
Although it is known that micmtubules are polymers in a dynamic equilibrium with a pool of soluble subunlts (17), the
intrinsic dynamic characteristics of pure micmmbules have
been studied only recently in vitro and called dynamic instability (16, 20, 25, 26). Microinjection of labeled tubulin or
microtubule-associated proteins (MAPs) into living cells has
also shown that most cytoplasmic microtubules in fibroblast
MAP, microtubule-associatedprotein; MTOC, microtubulc-organizing
center; tyr-tubulin,tyrosinateda-tubulin.
cells turn over with a half-lifeof '~I0 min (30, 31, 34). A
smaller fraction of the microtubules is very stable with a
half-lifeof several hours (34). The dynamic instabilityof
microtubules has severalpotentiallyimportant implications
for understanding how differentsubclasses of micmmbule
networks are generated during the cell cycle and cell differentiation.Kirschner and Mitchison propose that during
morphogenetic processes (polarizationor any sort of cell
shape remodeling) microtubules, although globally dynamic,become more stablein a restrictedarea of the cell,thereby
providing a basis for cytoplasmic asymmetry (20). This
selectivestabilizationhypothesis predicts that: (a) microtubules with differentstabilitycan coexist in a given cell; (b)
microtubule-stabilizing factors could be distributed nonhomogeneously in the cytoplasm; and (c) external signals
9 The Rockefeller University Press, 0021-9525/87109/1283/14 $2.00
The Journal of Cell Biology, Volume 105, September 1987 1283-1296
1283
1. Abbreviations used in this paper: glu-tubulin, detymsinated a-tubulin;
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Abstract. The micmtubnle-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in
epithelial cells (PtK2, Madin-Darby canine kidney
[MDCK]) by double-immunofluorescence labeling with
anti-centrosome and antitubnlin antibodies. Most of
the micmtubules emanated from the centrosomes in
Vero ceils, whereas the micmtubule network of MDCK
cells appeared to be noncentrosome nucleated and randomly organized. The pattern of micmtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibmblastic and epithelial cells.
The two centriole cylinders were tightly associated and
located close to the nucleus in Veto and PtK2 cells. In
MDCK cells, however, they were clearly separated and
electron microscopy revealed that they nucleated only
a few microtubules.
The stability of centmsomal and noncentrosomal
micmtubules was examined by treatment of these
different cell lines with various concentrations of
nocodazole. 1.6 gM nocodazole induced an almost
complete depolymerization of micmtubules in Vero
ceils; some centrosome nucleated micmtubnles remained in PtK2 cells, while many noncentrosomal
micmtubules resisted that treatment in MDCK ceils.
Materials and Methods
Cells
Madin-Darby canine kidney epithelial cells (MDCK) strain II were grown
in Eagle's minimal essential medium with Eagle's salts supplemented with
10 mM Hepes pH 7.3, 1% L-gintamine, 5% FCS, penicillin (110U/mt), and
streptomycin (100 l~g/ml). African green monkey kidney cells (Vero) and
Potoroo kidney epithelial ceils (PtK2) were grown as previously described
(22). The cells were seeded on glass coverslips and incubated in a humidified atmosphere equilibrated with 5% CO2 in air at 37~
Drug 1~eatments
Cells were seeded at a density that normally allows conflncncy between day
2 and 3 (36). All hhe experiments described in this paper were carried out
on subconfluent MDCK cells taken 24 h after seeding. Preliminary experiments indicated that the nocodazole concentration required for intermediate
de.polymerization in MDCK cells was 1.6 I~M, To induce an almost total
depolymerization, a concentration of 33 ttM was required for at least 3 h.
Nocodazole (Sigma Chemical GmbH, Deisenhofen, Federal Republic of
Germany) was kept as a stock solution in DMSO at -20"C (33 mM) and
diluted in culture medium at the appropriate concentration just before use.
Experimental protocols are described in the figure legends. Cells were
treated at 37~ with taxol at 5 ~tM using a stock solution in DMSO (10 mM)
kept at -20~ (Taxot was supplied by Dr. M. Suffness, Natural Product
Branch, Division of Cancer Treatment, National Cancer Institute, Betbesda, MD.) Experimental protocols are described in the figure legends.
Antibodies
Human anti-centrosome antibodies (505t) are a gift of "1".Mitchison and
M. Kirschner (26).
lmmunofluorescence
All the results presented here have been obtained on cells preextracted for
10 s in microtubule-stabilizing medium (80 mM K-Pipes pH 6.8, 5 mM
EGTA, 1 mM MgCI2, 0.5% Triton X-100) and fixed in methanol at -20~
for 5 rain as previously described (19). In control experiments, cells were
fixed directly in methanol at -20*(2. These and other fixation procedures
involving the use of glutaraldehyde combined with extraction buffer gave the
same results. ~ t r a c t i o n is useful because the backgrmmd due to free
tubulin subunits is elinfinated, making the images sharper. Immunofluorescence was carried out as previously described (19) including 0.1% Triton
X-100 in the PBS washes. Coverslips were covered with 20 ktl rabbit
anti-glu-tubalin (1:10 dilution) for 10 min. 20 ttl of a monoclonal anti-tyrtubulin (1:400) were added for a further 10 min incubation, Directly mixing
the two antibodies gave similar results. After three washes (5 rain each),
the coverslips were covered for 10rain with 20 ~tl of fluorescein-labeled goat
anti-rabbit and Texas Red-labeled goat anti-mouse antibodies (1:I00 dilution; Dianova GmBH, Hamburg, FRG). After further washes, the coverslips were cleaned quickly in ethanol and mounted in Mowiot.
In some experiments, cells were double stained with a monocloual mouse
anti-t3-tubulin 0:500 dilution) and a human anti-centrosome antibody
(1:100). In this case, secondary antibodies were a goat anti-mouse labeled
with fluorescein (1:50) and a goat anti-human labeled with Texas Red (1:100;
Dianova GmBH). All antibodies were diluted in PBS containing 0.1% Triton
X-100, 3% BSA, and 0.1% sodium azide.
lmmunogold Labelingfor Electron Microscopy
Ceils grown on plastic coverslips were briefly washed in PBS, preextracted
with the stabilizing medium, and fixed with 0.3 % glutaraldehyde in the same
medium at 37~ for 10 rain. Free aldehyde groups were fitrated by sodium
borohydride (1 nag/nil in PBS) for 7 rain. After washing in PBS, the coverslips were covered with a polyclonal rabbit anti-tubulin antibody for 30 rain
and washed five times (15 rain each). The coverslips were then incubated for
30 rain with gold-labeled protein A (8 nm size). After an overnight wash
in PBS containing 0.1% Triton X-100, the cells were postfixed in 1%
glutaraldehyde in 80 mM K-Pipes pH 6.8, 5 mM EG'I'A, and 1 mM
MgCl2. The specimens were then processed for electron microscopy and
embedded in Epon. Serial thick (0.I5-0.25 ltm) and thin sections were cut
and observed in a Philips 400 electron microscope.
Semiquantitative Analysis of Tyrosinatedand
Detyrosinated Tubulin by Immunoblotting
Cells were seeded on 3-cm petri dishes so as to reach confluency 24 h later
when they were treated with nocodazole or taxol as described in the figure
legends. At the desired time, the cells were lysed directly in hot Laemmli's
sample buffer (0.5 nil) (23a). Care w~s taken to recover the whole cell layer
from each dish. Samples were further treated at 950C for 5 min, souicated
for 3 rain in a water bath sonicator, spun 5 rain in a microfuge (Eppendorf;
Brinkmann Instruments Inc., Westbury, NY), divided into aliquots of 50 I~l
each, and kept frozen at -70~ until use. 5 I~l of each sample were applied
on 10% Laemmli's polyacrylamide minigels, or 50 [tl on normal 10% polyacrylamide gels. After transfer onto nitrocellulose according to Towbin et
al. (39), tyrosinated and detyrosinated tubulins were detected with the
monoclonal anti-tyr-antibody (1:6,000) and the polyclonal anti-glu antibody (1:1,000). PeroxidaseAabeled rabbit anti-mouse (1:t,000) and goat
anti-rabbit (1:t,000) from Tag., Inc., (Burlingame, CA) were used as secondary antibodies. Diaminobenzidine (5 mg/ml in.50 mM Tris-HCt pH 7.6,
0.01% H:O2) was used as the substratc for the peroxidase reaction. The
reaction was carefully controlled and allowed to proceed for the same time
for a given set of bints (1-3 min). The reaction was arrested by a quick wash
in distilled water followed by a wash in 5 % TCA.
Affinity-purified rabbit anti-glu tubulin antibodies was obtained by immunizing rabbits with synthetic peptide containing the 12 carboxy-terminal
amino acid of ct-tubulin without the last tyrosine. The monoclonal anti-tyr
tubulin was obtained by immunizing mice with the carboxy-terminal 13
amino acid of et-tubulin (including the last tyrosine residue). The preparation and complete characterization of these antibodies wilt be described
elsewhere (22a). Each antibody was preabsorbed with the other peptide before use in order to eliminate any possible cross-reaction. The monoclonal
anti-[3-tubulin was from Amersham Buchler GmbH, Braunschweig, FRG.
After trypsinization, l-d-old MDCK cells were washed twice with 50 mM
NaCI, 50 mM "IVIs-HCLpH 7,2, and 1 mM EDTA at 4~ An aliquot of
cells was resuspended directly in hot Laemmli's sample buffer at 2 x 106
cells/ml and boiled for 5 rain. Another aliquot was resuspended, at 4~
in 50 mM K-Pipes pH 6.9, I mM EG"rA, and 0,t% Triton at 4 x 10*
ceUs/ml. This aliquot was then treated for 20 rain at 3"/~ with 0.25 ttg/ml
The Journal of Celt Biology, Volume 105, 1987
1284
Carboxypeptidase A Treatment of Cell Lysates
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could influence the regional distribution of microtubulestabilizing factors.
Another important level of control over microtubule organization concerns the mechanism of polymer nucleation in
vivo. It is currently assumed that radial arrays of microtubules are nucleated off the centrosome by the pericentriolar
material (2-4, 11, 12, 32). However, the pericentriolar material sometimes dissociates from the centrioles and other
microtubule patterns are then generated (37). Moreover,
spontaneous nucleation could occur in the cytoplasm if the
tubulin concentration were high enough or if microtubulestabilizing factors were present (7, 19). The proportion and
dynamics of centrosome-nucleated microtubules may therefore be affected by the number and activity of other potential
MTOCs, active tubulin concentration, and microtubulestabilizing factors.
In this paper we compare the microtubule patterns in
fibroblasts (Vero cells), epithelial-like (PtK2), and epithelial
cells (MDCK) as well as the relative stability of microtubules
in these cell lines. The relationship between microtubule resistance to nocodazole, age, and a-tubulin detyrosination has
also been investigated. We show that stable noncentrosomal
microtubules exist in MDCK cells. We discuss the mechanism of generation and stabilization of these microtubules,
and we present experimental evidence supporting the general
hypothesis ttiat ct-tubulin is detyrosinated when associated
with long-lived microtubules (13, 14, 21).
of carboxypeptidaseA (phenylmethylsulfonylfluoridetreated; ServaCo.,
Heidelberg,FRG)preparedas previouslydescribedO8). The reactionwas
stoppedby addingan equalvolumeof hot samplebufferto the reactionmixture and furtherboiling for 5 min. 105-eellequivalents(50 Ial)wereloaded
on 10% polyacrylamideLaemmli'sgels and analyzedby immunoblotting.
Results
Centrosomes Are Split and Not Associated
with the Nucleus in Subconfluent MDCK Cells
In most fibroblastic cells, the two centriole cylinders are
tightly associated and bound to the nucleus (1, 2, 10, 27). By
immunofluorescence microscopy, using scleroderma human
anti-centrosome serum (42), they appeared as one bright dot
or two smaller dots tightly associated with each other both
in Vero and PtK2 ceils (Fig. 1 a). In subconfluent MDCK
cells they were separated, sometimes by distances as large
as 10 txm (Fig. 1 b). No specific association with the nucleus
was found by electron microscopy (Fig. 2 a).
The aspect of the microtubule network in MDCK, Veto, and
PtK2 cells stained with a monoclonal anti-13-tubulin antibody is shown in Fig. 3. The microtubule network was
clearly centered on the centrosome in most Vero cells. This
was less obvious in PtK2 cells. In MDCK cells, the microtubule network was randomly organized. There was no obvious correlation between the position of the split centrosome
and the microtubule pattern. The cells shown in Fig. 3 have
been double labeled with the centrosome antibody (data not
shown). The positions of centrosomes are shown by arrows.
We have observed serial thick sections of these cells by electron microscopy after immunogold staining of the microtubules. Between 0 and 15 microtubules were found to radiate
from the pericentriolar area in 12 pairs of centrioles observed. Fig. 2 b shows one of these sections in which only
2-3 microtubules were found to radiate from the centrioles.
Most of the Noncentrosomal Microtubules Are Stable
To determine the relative stability of the noncentrosomal
microtubules in MDCK cells, we treated the cells with a concentration of nocodazole (1.6 IxM) known to depolymerize
almost all microtubules in a few minutes in I.~9 cells (19).
This actually induced an almost complete depolymerization
in Vero cells (Fig. 3) fixed 30 min after addition of the drug.
Only occasional, long and curly stable microtubules remained, usually deriving from the centrosomes. In PtK2
cells, many more similar microtubules were left, all of them
emanating from the centrosome area (Fig. 3). In MDCK
cells, a large number of extremely tortuous microtubules
persisted 30 min after addition of the drug (Fig. 3). Most of
these microtubules apparently did not radiate from the centrosomes (Fig. 3, arrows). Upon prolonged incubation in the
same drug concentration, the stable microtubules found in
all three cell lines progressively disappeared. Short microtubules started to grow and elongate off the centrosomes between 1 and 4 h after addition of this concentration of the
drug. The strongest regrowth was observed in PtK2 cells. At
higher nocodazole concentration (33 ~tM, not shown), the
kinetics of depolymerization was essentially the same al-
Br6 et al. StableNoncentrosomalMicrotubules
Figure 1. Centrosome splitting in subconfluent MDCK cells. (a)
Centrosomes detected by the human anti-centrosome antibody
(diluted 1:100)are visible as one large dot or two closely associated
smaller dots in Vero cells. The brighter dots correspond to a centrosome in early prophase (arrow). (b) Each MDCK cell contains two
individual structures decorated by the same antibody and sometimes separated by a large distance. Bar, 5 Ixm.
though fewer microtubules remained. Microtubule regrowth
from centrosomes was never observed after 4 h of treatment.
These results show that by adding nocodazole at an intermediate concentration it is possible to induce a shift from a
mainly noncentrosomal pattern to an exclusively centrosomal pattern of microtubules in MDCK cells. A large fraction of the rnicrotubules in MDCK cells does disappear after
nocodazole addition. Therefore, the population of stable
microtubules coexist with more labile microtubules. The ratio of stable versus unstable microtubules seems to be high
in MDCK, intermediate in PtK2, and very low in Vero cells.
Inversely, the ratio of centrosomal versus noneentrosomal
microtubules seems to be low in MDCK, intermediate in
PtK2, and very high in Vero cells.
Most Stable Microtubules Contain
Detyrosinated Tubulin
It has been recently reported that a specific class of long sinuous microtubules is enriched in the posttranslationally
modified form of a-tubulin, the detyrosinated a-tubulin (glutubulin) (15). A monoclonal anti-tyrosinated tubulin (antityr) and an affinity-purified rabbit anti-detyrosinated tubulin
(anti-glu) have been prepared. These antibodies have been
1285
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Centrosomes of MDCK Cells Are Not Preferred
Sites of Nucleation
cells grown on plastic were immunostained for tubulin as described in Materials and Methods and thick sections screened for centrioles.
(a) Two centrioles (arrows)'~3 l~m apart from each other, not associated with the nucleus, are visible. (b) Higher magnification of the
centfioles shown in a. Among all the microtubulesdecorated by gold particles, only a few actually emanate from the centrioles (arrowheads)
Bars, 1 lma.
raised against the synthetic carboxy-terminal peptides of
a-tubulin and found to be highly specific for each form of
a-tubulin (22a).
The microtubule pattern revealed by the anti-tyr antibody
(tyr-microtubules) in MDCK cells was similar to the pattern
obtained after staining with the monoclonal anti-I~-tubulin
(Fig. 4). The anti-glu antibodies stained a distinct subset of
long, wavy microtubtdes as previously reported for TC7
cells (glu-microtubules; Fig. 4, b and d) (15). However,
MDCK cells appeared to contain many more of these
microtubules. Most of the microtubules remaining 30 min
after addition of nocodazole (1.6 I~M) were decorated by the
glu-antibodies, although to a variable extent (Fig. 4 d).
These microtubules were also decorated by the tyr-antibody
but very few microtubnles were stained only by the tyrantibody (Fig. 4 c). We will call "glu" any microtubule that
is clearly stained by the glu-antibody. This does not mean
that it is not detected by the tyr-antibody, inasmuch as some
of them being more strongly labeled by this antibody than
by the glu-antibody. The number of glu-microtubules slowly
decreased with time of incubation in the drug. The short centrosomal microtubules regrowing slowly between 1 and 4 h
were first exclusively stained by the anti-tyr-antibody and
became stained by the glu-antibody at 4 h, in the vicinity of
At the periphery of MDCK cells we occasionally found glumicrotubules with ends exclusively stained by the tyrantibody. Unfortunately, microtubule density hampered our
attempts to determine what happened at the other end of
these microtubules. It was easy, however, to observe microtubules double stained with tyr- and glu-antibodies over their
entire length in cells treated with nocodazole for 1 and 2 h
since only a few microtubules remained (Fig. 5). 1 h after
addition of nocodazole (1.6 ~tM), very few glu-microtubules
contained ends stained only by the tyr-antibody (not shown).
However, at 2 h, when tyr-microtubules start to appear
around the centrosomes, virtually all the remaining noncentrosomal glu-microtubules had only one end exclusively
stained by the tyr-antibody (Fig. 5, a and b). This staining
was often much brighter than over the rest of the microtubules and was rarely observed on the glu-microtubules of
cells treated with 33 txM nocodazole (Fig. 5, c and d). This
concentration prevented the regrowth of tyr-microtubules
The Journal of Cell Biology, Volume 105, 1987
1286
the centrosome (Fig. 4 f ) . At this time point, occasional,
long glu-microtubules were found associated with the centrosomes. These were probably "old microtubules".
One End of Noncentrosomal Glu-Microtubules
Can Be Exclusively Composed of 7l~r-Tubulin
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Figure2. Thick section of MDCK cells observed by electron microscopy after immunogoldstahtingof microtubules. SubconfluentMDCK
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Figure 3. Microtubule pattern and stability in MDCK, Vero, and PtK2 cells. MDCK (/eft), Veto (center), and PtK (right) cells were stained
by double immunofluoreseencefor microtubules and centrosomes as described in Materials and Methods. A mouse monoelonal anti-I[~tubulin was used for mierombule staining. The centrosome staining is not shown, but the position of each centrosome is indicated when
not obvious (arrows). Cells were fixed before or after various times of incubation in 1.6 IxM nocodazole. Numbers (left) indicate the time
in minutes of incubation of each cell line in nocodazole.
Br~ et al. Stable Noncentrosomal Microtabules
1287
MDCK cells were fixed before (a and b), 30 rain (c and
d), and 4 h (e andf) after incubation in 1.6 ~tM nocodazole. Double immunostainingwas performed with anti-tyr- (a, c, and e) and anti-glu(b, d, and f ) antibodies. Secondary antibodies were labeled with rhodamine (a, c, and e) and fluoreseein (b, d, and f ) (Bar, 5 txM).
from the centrosomes during the treatment. In this case,
however, different staining intensities were occasionally observed with the glu-antibodies along the length of a given
microtubule (Fig. 5 d). It seems that newly formed microtubules or microtubule fragments contain mainly tyrosinated
ct-tubulin.
Microtubule Network Reassembly Precedes
Tubulin Detyrosination after Nocodazole Removal
antibodies (Fig. 6). Microtubules detected by the tyrantibody elongated, and at 30 min an apparently normal
microtubule pattern was reformed. It was only then that
some mierotubnles became reactive to the glu-antibodies,
and only in a fraction of the cell population. A glumicrotubnle pattern comparable to what was found in the
normal population was observed at 1 h after nocodazole
removal. The ratio of noncentrosomal to centrosomal microtubules increases with time after nocodazole removal.
To investigate further the relationship between microtubule
polymerization and detyrosination of ct-tubulin in vivo, we
examined the kinetics of tyr- and glu-microtubule regrowth
after nocodazole reversal (Fig. 6). After 4 h of incubation in
33 ttM nocodazole, most microtubules were depolymerized.
Only a few, long centrosomal microtubules containing glutubulin remained (Fig. 6). Centrioles could be identified
with the glu-antibodies, which seemed to strongly stain the
centrioles (Fig. 6). Interestingly, the tyr-antibody stained
numerous dots, scattered in the cytoplasm, which were not
stained by the glu-antibodies. This was always found and
does not seem to be an artifact of fixation. Indeed, these dots
were also detected by the anti-13-tubulin in MDCK cells but
not in other cells (Fig. 3).
3 min after nocodazole removal, many short microtubules
were detected by the tyr-antibody, growing as small asters from
the centrosomes as small foci or as short individual segments
scattered throughout the cytoplasm (Fig. 6). With the exception of long (probably old) microtubules, none of the short
(newly formed) microtubules were stained with the glu-
The low abundance of centrosomal microtubules at steady
state in MDCK cells is very surprising in view of the large
amount of microtubules regrowing from centrosomes at early
time points after nocodazole removal. This means that noncentrosomal microtubules eventually predominate in this
cell line. This is different than what has been found in fibroblastic cells where centrosomal microtubules predominate.
To investigate the potential correlation between the appearance of noncentrosomal microtubules and polymer stabilization, we incubated cells for 4 h in 33 ~tM nocodazole, then
removed the drug, and at various times during microtubule
regrowth we further added nocodazole at 1.6 or 33 ttM for
30 min. The cells were then fixed and double stained with
the anti-glu and anti-tyr antibodies (Fig. 7). Only tyrmicrotubules are shown. Long centrosomal and short non-
The Journalof Cell Biology,Volume105, 1987
1288
Kinetics of Centrosomal and Noncentrosomal
Microtubule Stabilization during Regrowth after
Nocodazole Removal
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Figure4. Stable microtubules in MDCK cells contain detyrosinated r
centrosomal microtubules were found to be resistant to 1.6
lxM nocodazole 15 rain after release from the first drug treatment (Fig. 7 a). They were not stained by the glu-antibodies
(not shown). Long noncentrosomal mierotubules became
resistant to this drug concentration only after 30 min of
regrowth. They were predominant by 1 h (Fig. 7, b and c).
This coincides with the first appearance of rnicrotubules
strongly stained by the glu-antibodies (Fig. 6). The use of 1.6
~M nocodazole for 30 min as a test for the presence of relatively stable micrombules is fully valid since this treatment
induces a complete depolymerization in fibroblasts. However, we were concerned that the %table" centrosomal
microtubules observed after 15 min of regrowth arose by
elongation during the further 30 min of treatment in 1.6 ltM
nocodazole. To eliminate this possibility, we repeated the kinetics of polymer stabilization during regrowth by using a
high concentration of nocodazole (33 ~tM) (Fig. 7, d-h). The
result was striking: many centrosomal microtubules were
stabilized immediately after being nucleated (as soon as
3 min; Fig. 7 d). Moreover, the length of stabilized centrosomal microtubules increased with time of regrowth
while their number seemed to drop. Stable, short noncentrosomal microtubules arose between 8 and 15 min. Long
stable noncentrosomal micrombules were much less abundant at 30 and 60 rain than in cells treated with 1.6 IxM
nocodazole (Fig. 7, b, c, g, and h).
We conclude from these experiments that: (a) Some centrosomal microtubules are stabilized immediately after being
nucleated. (b) The number of stable centrosomal microtubules drops and their length increases with time of regrowth.
Br6 et al. StableNoncentrosomalMicrotubutes
(c) Noncentrosomal micrombules are stabilized later than
centrosomal microtubules. (d) Many of the noncentrosomal
micrombules, which are stable in 1.6 I~M, are unstable in 33
I~M nocodazole. (e) Microtubule stabilization is reversible
since the pattern of stable microtubules evolves with time of
regrowth. ( f ) Tubulin detyrosination occurs after partial
microtubule stabilization.
Microtubule Stabilization by Taxol Promotes
Tubulin Detyrosination
The results reported above strongly suggest that detyrosination of a-mbulin occurs primarily on stabilized polymer in
vivo. To strengthen this correlation, we examined the microtubule staining with the glu-antibodies in cells treated with
taxol, a drug known to reduce micrombule dynamics (Fig.
8). There was a complex rearrangement of the microtubule
network over the first 2 h of incubation in taxol. No obvious
increase in the number of glu-microtubules could be detected
during the first 30 rain of incubation. By 2 h, however, while
the microtubule pattern seemed to be relatively stable, most
microtubules appeared to be stained by the glu-antibodies.
Amount of Glu-Tubulin Is Decreased in
Nocodazole and Increased in Taxol-treated Cells
The previous set of experiments clearly shows that newly
formed microtubules are mostly composed of tyrosinated
tubulin. On the other hand, stable microtubules or taxolstabilized microtubules are clearly enriched in glu-tubulin.
This strongly suggests that most of the free tubulin is tyrosi-
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Figure5. In MDCK cells glu-microtubule ends are exclusivelystained by the tyr-antibody during depolymerizationin 1.6 IxM nocodazole.
2 h after the addition of noeodazole at 1.6 lxM (a and b) and 1 h aP~eraddition of noeodazole at 33 IxM (c and d) MDCK cells were fixed
and double stained with anti-tyr- (a and c) and anti-glu- (b and d) antibodies. (Arrows)Mierotubule ends exclusivelystained by the anti-tyrantibody. (Large arrowheads) Centrosomes. (Small arrowheads) Ends weakly labeled by the anti-glu-antibody in a microtubule that is
strongly stained. The large spots produced by the anti-tyr-antibody around centrosomes are in fact numerous, short microtubules not
resolved on the picture. Bar, 5 $tm.
The Journal of Cell Biology, Volume 105, 1987
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Figure 6. Kinetics of tyr- and glu-micmtubule regrowth in MDCK cells after nocodazole removal MDCK ceils were treated for 4 h with
33 gM nocodazole. The drug was removed, the ceils were fixed at the time (in minutes) indicated on the left of the figure, and double
stained for tyr- (left) and glu- (right) a-tubulin. (Arrows) Centrosomes; (arrowheads) noncentrosomal microtubule loci. Bar, 5 Ixm.
Discussion
Centrosome and Centrioles in Subconfluent
MDCK Cells
The term centrosome in mammalian cells usually refers to
the complex formed by the two centriole cylinders and the
pericentriolar material that nucleates microtubules (2). We
have found that in subconfluent MDCK cells, the two
cylinders are largely independent from each other and from
the nucleus. A detailed study is underway. This unusual condition of the centrosome has been found in other situations
(32, 33, 35). In MDCK cells, the two cylinders migrate up
to the apical and central part of the cell at a relatively constant distance from each other during cell polarization (Simons, K., M. H. Br6, and E. Karsenti, unpublished data).
This provides us with a unique model to study the control
of centrioles and pericentriolar material location during cell
Br6 et al. Stable Noncentrosomal Microtubules
differenfiaton and polarization. Several examples suggest,
indeed, that centrioles are relocated or eliminated during cell
differentiation (2, 8, 9, 32, 37, 40, 41, 45).
Origin and Persistance of Noncentrosomal
Microtubules in MDCK Cells
The microtubule pattern in MDCK cells appears to be much
more disorganized than in Vero and PtK2 cells, where most
microtubules originate at the centrosome. One could argue
that this is due to the fact that in MDCK cells the two centrioles are separated. Indeed, by being nucleated in two different locations, the microtubules could form a network that
would appear less organized. However, in the electron microscope, the centrioles were found to nucleate only a few
microtubules. Therefore, in MDCK cells, many microtubules are generated by a mechanism other than nucleation by
the centrosome. One possibility would be that the microtubule-nucleating material that is associated with the centrioles in fibroblasts is more dispersed in MDCK cells. The
balance between centrosomal and noncentrosomal microtubules could also be controlled by modulating the dynamics
of microtubule assembly and disassembly. Mitchison and
Kirschner have shown that microtubule asters formed on
purified centrosomes in vitro are very dynamic (25). Microtubules grow and then start to depolymerize catastrophically
with a probability that increases with decreasing concentrations of tubulin. The nucleation sites left unoccupied on the
centrosome can then nucleate new rnicrotubules below the
steady state tubulin concentration. This behavior in vitro
may explain why centrosomal microtubules predominate in
some cell lines. Indeed, during microtubule re.growth after
removal of depolymerizing drugs like nocodazole, both centrosomal and noncentrosomal microtubules appear in the
cytoplasm of these cells. However, the noncentrosomal
microtubules eventually disappear and at equilibrium a
centrosome-nucleated aster of microtubules predominates.
In the context of the dynamic instability model, this behavior
is easy to explain: Upon removal of nocodazole, the free
tubulin concentration in the cell is very high. Spontaneous
nucleation will be frequent and compete efficiently with centrosome nucleation. However, with time, more polymer assembles and since the cell is a closed system the free tubulin
concentration drops. A steady state situation is reached
where the tubulin concentration is too low to allow spontane-
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nated while detyrosination occurs on the polymer. If this
were the case, most of the cellular tubulin in nocodazoletreated ceils should be tyrosinated (since it is soluble) while
in taxol-treated cells, tubulin should be enriched in the gluform. To test this possibility, we carried out immunoblots
with the anti-glu and anti-tyr-antibodies on total extracts of
cells after nocodazole and taxol treatments (Fig. 9). The
specificity of the antibodies for tyrosinated and detyrosinated
tubulin has been checked on immunoblots made with total
cell extracts before and after treatment with carboxypeptidase A (Fig. 9 a). The reactivity of the tyr-antibody was decreased (Fig. 9 a, lane 2) and the reactivity of the gluantibodies dramatically increased (lane 4) after action of
carboxypeptidase A. Cells incubated for 4 h in 33 txM
nocodazole and lysed directly in sample buffer contained a
very low amount of glu-tubulin. Glu-tubulin became detectable between 15 and 30 rain after nocodazole removal. The
amount of tyr-tubulin remained constant throughout this
period, demonstrating that the lack of detection of glutubulin after nocodazole treatment is not due to a different
amount of total tubulin present in these samples (Fig. 9 b).
In cells treated with taxol, the amount of glu-tubulin increased progressively with the time of treatment. Here, the
amount of tyr-tubulin remained constant or dropped slightly
at longer times of incubation in taxol (Fig. 9 c).
ous nucleation and therefore only centrosome-nucleated microtubules remain (25). This situation implies that the centrosomal aster is a very dynamic structure and one would
expect it to disappear quickly in response to nocodazole.
This is what was found for L929 cells and we report a similar situation for Vero cells. In PtK2 cells, many centrosomal
microtubules are apparently stable since they are resistant to
a similar treatment.
In MDCK cells, the situation is different. Most stable
microtubules (which are numerous) appear to be noncentrosomai. In a way microtubules behave naturally in these cells
as they do in other cells (PtK2, 3T3, human fibroblasts) that
have been treated with taxol. De Brabander et al. (6) have
shown that taxol induces spontaneous microtubule assembly
in the cytoplasm of interphase cells without spatial relation
to the centrosome. Moreover, the preexisting centrosomal
microtubules gradually disappear. This effect was explained
by the properties of taxol that decrease the critical concentration for tubulin polymerization and increase microtubule stability. Therefore, it seems that MDCK cells contain factors
capable of stabilizing microtubules as taxol does. These may
be specific MAPs. The exact molecular mechanism by
which such factors would function is still unclear. Brain
MAPs, for example, bind quickly and tightly to microtubules
The Journal of Cell Biology, Volume 105, 1987
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Figure 7. Kinetics of microtubule stabilization aRer nocodazole removal. MDCK cells were treated for 4 h with 33 gM nocodazole. The
drug-containing medium was then removed and the cells were quickly washed three times and incubated with fresh, warm medium. After
15, 30, and 60 min of regrowth, nocodazole (1.6 ~tM) was further added for 30 min, and the cells fixed (a-c). After 3, 8, 15, 30, and 60
rain of regrowth, nocodazole (33 ~tM) was further added and the cells fixed (d-h). Double immunofluorescence with anti-tyr- and anti-gluantibodies was carried out. Only the tyr staining is shown. The positions of centrosomes are indicated (arrows). Bar, 5 ~tm.
in vitro, leading to a rapid and almost irreversible stabilization of microtubules (18). The presence of such molecules in
saturating amounts would not explain the progressive predominance of noncentrosomal microtubules in MDCK cells
during regrowth after nocodazole removal. However, Job et
al. (18) discuss the possibility that MAPs present in nonsaturating amounts could bind in discrete sites along
microtubules because of their fast association rate. They argue that microtubule stability subclasses could be generated
in this way. Moreover, they suggest that a given microtubule
could have different levels of stability along its length depending on the amount of MAPS bound locally. The presence
of limiting concentrations of MAPS in MDCK cells could explain the progressive predominance of noncentrosomal
microtubules according to the following mechanism: Initially after nocodazole removal, numerous short microtubules are nucleated in the cytoplasm. Both on free and
centrosome-nucleated microtubules, very short segments are
stabilized by the binding of MAPs. The existence of such stable segments along noncentrosomal microtubules would prevent them from complete depolymerization when the steady
state free tubulin concentration is approached. Such segments could then act as seeds for polymer elongation below
the free tubulin concentration required for spontaneous
nucleation. If many stable seeds exist, they will compete
with the nucleation activity of the centrosome.
We find that stable centrosomal microtubules arise early
and before the noncentrosomal ones during regrowth. This
Br6 et al. Stable Noncentrosomal Microtubules
probably occurs because they are capped at one end by the
centrosome. This indeed increases the probability of stabilization of a visible microtubule stretch. Interestingly, during
regrowth, the length of stable centrosomal microtubules increases in parallel with microtubule elongation. While this
occurs the number of stable centrosomal microtubules
drops. This behavior fits well with an initial rapid and reversible stabilization due to soluble factors, followed by their random redistribution. Short, stable noncentrosomal microtubules become visible after 8-15 min of regrowth; this occurs
when they happen to bind stabilizing factors in two different
points. In other words, a stable microtubule could be composed of a long nonstabilized region flanked by two short stabilized segments. The probability for this to occur increases
with the length of the microtubule and with its life span.
This mechanism would produce microtubules with segments of different ages as previously suggested (18) and with
dynamic extremities. Several observations argue in favor of
this idea: (a) We found micrombules strongly stained by the
glu-antibodies in their middle part with a weaker staining on
each side. Since we assume that the extent of r
detyrosination is representative of the microtubule age, this
suggests that the microtubule ends are not as "old" as the
rest of the microtubule. (b) After 1 h of mierotubule depolymerization in the presence of 1.6 lxM nocodazole, tyrmicrotubules regrow slowly from the centrosomes. In the
same time, short microtubule segments exclusively stained by
the tyr-antibody appear at one end of the remaining noncen-
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Figure 8. Stabilization of micrombules by taxol increases the proportion of detyrosinated microtubules. MDCK ceils were treated with
taxol (5 ~tM) for 30 min (a and b) and 2 h (c and d). After fixation the cells were processed for double immunofluorescencewith the
anti-tyr- (a and c) and anti-glu- (b and d) antibodies. Bar, 5 ltm.
Microtubule Stability and a-Tubulin Detyrosination
trosomal glu-microtubules. These are probably newly polymerized segments. Since most noncentrosomal microtubules
have disappeared by 4 h, we must conclude that during depolymerization in the presence of 1.6 BM nocodazole they grow
and shrink (the overall shrinking rate being higher than the
growth rate). The tyr-containing end might be the active end
(+ end) in these microtubules.
It has been shown recently that cells contain two classes of
microtubules that can be distinguished by antibodies directed
against the tyrosinated and detyrosinated forms of ct-tubulin
(15). The most abundant class of microtubules contains
mainly tyrosinated vt-tubulin. A less abundant class in many
cells is composed of long sinuous microtubules containing
considerable amounts of detyrosinated a-tubulin. As recently shown, there seems to be a continuum in the ratio of
tyrosinated and detyrosinated ct-tubulin in all microtubules
(13). It has been suggested that the microtubules strongly
stained by the anti-glu antibodies are long lived, stable
microtubules (13, 21). In the present paper we have accumulated evidence that this is indeed the case. In fact, it is likely
that the turnover of the caboxy-terminal tyrosine of a-tubulin
is tightly coupled to microtubule dynamics as proposed by
Thompson (38). In vivo as in vitro (28, 29), the tubulin tyrosin ligase seems to function only on the dimer. In vitro the
tubulin carboxypeptidase appears to function preferentially
on the polymer (23). If in vivo the carboxypeptidase functions on the dimer, we must assume that this reaction is
much slower than the tyrosination reaction since after
microtubule depolymerization by nocodazole we detect almost no detyrosinated u-tubulin on immunoblots of total cell
lysates. Moreover, three lines of evidence suggest that tubulin detyrosination occurs only on long-lived stabilized microtubules: (a) After nocodazole release, the first microtubules to regrow are tyrosinated. (b) The first detyrosinated
microtubules appear after 30 rain of regrowth while stable
microtubules already exist 3 min after nocodazole removal.
This time lag between microtubule polymerization and the
appearance of the first detyrosinated microtubules is in
agreement with results obtained in vitro (5). (c) Stabilization
of microtubules with taxol leads to an increase in the number
of detyrosinated microtubules and also to an increased amount
of total detyrosinated tubulin.
This shows that anti-glu antibodies are good markers
of long-lived microtubules and raises intriguing questions as
to how the tubulin carboxypeptidase functions. In any case,
The Journal of Cell Biology, Volume 105, 1987
1294
Figure 9. Immunoblot analysis of tyrosinated and detyrosinated
tubulin in MDCK cell lysates. (a) Cell lysates were prepared and
treated by carboxypeptidase A as described in Materials and
Methods. The proteins were run on a 10% Laemmli gel, transferred
to nitrocellulose, and stained with the anti-tyr (lanes 1 and 2) or
anti-glu (lanes 3 and 4) antibodies. Carboxypeptidase-treated sampies are in lanes 2 and 4. (b) Cells were treated with 33 gM nocodazole for 4 h, lysed in hot gel sample buffer at the indicated times
(in minutes) after nocodazole removal, and loaded on one 10%
Laemmli minigel in duplicate, n, cells not treated by nocodazole.
After transfer, the nitrocellulose sheet was split in two parts: onehalf was stained with the anti-tyr antibody (tyr) and the other half
with the anti-glu antibody (glu). (c) Ceils were treated with 5 IxM
taxol, lysed in hot gel sample buffer at the indicated times (in
minutes), and loaded on one 10% Laemmli minigel in duplicate.
After transfer, the nitrocellulose sheet was split in two parts: onehalf was stained with the tyr-antibody (tyr) and the other with the
glu-antibody (glu). In all cases, the secondary antibody was labeled
with peroxidase. See Materials and Methods for details.
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The regrowth of small centrosomal asters from centrosomes
after 4 h of incubation in 1.6 BM nocodazole shows that some
active tubulin remains under such conditions. The absence
of noncentrosomal microtubules in the same conditions suggests that the mechanism of microtubule stabilization is
different from the mechanism of nucleation by the centrosome. The centrosome probably caps irreversibly the "minus" ends of microtubules, while stabilizing factors bind
along the microtubule wall. Therefore, noncentrosomal microtubules having two ends free would be more sensitive to
very low tubulin concentrations. This interpretation is also
supported by the observation that stable centrosomal microtubules resist equally well to 1.6 and 33 BM nocodazole,
while many noncentrosomal microtubules do not (Fig. 7).
Although the model discussed above for the generation of
noncentrosomal microtubules fits well with our experimental
observations, other explanations could be proposed: Noncentrosomal microtubules could be stabilized by local interactions with insoluble structures such as intermediate filaments or membrane sites for example (41). Some stable
noncentrosomal microtubules could also arise by falling off
the centrosome (43).
We thank Shamsa Faruki and Heike Wilhelm for expert technical assistance.
We thank Gareth Griffith for efficient help with the electron microscopy and
critical reading of the manuscript, Kai Simons and Jean Davoust for stimulating discussions and suggestions during the preparation of this manuscript. We thank Alan Summerfield for his photographic work, and Annie
Steiner and Anne Walter for typing the manuscript.
Received for publication 18 February 1987, and in revised form 14 April
1987.
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our results demonstrate that detyrosination is not the cause
of reversible microtubule stabilization. Rather, micrombule
stabilization appears to be a prerequisite to tubulin detyrosination, at least in MDCK cells. This may mean that the tubulin carboxypeptidase functions slowly. If a micrombule is
short lived, the proportion of detyrosinated subunits may remain too low to be visualized by immunofluorescence. The
amount of detyrosinated mbulin in a given micrombule will
depend on its age. This may explain why nocodazole-resistant (stable) microtubules contain variable amounts of detyrosinated tubulin (Figs. 4 and 5). Indeed, the age of these
stable microtubules could vary broadly. The sort of microtubule stabilization we term "reversible" in this discussion
could be better named "short term: It is possible that
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The respective functions of dynamic and stable microtubules are unknown. Our results would imply that in an interphase cell at steady state many dynamic microtubules are
centrosome nucleated. Since the centrosome could be located in various places in the cell, it is tempting to speculate
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