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Cell Motility and the Cytoskeleton 3549-58 (1996)
Differential Expression of Tubulin lsotypes
During the Cell Cycle
Charles Dumontet, George E. Duran, Katherine A. Steger, Gloria L. Murphy,
Howard H. Sussman, and Branimir 1. Sikic
Departments of Medicine and Pathology, Stanford University School of
Medicine, Stanford, California
Microtubules play an essential role in cell division. Little is known about possible
variations of total tubulin and tubulin isotype expression during the cell cycle. We
analyzed the total tubulin content, tubulin polymerization status and tubulin isotype content in resting and dividing human K562 leukemic cells and human
MES-SA sarcoma cells. Although the total cellular tubulin content increases as
the cells progress toward mitosis, the total tubulidtotal protein ratio is stable
during the cell cycle. Reverse transcriptase-polymerase chain reaction was applied to analyze the levels of expression of a,p, and y-tubulin isotypes. Whereas
a-tubulin isotype and y-tubulin transcripts were found to be expressed at constant
levels throughout the cell cycle, some of the P-tubulin isotype transcripts were
found to be more highly expressed in dividing then in resting cells. Both of the
class IV P-tubulin isotype transcripts (human 5p and p2, Class IVa and IVb,
respectively) were expressed in dividing K562 and MES-SA cells at twice the
levels found in resting cells. Increased expression of the class IV isotype proteins
in dividing cells was confirmed by immunoblotting, both in K562 and in MES-SA
cells. A larger fraction of total cell tubulin was found to be polymerized in
dividing cells (36-40%) than in resting cells (27-30%). The degree of polymerization of class IV tubulin in dividing and resting cells was similar to that of total
tubulin. These results show that total tubulin is expressed as constant levels
throughout the cell cycle but that the degree of polymerization is increased as cells
are committed to division. The relative overexpression of the two class IV p-tubulin isotypes in dividing cells suggests functional specificity for these isotypes
and a regulatory role of these isotypes on the microtubule network during
mitosis. 0 1996 Wiley-Liss, Inc.
Key words: cell cycle, tubulin isotypes, microtubule, polymerization
INTRODUCTION
Microtubules are dynamic polymeric organelles involved in numerous cellular processes including mitosis
[Borisy and Taylor, 1967; Kirschner, 19781. Tubulin,
first described as a “colchicine-binding protein” was
shown to be the building block of all microtubular structures [Wilson and Meza, 19731. The demonstration that
tubulin dimers were heteropolymers of a and p subunits
[Luduena et al., 19771 was later followed by the demonstration that both of these subunits existed under the
form of various isotypes [Cleveland et al., 19801.
Thanks to the work of Cowan and coworkers in mam0 1996 Wiley-Liss, Inc.
mals and Cleveland and coworkers in chickens, a number
of isotypes of each subunit have been identified, including seven a-tubulin and seven P-tubulin isotypes [Cowan
et al., 1986; Hall et al., 1983; Lewis et al., 1985; Sullivan, 19881. Tubulin isotypes in humans belong to a
multigene family of 15 to 20 genes, many of which are
pseudogenes [Lewis et al., 1987; Wilde et al., 19821.
More recently a less abundant form of tubulin has been
identified, termed y-tubulin, which appears to be inReceived December 19, 1995; accepted May 20, 1996.
Address reprint requests to Dr. B.I. Sikic, Room M-211, Oncology
Division, Stanford Medical Center, Stanford, CA 94305-5306.
50
Durnontet et al.
volved in nucleation of microtubules from the centrioles
[Zheng et al., 19911. Presently only one isotype of y-tubulin has been reported [Oakley and Oakley, 19891.
Tubulin isotypes differ essentially by their terminal
carboxy portion [Sullivan, 19881. Tubulin isotypes, although quite different among themselves in a given species, have been remarkably conserved throughout evolution [Little and Seehaus, 19881. It has thus been tempting
to attribute functional specificities to some or all of these
isotypes. A number of attempts to identify functional
characteristics specific for tubulin isotypes have been
made. Although the question of the functional specialization of tubulin isotypes remains controversial, there is
definite evidence that some isotypes are differentially
expressed and required for some specific processes [Luduena, 1993; Raff, 19941. Some of the most compelling
evidence for functional specialization of isotypes has
been provided by the demonstration that some isotypes
are critically involved in meiosis and neurite outgrowth
[Gard and Kirschner, 1985; Joshi and Cleveland, 1989;
Kemphues et al., 19801. More recently, analysis of the
effect of isotype composition on microtubule stability in
vitro has shown that different isotypes form microtubular
structures with different dynamic properties [Banerjee et
al., 1992; Lu and Luduena, 1993; Panda et al., 19941.
In the present work we have analyzed the expression
of total tubulin and tubulin isotypes during the cell cycle.
We have chosen as models the human erythroleukemic
cell line K562 which grows in suspension and the adherent
human sarcoma cell line MES-SA. Total tubulin was
analyzed by immunoblotting and fluorescence-activated
cell sorter (FACS) analysis of viable cells. Tubulin isotype transcript levels were analyzed by semi-quantitative
reverse transcriptase-polymerase chain reaction (rtPCR) , using specific primers which we designed and
immunoblotting of class IV P-tubulin. Our results demonstrate that in both cell lines, the two class IV P-tubulin
isotypes, which share a common carboxy-terminal portion, are expressed at higher levels in dividing cells than
in resting cells, suggesting a role of these isotypes in
mitosis.
in a humidified atmosphere containing 5% CO,. Cell
stocks were screened routinely for Mycoplasma by the
DNA hybridization method (Gen-Probe, Inc., San Diego, CA) and by rt-PCR. Log phase cells were obtained
by seeding the cells at low concentration (5 X lo4 K562
cells/ml; 1 X 106 MES-SA cells in a 80-cm2 flask) and
collecting them after 48 hours of culture. Plateau cells
were obtained by collecting cells 24-48 hours after they
had reached plateau phase of growth (K562 cells) or
confluence (MES-SA).
Analysis of Tubulin Content and Cell Cycle
by FACS
To determine the percentages of cells in each phase
of the cell cycle, cells were collected, fixed with ice-cold
methanol containing 2 mM EGTA, washed, and resuspended in propidium iodide (50 pg/ml), then analyzed
by FACS. Percentages of cells in G1, S, and G2/M phase
were determined using CellFit@Software (Becton-Dickinson, San Jose, CA). For analysis of tubulin content in
the cell cycle, cells were first labeled with murine monoclonal antibody directed against all P-tubulin isotypes
and a secondary fluoresceinated antibody, then resuspended in propidium iodide, as previously described
[Jaffrezou et al., 19951. Briefly, cells were permeabilized prior to staining with cold (-20°C) methanol, then
exposed to pan-P-tubulin antibody (Sigma, 1:1,OOO) for
1 hour at room temperature, washed thrice, and exposed
to FITC-labeled goat-antimouse antibody for 1 hour at
room temperature. Cells were washed and resuspended
in a solution of propidium iodide (50 pg/ml), then analyzed by FACS.
Evaluation of Total Tubulin and Class IV
p-Tubulin lsotype Protein by lmmunoblotting
Cells were harvested in log or plateau phase of
growth, and pellets were resuspended in lysis buffer containing Tris-HC1 pH 6.80, 1 mM MgCl,, 2 mM EGTA,
and 0.2% Tween 20 and protease inhibitors (phenylmethylsulfonyl fluoride [PMSF] 1 mM, leupeptin 50 pg/
ml, pepstatin 1 pg/ml, trypsin inhibitor 1 mg/ml, and
aprotinin 20 kg/ml [Sigma, St. Louis, MO]). Total protein was quantified by the Lowry assay [Lowry et al.,
MATERIALS AND METHODS
19511, and samples were prepared in sodium dodecyl
Cell Culture
sulfate (SDS), then boiled before being applied to a 12%
The human erythroleukemic cell line K562 was polyacrylamide gel, as previously described. Proteins
purchased from the American Type Culture Collection. were then blotted onto a Hybond-ECL nitrocellulose
The MES-SA cell line was derived in our laboratory membrane (Amersham, Buckinghamshire, UK) using a
from sarcomatous elements of a uterine mixed mullerian Sartorius apparatus (Hayward, CA). The membrane was
tumor [Harker and Sikic, 19851. All cell lines were cul- blocked with buffer containing 5% milk and 1% bovine
tured in McCoy 5A medium supplemented with 10% albumin, then incubated 2 hours at room temperature
newborn calf serum, 2 mM glutamine, 200 unitdm1 pen- with pan+ monoclonal antibody (Sigma Immunochemicillinlml, and 100 pg streptomycin (all from Irvine Sci- icals, 1:3,000 dilution) or anti-class IV P-tubulin isotype
entific, Santa Ana, CA). Cells were maintained at 37°C monoclonal antibody (Biogenex, San Ramon, CA, 1:
Tubulin Isotypes and the Cell Cycle
1,000 dilution), washed, incubated in goat anti-mouse
antibody, washed, incubated in streptavidin-biotin,
washed, and processed in ECL reagents. Phosphocellulose-purified tubulin prepared from bovine brain (generously provided by M.A. Jordan, University of Santa Barbara) was used as a control. Preliminary experiments
were performed to determine the dilution of antibodies
allowing quantification of tubulin content.
0
0
0
0
0
Evaluation of Polymerized and Soluble
Tubulin Protein
Polymerized and soluble tubulin were distinguished using a modified version of the method reported
Minotti et al., [1991]. Briefly, cells were harvested,
washed, and lysed as described above. Cell lysates were
then incubated for 5 min at 37°C in the dark, and the
cellular residues were mixed briefly and centrifuged at
14,000 rpm for 10 min at room temperature. The resultant supernatants were transferred to a separate centrifuge
tube and kept on ice. The pellet was resuspended in
Ling's Lysis buffer (10 mM Tris pH 7.5, 1.5 mM
MgCl,, and 10 mM KC1) in a volume equal to the supernatant. A volume equivalent to 100 kg of sample
protein was incubated in 4 x Laemmli buffer, vortexed,
and boiled for 10 min prior to loading onto a 12% acrylamide-SDS gel. Gels were then processed for immunoblotting as described above. The percent of polymerized
tubulin was calculated by comparing the relative ratio of
polymerized tubulin (pellet fraction) to the unpolymerized tubulin dimers (supernatant) by densitometry.
rt-PCR
0
0
0
0
0
0
0
0
0
0
51
P4 reverse primer: (223, 243) AAA GGC CCC
TGAGCGGACACT
5P (Class IVa) forward primer: (-85, -68) TCT
CCGCCGCATCTTCCA
5p reverse primer: (167, 186) TCT GGG GAC ATA
ATT TCC TC
p2 (Class IVb) forward primer: (-42, -22) GTC
TACTTCCTCCTCTTCCC
P2 reverse primer: (291, 300) GTT GTT CCC AGC
ACC ACT CT
B a l forward primer: (1003, 1020) ATC AAG ACC
AAG CGT ACC
B a l reverse primer: (1363, 1380) CAG CAC CTT
TGT GAC GTT
K a l forward primer: (1000, 1017) ACC ATC AAA
ACC AAG CGC
K a l reverse primer: (1363, 1380) TGC AGG GCC
AAA AGG AAT
Ha44 forward primer: (139, 158) CCT TCA CCA
CCT TCT TCT GT
Ha44 reverse primer: (230, 149) TCG GTA TGG
GCC ATT TCG GA
H2a forward primer: (1059, 1075) GTG GGC ATT
AAC TAC CAG
H2a reverse primer: (1383, 1400) CAA CGT GGA
AGC AGC CAT
y forward primer: (1055, 1072) AGT TGG CCA
ACT TCA TCC
y reverse primer: (1349, 1367) TGC CCC AGG
AGA TGT AGT
Primers for a-tubulins were designed using published
sequences [Cowan et al., 1983; Dobner et al.,
Total RNA extraction and rt-PCR were performed
1987;
Villasante
et al., 19861. Primers for y-tubulin were
as previously described [Chen et al., 19941. PCR was
designed
using
the
sequence available from GeneBank
performed in a Perkin-Elmer Cetus DNA Thermal Cycler
(accession
number
M61764).
The P-tubulin isotype clas(Norwalk, CT) using the following profile: 10 s at 94"C,
sification
used
is
the
one
described
by Sullivan [Sullivan,
30 s at 55"C, and 30 s at 72°C. The amplimers used in
19881.
Primers
for
P-tubulin
isotypes
were designed usthis study were synthesized by Operon Technologies
I, class IVa, and
ing
published
sequence
data
for
class
(Alameda, CA).
class
IVb
isotypes
[Lewis
et
al.,
19851
or, in the case of
We designed the following primers for analysis of
I11
isotype
a
consensus
forward
primer
and partial
class
tubulin isotypes (in the case of P-tubulin isotypes, Arasequence
information
generously
provided
by
Kevin
Sulbic numerals refer to the gene, Roman numerals refer to
I,
livan
(Scnpps
Research
Institute,
La
Jolla,
CA).
Class
the tubulin protein isotype class):
111, IVa, and IVb P-tubulin primers were designed to
0 M40 (Class I) forward primer: (-42,
-22), CCA span introns. In the case of the class I1 P-tubulin isotype,
TAC ATA CCT TGA GGC GA
sequence was obtained from the EMBL GeneBank (ac0 M40 reverse primer: (226, 246) GCC AAA AGG
cession number X7 and 9353), and using the peptide
ACCTGAGCGAA
sequence previously reported by Cowan et al. [19861. In
0 P9 (Class 11) forward primer: (1 31, 1150) CGC
separate experiments the presence of tubulin pseuATC TCC GAG CAG TTC AC
dogenes was analyzed by performing PCR directly on the
0 P9 reverse primer: (1301, 1319) TCG CCC TCC
RNA, and by digesting RNA with DNAse (Gene Hunter
TCC TCC TCG A
Kit, Brookline, MA) before cDNA synthesis.
0 P4 (Class 111) forward primer: (1
15) ATG AGG
To allow semi-quantification, 28s ribosomal
GAA ATC GTG
cDNA was used as an endogenous control for PCR since
Dumontet et al.
52
TABLE I. Cell Cycle Distribution of Log and Plateau K562 and
MES-SA Cells*
K562 log phase
K562 plateau phase
MES-SA log phase
MES-SA Dlateau Dhase
24 + 4
88 k 8
18 5
86 k 9
*
70 k 6
9*3
72 2 5
10 -t 3
6 + 2
3 k l
10 k 2
4+ 1
~
*K562 and MES-SA log phase cells were collected 48 hours after
having been seeded at low concentration. Plateau phase cells were
collected 24 hours after having been seeded at maximal cell concentrations achieved by spontaneously growing cells.
the gene for rRNA is expressed at uniformly high levels
and may be used as a normalization factor for total RNA
content of cells. The amplimers used for ribosomal RNA
were the following: rRNA-A (1846-1826) TTA CCA
AAA GTG GCC CAC TA; rRNB-B (1501-1520) GAA
AGA TGG TGA ACT ATG CC. Furthermore, samples
were run at different cDNA concentrations and different
number of PCR cycles to ensure that the reaction was not
at the plateau phase. PCR samples were analyzed by 8%
polyacrylamide gel electrophoresis, stained with ethidium bromide, and analyzed by densitometric reading of
bands on an Alpha Innotech IS-1000 image analyzer
(San Leandro, CA).
RESULTS
Cell Cycle Distribution of Log and Plateau
Phase Cells
Analysis of cell cycle distribution, as determined
by propidium iodide staining and FACS analysis, of log
phase cells showed that, under the conditions of growth
used, both K562 and MES-SA cells were predominantly
in S phase (Table I). Conversely, in the plateau phase, 86
to 88% of the cells were in GO/Gl phase. These results
validate the use of log and plateau conditions of culture
to compare dividing and non-dividing cells.
Total Tubulin Content in Dividing and
Non-Dividing MES-SA and K562 Cells
Total tubulin content per cell was evaluated by double staining of perrneabilized log phase cells with propidium iodide and pan-p tubulin antibody and a secondary FITC-labeled anti-murine antibody. As shown in
Figure 1, total tubulin content per cell increased by approximately 6 0 4 0 % as the cells progressed from G1
through S phase and into G2/M phase. The percent increase was similar in K562 and MES-SA cells.
To determine whether the increase in tubulin content of dividing cells was specific to cells undergoing
mitosis or was due to growth of cell size and increased
protein content during cell division, we performed im-
munoblots comparing the amount of total p-tubulin relative to total cellular protein, in log and plateau phase
cells. Various protein loadings (25, 50, and 100 pg)
were compared, and protein loading was confirmed by
Coomassie staining of duplicate gels. Purified bovine
brain tubulin (5 pg) was used as a control. As shown in
Figure 2 (100 pg loading), the ratio of total p-tubulin to
total protein was similar in dividing and non-dividing
cells. This was found to be the case both in K562 cells
and in MES-SA cells.
Determination of total protein content per cell in log
and plateau phase cells demonstrated higher total protein
content in the log phase cells (443 pg/cell in K562 cells
and 164 pgkell in MES-SA cells) than in plateau phase
cells (160 pg/cell in K562 cells and 112 pgkell in
MES-SA cells). Given the fact that the majority of the
cells in the log and the plateau populations are in S phase
and in GO/Gl phase, respectively, these differences
should be interpreted as reflecting the total protein content
of resting cells and cells preparing for mitosis. The difference in total protein content between resting cells and
cells in M phase is probably even greater.
Expression of Tubulin lsotypes During the
Cell Cycle
a, p, and y-Tubulin isotype transcripts were analyzed by rt-PCR. In these experiments, isotype content
was normalized to 28s ribosomal RNA content. These
experiments were performed on three to six different
batches of cells. Given the caveats of semi-quantitative
PCR, samples were run at different number of cycles and
at different concentrations of cDNA in order to ensure
that the reaction had not reached saturation. Furthermore
to ensure comparability of samples, cDNA concentrations were chosen in order to obtain ribosomal PCR
products which did not differ in amount by more than
10%. Among the sets of primers analyzed, only H2a and
class IVa p-tubulin generated PCR products compatible
with the presence of pseudogenes. In both cases, however, the amount of PCR products produced were low
(less than 5% of the corresponding products obtained
from cDNA), and pretreatment with DNAse did not significantly modify the amount of PCR product obtained
(data not shown).
Tubulin isotype content of K562 and MES-SA cells
are shown in Table I1 and Figure 3 . The isotype profile of
the two cell lines is clearly different, in particular as
concerns the a-tubulin content. Both K562 and MES-SA
cells were found to express two of the four a-tubulin
isotypes. Both cell lines expressed the more abundant
K l a isotype. However, K562 cells were found to express Ha44 and MES-SA cells were found to express
B l a . None of the cell lines expressed H2a, which had
initially been reported to be a testis-specific isotype.
Tubulin Isotypes and the Cell Cycle
53
Phase of the Cell Cycle
Fig. 1. Analysis of total P-tubulin content during the cell cycle by
FACS analysis. K562 and MES-SA cells were collected in log phase
of growth, permeabilized with ethanol, and stained with a pan+
monoclonal antibody and secondary FITC-labeled antibody. Cells
were incubated in 50 Kglml propidium iodide and analyzed by FACS
on a Becton-Dickinson cytofluorometer.
Pan R tubulin
scripts, as evidenced by the number of cycles required
for amplification.
a-Tubulin and y-tubulin transcripts were expressed
at comparable levels in log and plateau cells. Among the
P-tubulin isotypes, class I, class 11, and class I11 transcripts were expressed at constant levels throughout the
cell cycle. Conversely, both of the class IV isotype tranFig. 2. Analysis of total P-tubulin content during the cell cycle by scripts were expressed approximately twofold more in
immunoblotting. Cells in log or plateau phase of growth were lysed,
and 100 Kg of total protein was electrophoresed on a 12% SDS- dividing cells than in non-dividing cells, both in K562
polyacrylamide gel. Total bovine brain tubulin (5 pg) was used as a and in MES-SA cells. In both cell lines, the class IVb
control. Samples were transferred onto nitrocellulose and immuno- isotype product was amplified at a lower number of cyblotting was performed with pan-P-tubulin monoclonal antibody.
cles than the class IVa isotype product.
In separate experiments (data not shown), limited
numbers of cells stained with propidium iodide were
Both cell lines expressed y-tubulin. Both cell lines ex- sorted into GO/G1, S, and G2/M populations, and impressed all classes of P-tubulin isotypes, but class I11 mediately processed for rt-PCR analysis of their tubulin
(considered to be a minor neuronal isotype) was detected isotype transcript levels. Results showed no difference in
in very small amounts in K562 cells. Class I transcripts class I P-tubulin isotype levels but a twofold increase in
appeared to be the most abundant among P-tubulin tran- class IV P-tubulin isotype levels between GO/G1 and S
K562
MES-SA
PC-TUB
54
Dumontet et al.
RIB0
K1 a
Bla
Ha44
H2a
class I
class II
class 111
class IVa
class IVb
Y
W
I-
Fi
m
(0
c
P
0
vl
Q,
0
I
x
7c
I-
(Q
m
m
v)
m
N
N
D
Fig. 3. Tubulin isotype PCR products in dividing and resting K562
and MES-SA cells. Log and plateau phase K562 and MES-SA cells
were analyzed for their tubulin isotype contents by rt-PCR. Samples
were run at various number of cycles using various concentrations of
cDNA. 28s ribosomal RNA was used as an endogenous control. Products were run on a 8% polyacrylamide gel, stained with ethidium
bromide, and quantified by densitometry.
phase, with no difference between S phase and G2/M
phase cells.
Overexpression of class IV P-tubulin was confirmed at the protein level by immunoblotting with a
monoclonal antibody which recognizes the C-terminal
portion common to both class IVa and Class IVb isotypes
(Fig. 4). Consistent results were found at different protein loadings (25 to 100 pg/lane), on at least three different batches of cells. Immunoblotting of cell lysates
with a polyclonal anti-y-tubulin antibody (generously
provided by Tim Steams, Stanford University) confirmed that there was no difference in y-tubulin protein
content in dividing and non-dividing cells (data not
shown).
TABLE 11. Expression Ratios of a,p, and y-Tubulin Isotypes
in K562 and MES-SA Cells*
Polymerization Status of Total Tubulin and Class
IV P-Tubulin
Comparison of polymerized (cytoskeletal) and soluble tubulin was performed by precipitation of polymer-
K562
MES-SA
Kla
Bla
Ha44
H2a
Class I p
Class I1 p
Class I11 p
Class IVa p
Class IVb f3
Y
*The ratios are expressed as log divided by plateau values for each cell
type. Tubulin isotype expression was analyzed by semi-quantitative
PCR, at different concentrations of cDNA and at different number of
PCR cycles. Values were obtained by densitometric reading of ethidium bromide gels and normalized to 28s ribosomal transcript levels.
The results are expressed as the ratios of log/plateau cells. Numbers in
parentheses represent the number of cycles used for amplification.
N.E., isotype not expressed at 44 cycles of PCR.
Tubulin Isotypes and the Cell Cycle
Class IV R tubulin
K562
MES-SA
PC-TUB
Fig. 4. Immunoblotting of K562 and MES-SA total cell lysates with
anti-class IV P tubulin monoclonal antibody. Log and phase cells were
collected, and total lysates were run on a 12% SDS-polyacrylamide
gel. Purified bovine brain microtubule protein was used as a control.
Samples were transferred onto nitrocellulose, and immunoblotting was
performed with anti-class IV P-tubulin monoclonal antibody.
ized tubulin and immunoblotting of both fractions with
pan+ monoclonal antibody and class IV-specific p-tubulin monoclonal antibodies. As shown in Table 111, a
larger fraction of total P-tubulin was found to be polymerized in dividing cells (36-40%) than in resting cells
(27-30%), both in K562 and in MES-SA cells. The polymerized fraction were quite similar in K562 cells,
which grow in suspension, and in MES-SA cells, which
are adherent. The fraction of class IV P-tubulin under
polymerized form in dividing and resting cells was comparable to that of total P-tubulin (Fig. 5).
DISCUSSION
Progression through the cell cycle is dependent
upon the assembly and dynamic behavior of the mitotic
spindle. Constitution of the mitotic spindle allowing
chromosome separation at anaphase is an essential step
of mitosis. A number of mitotic regulatory molecules,
such as cyclin B/p34cdc2, have been shown to bind both
to cytoplasmic and to spindle microtubules [Ookata et
al., 19931. Certain antimitotic drugs, including depolymerizing agents (such as vinca alkaloids) and stabilizing
agents (such as taxanes), are targeted toward the mitotic
spindle and inhibit cell division by modifying microtubular dynamics [Jordan et al., 1993; Wilson and Jordan,
19951.
Considerable data are available regarding the roles
of microtubules in mitosis [McIntosh, 1979; Wordeman
and Mitchison, 19941. However most of these data are
qualitative, and little is known concerning the variations
in total tubulin, specific isotype contents, and polymerization status during the cell cycle. In our experiments,
the total cellular tubulin content was found to increase as
cells progressed through mitosis, a phenomenon attrib-
55
utable to the increase in total protein content as the cells
increase in size before undergoing cytokinesis. However, a larger percentage of total cell tubulin was in the
polymerized form in dividing cells. Microtubules form
an essential component of the cytoskeleton, and these
data suggest that cells preparing for or undergoing mitosis are submitted to specific structural constraints.
Our results indicate that dividing cells have higher
transcript and protein levels of class IV P-tubulin than
non-dividing cells. This was found to be true both in the
leukemic cell line K562, which grows in suspension, and
in the adherent sarcoma cell line MES-SA. These results
suggest that the regulation of class IV P-tubulin during
the cell cycle is not organ specific. A number of investigators have analyzed the distribution of isotypes among
the different microtubular structures and shown that in
most cases all cellular microtubules are copolymers of all
available tubulin isotypes [Lewis et al., 1987; Lopata
and Cleveland, 19871. Immunofluorescence studies by
ourselves (unpublished data) and others have not demonstrated preferential distribution of the class IV isotype
with the mitotic spindle [Lopata and Cleveland, 1987;
Sawada and Cabral, 19891. However, the level of resolution in these experiments has not allowed the quantitative analysis of the different isotypes in individual subsets of spindle microtubules. Sisodia et al. [1990] have
isolated spindle and cytoplasmic microtubules from
CHO cells and reported that the ratio of isotypes is comparable in both fractions.
Altered isotype content may affect the spindle’s
dynamic behavior through a number of ways. To allow
chromosomal positioning and separation, the microtubules composing the spindle must be dynamic. It has
been shown that microtubule turnover is approximately
20-fold faster in mitotic than in interphase cells [Saxton
et al., 19841. Recent data suggest that different P-tubulin
isotypes, in particular class 111, differ in their dynamic
behavior [Panda et al., 19941. Thus a modification in the
ratios of P-tubulin isotypes may play an important role in
the regulation of the spindle microtubule dynamics during mitosis. Another possible mechanism of dynamic
regulation may rely on the interactions between given
isotypes and specific microtubule-associated proteins
(MAPs). P-Tubulin isotypes differ primarily between
their C-terminal region. This region has been shown to
be the binding region for a number of MAPs [Littauer et
al., 1986; Paschal et al., 1989; Serrano et al., 19851.
Certain MAPs such as tau and MAP2 have been shown
to modulate microtubule dynamics as well as flexibility
[Dye et al., 1993; Lee and Rook, 19921. Furthermore,
coordinated regulation of MAP and tubulin genes has
been reported [Oblinger and Kost, 19941. It is possible
that microtubules with different isotype contents may
display various affinities for the binding of MAPs, and
Dumontet et al.
Total R Tubulin
Soluble
Polymerized
ao
I
70
60
50
40
30
20
10
0
K562 log
MES-SA log
K562 plateau
MES-SA plateau
Class IV t3 Tubulin
f
52
70
w2
60
a
.
3
50
W
40
I
80 I
I
0
30
$
8
a
20
10
0
K562 log
K562 plateau
Fig. 5. Analysis of polymerized (cytoskeletal) and soluble tubulin in
MES-SA and K562 cells in log or plateau phase of growth by precipitation of both fractions with pan+ and Class IV-specific P-tubulin
monoclonal antibodies. Fractions were electrophoresed on a 12%
consequently be more or less susceptible to their modulating effects.
One notable aspect of our findings is that the isotypes which are differentially regulated during the cell
cycle are present in relatively small amounts, as suggested by the number of cycles of PCR required to amplify products. Considering the functional multiplicity of
microtubules in cells, as well as their mechanistic contraints during mitosis, it should not be considered surprising that an isotype-based regulation of spindle behavior would depend on modifications of a small but
functionally important class of regulatory isotypes,
rather than on the bulk of structural tubulin isotypes.
Quantification of isotypes in different non-neuronal cell
-
MES-SA log
MES-SA plateau
SDS-polyacrylamide gel, and transferred onto nitrocellulose. Immunoblotting was performed with the appropriate monoclonal antibody,
and quantified by densitometry.
TABLE 111. Percentages of Polymerized Tubulin in K562 and
MES-SA Cells*
K562
Total P-tubulin
Class IV 13-tubulin
MES-SA
Dividing
Resting
Dividing
Resting
40kl
35 k 4
30k3
30 f 6
36kl
39 ? 6
27k4
32 k 3
*Total and polymerized P-tubulin and class IV P-tubulin were evaluated by immunoblotting in dividing and resting K562 and MES-SA
cells. Values shown are the average (2 SD) of the percentages of
polymerizedkotal tubulin.
Tubulin Isotypes and the Cell Cycle
types has shown the class I isotype to be most abundant
[Lopata and Cleveland, 1987; Sisodia et al., 19901, with
the class IV isotype representing 10-30% of all P-tubulin. In our cell lines, PCR data also suggest that class I is
the most abundant, with class IVb representing a smaller
contingent and class IVa being a minor fraction. The fact
that class IV accounts for only a fraction of total tubulin
explains why the total tubulidtotal protein ratio is not
different in dividing and resting cells, in spite of the
overexpression of class IV tubulin protein. Of note is the
fact that the “isotypic profile” of 01 and P-tubulins differs among cells, with some of the isotypes not being
expressed at all in a given cell type. y-Tubulin, reported
to be associated with the centrosome, was found to be
expressed at constant levels throughout the cell cycle, a
finding consistent with the fact that centrosome function
is independent of the cell cycle [Tournier and Bornens,
19941.
It is remarkable that the two isotypes which are
modulated during the cell cycle share the same C terminus and are both considered as belonging to class IV in
the Sullivan classification [Sullivan, 19881. These two
isotypes differ only by ten amino acid residues, spaced
out along the amino acid sequence. This functional redundancy, as well as the strong conservation of these two
isotypes during evolution, suggests that this class plays
an important role in cell physiology. However, although
class IVb has been reported to be present at low levels in
a number of tissues, the class IVa isotype has previously
been reported to be present in neuronal tissues only [Lee
et al., 19841. The fact that class IVa isotype transcripts
have not yet been described in other tissues may be due
to the fact that these transcripts are present in very small
amounts, as evidenced by the number of cycles of PCR
required to display specific products, and may not have
been detected by traditional RNA analysis methods such
as Northern blotting. The coordinate regulation of these
two redundant isotypes sharing the same C-terminal portion suggests a common transcriptional regulatory mechanism. The simultaneous variations in both isotypes may
be interpreted either as a redundant way for the cells to
ensure the presence of sufficient amounts of class IV
P-tubulin protein, or may be due to discrete functional
differences between these two isotypes which we have
yet to identify.
In conclusion, we report that cells undergoing mitosis contain larger amounts of total tubulin, a larger
fraction of polymerized tubulin, and a selectively increased content of class IV P-tubulin isotypes. Our results suggest that these cell cycle-specific phenomena
are not tissue-specific. These data support the hypothesis
of functional specificity of P-tubulin isotypes, as has
been suggested by the strong conservation of these isotypes during evolution.
57
ACKNOWLEDGMENTS
This work was supported by American Cancer Society grant DHP-76, Department of the Army grant
DAMD 17-94-5-4352, NIH grant R01 CA 68217
(B.I.S.), 1’Association pour la Recherche sur le Cancer,
the Ligue Contre le Cancer de la Drdme, and the Philippe
Foundation, Inc. (C.D.). We thank S.A. Lewis and N.
Cowan for providing vectors and helpful advice and information concerning tubulin isotypes.
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