Download T. brucei basal body component - Journal of Cell Science

4687
Journal of Cell Science 112, 4687-4694 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0773
Characterization of a coiled coil protein present in the basal body of
Trypanosoma brucei
Vincent Dilbeck1, Magali Berberof1, Anne Van Cauwenberge2, Henri Alexandre2 and Etienne Pays1,*
1Laboratory
2Laboratory
of Molecular Parasitology, University of Brussels, 67 rue des Chevaux, B-1640 Rhode St Genèse, Belgium
of Biology and Embryology, University of Mons-Hainaut, 6 Avenue du champ de Mars, B-7000 Mons, Belgium
*Author for correspondence (e-mail: [email protected])
Accepted 2 October; published on WWW 30 November 1999
SUMMARY
TBBC (for Trypanosoma brucei basal body component) is a
unique gene transcribed in a 4.8 kb mRNA encoding a 1,410
amino acid protein that consists almost entirely of a coiled
coil structure. This protein appeared to localize in the basal
body, with an accessory presence at the posterior end of the
cell, the nucleus and over the flagellum. Since the two other
known components of the trypanosome basal body are γtubulin and an uncharacterized component termed BBA4
we performed double immunofluorescence experiments
with anti-TBBC and either anti-BBA4 or anti-γ-tubulin
antibodies. These three components did not colocalize but
were very closely associated, BBA4 being the most proximal
to the kinetoplast DNA. Anti-TBBC antibodies detected a
170 kDa protein in western blots of total HeLa cell extracts.
Moreover, these antibodies stained the centriole of HeLa
and COS cells as well as the centriole of mouse spermatozoa,
indicating that a TBBC-like centriolar component has been
conserved during the evolution of eukaryotes.
INTRODUCTION
the basal body seems to play at least three different roles.
During cell division this structure directs the assembly of the
new axoneme and is involved in mitochondrial DNA
(kinetoplast) segregation (Robinson and Gull, 1991). In
addition, it is believed to play a role in the organisation of four
particular microtubules from the corset which unlike other
microtubules arise from the basal body region and probably
show inverted polarity (Khol and Gull, 1998). These
microtubules are a part of the flagellar attachment zone (FAZ)
and are associated with the smooth endoplasmic reticulum
(Vickerman, 1969; Sherwin and Gull, 1989).
Here we describe the serendipitous discovery of a new basal
body component. In an attempt to identify the gene for a T. brucei
immunosuppressive factor, we cloned a gene encoding a 212 kDa
cytoskeletal protein which is expressed in the three major
developmental stages of the parasite. Immunofluorescence
microscopy showed that this protein is mainly located in the basal
body and also in the posterior end of the cell, the flagellum and
the nucleus. Anti-TBBC antibodies detected a TBBC-like
component in the centrosome of mammalian cells such as HeLa
cells and mouse spermatozoa.
Trypanosoma brucei is a protozoan flagellate responsible for
several severe diseases in various mammals, including the
nagana disease in cattle and human sleeping sickness. This
parasite is transmitted by the tsetse fly, where it develops in the
midgut as the so called procyclic form which proliferates rapidly.
Once the trypanosomes have reached the fly salivary glands they
transform into quiescent metacyclic forms which are preadapted
to the bloodstream environment. After injection into the
mammalian host upon biting by infected flies, trypanosomes
proliferate as bloodstream slender forms until they stop dividing
and differentiate into stumpy forms which are preadapted to the
fly (Vickerman, 1985; Vickerman et al., 1988).
The cytoskeleton of T. brucei is composed essentially of a
microtubule cage which is responsible for maintaining the
shape and the form of the cell, and a flagellum used for motility
and attachment to the membranes of host cells (Hemphill et al.,
1991; Kohl and Gull, 1998). The flagellum is composed of the
classical axoneme together with a structure identified in
Euglenoids, Dinoflagellates and Kinetoplastids termed the
paraflagellar rod (Schlaeppi et al., 1989; Deflorin et al., 1994;
Bastin et al., 1996), and the basal body. The basal body is a
centriole-like structure located at the base of the flagellum. The
role of centrioles and the pericentriolar material as microtubule
organizing centers (MTOC) has been progressively clarified
(Marshall and Rosenbaum, 1999) but the MTOC activity of the
basal body is still poorly understood. Like the centrioles, the
basal body is complexed with accessory structures. In T. brucei,
Key words: Trypanosoma brucei, Basal body, Coiled coil protein
MATERIALS AND METHODS
Trypanosomes
The pleomorphic AnTat 1.1E clone was used for the characterization
of the T. brucei bloodstream form. Procyclic forms were derived from
this clone and were grown in SDM79 as described by Brun and
Schoënenberger (1979).
4688 V. Dilbeck and others
Isolation of TBBC
Different cDNA clones originating from the same gene subsequently
termed TBBC were selected following the screening of a λgt11
expression library with polyclonal antibodies raised against an
immunosuppressive fraction of T. brucei. All clones appeared to
contain an incomplete sequence of this gene, lacking the 5′ extremity.
PCR probes from the 5′-region, obtained by inverse PCR (Huang,
1997) with the PWO polymerase (Boerhinger) from the 5′-region
allowed the cloning of genomic fragments encompassing the entire
open reading frame from a library of Sau3A partially digested AnTat
1.3A DNA cloned into the lambdaGEM-12 vector.
DNA sequencing
cDNA and genomic fragments were subcloned in the pUC 18 vector
(Pharmacia) and the DNA sequence was determined on both strands
using the Thermo Sequenase radiolabeled terminator cycle
sequencing kit (Amersham Pharmacia Biotech). Sequence alignment
and pairwise evaluations of homology were performed with the Blast
2.0 software (http//www.ncbi.nlm.nih.gov/BLAST/).
DNA and RNA analysis
Southern and northern blot hybridizations were performed as
previously described (Pays, 1980). The northern blot was normalised
with a probe against the 18S rRNA.
Western immunoblotting
50 µg of proteins from each cell lysate supernatant were used for
western immunoblotting. In summary, lysates were heated for 5
minutes at 95°C in SDS-β mercaptoethanol-dithiothreitol (DTT)containing sample buffer, separated in a 7% polyacrylamide gel and
transferred onto a nitrocellulose membrane. TBBC was detected with
rabbit polyclonal anti-TBBC antibody (1/2000 dilution). Antibodies
were detected with alkaline phosophatase-conjugated antibody
(Promega). Anti-actin antibodies were used as probes for the control
of sample loading.
Expression of TBBC fusion protein and antibody
production
A 2.3 kb BamHI-EcoRI PCR fragment of the TBBC gene was cloned
in frame in the E. coli expression vector pGEX-1λT (Pharmacia) to
generate a fusion protein containing a GST (glutathione S transferase)
domain linked to amino acids 457 to 1215 of TBBC.
This fusion protein was purified by preparative
electrophoresis and injected into rabbits to generate
specific antisera. The same fusion protein was also
injected into the footpad of a mouse to generate
monoclonal antibodies.
Affinity selection of the polyclonal
antibodies
Nitrocellulose membranes saturated with the TBBC
fusion protein were incubated for 1 hour at room
temperature with 5 ml of the TBBC polyclonal
Fig. 1. Organisation and expression of the TBBC
gene. The map at the bottom shows the genomic
organisation of TBBC, with the open reading frame
indicated by the boxed area and the probe employed
for hybridization experiments also indicated below
this region. M: MluI; X: XbaI; H: HindIII; D: DraIII.
(A) Southern blot analysis of T. brucei genomic
DNA (1 µg/lane). (B) Northern blot analysis of
T. brucei polyA+ mRNA (10 µg/lane). P, procyclic
form; SL, bloodstream slender form; ST,
bloodstream stumpy form. (C) Western blot analysis
of T. brucei total protein extracts (50 µg/lane).
antibody. After thorough washing of the filters, the antibody was
eluted with 3 ml of 0.2 M glycine/1 M EGTA (pH 2.5) for 10
minutes at room temperature. After neutralization with 1 M TrisHCl (pH 8.2), bovine serum albumin was added to a final
concentration of 100 µg/ml, and the final solution was dialysed
against two changes of PBS (0.14 M NaCl, 2.5 mM KCl, 8 mM
Na2HPO4, 1 mM KH2PO4) at 4°C.
Immunofluorescence of T. brucei cytoskeletons
T. brucei cells were harvested by centrifugation. The pellet was
resuspended in PEME (2 mM EGTA, 1 mM MgSO4, 0.1 mM EDTA,
0.1 M Pipes, pH 6.9) and settled onto poly-L-lysine coated slides. The
slides were incubated with 1% NP40 in PEME on ice for 5 minutes.
The isolated cytoskeletons were then fixed in 100% methanol
for at least 10 minutes at −20°C. Further processing for
immunofluorescence was done essentially as described by Sherwin et
al. (1987) using a 1/800 dilution of anti-TBBC antibodies. The mouse
monoclonal antibody against the TBBC fusion protein was not diluted
and was revealed with an anti-mouse fluorescein isothiocyanate
(FITC)-conjugate (dilution 1/100) raised in sheep (Amersham Life
Science).
Preparation of flagella
Isolated cytoskeletons were pelleted, then resuspended in 1% NP40,
1 M NaCl in PEME and incubated on ice for 20 minutes. This
preparation was centrifugated and resuspended in PEME alone.
Immunofluorescence of mammalian cells
HeLa cells were cultured overnight on sterile glass coverslips,
washed in PBS and fixed in methanol at −20°C. The rabbit
polyclonal antiserum against the TBBC fusion protein was diluted
1/800 in PBS and revealed with an anti-rabbit fluorescein
isothiocyanate (FITC)-conjugate (dilution 1/100) raised in donkey
(Amersham Life Science). The γ-tubulin monoclonal antibody was
diluted 1/500 (Sigma) and visualised with an anti-mouse Texas redconjugated sheep antibody (Amersham Life Science). DNA was
visualised using the DNA intercalating dye 4,6-diamindino2–phenylindole (DAPI). The slides were mounted in Vectashield
mounting medium (Vector)
Uncapacited caudal epididymal spermatozoa were collected from
10-week-old male NMRI mouse and incubated for 2 hours at 37°C in
T. brucei basal body component 4689
TTTTCCGACTTCAGCCCCTGTCACAGCGTGTAACTACGGGCGTTGCATATCCTTTCCACTTGTCTCTTCATTATGTTACGTTATGTTTCTTCTTTCTTTT
1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100
CCCTCCCTTTTGATGTGTTGATATGTGTTCGTTCAGGATCGTGCCCTCCTCTAGGCGGCACACACACACAAAAGAAGGAAGAAAAACAAAGGGACACCAC
101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200
ACCGCGTCGTTACCTGTAGGTTTTGATTTGTTGATTTTTTCCCCCTGATCTGTAGCTTTGTTTATTAACTGTGAGCCCACCGCGTCGCCGTGAAGGGCTC
201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300
CCTCTCCGTGGACTGGCGTGAACCTCTTCCCTTCCGTTCGTAAACGGTATGGCACAACTTGACCAGTTGCTTGAGGAGTATCTTCGCGTTATTAACGCGA
301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400
M A Q L D Q L L E E Y L R V I N A K AGGATGGCGAAATCCACGAACTCATCCGCGTGCTTGAGGAGCTACGGTGTGAAGGGAAGAAGAAGGATGAGGCTCACAAATCACATGCCCAAAAGATTAG
401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500
D G E I H E L I R V L E E L R C E G K K K D E A H K S H A Q K I S CAACGAGTTAGATAAAGTTATGAGGAAGGCGAAGAAGTTGGAACAAAAGAACCGTACATGCGAAGATCAGATAGATAAACTTGTAAAGGAGAATGAAGCT
501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600
N E L D K V M R K A K K L E Q K N R T C E D Q I D K L V K E N E A
TTGAGTATAGACTTTCAAAAAGTGCAACACAGTTTAATAGATGCCGAAAAGATCATAGAGGAAAAGAAGAGAGCGCATGAGGCCGCGCTTGAGGAACTCC
601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700
L S I D F Q K V Q H S L I D A E K I I E E K K R A H E A A L E E L Q AGCAGACTTATAACGGTATGGTCTCCAAATTTTCGACGGAGCGGGAAGAGTTGAAAGCCCATCAATCGGAACTTCAGAAAGAGCTTGAGGAAGCTCTGAA
701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800
Q T Y N G M V S K F S T E R E E L K A H Q S E L Q K E L E E A L K ATCTGTGGATGAATGTAGGCTGAAGGAGGAAGAAATTCGAAAACTGGAAGTGAGGATTGCCGCGTTAACGCAGTCAGTGGAAGATGCACGGGGGGAAATG
801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900
S V D E C R L K E E E I R K L E V R I A A L T Q S V E D A R G E M
CAGTCGATGGTTCCTGCGGAGCGCGTTAAGCAAATCGTAAGTGAACATGAAGAGGAGTTGCGCACAGTAAGGAAAGCCTGTGCCGAAGAGTTTGACGAAG
901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000
Q S M V P A E R V K Q I V S E H E E E L R T V R K A C A E E F D E V TTTCCGCGCAGTTATCTGATGCTCAGAAATCGGGACGCAAAATGAAAGAAAAACTCAAAGAACTGAAAGAATCATATGGTCAGTTGAAGGACAAGCTGGA
1001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1100
S A Q L S D A Q K S G R K M K E K L K E L K E S Y G Q L K D K L D CGAAACTACTTGCGAGCTGGAAGAAGTTCGCAAACTTCGTCAGAAGGAGCAAGAAACTCACAACGAGGTGCGATCCAGGCAGCAGGAAGAAATTGAGCAG
1101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1200
E T T C E L E E V R K L R Q K E Q E T H N E V R S R Q Q E E I E Q
GCTATTCACGCGGCCAAGTCATCGACAGAGAAGCTGTGTGCCATGACTGGCCAATTGCGGCAGTGCGAAGTTGGCGCGCAAACTATGGAGCAGCGATGGA
1201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1300
A I H A A K S S T E K L C A M T G Q L R Q C E V G A Q T M E Q R W K AGGAAGTGTCGGCGACTCTTGAGCAGGAGCGCAGCCGCAACACACGCGATCGTGAGCAGAT GAATTCCCAACTGGAGGCGTCTCAGGCTCAGGTGACGGA
1301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+
1400
E V S A T L E Q E R S R N T R D R E Q M N S Q L E A S Q A Q V T E AATCAAGGCGGAGATGAGTCGGCTGCGCGTCCAACTGGAGCAGGGCGCAACCAAACTGAAGGAATGCCAAGATGCTCTTGCTTCGTCTAAAGAAGCGAGC
1401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+
1500
I K A E M S R L R V Q L E Q G A T K L K E C Q D A L A S S K E A S
AGCCGAGCAGCTGCGGATTCGCGAGAGTCAATTGCACTCATCGCCTCTGATCGGGACCGCTTAAAGGAGGACAGGGATCGGGTGGCCTTTGAGTTAAAGG
1501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+
1600
S R A A A D S R E S I A L I A S D R D R L K E D R D R V A F E L K E AAGCTGAACATCGGCTAAGCATGGAAAGAGATCGTGCTTCCGATGCGAAAAGAGAGTTATCAAGGCGTCTTGACGATGCTGCGCACACCATAGAGCGGAT
1601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+
1700
A E H R L S M E R D R A S D A K R E L S R R L D D A A H T I E R M -
↓
GCGGGATCAGCTGAAGGA CAAGGAGCATCAACTGCAACTGTTGTCAACCGCGCATGAGAAAAAAATCCAGGAACTCGCATTTGAGCATAACAATAAGTTG
1701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1800
R D Q L K D K E H Q L Q L L S T A H E K K I Q E L A F E H N N K L
GGCGACTGCAAGTCCCAGAAGAAAAATGCTATTGATGATGTGCGCAGGCAGCTTGAAGCCGCGAATCTTCGCTTGACCGAAGAAATGTCGGGTAACAAGG
1801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1900
G D C K S Q K K N A I D D V R R Q L E A A N L R L T E E M S G N K A CGCTTCAATGTGAACTTAACTCCGCCCGTGAGGCACTGGCCAATGTGCGTGACGAATGCGAAAGGTGGGCCAAGGAAGCCAAGGAAAGCGCACGGCGACT
1901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2000
L Q C E L N S A R E A L A N V R D E C E R W A K E A K E S A R R L GGAAGCGGCGACATCTGAGGCATCAAGCTTGCGCGCGTCTTTAGCAAAGCAACAGGAGCTCCTTGCGGCTGCCGCTGAGCGTGAGAAAACCCTCTGTAAA
2001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2100
E A A T S E A S S L R A S L A K Q Q E L L A A A A E R E K T L C K
GTAGCAGAGCATGCCAACGCAGAAAAAGAAATGGAGATCAAGCGACGTGAGCTCCTTGAACGCACCCTTGAAGACACTAAGAGAGAGGTCGTTGCCAGGC
2101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2200
V A E H A N A E K E M E I K R R E L L E R T L E D T K R E V V A R R GGGATGAGGTGCAGGAGCTGCGCACAAGGATTGATAAGGAGAATAACAACACTTTAGCGAAAGAACTTATGGAATGCGAGGCAAGGTTCCGCGAGAGCCA
2201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2300
D E V Q E L R T R I D K E N N N T L A K E L M E C E A R F R E S Q GCGCTCCCTCGAGCGGACACAGCGGGAGATGGTTGATGTGCAACGTTGTGGGGAAACGCTACAAGCGACCAATAAGGCGCTTGAAGAAAAATGTCGAGTG
2301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2400
R S L E R T Q R E M V D V Q R C G E T L Q A T N K A L E E K C R V
GCCGAGAGGTCCCAGCGGGAGGTTGAAGAGGAGCTTCGTCGGTTAAAAGGCGAAATTCTGTCGAAGGAGACGGAGTGTTGCGCGGGTACGCAGCATGCGC
2401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2500
A E R S Q R E V E E E L R R L K G E I L S K E T E C C A G T Q H A R GTGAAGCTGAAGATGCTGCGAAGCAGAGCTGTGAGCATATGGAGCGTGAAATAACCCAACGTGAGACTACCATTGCGGCGTTGCAACAGGAAATTTCTGC
2501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2600
E A E D A A K Q S C E H M E R E I T Q R E T T I A A L Q Q E I S A GCTCTCCGAGGAGCGAACCAAAGTGGCACTGTTGGAAGAACGGATGCAGCACCAGGTGGATATGGCCCGCCGTGACTCAGACAACTTGCAAGCTCGCGTA
2601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2700
L S E E R T K V A L L E E R M Q H Q V D M A R R D S D N L Q A R V
-
Fig. 2. For legend see p. 4691
4690 V. Dilbeck and others
GAGTTTCTCGAGAGGGAGGTCCAGGATCGTGAAGAGAAAATCCAGCAGAAACACAAGGAGATGTTGCAAACGGTGGATCGCTTACAAACCCTACAGGAGC
2701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2800
E F L E R E V Q D R E E K I Q Q K H K E M L Q T V D R L Q T L Q E R GCGCTGTGGAATTGGAAGAGGCGATGGCGCCGAAGGAGAAGAAACACACCATGCGAAAGGAGGCGCTTCGTAAGGCGCTTCAGCAAGTGGATGAGGTGAA
2801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 2900
A V E L E E A M A P K E K K H T M R K E A L R K A L Q Q V D E V N CAAGCTCCGGTCAGAACTGGAGCGCCACCTGGAAAAGGTAAAGGCGTCGCGAGAAGAGGAATCTAGAATTTACAAAGCACAAATTCACCAACAGGATGAA
2901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3000
K L R S E L E R H L E K V K A S R E E E S R I Y K A Q I H Q Q D E
CGAATGAGGGTACTCCTGGAGAAACATCGGGAAATGGAGCGCCAACTAGTGGCTCAAGAGAGAGATCTCAAAGCAGCAGCAAATGAGCAGATGACGTTGC
3001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3100
R M R V L L E K H R E M E R Q L V A Q E R D L K A A A N E Q M T L Q AGCAACGGTTGGCGGTGATACGCGACAGAGAGCAAGTTAACGTTGGTAAGCACAGCGAGGAGCAACAGAAGATGCAGGAAAAGTTGGATGCAATGAGCAG
3101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3200
Q R L A V I R D R E Q V N V G K H S E E Q Q K M Q E K L D A M S S CGAACTCGCGCGTGCCGCCACAATCAAAAGTGTGGAGGAGGAGAAAAACAATTCAGTGTGTGAAGCGAGTGACGTGCAACGTAGGACGGCGGTAAACTCT
3201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3300
E L A R A A T I K S V E E E K N N S V C E A S D V Q R R T A V N S
AACCGCCTACTTATCACCTATGCTGATGACGCATTAATGACCAAAGAGATGTTTGGTGAGTTTGCCATTAGCATCGCCACTCGCCTCTTACGAGGTGTTA
3301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3400
N R L L I T Y A D D A L M T K E M F G E F A I S I A T R L L R G V N ATAGTATCGCCAACAAGGGGTGTGACAGCGCCCTTTTATGCATGAGAGAGTACACTGAAGAGGCTGAGAAGCAGCAACTGAGACTTAAACGTGAGATTGA
3401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3500
S I A N K G C D S A L L C M R E Y T E E A E K Q Q L R L K R E I D TGATCTAAAATACGCCACGGAGCGTGTTCGAAAACTTGAGGAGGAGAATAGTAAGGAAATGGCAAAACAGGCGCAACATCACGCTGCAGAGTTGGCAAAA
3501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3600
D L K Y A T E R V R K L E E E N S K E M A K Q A Q H H A A E L A K
CTACGTCAGGAATTATCCGATGCATCGGCACGTGCAGGCCAAGAGATAGAAAACCAGCTGAAGGATTACCGCCGCAAAGAGGAGCAATTCCACCGTGAGA
3601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3700
L R Q E L S D A S A R A G Q E I E N Q L K D Y R R K E E Q F H R E K AGACGGAGTTAGAGTGCGCACGTGTTGAGATGGCTCATCAAATGGGTCAAATCTCTTCACTCAAGGATCAGCTTCGGACCCTTGAGCAGCGTATGGATAC
3701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3800
T E L E C A R V E M A H Q M G Q I S S L K D Q L R T L E Q R M D T CGAACGTATTGCTCTTGAGCAGGAGAAACGCGTAATACAACAACAGTACGATCGGGCTAGTCGGCGCTTGGATGAGTGCGCAACGGTACAGAGCCAAAGT
3801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 3900
E R I A L E Q E K R V I Q Q Q Y D R A S R R L D E C A T V Q S Q S
-
↓
GAGGTAGAACTGCGTGAGCTCCGGAGCGAACTGACAAATGCACAACAGGCATTGCACGAGAAAGAGAAAGCTTTGCTTGTTGAGCGTGAAAAGAATTCTC
3901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4000
E V E L R E L R S E L T N A Q Q A L H E K E K A L L V E R E K N S Q AGGCAGCTTACCAACTGGAAGCCGTTAACACTGCGAAGGAGATGCTGTACACACAATTAAATGATGCAAATAGGAAAGCGCTTAATATAAAGCAGCAGCT
4001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4100
A A Y Q L E A V N T A K E M L Y T Q L N D A N R K A L N I K Q Q L GCTGTCTGCTCGGCAGGAGGCACAACAAGCAACTGCAGCAGCTACAGCAGAAAGGCAGAGATTGGAGGAGCGGATATCTGATCTTGCGAAGGCGGTTAGC
4101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4200
L S A R Q E A Q Q A T A A A T A E R Q R L E E R I S D L A K A V S
GCCCACGACATGGAGAATCGCCGCCTTGACGGTCAGATACGCAGTGATGAGAAGAAGTTTATTGCTCTTGAGAGAGAACTGGCGGAATCTCGCAAGCGGG
4201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4300
A H D M E N R R L D G Q I R S D E K K F I A L E R E L A E S R K R E AGGCGGAGATGTCCTGTCAACTGCAGGCTCGAAGGTTAGAAAATTCATCCCTGAGGGAGCGGTGCGCAAACTTGGAGTCACTCAAAAATATTAATGACGT
4301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4400
A E M S C Q L Q A R R L E N S S L R E R C A N L E S L K N I N D V TACCCTTGCTGAGACTCGTATGCGGGAGAAAGATCTCTTTGAAAAAATAGAAGAAATGCGCAGCGCACAACAACTGATGCAATTGTGTTTCGACAAGCAG
4401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4500
T L A E T R M R E K D L F E K I E E M R S A Q Q L M Q L C F D K Q
CAGGAACAACTGGAGGCAGGACGACGCATGCACGAAGAAGACACAGGTACAAACGAGTTCATGTTTGGAGGTGTCGATTGAAGTGGATGAATTTGTAGTT
4501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4600
Q E Q L E A G R R M H E E D T G T N E F M F G G V D *
CAGGTAGTTTCTTCTAGTTGTTTGCCTTGTTTTTTGTTGTTGTTGTTGCGCCCCTTCTATGAACATAGATATACAGCATAAATTTTTGTGTGCAACCACT
4601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4700
CGGTTGTGCTTTCTTTCTCTTTTCTGCGGCATTTTTTCATCTTTTGAGGGATGGGGCGGAGTGGACCTTAGCGCACTCTCTCTTTACTTTTGTTTCCATT
4701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4800
ATTATTATTATTATTATTATTATTTTGTTCCTTACACTGTTTACTCCTTCTTTTCATCGTATGAGATAATACGGGACGCAGCGTGGCTATATATACAATT
4801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 4900
(A)20
䉲
TTTTTTTTGAGGTTGTAATCTTTCCGCAAAATCAACCGGAAGAGGGGGTGGTTTCTCCACAGCGTTGTTGGGGTGGTTTCTCCACAGCGTTGTTATTTGT
4901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 5000
CAGATCGAGAATTCCCGGGATCCGCGGCCGCAGCTCTTCTATAGTGTCACCTAATGGCCATTTAGGCTAGAGTCGACCCAGATCCACTCGTTATTCTCGG
5001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 5100
ATGAGTGTTCAGTATGACCTCTGGAGAGAACCATGTATATGATCGTATCTGGTGACTCTGCTTTAAGCCAGATAACTGGCTGAATATGTTAATGAGAGAA
5101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 5200
TCGGTATTCCTCATGTGTGGCATGTTTTCGTCTTTGCTTTGCATTTTCGCTAGCAATTAATGTGCATCGATTATCAGCTATTGCCAGCGCCAGATATAAG
5201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 5300
CGATTTAAGCTAAGAAAACGCATTAAGATGCAAAACGATAAAGTGCGATCAGTAATTCAAAACCTTACAGAAGAGC
5301 ---------+---------+---------+---------+---------+---------+---------+------ 5376
T. brucei basal body component 4691
Fig. 2. Nucleotide sequence and deduced amino acid sequence of
TBBC (EMBL accession number: AJ242745). The sequence is
derived from a cDNA inserted in a λgt11 expression vector (the 3′
end of the gene), a PCR product (the sequence in the box) and a
genomic clone which comprises the full length coding sequence
together with partial 5′ and 3′ untranslated regions. The sequence
between the two arrows was inserted in an E. coli expression vector
(pGEX-1λT) to produce a fusion protein. The start and stop codons
of the TBBC ORF are shown in bold and the polyadenylation site of
the TBBC mRNA is indicated by an arrowhead. The underlined
stretch shows the putative 3′ splice site of the TBBC mRNA.
RESULTS
Cloning and characterization of the TBBC gene
In an attempt to characterize a trypanosomal
immunosuppressive factor (Darji et al., 1992), a T. brucei
cDNA expression library was screened with antibodies raised
against trypanosome extracts enriched in immunosuppressive
activity. Eight clones were selected, five of which turned out
to contain fragments of the same gene. Southern blot analysis
indicated that TBBC is a single copy gene, since a single DNA
fragment was detected by TBBC probes in various restriction
digests of genomic DNA (Fig. 1A). This gene appeared to be
a fertilization medium (Hogan et al., 1994). The immunofluorescence
transcribed into a mRNA of approximatively 4.8 kb which was
was done essentially as described by Morte et al. (1988).
developmentally regulated, being more abundant in the two
major bloodstream-specific stages of the parasite life-cycle, the
Capturing of digital images
proliferative slender form and the quiescent stumpy form (SL
Immunofluorescence was observed on a Leica DMRBE and on a
and ST in Fig. 1B), than in the insect-specific procyclic form
Zeiss AxioskopII microscope for Fig. 5 and Figs 4 and 6,
(P in Fig. 1B). Specific anti-TBBC antibodies were produced
respectively. Images were captured by a Quantix camera (Roper
by injecting a E. coli-synthesized TBBC polypeptide into
scientific) and a spot camera (DIAGNOSTIC instruments). The
rabbits. As shown in Fig. 1C, these antibodies detected a single
mouse spermatozoon images were realised with the TCS 4D Leica
protein with an apparent molecular mass of 212 kDa in the
confocal microscope.
three developmental stages of the parasite. Although
this protein was more abundant in the bloodstream
stages than in procyclic forms, it was clear that the
difference in the protein levels did not match that of
Identity (%) Gaps (%) Overlap (aa)
the mRNA levels, pointing to possible translational
controls.
Liver stage antigen-1
21
9
1410
The entire ORF of the TBBC gene was cloned by
Plasmodium falciparum
a combination of PCR and DNA hybridization
Plectin
screening of a genomic DNA library. The nucleotide
21
12
1410
sequence of a 5,376 bp fragment encompassing the
Rattus norvegicus
TBBC ORF is shown in Fig. 2. The polyadenylation
Trichohyalin
22
9
1201
site of the mRNA was found to be 290 bp downstream
Oryctolagus cuniculus
from the stop codon but we were unable to clone
cDNAs containing the 5′-spliced leader so that the
C-Nap1
21
15
1410
size of the 5′ untranslated region is not known.
Homo sapiens
However, in Fig. 2 the sequence upstream from
position 348 is interrupted with stop codons and a
NuMa
18
15
1410
possible 3′-splice site (AG preceded by a
Homo sapiens
polypyrimidine stretch) is present at position 255
Myosin heavy chain
(underlined in Fig. 2). Splicing at this position would
19
13
1169
confer a size of 4.67 kb to the mRNA, which is in
Dugesia japonica
agreement with the size estimated from the northern
blot analysis.
B
Probability of coiled coil
A
TBBC is predicted to be a coiled coil protein
The TBBC ORF contains 1,410 amino acids (Fig. 2).
This ORF did not show extensive sequence homology
with known proteins and did not contain hydrophobic
stretches susceptible to behave as transmembrane
spanning regions. The highest homology (identity
Residue number
Fig. 3. Amino acid sequence analysis of TBBC. (A) Best
sequence homology between TBBC and proteins found in
current databases as determined by the Blast 2.0 program
(http://www.ncbi.nlm.nih.gov/BLAST/). All selected
proteins contain at least one coiled coil domain which
accounts for the homology with TBBC. (B) Probability of
the TBBC sequence to form a coiled coil structure, as
determined by the program COILS (Lupas et al., 1991;
http://www.ch.embnet.org/software/coils_form.html).
4692 V. Dilbeck and others
Fig. 4. Localization of TBBC using antiTBBC polyclonal antiserum. Anti-TBBC
staining was detected by indirect
immunofluorescence using an FITCconjugated second antibody. No staining was
obtained with the preimmune serum under
the same conditions (data not shown). (A to
C, respectively) A procyclic form, two
slender forms, a non dividing above and a
dividing below (two kinetoplasts) and a
stumpy form. (D and E, respectively) A
procyclic form stained with an anti-TBBC
monoclonal antibody and isolated flagellum
stained with anti-TBCC polyclonal
antiserum. The blue DAPI staining shows the
position of the nuclear and kinetoplastic DNA
(large and small spot, respectively).
p, posterior end of the cell; t, TBBC.
around 20% and similarity around 40%)
was shared with various proteins
including myosins, trichohyalin and a
centrosomal protein termed C-Nap1
(listed in Fig. 3A), whose common
feature is the presence of extensive coiled coil regions.
Significantly, the homology with trichohyalin, which is not
totally organized in coiled coils, was only restricted to the
coiled coil region (data not shown). In accordance with these
observations, sequence analysis of TBBC predicted a highly
probable coiled coil structure over almost the entire length of
the polypeptide (Fig. 3B). This finding provides an explanation
for the discrepancy between the apparent molecular mass of
TBBC on SDS-PAGE, 212 kDa, and the predicted size of 163
kDa, since a similar difference was also observed for coiled
coil proteins such as dynein and tropomyosin. Finally, typical
motives such as ATP binding sites and the ‘head’ present in
myosins were not detected in TBBC. In
conclusion, the only characteristic that
emerged from studies on the TBBC
sequence was an extensive coiled coil
structure and no specific function could be
ascribed to the protein.
TBBC is a component of the basal
body
The anti-TBBC antibodies were used to
determine its subcellular localization by
immunofluorescence. As shown in Fig. 4AC, in the three main stages of the parasite
Fig. 5. Immunofluorescence of basal body
components in an early dividing T. brucei
procyclic form.The localization of TBBC (t, A)
and γ-tubulin (γ, B) was revealed using antiTBBC and anti-γ-tubulin monoclonal antibodies,
respectively. In C, the double
immunofluorescence shows the close association
of the two proteins. (D) Double
immunofluorescence obtained with BBA4
monoclonal (b) and TBBC polyclonal (t)
antibodies. The blue DAPI staining shows the
position of the nuclear and kinetoplastic (k) DNA.
development TBBC was found to be present in several
locations such as at the posterior end of the cell, the nucleus
and over the flagellum, but clearly the intensity of staining was
the greatest over the basal body. Polyclonal and monoclonal
antibodies gave similar results (A and D), although the staining
consistently appeared to be only restricted to the basal body in
the latter case. The association with the basal body was
evidenced by the close proximity to the kinetoplast DNA, well
apparent when the cell is initiating division (A,B,D), and by
the physical attachment to the flagellum when the latter is
isolated from the cell (E). In the basal body TBBC was readily
detectable irrespective of the moment in the cell cycle
T. brucei basal body component 4693
Fig. 6. Localization of a TBBCrelated protein in the centrosome of
HeLa. (A and B, respectively)
Staining of a dividing HeLa cell with
anti-γ-tubulin (γ) and anti-TBBC (t)
antibodies. (C) Colocalization of
both proteins (arrows). In this panel,
the blue DAPI staining shows the
localization of the DNA. (D) The
results of western blot analysis with
anti-TBBC polyclonal antibodies.
Lanes 1 and 2, respectively, contain
20 µg and 50 µg of total protein
extracts from HeLa cells and T.
brucei bloodstream forms. The
TBBC preimmune serum did not
stain the blot (data not shown).
(compare the dividing and nondividing bloodstream forms in B).
So far two distinct components
of the basal body have been
identified
in
trypanosomes,
namely γ-tubulin (Scott et al.,
1997) and an uncharacterized
component
termed
BBA4
(Woodward et al., 1995). Double
immunofluorescence experiments
were conducted on early dividing
procyclic forms with anti-TBBC
and either anti-γ-tubulin or antiBBA4 antibodies. As shown in
Fig. 5, these three components did
not colocalize (A to C), but were
very closely associated, BBA4
being the most proximal to the
kinetoplast DNA (D).
A TBBC-like protein is
present in the centriole of
higher eukaryotic cells
Given that the basal body is
functionally analogous to the
centrosome of higher eukaryotes,
we evaluated the presence of
TBBC-like components in HeLa
cells. In accordance with the
prediction,
the
anti-TBBC
antibodies where found to
recognize the centrosome of HeLa cells as revealed by the
colocalization of anti-TBBC and anti-γ-tubulin antibodies (Fig.
6A-C). Accordingly, western blotting of HeLa cell extracts
reavealed the presence of a 170 kDa component specifically
detected with the anti-TBBC antibodies (Fig. 6D, lane 1). In
addition, in mouse spermatozoa the anti-TBBC antibodies also
stained the centrosome, the distal centriole of which plays a
role of basal body (Fig. 7). In this case, the anti-TBBC
antibodies also recognized the distal part of the flagellum
where, in contrast to the proximal region, the axoneme is
surrounded by outer fibres and a fibrous sheath of unknown
composition (Fig. 7).
Fig. 7. Localization of a TBBC-related protein in the centrosome of a
mouse spermatozoon. In the distal part of the flagellum (d), the
axoneme is surrounded by outer fibres and a fibrous sheath of
unknown composition, whereas in the proximal part (p) the fibrous
sheath disappears and is replaced by a mitochondrial envelope.
Immunofluorescence was detected with anti-TBBC antibodies on the
centrosome (tc) as well as on the distal part of the flagellum (t). Two
different magnifications are shown (A and B). The DNA was stained
in red with To-Pro-3 (Molecular Probes). The preimmune serum did
not stain the cell (data not shown).
DISCUSSION
We describe a new structural component of the basal body of
T. brucei. This component, termed TBBC for T. brucei basal
body component, was identified as a major antigen (5 out of 8
clones selected) while screening for a trypanosome
immunomodulatory factor using antibodies raised against an
immunossuppressive fraction of the parasite. However, no
evidence for the involvement of TBBC in immunosuppression
has been found (data not shown). Since the only characteristic
of TBBC is a probable arrangement as a long coiled coil, it is
possible that this protein was detected because it shares coiled
coil regions with the immunosuppressive factor. However, in
this respect it may be worth stressing that other trypanosome
coiled coil proteins were not selected in this screening whereas
the other clones selected did not show obvious coiled coil region
(data not shown).
The localization of TBBC in the basal body adds to the list
of different coiled coil proteins known to be present in the
centrosome and spindle pole body in eukaryotic cells (Stearns
and Winey, 1997). Although the exact function of these
components is not known, their nature strongly suggests
4694 V. Dilbeck and others
association into filaments involved in structural organization. In
particular, the cell-cycle-regulated phosphorylation of one of
them, C-Nap1, has been implicated in connecting the proximal
ends of centrioles to each other during interphase (Fry et al.,
1998). Interestingly, we found that a TBBC-like component
exists in centrosome of mammalian cells since in total HeLa
cell extracts anti-TBBC antibodies clearly detected a 170 kDa
protein and immunofluorescence experiments revealed TBBCspecific material in the centrosome. The fact that a single band
was detected in western blots strongly argues against a
fortuitous recognition based only on the presence of a coiled
coil domain. Moreover, a TBBC-related component was also
detected in the centrosome region of mouse spermatozoon. It is
tempting to speculate that this component is the centrin-related
centrosomal protein of 165 kDa described by Baron and
Salisbury (1988). It was not possible to check this hypothesis
because the antibodies against this component are no longer
available (J. L. Salisbury, personal communication). In addition,
so far our attempts to clone this gene from either spermatozoon
or HeLa cDNA libraries have been unsuccessful.
In T. brucei, TBBC was found to localize close to the two
other markers of the basal body, γ-tubulin and BBA4. TBBC
appeared to be the most distal of these markers with respect to
the kinetoplast DNA. TBBC was also found to be physically
connected to the flagellum since it labelled the end of isolated
flagella. The inefficacy of the anti-TBBC antibodies in
immunogold electron microscopy prevented us from analyzing
this connection in more details. It may be worth mentioning
that, as opposed to C-Nap1, TBBC was detected in equal
amounts in both the resting and dividing stages of the cell cycle,
which suggests no drastic conformational changes such as those
occurring in C-Nap1 following phosphorylation. So far attempts
at eliminating both alleles of the TBBC gene either by standard
gene knock out approaches or abbrogation of functional
translation by an antisense strategy (Bastin et al., 1998) have
been unsuccessful which suggests that TBBC may be a vital
component of the cell.
We thank P. Bastin and K. Gull (School of Biological Sciences,
Manchester)
for
antibodies
and
invaluable
help
for
immunofluorescence and T. Seebeck, I. Hunger-Glaser (Instituut fuer
Allgemeine Mikrobiologie, Berne) and M. Radwanska for technical
help. The work in our laboratory was financed by the Belgian FRSM
and FRC-IM, a research contract with the Communaute Française de
Belgique (ARC), the Interuniversity Poles of Attraction Programme,
Belgian State, Prime Minister’s Office, Federal Office for Scientific,
Technical and Cultural Affairs, and by the Agreement for collaborative
Research Between ILRAD (Nairobi) and Belgium Research Centres.
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