articles
δ-Tubulin and ε-tubulin: two new human
centrosomal tubulins reveal new aspects
of centrosome structure and function
Paul Chang* and Tim Stearns*†
*Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA
†e-mail: [email protected]
The centrosome organizes microtubules, which are made up of α -tubulin and β -tubulin, and contains centrosomebound γ -tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that
they are associated with the centrosome. One is a homologue of the Chlamydomonas δ-tubulin Uni3, and the other is
a new tubulin, which we have named ε -tubulin. Localization of δ -tubulin and ε -tubulin to the centrosome is independent
of microtubules, and the patterns of localization are distinct from each other and from that of γ -tubulin. δ -Tubulin is
found in association with the centrioles, whereas ε -tubulin localizes to the pericentriolar material. ε -Tubulin exhibits a
cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated
pair and later associating with both centrosomes. ε -Tubulin thus distinguishes the old centrosome from the new at the
level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the
recruitment of ε -tubulin.
he microtubule cytoskeleton consists of a dynamic, highly
polarized network of microtubule filaments, microtubuleassociated proteins, microtubule motors, and microtubuleorganizing proteins. In animals and fungi, the organizing proteins
are concentrated at a microtubule-organizing centre or centrosome.
Microtubules are complex polymers of α-tubulin/β-tubulin heterodimers, and the polymerization of α-tubulin/β-tubulin into
microtubules is nucleated by the centrosome. Microtubule nucleation requires a third tubulin, γ-tubulin, which does not polymerize
with α-tubulin/β-tubulin, but is instead limited in distribution to
the centrosome and to the cytoplasm1–3. γ-Tubulin is found as part of
a large protein complex4 that contains at least five other proteins5–7
and has the shape of a ring that is of roughly the same diameter as a
microtubule5,8. The properties of the γ-tubulin complex indicate that
it may act directly to form a template for the polymerization of
microtubles.
The centrosome is about 1 µm in diameter, and is traditionally
described as being composed of a pair of centrioles surrounded by
pericentriolar material. γ-Tubulin-containing ring structures are
present in the pericentriolar material9,10, the site of microtubule
nucleation11. The centrioles in the centrosome are equivalent to the
basal bodies at the base of cilia and flagella, both having the exquisite nine-fold symmetry formed by the triplet microtubules that
make up their outer walls. Formation of the centriole/basal-body
triplet microtubules is defective in Chlamydomonas strains with a
mutation in the UNI3 gene, which encodes a fourth member of the
tubulin superfamily, δ-tubulin12. The identification of δ-tubulin
raises several important questions. Is δ-tubulin, like α-, β-, and γtubulin, present in all cells that have microtubule cytoskeletons, or
is it a molecule specialized for function in ciliated cells? The absence
of any recognizable homologue in the complete genome of the yeast
Saccharomyces cerevisiae would argue for the latter possibility, but
yeast may have a reduced set of tubulins for the limited functions
that it carries out. Is δ-tubulin associated with the microtubuleorganizing centre, or might it be a component of functionally distinct microtubules? Finally, are there other members of the tubulin
superfamily that remain to be discovered?
Here we identify two new human tubulins, one a homologue of
the Chlamydomonas δ-tubulin Uni3 (ref. 12) and the other a previously undescribed tubulin, which we name ε-tubulin. Both are conserved in vertebrates, but neither is present in the S. cerevisiae
T
30
genome. Most interesting is that both δ-tubulin and ε-tubulin
localize to the centrosome, with patterns of localization that are distinct both from each other and from all other known centrosomal
proteins, including γ-tubulin. The spatial and temporal information deriving from the localization of these new tubulins raises
interesting questions about centrosome duplication and structure.
Results
Identification of human δ-tubulin and ε-tubulin. We identified the
gene encoding human δ-tubulin in the human genome database on
the basis of its homology to the Chlamydomonas δ-tubulin gene12.
The complete human δ-tubulin nucleotide sequence was assembled
from expressed sequence tags (ESTs) and from a human genomic
sequence from chromosome 17 containing the entire δ-tubulin gene.
We used polymerase chain reaction (PCR) primers to isolate several
full-length δ-tubulin clones from human 293-cell complementary
DNA. These clones were sequenced and found to be identical to the
predicted cDNA sequence from the database sequences. The δ-tubulin cDNA sequence predicts a protein of 453 amino acids, with a relative molecular mass of 51,000 (Mr 51K), that is roughly 40%
identical to Chlamydomonas δ-tubulin. We identified the gene
encoding human ε-tubulin in the human genome database on the
basis of its homology to other tubulins. The complete ε-tubulin gene
is contained within a genomic clone that maps to band 21 of the long
arm of chromosome 6 (6q21). The full-length ε-tubulin cDNA was
cloned by PCR from 293-cell cDNA and sequenced. The ε-tubulin
cDNA sequence predicts a protein of 475 amino-acid residues, with
an Mr value of 53K.
We searched databases for other sequences homologous to
human δ-tubulin and ε-tubulin. The human δ-tubulin is 85% identical to a full-length mouse δ-tubulin reported in GenBank. Nucleotide sequence fragments corresponding to δ-tubulin were also
found among mouse and rat ESTs, and sequences corresponding to
ε-tubulin were found among mouse, rabbit and Xenopus ESTs. In
all cases, the predicted protein fragments were highly similar to the
human proteins, indicating a degree of conservation among mammals similar to that of α-, β-, and γ-tubulin. It was not possible to
identify clear homologues of these tubulins either in the S. cerevisiae
and Caenorhabditis elegans genomes, or in the Drosophila and Arabidopsis EST collections.
© 1999 Macmillan MagazinesNATURE
Ltd
CELL BIOLOGY | VOL 2 | JANUARY 2000 | cellbio.nature.com
articles
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DELTA
ALPHA
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A
A
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P
P
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R
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80
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90
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100
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110
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120
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130
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140
H C
K C
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EPSILON
DELTA
ALPHA
BETA
GAMMA
D
D
T
D
D
C
S
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C
S
L
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L
L
L
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150
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160
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170
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180
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190
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200
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L
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210
H A
S S
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N T
N A
EPSILON
DELTA
ALPHA
BETA
GAMMA
D
D
D
D
D
C
A
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C
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220
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230
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240
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260
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270
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280
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DELTA
ALPHA
BETA
GAMMA
F
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290
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310
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320
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330
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340
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D
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M
P
350
S D
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T Q
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DELTA
ALPHA
BETA
GAMMA
L
D
V
V
V
R
V
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360
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370
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380
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390
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400
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410
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420
L K
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EPSILON
DELTA
ALPHA
BETA
GAMMA
E
G
H
E
R
R
K
K
Q
Q
F
A
F
F
Y
M
W
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L
A
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L
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M
M
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A
A
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430
K K
S K
K R
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A
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Y
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F
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L
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440
V E
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G E
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G
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450
E A
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L
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L
L
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460
Q E
A S
K D
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Y
Y
Y
Y
I
D
C
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Q
D
Q
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Q
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L
L
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Y
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b
470
480
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Q D A T A E E E G E M Y E D D E E E S E A Q G P K
H A A T R P D Y I S W G T Q E Q
H.s. α-tubulin
C.r. α -tubulin
H.s. β-tubulin
C.r. β-tubulin
H.s. δ -tubulin
C.r. δ -tubulin
H.s. γ-tubulin
C.r. γ -tubulin
H.s. ε-tubulin
Figure 1 Comparison of human tubulin sequences. a, Alignment of amino-acid
sequences of human α-tubulin, β-tubulin, γ-tubulin, δ-tubulin and ε-tubulin. Positions at
which three or more amino-acid residues are identical or similar between proteins are
outlined. Identical residues are outlined in grey, and similar residues in black. The
inverted triangle marks the position of an insertion of the sequence
LGTTVKPKSLVTSSSGALKKQ in ε-tubulin; the diamond marks the position of an
insertion of the sequence QMLISNAKMEEGIDRHVWPPL in δ-tubulin; these sequences
do not show any similarity to the other tubulins, and were removed for alignment
purposes. b, ClustalW dendrogram of the relationship between the Homo sapiens
(H.s.) and Chlamydomonas reinhardtii (C.r.) tubulin proteins. The length of the lines
separating sequences indicates the degree of sequence similarity
We aligned the amino-acid sequences of members of the five
known tubulin families in Fig. 1a. δ- and ε-tubulin have the conserved GTP-binding domains of α-, β-, and γ-tubulin, as defined in
the three-dimensional structure of the α-tubulin/β-tubulin
heterodimer13. ε-Tubulin has a single large insertion of 26 aminoacid residues (position 236 in Fig. 1) relative to the other tubulins.
This region is between helices 6 and 7 of the α/β-tubulin structure13;
in α/β-tubulin, this region maps to the surface of tubulin facing the
microtubule lumen13,14. As aligned in Fig. 1, δ-tubulin has a smaller
insertion of 5 residues in the same position, as well as an insertion
of 21 residues at the end of helix 9 (position 316 in Fig. 1). In βtubulin, this region is involved in the lateral interaction between
protofilaments in the microtubule14. δ-Tubulin and ε-tubulin lack
the highly acidic carboxy termini that are characteristic of α-tubu-
lin and β-tubulin.
Tubulin-family members from divergent species typically share
~70% amino-acid-sequence identity. However, there are several
known exceptions for which functional criteria indicate that two
tubulins are homologues despite their sequences being considerably more divergent. For example, the S. cerevisiae γ-tubulin is only
about 40% identical to vertebrate γ-tubulins, yet it has a similar
function and localization, and is associated with homologous
proteins7,15,16, indicating that it is indeed a γ-tubulin rather than a
defining member of a distinct family, as initially suggested 17.
Because the δ-tubulin that we describe here is only ~40% identical
to the original Chlamydomonas Uni3 δ-tubulin12, we compared
members of the known tubulin families in human and
Chlamydomonas using the ClustalW algorithm (Fig. 1b). The length
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7S
18S
Alpha
Gamma
Epsilon
Figure 3 Sucrose-gradient sedimentation of human tubulins. Cytoplasmic
extracts of U2OS cells were fractionated on a 5–20% sucrose gradient and probed
by western blotting for α-tubulin (alpha), γ-tubulin (gamma), or ε-tubulin (epsilon). Note
that the majority of γ-tubulin, which sediments as a large 32S complex, is in the pellet
fraction. S-value markers are aldolase (7S) and ferritin (18S)
Figure 2 Localization of centrin, γ-tubulin, δ-tubulin and ε-tubulin to the
centrosome. a, b, U2OS human osteogenic sarcoma cells were labelled with
antibodies against centrin, γ-tubulin (gamma), δ-tubulin (delta) or ε-tubulin (epsilon).
The right-hand column shows merged pseudocolour images in which δ-tubulin (a) and
ε-tubulin (b) are shown in red, and centrin (a, b, top) and γ-tubulin (a, b, bottom) are
shown in green. Note that ε-tubulin preferentially localizes to one of the centrosome
pairs relative to γ-tubulin and δ-tubulin.
of the lines connecting the tubulins in the resulting dendrogram
represents the amount of sequence divergence. Human δ-tubulin is
most similar to Chlamydomonas δ-tubulin among all tubulins
tested, and human ε-tubulin is distinct from all others, justifying
the nomenclature that we have adopted for these new tubulins.
δ-Tubulin and ε-tubulin are centrosomal proteins. We generated
polyclonal peptide antibodies against specific sequences in δ-tubulin and ε-tubulin. We chose the peptide antigens to minimize crossreactivity with other tubulins, and used them to immunize rats (the
δ-tubulin peptide) or rabbits (two separate ε-tubulin peptides).
Immunofluorescence experiments with these antibodies showed
that both δ-tubulin and ε-tubulin localize to the centrosome, and
not to microtubule polymers. Figure 2 shows the centrosomes of
human osteogenic sarcoma cells (U2OS cells) labelled with antibodies against δ-tubulin, ε-tubulin, γ-tubulin and the centriolar
marker centrin. Centrosomal localization of δ-tubulin and ε-tubulin was also observed in cultured mouse, rat, hamster and frog cells,
and eliminated by competition with soluble peptide antigen. As
well as being found at the centrosome, ε-tubulin was present in the
midbody between dividing cells (data not shown), as has been
reported for γ-tubulin18; no other specific localization was observed.
The localization of δ-tubulin and ε-tubulin to the centrosome was
not affected by treating cells with nocodazole to depolymerize
microtubules (see below), indicating that both δ- and ε-tubulin,
like γ-tubulin, are integral components of the centrosome2.
The specific localization patterns for these two new tubulins are
unique among the known centrosomal proteins. δ-Tubulin par32
tially co-localized with both centrin and γ-tubulin, but was most
prominent between the centrioles within a centrosome, as defined
by the centrin staining, and between the centrosomes themselves, as
defined by the γ-tubulin staining (Fig. 2a). The localization of εtubulin appeared to be dependent on the cell-cycle stage. In cells
with only one centrosome, ε-tubulin roughly co-localized with γtubulin, but in cells with duplicated centrosomes ε-tubulin often
labelled only one of the pair whereas γ-tubulin homogeneously
labelled both (Fig. 2b); this difference is considered in depth below.
To confirm the δ- and ε-tubulin localization patterns, we created
stable cell lines expressing δ-tubulin–GFP (green fluorescent protein) fusion proteins and ε-tubulin–GFP fusion proteins. We studied these cell lines in vivo by examining fluorescence of the GFP
fusion protein, and in fixed cells by immunofluorescence using
anti-GFP antibodies. In both cases the localization was identical to
that observed with anti-δ-tubulin antibodies and anti-ε-tubulin
antibodies (data not shown).
ε-Tubulin is not part of the γ-tubulin ring complex. The similarity
in localization of ε-tubulin and γ-tubulin to the pericentriolar
material in the centrosome led us to test whether the two tubulins
are associated in vivo. More than half of the γ-tubulin in cells is soluble, in the form of the 32S γ-tubulin ring complex4,5. In extracts of
U2OS cells, ε-tubulin comprised ~0.02% of the total protein, a percentage similar to that of γ-tubulin in cells2,5, and substantial
amounts of soluble ε-tubulin were present (data not shown). We
tested for an association of γ-tubulin and ε-tubulin by fractionating
U2OS cell extracts (Fig. 3) or frog egg extracts (data not shown) on
a sucrose gradient, and probing the fractions with antibodies
against α-tubulin, γ-tubulin and ε-tubulin (Fig. 3). Under the conditions used, the 32S γ-tubulin complex pellets at the bottom of the
gradient, well separated from the 6S α/β-tubulin heterodimer. In
both human and frog cytoplasmic samples, ε-tubulin co-sedimented with the α/β-tubulin heterodimer, and not with the γtubulin complex. Although the resolution of this experiment is not
sufficient to determine whether the soluble ε-tubulin is monomeric
or part of a dimer with either another tubulin or other proteins, it
does show that ε-tubulin is not part of the soluble γ-tubulin ring
complex. Biochemical identification of components of the γ-tubulin complex has also failed to reveal evidence of either δ-tubulin or
ε-tubulin in the complex (S. Murphy and T.S., unpublished observations). A small portion of the γ-tubulin was consistently found to
sediment more slowly in the gradient than the rest of the γ-tubulin;
this small fraction probably corresponds to a subcomplex of the
large γ-tubulin complex, as defined in ref. 8, and, because of overlap
of the fractions, we have not ruled out the possibility that it is associated with ε-tubulin.
ε-Tubulin localization differentiates the old and new centrosomes.
The localization of ε-tubulin to the centrosome was cell-cycle
dependent. In cells having a single centrosome, ε-tubulin mostly
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Gamma
Epsilon
G1
Early
S/G2
Late
S/G2
Figure 5 ε-Tubulin localizes preferentially to the old centrosome. NIH 3T3
cells were labelled with antibodies against acetylated-α-tubulin to visualize the
primary cilium (alpha*) and co-stained for either γ-tubulin (gamma) or ε-tubulin
(epsilon). The right-hand column shows merged pseudocolour images, in which εtubulin (top) and γ-tubulin (bottom) are shown in red, and acetylated-α-tubulin (top and
bottom) is shown in green. The primary-cilium-associated centrosome stains more
intensely for ε-tubulin than does the other centrosome of the pair.
M
Figure 4 Cell-cycle localization of ε-tubulin in synchronized cell populations.
U2OS cells were synchronized at mitosis and assayed at 4 h (G1 phase), 6 h (early S/
G2), and 8 h (late S/G2) after plating, as well as at the subsequent mitosis (M). Cells
were fixed and stained with both anti-γ-tubulin antibodies (gamma) and anti-ε-tubulin
antibodies (epsilon). In the bottom panels, only the poles of the spindles are shown.
co-localized with γ-tubulin. However, in cells with duplicated
centrosomes, often only one of the pair of centrosomes was
labelled with anti-ε-tubulin antibodies, whereas both labelled
evenly with anti-γ-tubulin antibodies. The differential localization of ε-tubulin varied from barely detectable on the second centrosome to equal labelling of both centrosomes. This pattern was
similar in human, mouse and frog cell lines, including a primary
human diploid fibroblast line, and was observed with two different polyclonal anti-ε-tubulin antibodies and with an ε-tubulin–
GFP construct. To resolve the pattern of ε-tubulin localization
with respect to the cell cycle, we synchronized U2OS cells by
mitotic shake-off. At different time points after plating, cells were
stained with anti-ε-tubulin and anti-γ-tubulin antibodies (Fig. 4).
The cell-cycle state for each of the time points was determined by
labelling with bromodeoxyuridine (BrdU). In G1 phase, the single
centrosome stained equally for ε-tubulin and γ-tubulin. In early
S/G2 phase, soon after the centrosome has duplicated, one centrosome stained brightly for ε-tubulin and the other stained faintly;
γ-tubulin staining was homogenous. In late S/G2 phase and mitosis, both centrosomes stained brightly for ε-tubulin and γ-tubulin,
but the labelling patterns did not exactly coincide in most cases.
In particular, γ-tubulin was more prominent in the mitotic spindle than ε-tubulin (data not shown), consistent with previous
reports of γ-tubulin localization19.
To determine which of the two centrosomes ε-tubulin is associated with after duplication, we analysed ε-tubulin localization with
respect to the primary cilium. The primary cilium is a non-motile
cilium that grows from one of the centrioles during interphase in
otherwise unciliated animal cells20. Centriole distribution is semiconservative21, and the primary cilium always grows from the older
of the two centrioles in a cell with a single centrosome22. After centrosome duplication, the primary cilium remains associated with
the centrosome containing the older centriole. In mouse 3T3 cells
the primary cilium can be visualized by staining for acetylated-αtubulin, a post-translationally modified form of α-tubulin that is
associated with stable microtubule structures23, thus establishing
the lineage of the centrosomes24. To determine whether ε-tubulin
localizes to the new or the old centrosome, we stained 3T3 cells with
antibodies against acetylated-α-tubulin, ε-tubulin and γ-tubulin
(Fig. 5). In cells with two centrosomes that exhibit differential εtubulin localization and a primary cilium, ε-tubulin localized to the
primary-cilium-associated centrosome. Thus, ε-tubulin remains
associated with the old centrosome throughout the cell cycle, and is
only recruited to the new centrosome at some time after centrosome duplication.
Microtubule regrowth does not depend directly on ε-tubulin concentration. The main function of the centrosome is to nucleate
microtubule polymerization and to anchor the nucleated microtubules in an organized array. To determine whether newly duplicated centrosomes that differ in ε-tubulin content also differ in
nucleation capacity, we depolymerized microtubules in U2OS cells
by treatment with nocodazole, then allowed the microtubules to
regrow after washing out the nocodazole. Cells were fixed at different times after nocodazole washout and stained for α-tubulin and
ε-tubulin. The nocodazole treatment completely depolymerized
the microtubules (Fig. 6a), so any new growth of microtubules from
the centrosomes should be due to de novo microtubule nucleation.
In cells with two centrosomes that exhibited differential labelling of
ε-tubulin, microtubules regrew to the same extent and with the
same kinetics from both centrosomes (Fig. 6b). Both the differential
staining for ε-tubulin and the microtubule-nucleation capacity
were independent of the distance between the duplicated centrosomes. These results indicate that ε-tubulin, unlike γ-tubulin, is not
directly required for nucleation of microtubules, and that the maturation event associated with the recruitment of ε-tubulin is not
associated with any obvious changes in microtubule-organizing
ability of the centrosome.
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Figure 6 Centrosomal nucleation of microtubules is independent of ε-tubulin
localization and centrosome separation. a, Merged pseudocolour images.
U2OS cells were either treated with nocodazole to depolymerize microtubules (+
nocodazole) or mock treated (– nocodazole). Cells were stained for α-tubulin (green)
and ε-tubulin (red). b, U2OS cells treated with nocodazole were washed and allowed
to recover for 10 min. The localization of ε-tubulin (epsilon) is shown on the left, and
merged pseudocolour images showing ε-tubulin in red and α-tubulin in green are on
the right (epsilon + MT (microtubule)). Magnification of the images in b is × 28
relative to those in a.
Discussion
We have described two new human tubulins, δ-tubulin and ε-tubulin. Remarkably, both are localized to the centrosome, bringing the
number of non-microtubule tubulins at the centrosome to three.
However, the localization of δ-tubulin and ε-tubulin within the
centrosome is distinct from that of γ-tubulin and from each other;
thus it seems likely that each of the centrosomal tubulins is performing a different function. α-Tubulin, β-tubulin and γ-tubulin
are conserved in fungi, plants and animals, but neither δ-tubulin
nor ε-tubulin is encoded in the genome of the budding yeast S. cerevisiae, indicating that these proteins might be involved in functions of the centrosome that are distinct from those of the yeast
spindle pole body.
The tubulin superfamily now consists of the five tubulins known
to exist in vertebrates, and the distantly related ftsZ proteins of bacteria. The tubulins have in common regions of homology that are
involved in GTP binding25, although a functional role for GTP
hydrolysis has been described only for β-tubulin. The tubulin proteins have similar relative molecular masses, and share about 25–
30% homology between families. Despite having information
about the structure of the α/β-tubulin heterodimer13, it is difficult
to predict how the differences in primary sequence in δ-tubulin and
ε-tubulin would alter their properties. Although their localization
in cells would seem to indicate that they do not form long polymers,
it is possible that they form small oligomers, either by lateral interaction, as in the protofilament–protofilament interaction within a
microtubule14, or longitudinally to make short filaments. Indeed, it
may be that an ability to form oligomers in a manner controlled by
nucleotides is the fundamental property of all tubulins.
It is interesting that the other major cytoskeletal polymer, actin,
also has a family of proteins related to the major subunit of the
34
polymer26. The actin-related Arp2 and Arp3 proteins are involved in
actin-filament nucleation27, a function similar to that of γ-tubulin
in the polymerization of α/β-tubulin heterodimers. The other Arps,
where studied, seem to have functions that are less involved with
the control of the actin cytoskeleton. In contrast, the localization of
all known tubulins to parts of the microtubule cytoskeleton indicates that they may all be involved in microtubule function.
The first δ-tubulin described, Uni3 from Chlamydomonas, was
identified from a mutant with defects in flagellar biogenesis and in
the structure of the triplet microtubules of the basal body/
centriole12. The localization of human δ-tubulin to the region
around the centrioles points to a potentially similar role for this
protein in animal cells; however, the prominent intercentriolar
localization of δ-tubulin suggests other possible functions as well.
For example, Paintrand et al.28 observed fibres linking the two centrioles within the centrosome, and in vitro studies of centrosome
duplication indicate that separation of the centrioles, and thus dissolution of the fibres, may be an early regulated step in the process29.
The unusual localization pattern of δ-tubulin resembles in several
respects that observed for Skp1, a component of the SCF ubiquitin
ligase, which is required for centriole separation30.
ε-Tubulin has not been described previously, and the only clue
as to its function comes from the cell-cycle-specific localization that
we have observed. Given that the single centrosome of G1 cells
begins the cycle having ε-tubulin, the simplest interpretation of the
differential localization in S and early G2 is that the pericentriolar
material of one centrosome is different from that of the other after
duplication. The newer centrosome, defined by the age of the
parental centriole within the centrosome, acquires ε-tubulin only
after some maturation process. This maturation is independent of
the acquisition of nucleating capacity by the new centrosome, but
might reflect some other function that varies with the cell cycle. The
differential localization of ε-tubulin to old and new centrosomes is
particularly interesting in light of the findings of Lange and Gull24,
who identified cenexin as a protein that associates only with the
older centriole within a centrosome. Centriole distribution is semiconservative21 and, after centriole separation, the newer centriole of
the original pair does not acquire cenexin until the G2/M transition,
roughly at the same time that the new centrosome has acquired a
full complement of ε-tubulin. The ε-tubulin results also indicate
that duplication of the centrosome does not involve merely partitioning of the existing pericentriolar material around the two pairs
of centrioles resulting from duplication, but rather that the centrosome containing the newer centriole also has ‘new’ pericentriolar
material. The functional consequences of the difference between
the duplicated centrosomes remain to be determined, but an attractive possibility is that it might be involved in the regulation of a cellcycle-specific function, such as centrosome duplication, or cellcycle-specific recruitment of other molecules to the centrosome31.h
Methods
Molecular biology.
PCR primers for δ-tubulin were designed from sequence upstream of putative start, and downstream of
putative stop, sequences derived from the δ-tubulin genomic sequence (primers 5′CGAAGCTTGAGGTAATACACCTAGTT and 5′-CGGTCGACTTAGTCCAGAAATGCCTT).
PCR primers for ε-tubulin were designed from sequences upstream of putative start, and
downstream of putative stop, sequences derived from ε-tubulin ESTs (primers 5′CGAAGCTTTAGCAAGCTCCCGGAGCC and 5′-CGGTCGACCACATTGTATTACCAAC).
Human 293-cell total RNA was obtained through standard cell lysis and RNA-isolation techniques32.
The RNA and primers were used in a one-step reverse transcription with PCR (RT-PCR) system (Gibco)
to isolate several full-length ε-tubulin and δ-tubulin cDNA clones. Several clones were sequenced and
found to be identical to the sequence predicted from the contigs assembled from the EST and human
genomic DNA databases.
Full-length δ-tubulin and ε-tubulin cDNAs were subcloned into pEGFP-N1 GFP (Clontech), and
pET15b (Stratagene) bacterial expression vectors. The δ-tubulin–GFP and ε-tubulin–GFP constructs
were used to transfect U2OS cells using Lipofectamine Plus (Gibco) and stable cell lines were selected
with G418. Full-length δ-tubulin and ε-tubulin were expressed in Escherichia coli BL21DE3 (pLysS) cells
from the pET15b constructs; the resulting insoluble protein was purified as inclusion bodies33.
Antibodies and immunofluorescence.
The following peptides were synthesized (Research Genetics) and either injected directly (multiple
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antigenic peptides (MAPs)) or first conjugated to KLH. Immunogens were injected into rabbits
(ε-tubulin peptides) or rats (δ-tubulin peptide) to generate polyclonal antibodies, as follows: ε-1 (aminoacid sequence CNVQISDLRRNIERLKP); ε-2 (DMEEGVVNEILQGPLR) MAP; δ-1
(SLKMNQIIWPYGTGEV) MAP. Anti-ε-tubulin serum was affinity-purified against bacterially
expressed protein that had been transferred to nitrocellulose33.
For immunofluorescence, all cell types were fixed in –20 °C methanol for 5 min and stained with the
following antibodies: anti-δ-tubulin, anti-ε-tubulin, anti-γ-tubulin monoclonal antibody GTU-88
(Sigma), anti-α-tubulin monoclonal antibody DM1α (ref. 34), anti-acetylated-α-tubulin monoclonal
antibody 6-11b-1 (ref. 23), or anti-centrin monoclonal antibody 20H5 (ref. 35). Cell lines expressing δtubulin–GFP and ε-tubulin–GFP were fixed in 4% paraformaldehyde for 20 min, permeabilized in celllysis buffer (HBS + 0.5% Triton X-100) for 10 min, and stained with polyclonal anti-GFP antibody (a gift
from J. Kahana). All secondary antibodies were from Molecular Probes. Samples were analysed on a Zeiss
Axioskop microscope equipped with a cooled charge-coupled-device (CCD) camera (Princeton
Instruments) controlled by OpenLab software (Improvision).
Cell-culture experiments.
U2OS cells were synchronized by growing to three-quarters confluency followed by shaking-off of
mitotic cells36; these were fixed and processed for immunofluorescence at 2-h intervals. Two pre-shakes
were performed to improve synchronization in subsequent samples.
Microtubule regrowth was assayed by first depolymerizing microtubules in U2OS cells with 10 µg ml–1
nocodazole (US Biological) in growth medium for 2 h at 37 °C. Cells were washed with PBS and allowed to
recover in normal media at 37 °C for 10 min, then processed for immunofluorescence.
U2OS cells were treated with 5-bromo-2′-deoxyuridine (BrdU) for 30 min at 37 °C and processed for
BrdU detection using the BrdU detection and labelling kit II (Boehringer Mannheim). BrdU-treated cells
were then stained with anti-ε-tubulin antibody, GTU-88 or 20H5 (see above).
Sucrose sedimentation gradients.
U2OS cells were lysed in HBS (50 mM HEPES, pH 7.4, 15 mM NaCl, 1 mM EGTA) plus 0.5% Triton X100 and 1 mM dithiothreitol on ice for 10 min, and cleared by centrifugation in an Eppendorf microfuge
for 5 min at 12,000g at 4 °C. Extracts were layered over a 5–20% sucrose gradient in HBS and centrifuged
at 55,000 r.p.m. in a TLS-55 rotor (Beckman) for 6 h at 4 °C. Fractions were blotted for α-tubulin, γtubulin and ε-tubulin. Aldolase (7S) and ferritin (18S) size markers were run at the same time in a
separate, identical gradient.
Accession numbers.
Human δ-tubulin cDNA sequence, GenBank accession number AF201333; human ε-tubulin cDNA
sequence, GenBank accession number AF201334; human genomic clone containing the δ-tubulin gene,
GenBank accession number AC004686; human genomic clone containing the ε-tubulin gene, GenBank
accession number AC004686; mouse δ-tubulin cDNA sequence, GenBank accession number AC004686;
human α-tubulin amino-acid sequence used for Fig. 1, Protein Databank accession number AAA91576;
human β-tubulin amino-acid sequence used for Fig. 1, Protein Databank accession number CAA56071;
human γ-tubulin amino-acid sequence used for Fig. 1, Protein Databank accession number P23258.
RECEIVED 14 OCTOBER 1999; REVISED 24 NOVEMBER 1999; ACCEPTED 24 NOVEMBER 1999;
PUBLISHED 3 DECEMBER 1999.
1. Oakley, B. R., Oakley, C. E., Yoon, Y. & Jung, M. K. Gamma-tubulin is a component of the spindle
pole body that is essential for microtubule function in Aspergillus nidulans. Cell 61, 1289–1301
(1990).
2. Stearns, T., Evans, L. & Kirschner, M. Gamma-tubulin is a highly conserved component of the
centrosome. Cell 65, 825–836 (1991).
3. Zheng, Y., Jung, M. K. & Oakley, B. R. Gamma-tubulin is present in Drosophila melanogaster and
Homo sapiens and is associated with the centrosome. Cell 65, 817–823 (1991).
4. Stearns, T. & Kirschner, M. In vitro reconstitution of centrosome assembly and function: the central
role of gamma-tubulin. Cell 76, 623–637 (1994).
5. Zheng, Y., Wong, M. L., Alberts, B. & Mitchison, T. Nucleation of microtubule assembly by a gammatubulin-containing ring complex. Nature 378, 578–583 (1995).
6. Detraves, C. et al. Protein complexes containing gamma-tubulin are present in mammalian brain
microtubule protein preparations. Cell Motil. Cytoskeleton 36, 179–189 (1997).
7. Murphy, S. M., Urbani, L. & Stearns, T. The mammalian gamma-tubulin complex contains
homologues of the yeast spindle pole body components spc97p and spc98p. J. Cell Biol. 141, 663–674
(1998).
8. Oegema, K. et al. Characterization of two related Drosophila gamma-tubulin complexes that differ in
their ability to nucleate microtubules. J. Cell Biol. 144, 721–733 (1999).
9. Moritz, M., Braunfeld, M. B., Sedat, J. W., Alberts, B. & Agard, D. A. Microtubule nucleation by
gamma-tubulin-containing rings in the centrosome. Nature 378, 638–640 (1995).
10. Vogel, J. M., Stearns, T., Rieder, C. L. & Palazzo, R. E. Centrosomes isolated from Spisula solidissima
oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin. J Cell Biol 137, 193–
202 (1997).
11. Gould, R. R. & Borisy, G. G. The pericentriolar material in Chinese hamster ovary cells nucleates
microtubule formation. J. Cell Biol. 73, 601–615 (1977).
12. Dutcher, S. K. & Trabuco, E. C. The UNI3 gene is required for assembly of basal bodies of
Chlamydomonas and encodes delta-tubulin, a new member of the tubulin superfamily. Mol. Biol.
Cell 9, 1293–1308 (1998).
13. Nogales, E., Wolf, S. G. & Downing, K. H. Structure of the alpha beta tubulin dimer by electron
crystallography. Nature 391, 199–203 (1998).
14. Nogales, E., Whittaker, M., Milligan, R. A. & Downing, K. H. High-resolution model of the
microtubule. Cell 96, 79–88 (1999).
15. Knop, M., Pereira, G., Geissler, S., Grein, K. & Schiebel, E. The spindle pole body component Spc97p
interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule
organization and spindle pole body duplication. EMBO J. 16, 1550–1564 (1997).
16. Geissler, S. et al. The spindle pole body component Spc98p interacts with the gamma-tubulin
like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 15,
3899–3911 (1996).
17. Burns, R. G. Identification of two new members of the tubulin family. Cell Motil. Cytoskeleton 31,
255–258 (1995).
18. Julian, M. et al. Gamma-tubulin participates in the formation of the midbody during cytokinesis in
mammalian cells. J. Cell Sci. 105, 145–156 (1993).
19. Lajoie-Mazenc, I. et al. Recruitment of antigenic gamma-tubulin during mitosis in animal cells:
presence of gamma-tubulin in the mitotic spindle. J. Cell Sci. 107, 2825–2837 (1994).
20. Albrecht-Buehler, G. & Bushnell, A. The ultrastructure of primary cilia in quiescent 3T3 cells. Exp.
Cell Res. 126, 427–437 (1980).
21. Kochanski, R. S. & Borisy, G. G. Mode of centriole duplication and distribution. J. Cell Biol. 110,
1599–1605 (1990).
22. Rieder, C. L. & Borisy, G. G. The centrosome cycle in PtK2 cells: asymmetric distribution and
structural changes in the pericentriolar materiel. Biol. Cell 44, 117–132 (1982).
23. Piperno, G. & Fuller, M. T. Monoclonal antibodies specific for an acetylated form of alpha-tubulin
recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085–2094
(1985).
24. Lange, B. M. & Gull, K. A molecular marker for centriole maturation in the mammalian cell cycle. J.
Cell Biol. 130, 919–927 (1995).
25. Nogales, E., Downing, K. H., Amos, L. A. & Lowe, J. Tubulin and FtsZ form a distinct family of
GTPases. Nature Struct. Biol. 5, 451–458 (1998).
26. Frankel, S. & Mooseker, M. S. The actin-related proteins. Curr. Opin. Cell Biol. 8, 30–37
(1996).
27. Welch, M. D., Iwamatsu, A. & Mitchison, T. J. Actin polymerization is induced by Arp2/3 protein
complex at the surface of Listeria monocytogenes. Nature 385, 265–269 (1997).
28. Paintrand, M., Moudjou, M., Delacroix, H. & Bornens, M. Centrosome organization and centriole
architecture: their sensitivity to divalent cations. J. Struct. Biol. 108, 107–128 (1992).
29. Lacey, K. R., Jackson, P. K. & Stearns, T. Cyclin-dependent kinase control of centrosome duplication.
Proc. Natl Acad. Sci. USA 96, 2817–2822 (1999).
30. Freed, E. et al. The SKP1 and CUL1 ubiquitin ligase components localize to the centrosome and
regulate the centrosome duplication cycle. Genes Dev. 13, 2242–2257 (1999).
31. Bobinnec, Y. et al. Centriole disassembly in vivo and its effect on centrosome structure and function
in vertebrate cells. J. Cell Biol. 143, 1575–1589 (1998).
32. Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal. Biochem. 162, 156–159 (1987).
33. Harlow, E. & Lane, D. Antibodies: a Laboratory Manual (Cold Spring Harb. Lab. Press, Cold Spring
Harbor, NY, 1988).
34. Blose, S. H., Meltzer, D. I. & Feramisco, J. R. 10-nm filaments are induced to collapse in living cells
microinjected with monoclonal and polyclonal antibodies against tubulin. J. Cell Biol. 98, 847–858
(1984).
35. Sanders, M. A. & Salisbury, J. L. Centrin plays an essential role in microtubule severing during
flagellar excision in Chlamydomonas reinhardtii. J. Cell Biol. 124, 795–805 (1994).
36. Mariani, B. D., Slate, D. L. & Schimke, R. T. S phase-specific synthesis of dihydrofolate reductase in
Chinese hamster ovary cells. Proc. Natl Acad. Sci. USA 78, 4985–4989 (1981).
ACKNOWLEDGEMENTS
We thank A. Sidow for advice on sequence comparisons, and S. Murphy, K. Lacey and J.C. Zabala for
comments on the manuscript. This work was supported by grants to T.S. from the NIH (GM52022) and
from the Searle Scholars Foundation.
Correspondence and requests for materials should be addressed to T.S.
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