Different roles of two c-tubulin isotypes in the
cytoskeleton of the Antarctic ciliate Euplotes focardii
Remodelling of interaction surfaces may enhance microtubule
nucleation at low temperature
Francesca Marziale
1
, Sandra Pucciarelli
1
, Patrizia Ballarini
1
, Ronald Melki
2
, Alper Uzun
3
,
Valentin A. Ilyin
3
, H. W. Detrich III
3
and Cristina Miceli
1
1 Dipartimento di Biologia Molecolare, Cellulare e Animale, University of Camerino, Italy
2 Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
3 Department of Biology, Northeastern University, Boston, MA, USA
Microtubule assembly in metazoan cells is nucleated
by organizing centers, which include centrioles, basal
bodies, and other structures. Mitotic centrosomes con-
tain a pair of centrioles and associated pericentriolar
material, whereas basal bodies recruit other accessory
structures [1,2]. Both centrioles and basal bodies
require c-tubulin, the ubiquitous third member of the

and (c) in the M loop that forms lateral interactions. Relative to c-T1, the
c-T2 gene is amplified by approximately 18-fold in the macronuclear gen-
ome and is very strongly transcribed. Using confocal immunofluorescence
microscopy, we found that the c-tubulins of E. focardii associate throughout
the cell cycle with basal bodies of the non-motile dorsal cilia and of all of
the cirri of the ventral surface (i.e. adoral membranelles, paraoral mem-
brane, and frontoventral transverse, caudal and marginal cirri). By contrast,
only c-T2 interacts with the centrosomes of the spindle during micronuclear
mitosis. We also established that the c-T1 isotype associates only with basal
bodies. Our results suggest that c-T1 and c-T2 perform different functions
in the organization of the microtubule cytoskeleton of this protist and are
consistent with the hypothesis that c-T1 and c-T2 have evolved sequence-
based structural alterations that facilitate template nucleation of microtu-
bules by the c-tubulin ring complex at cold temperatures.
Abbreviations
qPCR, quantitative PCR; RATE, rapid amplification of telomeric ends; TuRC, tubulin ring complex.
FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5367
other proteins to form two macromolecular structures,
the c-tubulin small complex, which possesses a weak
microtubule nucleating activity [10,11], and the c-tubu-
lin ring complex (TuRC) [12], which nucleates
strongly. c-TuRC resembles a lock washer and is con-
sidered to be the fundamental unit required for micro-
tubule nucleation. Two models have been proposed to
explain microtubule nucleation by c-TuRC: (a) the
‘protofilament’ model, in which the c-tubulin subunits
of c-TuRC associate longitudinally with ab-tubulin
dimers [13], and (b) the ‘template’ model, in which the
c-TuRC ring mimics the end of a microtubule, and
c-tubulin interacts both longitudinally and laterally

c-tubulin to perform efficient microtubule nucleation
at cold temperatures reflects evolved molecular altera-
tions to its interaction surfaces.
Psychrophilic ciliated protozoa are uniquely suited
to an investigation of this issue. As single cells, ciliates
are directly exposed to environmental factors through-
out their life cycle, and modifications of the primary
sequences of many of their proteins are likely to reflect
adaptive mutations that increase the fitness of the
organism at cold temperatures. In ciliates, microtubule
nucleation is promoted mainly by basal bodies, which
are positioned precisely in organized rows in the
somatic cell cortex and in the oral apparatus [8]. The
assembly and maintenance of basal bodies were both
shown to require c-tubulin [7,8].
The ciliate Euplotes focardii, which is endemic to Ant-
arctic coastal seawaters, shows strictly psychrophilic
phenotypes, including optimal survival and multiplica-
tion rates at 4–5 °C [27], the lack of a transcriptional
response of the Hsp70 genes to thermal shock [28], and
modifications in the primary structures of the a- and
b-tubulin [29–31] and of the proteins that form the ribo-
somal stalk [32]. In the present study, we characterized
the two c-tubulin isotypes, c-T1 and c-T2, of E. focardii ,
model their 3D structures, and examined their differen-
tial expression and cellular localization. We suggest that
novel amino acid substitutions located at the plus ends,
near the GTP-binding sites, and within the M loops of
the E. focardii c-tubulins, preserve their microtubule-
nucleating activities at cold temperatures and ⁄ or confer

c-Tubulin isotypes in E. focardii F. Marziale et al.
5368 FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS
The coding sequences of the E. focardii c-T1 and
c-T2 nanochromosomes were interrupted by two
introns located in identical positions (Fig. 1B). The first
intron included nucleotides 50–96 and the second intron
included nucleotides 210–253 in each gene. Excluding
introns and stop codons, the c-T1 and c-T2 coding
regions were each 1383 bp in length and predicted
proteins of 461 amino acids. The nucleotide sequence
identity between c-T1 and c-T2 was 94.6%. Two
in-frame UGA codons, which are known to code for
cysteine in other Euplotes species [33,34], were present at
residue positions 109 and 185 in each of the genes.
Comparative structural modelling of E. focardii
c-tubulins to human c-tubulin
The 3D structures of the E. focardii c-tubulins were
modeled comparatively with respect to human c-tubu-
lin [35]. The predicted structures of c-T1 and c-T2
were remarkably similar to that of the human protein
(Fig. S1).
Structural features of E. focardii c-tubulin
isotypes
Plus ends
The deduced amino acid sequences of the c-T1 and
c-T2 isotypes were aligned with respect to a Euplotes
c-tubulin consensus sequence and mapped onto the
consensus secondary structure of the tubulin mono-
mer [35,36] (Fig. 2). c-T1 and c-T2 were 95.4% iden-
tical in amino acid sequence. The main differences of

Y398F, D400T, N401T, and K403Q (c-T1 ⁄ residue
position ⁄ c-T2). This suite of residue substitutions may
confer unique functions upon each isotype.
Fig. 1. The macronucleus of Euplotes focardii contains two differ-
ent c-tubulin nanochromosomes. (A) Southern blot analysis of the
two c-tubulin genes of E. focardii using the c-T2 gene as probe.
Lane 1, undigested DNA; lane 2, EcoRI- and HindIII-digested DNA.
The sizes (bp) of DNA standards are indicated on the left. The sizes
of the two c-tubulin nanochromosomes (1600 bp) and their diges-
tion products are indicated on the right. (B) Structural features and
EcoRI ⁄ HindIII restriction maps of the E. focardii c-T1 and c-T2 nano-
chromosomes. Coding, noncoding regions, introns, and telomeres
(C
4
A
4
⁄ G
4
T
4
) are indicated in the key.
F. Marziale et al. c-Tubulin isotypes in E. focardii
FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5369
Nucleotide-binding sites
Human c-tubulin binds GTP in a plus-end cleft
enclosed by residues G11, Q12,
C13, Q16, G101,
N102, S140, A142, G143, G144, T145, V171, P173,
N207, F225, I228, and N229 (where the residues
shown underlined form main- and ⁄ or side-chain

MODELLER, version 9.1 (http://www.
salilab.org/modeller/) [64]. Residues that distinguish the E. focardii c-tubulins from the Euplotes consensus are shown in red and annotated
as consensus residue ⁄ position ⁄ c-T1 or c-T2 residue. The GTP molecule is shown in green. The plus and minus ends, the H3 helix, and the
M-loop are indicated.
F. Marziale et al. c-Tubulin isotypes in E. focardii
FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5371
M loops
The ‘extended’ M loop, which we define as encom-
passing the S7-H9 (M) loop, H9, and the H9-S8
loop, and the H3 surfaces of c-tubulin are involved
in lateral contacts [35–37]. Amino acid substitutions
with respect to the Euplotes consensus were found in
the extended M loop in E. focardii c-T1 and c-T2
(Figs 2 and 3). Two hydrophobic-for-hydrophobic
changes occurred near position 280 (F279L, V282I,
consensus ⁄ position ⁄ c-T1 and c-T2) and c-T1 pos-
sessed an alanine at 280 in place of consensus
threonine. The changes in the H9-H9¢ loop were
more dramatic. Both c-T2 and c-T1 contained pro-
line-for-hydroxyl substitutions (T297P and T303P,
respectively).
Tertiary structural differences between E. focardii
c-T1 and c-T2
Figure 4 shows the superimposition of the 3D struc-
tures of c-T1 and c-T2 from the side and the plus end,
respectively. The comparison demonstrates that the
differences between c-T1 and c-T2 (c-T1 ⁄ residue posi-
tion ⁄ c-T2) mapped largely to exposed areas (plus-end
loops and helices, extended M loop) of the polypep-
tides. The valine at S3 position 93 of c-T2 appears to

the longitudinal interactions formed by c-T1 and c-T2.
Finally, c-T2 possesses a glycine at position 72 in place
of consensus ⁄ c-T1 arginine (Fig. 4B¢). This substitution
may ‘open’ the nucleotide-binding site to facilitate
exchange.
We have not attempted to quantify the loop dis-
placements because the T3 and M loops of the 3.0 A
˚
crystal structure of GTP-bound tubulin are disordered
[35]. Hence, we consider the modeled loop displace-
ments of the E. focardii c-tubulins to be provisional
and to require future validation.
Transcription of the E. focardii c-T1 and c-T2
nanochromosomes
To gain insight into the roles of the E. focardii c-tubu-
lin isotypes, we measured the steady-state levels of
macronuclear mRNAs transcribed from the c-T1 and
c-T2 nanochromosomes of starvation-synchronized
cultures by quantitative PCR (qPCR). During starva-
tion, the transcript levels for both isotypes were low
(Fig. 5A). After feeding, the amounts of c-T1 and
c-T2 mRNAs increased, with the latter being two- to
three-fold higher than the former at 18 h. At 36 h
post-feeding, c-T2 mRNA increased 16-fold relative to
its abundance at 18 h (53-fold increase with respect
to t = 0 h), whereas the level of the c-T1 transcript
remained unchanged. Ninety-eight percent of the cells
were undergoing mitosis ⁄ cytokinesis at this time (as
determined by counting of cells using a stereomicro-
scope). By 54 h, c-T2 mRNA returned to a value simi-

scription of Euplotes focardii c-T1 and c-T2
genes. (A) Cell-cycle-dependence of steady-
state c-T1 and c-T2 mRNA levels deter-
mined by qPCR. Values are the mean ± SD
(n = 4). (B) Determination of macronuclear
gene copy-number of c-T1 and c-T2 by
qPCR. Values are the mean ± SD (n = 4).
(C) Sequences of the 5¢- and 3¢ noncoding
regions of c-T1 and c-T2 putative GATA tran-
scription factor-binding motifs are shown in
gray.
F. Marziale et al. c-Tubulin isotypes in E. focardii
FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5373
location. GATA-binding transcription factors are
known to regulate the transcription of some genes in
protists [39]. The 3¢-UTR of c-T2 was three nucleo-
tides shorter than that of c-T1 but, otherwise, these
two sequences were quite similar. We have not yet
investigated the role of message degradation with
respect to the control of c-tubulin transcript abun-
dance. With the latter caveat, we propose that the
quantities of c-T1 and c-T2 mRNAs are regulated, at
least in part, by differential gene amplification and by
the number of GATA-factor promoter motifs.
Distribution of c-tubulins in E. focardii cells
To examine the cellular distribution of c-tubulin, we
used polyclonal anti-(human c-tubulin) serum [40] and
a polyclonal antibody that we prepared against the
most divergent peptide [(390)RIFRRRNAYIDNYK
(403)] of E. focardii c-T1. Figure 6 presents confocal

duplicated basal bodies nucleate new ciliary micro-
tubules of the nascent cirri, as shown by the DMIA
staining in Fig. 6I.
To determine the subcellular localization of c-T1
and c-T2, we attempted to prepare rabbit polyclonal
antibodies specific for the two peptides that clearly dis-
tinguish c-T1 [(390)RIFRRRNAYIDNYK(403)] and
c-T2 [(390)KKLRSNNAFITTYQ(403)]. We obtained
an antibody specific for c-T1. The c-T2 peptide was,
however, not immunogenic. Fig. 6M–O shows that the
anti-c-T1 serum gave staining identical to that
observed with anti-(human c-tubulin), with the excep-
tion that the micronuclear spindle poles were not rec-
ognized. Therefore, we conclude that c-T2, but not c-
T1, participates in the assembly of the mitotic spindle
of E. focardii and that both isotypes are involved in
the nucleation of other microtubule structures.
Finally, we examined the distribution of E. focardii
c-tubulins in total cell extracts and in subfractions
enriched in basal bodies or in micronuclei using anti-
(human c-tubulin) and anti-
c-T1 sera. Figure 7 shows
that the human antibody recognized c-tubulins in all
three samples, whereas the c-T1 antibody gave positive
signals only for the total cell extracts and basal bodies.
These results confirm that c-T2 alone nucleates micro-
tubules in the micronucleus.
Discussion
In the present study, we have shown that the psychro-
philic ciliate E. focardii possesses two c-tubulin genes

Dorsal
Equatorial
area
Newly
formed
basal bodies
cirri
F. Marziale et al. c-Tubulin isotypes in E. focardii
FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5375
M loop, which participates in lateral bonding. The
extensive alterations of sequence elements that form
these surfaces are likely to be adaptations that preserve
c-tubulin function at cold temperatures. Moreover,
c-T1 and c-T2 differed substantially in their sequences
at these locations, consistent with the possibility that
the functions of the single c-tubulin found in most
organisms may be partitioned between the two protis-
tan isotypes. Together, our results suggest strongly
that E. focardii has evolved c-tubulins that are able to
nucleate microtubule structures at low temperature
while individually performing specialized subfunctions.
The E. focardii c-tubulin gene family – regulation
of expression
The two c-tubulin genes of E. focardii appear to be a
feature characteristic of this protistan genus. Two
c-tubulin genes have also been reported for
Euplotes octocarinatus [44] and for E. crassus [34]. In
the former case, the c-tubulin genes produce identical
proteins, whereas, in the latter, they encode two differ-
ent isotypes whose functional differences, if any, are

chemically from each other and from that of the
mesophilic Euplotes consensus is clear. The extended
M loops of c-T1 and c-T2 are more hydrophobic
than the Euplotes consensus and contain proline sub-
stitutions whose role may be to constrain the lateral
contact residues to a conformation that is favorable
for formation of the c-TuRC nucleation complex
from multiple c-tubulin small complexes [12].
Similarly, the lateral surfaces of
a-tubulins from
E. focardii and from two psychrophilic algae of the
genus Chloromonas contain hydrophobic substitutions
with respect to the corresponding a-isotypes of tem-
perate congeners [31,46]. Detrich et al. [27] have
shown that a small number of hydrophobic substitu-
tions in Antarctic fish tubulins appear to be impor-
tant for compensatory adaptation of microtubule
assembly at cold body temperatures; one such
change, F200Y (Antarctic fish residue⁄ position ⁄ meso-
philic residue), which is located at the interface
between the nucleotide-binding and intermediate
domains of b-tubulin, clearly affects microtubule
dynamics when mutated in Schizosaccharomyces
pombe [47]. Thus, increased hydrophobicity of tubu-
lins, both at surface interaction sites and at internal
domain interfaces, emerges as a common theme for
psychrophilic organisms.
Sequence alterations near the nucleotide-binding
pocket of E. focardii c-tubulins are also candidates
for adaptive compensation. Amino acid changes

has been proposed to act as a ‘synergistic’ loop in
nucleotide cleavage [37,48,49].
Functions of the E. focardii c-tubulin isotypes
The results obtained in the present study demonstrate
that one or both of the c-tubulins of E. focardii associ-
ate permanently with basal bodies, consistent with
prior observations that c-tubulin is present in these
microtubule organizing centers in the ciliates E. octoca-
rinatus [50], T. thermophila [8], Tetrahymena pyriformis
[51], and Paramecium tetraurelia [52]. Furthermore, we
show that duplication of the basal bodies of the dorsal
surface during cell division in E. focardii is associated,
perhaps causally, with disassembly of the equatorial
cilia and their microtubules. It is tempting to speculate
that the ciliary tubulins are recycled to form the cyto-
plasmic microtubule bundles [42,43] that guide the
positioning of basal bodies in the nascent daughter
cells.
During cell division, the poles of the micronuclear
mitotic spindle of E. focardii stained with an antibody
prepared against human c-tubulin but not with an
antibody specific for the c-T1 isotype, which recog-
nized only basal bodies. Thus, we conjecture that
E. focardii c-T2, whose mRNA levels peak in mitosis,
is the only isotype required for centrosome function in
the closed orthomitosis of the micronucleus. We did
not detect c-tubulin or microtubules in the macronu-
cleus of E. focardii, in contrast to reports that c-tubu-
lin and microtubules are present in the amitotically
dividing macronucleus of T. thermophila [8,53]. This

How do the unique features of the E. focardii c-tubu-
lins enhance microtubule nucleation by the c-TuRC
complex at low temperature, and what do these changes
imply regarding the mechanism of nucleation? Based on
the evidence presented in the present study, we propose
that the conformational differences and increased
hydrophobicity of the M loops of c-T1 and c-T2 pro-
mote the lateral interactions necessary to form the
c-TuRC ring template at cold temperatures [12,35]
and that the residue substitutions and conformational
changes at the plus-end facilitate longitudinal inter-
action with the a-tubulin subunit located at the
minus-end of the tubulin heterodimer [12] or lateral
interactions with the b-tubulin subunit [61]. These
hypotheses are readily amenable to testing via site-direc-
ted mutagenesis and functional analysis of c-tubulins in
several model systems, including the yeasts Saccharo-
myces cerevisiae and Schizosaccharomyces pombe. Thus,
our comparative analysis of the c-tubulins of psychro-
philic and mesophilic Euplotes species should contribute
to resolving the validity of the template versus pro-
tofilament models of c-TuRC-mediated nucleation of
microtubules.
Experimental procedures
Cell culture and cell cycle synchronization
Cultures of E. focardii strains TN1 and TN15 were used
[27]; they represent type-species material chosen from a
number of wild-type strains isolated from sediment and
seawater samples collected in Antarctica (Terra Nova Bay
F. Marziale et al. c-Tubulin isotypes in E. focardii

4
A
4
[62]. This stereotypic organi-
zation facilitated obtaining the sequences of the C-terminal
coding region and the 5¢- and 3¢-UTRs using the RATE-
PCR technique as described previously [31,32]. We used the
forward and reverse primers (see above) individually in
combination with the telomeric oligonucleotide 5¢-(C
4
A
4
)
4
-
3¢. Amplified products were cloned into the pCR2.1-TOPO
vector of the TOPO TA Cloning
Ò
kit (Invitrogen, San
Diego, CA, USA) following the manufacturer’s recommen-
dations. Colony blotting and double-strand DNA labeling
by the random priming method were performed as
described previously [63]. Clones containing c-tubulin-
recombinant plasmids were sequenced in both strands (ABI
Prism sequence analyzer Model 373A and Big Dye Termi-
nator Methodology; PE Applied Biosystems, Foster City,
CA, USA).
DNA sequence analysis of c-tubulin
nanochromosomes and prediction of the
encoded amino acid sequences

Sciences, Milan, Italy). For cDNA synthesis, the poly[A] +
RNA (4 lg) was treated with 10 U of RNase-free DNaseI
(Bethesda Research Laboratories, Bethesda, MD, USA), in
the presence of 40 U of RiboLock
Ò
(Fermentas, Milan,
Italy) and 4 mm MgCl
2
for 1 h at 37 °C. DNase-treated
RNA was incubated with Moloney murine leukemia virus
reverse transcriptase (Bethesda Research Laboratories) as
recommended by the manufacturer. The resulting cDNA
was then precipitated in ethanol, collected by centrifuga-
tion, resuspended in distilled water, and used as template
for PCR; the program was 30 cycles at 94 °C for 50 s,
48 °C for 1 min, and 72 °C for 1 min. A final incubation at
72 °C for 7 min was added to the last cycle. cDNA was
cloned into pCR2.1-TOPO as described above.
Southern and northern blotting were performed accord-
ing to standard procedures on Hybond-N filters from
Amersham (Milan, Italy). Filters were prehybridized,
hybridized to DNA probes, and washed to remove nonspe-
cifically bound probe according to the manufacturer’s rec-
ommendations. Filters were stripped for reuse by boiling in
distilled water for 15 s.
Estimation of macronuclear gene copy number and
gene transcription
To estimate gene copy number, qPCR was performed on
total macronuclear DNA using the SYBR green DNA-
binding method (TaKaRa Biotech, Dalian, China). The

NYK(403)] and c-T2 [(390)KKLRSNNAFITTYQ(403)]
were synthesized by Sigma Genosys (Milan, Italy) and used
as antigens to obtain rabbit polyclonal anti-peptide sera.
The c-T1 peptide was immunogenic, whereas the c-T2
peptide was not.
Immunofluorescence microscopy
E. focardii cells in logarithmic phase were washed, placed
on a polylysine-coated (0.5 mgÆmL
)1
) coverslip, and per-
meabilized with 0.2% Triton X-100 in PHEM buffer
(60 mm Pipes, 25 mm Hepes, 10 mm EGTA, 2 mm MgCl
2
,
final pH adjusted to 6.9 with NaOH). Cells were fixed with
2% paraformaldehyde in PHEM for approximately 30–
60 min, washed once with NaCl ⁄ P
i
(130 mm NaCl, 2 mm
KCl, 8 mm Na
2
HPO
4
,2mm KH
2
PO
4
, pH 7.2), and then
twice with NaCl ⁄ P
i

(w ⁄ v) Percoll was then added, and the mixture was centri-
fuged for 30 min at 14 500 g in a fixed-angle rotor. The
fraction containing the basal bodies was recovered from the
interface between the Percoll and aqueous phases, diluted
in MT buffer, and centrifuged for 15 min at 14 500 g. The
resulting pellet was washed twice in MT buffer.
Nuclei were prepared by resuspension of E. focardii cell
pellets in two volumes of lysis buffer [10 mm Tris–HCl
(pH 6.8), 0.25 m sucrose, 10 mm MgCl
2
,1mm phen-
ylmethanesulfonyl fluoride, 0.5% NP-40] with gentle stirring
on ice for 2 min; two volumes of washing buffer (0.25 m
sucrose, 10 mm MgCl
2
) were added to stop lysis. The nuclear
suspension was centrifuged at 1000 g for 1 min (4 °C), the
supernatant was transferred to glass tubes, and nuclei were
collected by centrifugation (9000 g, 2 min, 4 °C).
SDS ⁄ PAGE and immunoblotting
Denaturing SDS ⁄ PAGE was performed according to the
method of Laemmli [67]. After electrophoresis, gels were
subjected to immunoblotting as described previously [29].
Blots were incubated either with a rabbit polyclonal anti-
(human c-tubulin) primary serum [40] at a 1 : 1000 dilution
or with a rabbit polyclonal antibody directed against the
c-T1 peptide (see above) at the same dilution. Blots were
washed extensively in NaCl ⁄ Tris ⁄ Tween buffer [5 mm Tris–
HCl (pH 7.5), 0.138 m NaCl, 0.1% Tween-20] and then
incubated with peroxidase-conjugated secondary anti-rabbit

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