1 Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am
Fassberg 11 37070 Göttingen, Germany
2 Department of Biochemistry and Pathobiochemistry, Medical Faculty of the
University of Halle, 06097 Halle/Saale, Germany
Author for correspondence (e-mail:
mechthild.hatzfeld{at}medizin.uni-halle.de)
Accepted 3 August 2002
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Summary |
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Key words: Astrin, Coiled coil, Microtubules, Mitotic spindle, RNA interference
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Introduction |
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The primary function of the mitotic spindle is to segregate chromosomes
such that a complete set of chromosomes ends up at each spindle pole. The
process of segregation depends on a complex interplay between forces generated
by motor proteins associated with spindle microtubules, kinetochores and
chromosome arms as well as dynamic instability of spindle microtubules
(Howell et al., 2001;
Maney et al., 2000
;
Nicklas et al., 1995
;
Rieder et al., 1995
;
Rieder and Salmon, 1998
).
Chromosome separation also depends on attachment of chromosomes to spindle
microtubules via their kinetochores, and it has been shown that cytoplasmic
dynein as well as several kinesin family members localize at the kinetochore
(Banks and Heald, 2001
). CENP-E
is another kinesin-like protein that is part of a kinetochore-associated
signalling pathway that monitors kinetochore-microtubule attachment and
ensures high segregation fidelity. Depletion of CENP-E from mammalian
kinetochores leads to a reduction of kinetochore-microtubule binding and
mitotic arrest, producing a mixture of aligned and unaligned chromosomes
(Abrieu et al., 2000
;
Lombillo et al., 1995
;
Yao et al., 2000
;
Yen et al., 1992
).
In order to gain more insight into spindle organization, Mack and Compton
used mitotic microtubules prepared from Hela cell extracts to identify
spindle-associated proteins in an elegant mass spectroscopic analysis
(Mack and Compton, 2001).
Several proteins with functional roles in spindle assembly and a novel
non-motor coiled-coil protein named astrin were described. Using
immunofluorescence and ectopic expression of the GFP-tagged astrin, its
spindle localization during mitosis was confirmed
(Mack and Compton, 2001
). We
originally identified an astrin cDNA clone in a two-hybrid screen with a
keratin 18 bait although this interaction could not be confirmed using other
methods. We named the full-length 3,793 bp cDNA DEEPEST because the
predicted protein contains this sequence motif. Antibodies located the protein
to the spindle, and sequence predictions indicated two long coiled coils. When
we entered the cDNA sequence into the EMBL/GenBank databases in May 1998 we
added information concerning a coiled-coil protein associated with the mitotic
spindle apparatus (accession number AF 063308). Owing to a sequence error at
position 3341, our predicted protein sequence lacked the C-terminal 101 amino
acids provided by Mack and Compton (accession number AF 399910). The same
protein sequence including the sequence error of our original DEEPEST-sequence
was also provided by Chang et al. (Chang et
al., 2001
) (accession number P33176 and NM 006461), who called
this protein hMAP126. hMAP126 has been described as a
mitotic-spindle-associated protein that is post-translationally modified by
cdk1 phosphorylation. To avoid confusion we have dropped the name DEEPEST and
use instead the name astrin.
Here, we describe a more detailed characterization of astrin's domain organization and function in spindle pole organization. Astrin has a domain structure resembling that of motor proteins with a large head domain, which lacks sequence similarity to motor domains, and a coiled-coil domain responsible for formation of parallel dimers. Under physiological conditions, recombinant astrin dimers oligomerize via their head domains into aster-like structures. Moreover, we show that astrin is essential for progression through mitosis. Depletion of astrin by RNA interference resulted in the formation of multipolar and highly disordered spindles and lead to growth arrest and apoptotic cell death. These results indicate that astrin has a critical role in assembly or orientation of the bipolar structure of the spindle.
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Materials and Methods |
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Astrin cDNA expression constructs were generated by rt-PCR with total RNA from MCF-7 cells. Suitable restriction sites were included in the primer sequences. The following constructs were used in this study: (1) Astrin DN: aa 1-356. Fragment DN was cloned into BamHI/HindIII sites of pET-23a. (2) Astrin DC: aa 707-1027. Fragment DC was cloned into pGEX-2T (Pharmacia Biotech). This vector contains an N-terminal glutathione-S-transferase (GST)-tag sequence. (3) Astrin 1-1,123 was cloned into pET23a for prokaryotic expression (astrin-T7). This construct lacks the C-terminal 70 amino acids of full-length astrin. Fig. 1B gives an overview of constructs used in this study.
|
Expression and purification of recombinant polypeptides
Constructs in pET-23a (astrin-T7, aa 1-1,123 and DN aa 1-356) were
expressed in Escherichia coli BL21 (DE3) pLysS. GST-DC (aa 707-1027)
was expressed in E. coli strain Xl1 blue.
Astrin-DN was soluble and purified from bacteria by anion exchange
chromatography on a MonoQ column and gel filtration on Sephadex 75. Astrin-T7
aggregated into inclusion bodies, which were prepared according to Hatzfeld
and Weber and solubilized in urea buffer (8.5 M urea, 10 mM Tris-HCl pH 8, 5
mM EDTA, 2.5 mM DTT) (Hatzfeld and Weber,
1990). Astrin-T7 was purified to homogeneity from this solution by
anion exchange chromatography on MonoQ column. The GST-DC fusion protein was
soluble and purified on glutathione sepharose matrix (Pharmacia Biotech) in
PBS (8 mM Na2HPO4, 1.5 mM KH2PO4,
140 mM NaCl, 3.7 mM KCl). The GST-tag was cleaved by thrombin digestion, and
astrin was eluted in PBS.
CD spectroscopy
Purified recombinant astrin-T7 protein at a concentration of about 0.2
mg/ml was dialyzed against 8 mM Na2HPO4, 1.5 mM
KH2PO4, 0.5 mM DTT (pH 7.5) at 4°C overnight. The
extinction at 280 nm was measured. Protein concentration was determined using
the corresponding extinction coefficient calculated from the deduced
amino-acid sequence of the construct. CD spectra from 200 to 250 nm (stepsize
0.5 nm, speed 50 nm/minute) were measured with the polarimeter J-720 (Jasco,
Groß-Umstadt, Germany) in a cuvette of 0.1 cm thickness. For comparison,
-tropomyosin from bovine brain (which has an
-helix content
greater than 90%; the protein was kindly provided by N. Geisler) was measured
in the same buffer. CD spectra of poly-L-lysine were measured under the
following conditions: 0.1 N KOH (100%
-helix), 0.1 N KOH after
incubation at 60°C for 2 hours (100% ß-strand) and 0.1 N HCl (100%
random coil). The relative
-helix content of recombinant astrin-T7 was
determined by comparing its molecular ellipticity at the isosbestic point of
poly-L-lysine ß-strand/random coil (208 nm) with the molecular
ellipticity of 100%
-helical poly-L-lysine at the same wavelength.
Electron microscopy
Recombinant astrin-T7 protein was microdialyzed for 3 hours at room
temperature either against PBS containing 2.5 mM DTT or phosphate buffer
without salt (8 mM Na2HPO4, 1.5 mM
KH2PO4, 2.5 mM DTT). After dialysis, glycerol was added
to 33% of the volume, samples were sprayed onto mica sheets, rotary shadowed
at an angle of 4° first with platinum/carbon at 700 Hz and then with
carbon at 60 Hz in a vacuum rotary shadowing device (Baltzers, Liechtenstein)
and inspected with a CM-12 transmission electron microscope (Phillips,
Eindhoven, The Netherlands).
Antibody to astrin
Recombinant astrin fragments covering amino-acid residues 1-363 (astrin-DN)
and 707-1027 (astrin-DC) were purified from E. coli and used to
elicit rabbit antibodies. An aliquot of the truncated astrin was coupled to
Sepharose 4B and used to obtain antigen affinity-purified rabbit antibodies.
Purified astrin antibodies eluted with low pH buffer were immediately
neutralized with Tris-base.
Gel electrophoresis and western blot analysis
SDS gel electrophoresis and western blotting were performed according to
standard protocols.
A monoclonal antibody against the T7-tag was obtained from Novagen (Calbiochem-Novabiochem, Schwalbach FRG), and affinity-purified horseradish-conjugated rabbit anti-mouse and swine anti-rabbit immunoglobulins were from Dako, (Copenhagen, Denmark). Bands were detected using ECL (Amersham Pharmacia).
Silencing of astrin by siRNA
RNA interference mediated by duplexes of 21-nt RNAs was performed on human
HeLa cells as described previously
(Elbashir et al., 2001;
Harborth et al., 2001
). The
siRNA sequence for targeting astrin was from position 2639 to 2661, relative
to the first nucleotide of the start codon (GenBank accession number AF
399910). As an siRNA control, a sequence targeting firefly (Photinus
pyralis) luciferase (accession number X 65324) at positions 153 to 175
was used (pGL2 siRNA). The 21-nt RNAs were chemically synthesized by Dharmacon
(Lafayette, CO, USA) and delivered in a salt-free and deprotected form.
Duplexes were formed as before. Alternatively, duplexes were obtained
commercially. Transfection with Oligofectamine (Invitrogen; lot number
1122079) was as described previously
(Harborth et al., 2001
). Cells
were monitored by phase microscopy at intervals, and after 44 hours, they were
analyzed by immunofluorescence microscopy. Murine
-tubulin and
-tubulin monoclonal antibodies were from Sigma, the antigen
affinity-purified rabbit antibody to tubulin was kindly provided by M. Osborn.
DNA was visualized by Hoechst 33342 dye. Separate samples were processed for
immunoblotting with rabbit astrin antibody and peroxidase-conjugated swine
anti-rabbit immunoglobulins (Dako, Denmark) using the ECL technique (Amersham
Pharmacia Biotech). To confirm equal loading of gels used for astrin and
control siRNAs, the blots were stripped (Re-Blot Western Blot Recycling Kit,
Chemicon) and reprobed with the monoclonal vimentin V9 antibody
(Osborn et al., 1984
).
TUNEL test
For detection of apoptosis, a TUNEL test (In Situ Cell Death Detection Kit,
Roche) was performed. Transfected cells grown for 60 hours were fixed in
-20°C methanol for 6 minutes and treated with PBS containing 0.1% Triton
X100 and 0.1% sodium citrate on ice for 2 minutes. Free 3' ends of
fragmented DNA were enzymatically labeled with FITC-tagged deoxynucleotide
triphosphates using deoxynucleotidyl transferase (TdT). Labeled DNA fragments
were monitored by fluorescence microscopy.
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Results and Discussion |
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Structure and domain organization of astrin
As previously noted (Mack and Compton,
2001), the astrin sequence shows similarities to a partial mouse
clone of unknown function (Nehls et al.,
1995
) and to the rat Spag5 protein
(Shao et al., 2001
), which
interacts with an outer dense filament (odf) protein (odf1) from sperm
flagella (Shao et al., 2001
).
However, depletion of Spag5 revealed no phenotype, and Spag5-null mice were
viable and fertile, suggesting that if Spag5 plays a role in spermatogenesis
it is probably compensated for by other unknown proteins
(Xue et al., 2002
).
According to secondary structure predictions, the astrin sequence can be
separated into several distinct domains
(Fig. 1A). The N-terminal head
domain (aa 1-481) of astrin is rich in proline (7.5%) together with serine and
threonine residues, suggesting that astrin's function is regulated by
phosphorylation in vivo. This hypothesis is supported by the finding of Chang,
who showed that astrin is a substrate of cdk1 in vitro (Chang, 2001). In
addition, four PXXP motifs in the astrin head domain may represent putative
SH3-interaction sites, suggesting that astrin could be a target for regulatory
proteins (Alexandropoulos et al.,
1995; Yu et al.,
1994
). Three regions (amino acids 95 to 119, 287 to 330 and 463 to
477) are especially rich in Pro, Glu/Asp and Ser/Thr residues, respectively.
These so-called PEST sequences are frequently found in proteins underlying
rapid degradation through ubiquitin-protein ligase complexes. This degradation
is regulated through phosphorylation of the PEST sequence motifs
(Rechsteiner and Rogers, 1996
;
Rogers et al., 1986
). Rapid
protein degradation is an important regulatory mechanism in cell cycle
regulation. Cyclins contain PEST sequences of the cyclin destruction box type,
and phosphorylation of these motifs leads to ubiquitylation and degradation,
which is a prerequisite for cell cycle progression
(Rechsteiner and Rogers, 1996
;
Rogers et al., 1986
). Amino
acids 482 to 903 of astrin are predicted to be essentially
-helical
except for a short interruption. Most of this region shows the heptad repeat
pattern of coiled-coil proteins, suggesting dimerization and formation of a
rod-like structure. Amino acids 904 to 965 are predicted to be
non-
-helical owing to a high proline content (linker L). This linker
region joins the first coiled-coil domain to a second coiled-coil region
(amino acids 966 to 1175).
In order to analyze structure, domain organization and oligomerization of
astrin, we used the purified recombinant protein. Astrin-T7 aggregated into
inclusion bodies in E. coli and was purified under denaturing
conditions by anion exchange chromatography.
Fig. 2A shows a summary of the
purification. Purified astrin-T7 was dialyzed against phosphate buffer (pH
7.5, containing 0.5 mM DTT), and CD spectroscopy was used to compare the
actual and predicted -helix content to ensure renaturation
(Fig. 2B). The
-helix
content was determined to be 55%, which is in good agreement with the
predicted content (60%, compare Fig.
1A). Hence, we conclude that recombinant astrin refolded into its
native structure.
|
Electron microscopy of astrin molecules in phosphate buffer revealed a
`lolly pop'-shaped structure with a prominent globular head and a rod domain
with a flexible hinge (Fig.
3A). The length of the dimer in its extended version was
approximately 80 nm. On the basis of structural predictions and electron
microscopy, we propose a model according to which astrin forms parallel dimers
via its -helical coiled-coil domains. The N-terminal region forms a
globular head domain, giving the dimers a lollipop-like structure. The first
-helical region with a heptad repeat pattern (aa 481 to 903) is
predicted to form a rod domain of about 35 nm, which is in agreement with
measurements from electron microscope images. The flexible linker corresponds
to the L-domain, which contains the DEEPEST motif. Kinks of varying angular
degrees indicate that this region represents a flexible joint in the stiff rod
domain. The linker is followed by a second rod domain of 30 nm, representing
the
-helical region from aa 966-1123
(Fig. 1A, Fig. 3A). Since the globular
heads are on one side of the rods, the double-stranded coiled coils are
parallel. Although the overall structure of astrin dimers resembles that of
motor proteins, sequence analysis did not reveal any homology to motor
domains, which have a well characterized and conserved protein module
(Mack and Compton, 2001
;
Bloom and Endow, 1995
;
Hirokawa et al., 1998b
;
Hirose and Amos, 1999
;
Kim and Endow, 2000
;
Kirchner et al., 1999
;
Miki et al., 2001
;
Moore and Endow, 1996
).
|
In PBS (containing 2.5 mM DTT), the astrin dimers associated into higher order structures (Fig. 3B). Besides the dimers seen in low salt buffer, oligomers of two to five dimers were formed under physiological salt conditions. Oligomerization was exclusively mediated by the head domains, and higher oligomers resembled astral structures. These oligomers may have the potential to bundle microtubules and to crosslink other astrin-binding partners. Thus, astrin may provide a scaffold for crosslinking regulatory and structural components at the mitotic spindle.
Astrin is an essential protein of the spindle
Immunofluorescence analysis of endogenous and GFP-tagged astrin in HeLa
cells confirmed the results of Mack and Compton and Chang et al.
(Chang et al., 2001;
Mack and Compton, 2001
) and
showed a punctate staining in the cytoplasm of interphase cells and
colocalization with the spindle apparatus in mitotic cells (data not shown).
For RNA interference mediated by duplexes of 21-nt RNAs on HeLa cells, the
published protocol was used (Elbashir et
al., 2001
; Harborth et al.,
2001
). The siRNA sequence for targeting human astrin was from
position 2639 to 2661 relative to the first nucleotide of the start codon.
Control experiments used a siRNA sequence for firefly luciferase (pGL2). Phase
microscopy showed that with time the astrin-siRNA-treated cells became growth
arrested and that an increasing number of cells rounded up. Forty-four hours
after transfection, more than 50% of the cells had rounded up. Indirect
immunofluorescence microscopy was performed at this time using double staining
with affinity-purified rabbit antibodies to astrin and a murine monoclonal
-tubulin antibody (Fig.
4A). Astrin silencing resulted in a strong decrease in astrin
staining. Microtubule distribution and morphology were not affected in
astrin-silenced interphase cells. However, mitotic cells showed a remarkable
phenotype, with aberrant mitotic arrest and multipolar and highly disordered
spindles indicating that astrin function is essential for progression through
the cell cycle. Chromosomes did not congress to the spindle equator and
remained dispersed (Fig. 4A,B).
Staining of astrin-silenced cells with
-tubulin antibody to visualize
centrosomes clearly indicated fragmentation of centrosomes.
-tubulin
was present in all poles of the aberrant multipolar spindles after astrin
depletion (Fig. 4B). Astrin
silencing was also confirmed by immunoblotting experiments. Although cells
transfected with the luciferase siRNA revealed a strong astrin band at 140
kDa, the silenced cells lacked a recognizable signal
(Fig. 4C). The phenotype seen
with astrin siRNA was somewhat similar to that seen after RNAi of the
kinetochore-associated protein CENP-E
(Harborth et al., 2001
).
Ablation of this protein by antisense RNA revealed flattened spindles and
fragmentation of spindle poles, indicating that CENP-E contributes to the
geometry and stability of bipolar spindles
(Yao et al., 2000
). Moreover,
suppression of CENP-E leads to chronic activation of the mitotic checkpoint
machinery, suggesting that CENP-E contributes to checkpoint silencing
(Abrieu et al., 2000
;
Yao et al., 2000
). In the
astrin siRNA-treated cultures, the majority of the cells became apoptotic. A
TUNEL assay performed at 60 hours post-transfection with astrin siRNA showed
that 70% of the cells had entered apoptosis whereas control cells transfected
with luciferase siRNA had hardly any apoptotic cells
(Fig. 5).
|
|
The combined results show that astrin is an essential protein involved in early events of spindle formation at a stage prior to metaphase. Although it is currently not known how astrin contributes exactly to spindle organization, structural data suggest that astrin oligomers may function in microtubule bundling and/or providing a scaffold for crosslinking regulatory proteins to spindle microtubules. Since astrin is expressed not only throughout mitosis but also during interphase, and mRNA expression seems not to correlate with the proliferative activity of a tissue (e.g. relatively high expression in heart, data not shown), we assume that astrin has an additional function in interphase cells that remains to be determined.
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Acknowledgments |
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Footnotes |
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