代写留学生论文Centrosomes direct microtubule organization during cell division.
Aberrant number of centrosomes results from alteration ofits components and leads to abnormal mitoses and chromosome
instability. HOPS is a newly discovered protein isolated duringliver regeneration, implicated in cell proliferation. Here, we provideevidence that HOPS is an integral constituent of centrosomes.HOPS is associated with classical markers of centrosomes and
found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1Aand HSP70. These features suggest that HOPS is involved incentrosome assembly and maintenance. HOPS depletion generates
supernumerary centrosomes, multinucleated cells and multipolarspindle formation leading to activation of p53 checkpoint andcell cycle arrest. The presence of HOPS in cytosolic complexessupports that centrosome proteins might be preassembled in thecytoplasm to then be rapidly recruited for centrosome duplication.
Altogether these data show HOPS implication in the control ofcell division. HOPS contribution appears relevant to understandgenomic instability and centrosome amplification in cancer.
Introduction
In animal cell the centrosome is a small organelle formed by two
centrioles surrounded by different proteins indicated as pericentriolar
material (PCM).1,2 The strictly controlled presence of two centrosomes
at the beginning of mitosis guarantees the appropriate formation of
the mitotic spindle.3,4 The centrosome has been hypothesized to
be an essential guardian of cell cycle progression, although this role
has not been clearly defined at the molecular level.2,5 Centrosomes
start duplicating in the late G1/S phase, allowing the formation of
procentrioles. During the S and G2 phases the procentrioles recruit
the PCM proteins until the centrosomes are entirely duplicated.
The mechanism, by which the centrosome is rapidly duplicated
and activated during cell cycle, is presently unclear. Perturbations in
centrosome duplication lead to abnormal amplification of centrosomes,
multipolar spindles and genomic instability.6,7
Remarkably, proteins involved in the nucleus-cytoplasmic transport
play a pivotal role in spindle assembly.8,9 CRM-1 is one of the
most important exportin that, through its binding to the nuclear
export sites (NES) on cargo proteins, allows the export of specific
proteins to the cytoplasm.10 Localization of CRM-1 and Ran in
kinetochores and centrosomes11,12 as its role in mitotic spindle
assembly and centrosome duplication13,14 underlines the importance
of the association between nucleus-cytoplasmic shuttling and mitosis.
Moreover, several proteins shuttled by CRM-1 from the nucleus to
the cytoplasm are associated with the centrosome.11
HOPS (Hepatocyte Odd Protein Shuttling) is a recently identified
protein containing a putative NES and an ubiquitin domain.15
HOPS is overexpressed in residual hepatocytes following partial
hepatectomy and it changes localization in relation to the proliferative
state of the cell. HOPS shuttles from nucleus to cytoplasm via
CRM-1. Overexpression of HOPS induces arrest of proliferation.15
Following the demonstration of HOPS binding to γ-tubulin and
CRM-1, we aimed at unravelling the role of HOPS in centrosome
function. For this, we knocked down HOPS expression by small
interfering RNA (siRNA) in cells. Strikingly, our results show that
inhibition of HOPS expression leads to: (i) abnormal centrosome
amplification; (ii) multinucleated cells accumulation and (iii)
abnormal spindle formation.
Furthermore, we provided evidence that HOPS is associated to
CRM-1, γ-tubulin, eEF-1A and HSP70 in both the centrosome
and cytosolic complexes. Previous investigations suggested that
centrosomal proteins are not assembled de novo in centrosomes,
but they are associated in cytosolic complexes that can be rapidly
recruited for centrosome duplication.16
Our results assign to HOPS an important role in centrosome
duplication and maintenance of genomic stability.
Results and Discussion
HOPS in cell cycle. HOPS has been described as a protein
involved in liver regeneration.15 To analyze HOPS function during
cell proliferation, we studied HOPS localization and expression
throughout the cell cycle. Immunohistochemical analyses showed
that HOPS colocalizes with γ-tubulin, in H35 and NIH-3T3 cells,
specifically during the G0/G1 phase, but not during the S and G2/M
phases (Fig. 1A; Suppl. Fig. 1 online). Western blot analyses showed
a reduction of 50% (densitometric analysis, data not shown) of
HOPS expression in S-phase with respect to G2/M phase in cells
arrested by aphidicolin and nocodazole treatments, respectively
*Correspondence to: Giuseppe Servillo; Dipartimento di Medicina Clinica e
Sperimentale; Policlinico Monteluce; Perugia 06122 Italy; Tel.: 39.075.5720655;
Fax: 39.075.5726803; Email: [email protected]
Submitted: 02/21/08; Accepted: 03/07/08
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/article/5882
Report
HOPS is an essential constituent of centrosome assembly
Stefania Pieroni,1,† Maria Agnese Della Fazia,1,† Marilena Castelli,1 Danilo Piobbico,1 Daniela Bartoli,1 Cinzia Brunacci1,
Marina Maria Bellet,1 Mariapia Viola-Magni1 and Giuseppe Servillo1,*
Università degli Studi di Perugia; Dipartimento di Medicina Clinica e Sperimentale; Policlinico Monteluce; CEMIN; Perugia Italy
†These authors have contributed equally to this work.
Key words: centrosome, HOPS, cell cycle, CRM-1, mitotic spindle
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Running title
www.landesbioscience.com Cell Cycle 1463
(Fig. 1B). This is in support of the rapid decrease of HOPS
expression observed during the S-phase of cells blocked in
G0/G1 and released from the arrest.15 These data suggest a
dynamic participation of HOPS in the cell cycle.
HOPS is localized in centrosomes. Next, we performed
immunoprecipitation assays using H35 cell extracts to investigate
HOPS interaction with γ-tubulin. Interestingly, HOPS
antibody were able to immunoprecipitate γ-tubulin from
total cell lysates (Fig. 1C), indicating a specific interaction
between these two proteins.
Centrosomes were then isolated by a discontinuous
sucrose gradient from extracts of H35 cells treated with
aphidicholin/nocodazole.17 Fractions of centrosome preparations
analyzed by western blot revealed cosedimentation of
HOPS, γ-tubulin and CRM-1 in the same fractions (Fig.
1D). The association of centrosome proteins with HOPS, in
cells treated with nocodazole, strongly suggests that HOPS is
a centrosomal protein.
HOPS depletion leads to an abnormal centrosome
amplification. To assess the role played by HOPS in the
centrosome function, we performed siRNA experiments in
NIH-3T3 cells. Hops-siRNA treated cells were analyzed at 24,
48 and 96 hours after transfection. HOPS-depleted cells were
compared to untreated cells and scramble-siRNA transfected
cells. HOPS siRNA reduced HOPS expression by 70% while
control cells did not show any reduction (Fig. 2A and B).
Following HOPS silencing, we evaluated whether this
perturbation might interfere with centrosome formation.
To do this, we counted the number of centrosomes per cell
using γ-tubulin (Fig. 2C). In addition, to assess a centrosome
hyperamplification, we used two other centrosome markers,
centrin and pericentrin (Fig. 2D; Suppl. Fig. 2 online).
Centrosome’s amplification gives rise to cytokinesis failure,
errors in chromosomes segregation and aberrant mitosis.6,7
Interestingly, while 24 hours after HOPS depletion no effects
on centrosomes were observed, 48 and 96 hours later we
found a centrosome amplification as demonstrated by the
number of cells with more than two centrosomes which rose to 23%
and 21%, respectively. Untreated and scramble-siRNA transfected
cells displayed the same number of centrosomes as at 24 hours (10%
and 12%) (Fig. 2C).
We next analyzed the presence of multinucleated cells and
multipolar spindles formation. 96 hours after HOPS silencing,
multinucleated cells represented 7% with respect to 0.9% in control
cells. Immunofluorescence analyses showed that Hops-siRNA treated
cells exhibited abnormal cell division, resulting in the formation of
binucleated or multinucleated cells (Fig. 2E and F).
Daughter cells derived from cells with abnormal spindle are
aneuploid and present genomic instability.7,18,19 Thus we analyzed
spindle alteration and cell division arrest in Hops-siRNA transfected
cells versus untreated cells (Fig. 3A). Notably, the analysis of HOPSsilenced
cells exhibited a significant increase of multipolar spindle
number with respect to the control. In untreated cells multipolar
spindles represent 4.5%, while in Hops-siRNA treated cells this
number was dramatically increased from 8.6% at 24 hours to 23,7%
at 48 hours, than it decreased to 6% at 96 hours (Fig. 3B).
In these cells the number of mitosis counted at 48 and 96 hours
was 0.6% and 0.02%, respectively. Mitotic figures in untreated cells
and scramble-siRNA transfected cells were estimated at 1.3% at 48
and 96 hours (Fig. 3C). Remarkably, the increase of multipolar
spindles in HOPS depleted cells was independent from the number
of mitotic figures. These results show that HOPS depletion leads to
a striking arrest of cell proliferation.
Importantly, HOPS depleted cells arrested their proliferation in
the G0/G1 phase (62.7%) with a substantial reduction of cells in
G2/M phase (4.9%) 96 hours after silencing (Fig. 3D). Recently, it
has been described that the lack of proteins involved in centrosome
assembling activates p53-dependent cell cycle checkpoint, arresting
cells in G1 phase.20,21 To verify whether silencing of HOPS leads to
activation of the cell cycle checkpoint, we analyzed the expression of
p53 and p21 in Hops-siRNA treated cells. Overexpression of p53 was
observed 24 hours after HOPS depletion and progressively decreased
afterwards at 48 and 96 hours. p53 activation in turn induced
p21, which became overexpressed at 48 and 96 hours after HOPS
silencing (Fig. 3E).
Taken together, these observations reveal that HOPS depletion
leads to defects in centrosome assembly, with an increase of
centrosomes’ number, formation of multinucleated cells and multipolar
spindles. These alterations very likely might lead to errors during
Figure 1. HOPS localization throughout cell cycle and in centrosomes. (A) H35
cells were stained with HOPS and γ-tubulin antibodies in different phases of cell
cycle. Bar is 10 μm. (B) Western blot analysis of H35 cells arrested in S and G2/M
phases by aphidicholin/nocodazole treatments. CyclinA and Ser10 phosphorylated
H3 histone (H3S10ph) antibodies were used as S phase and G2/M phase control
respectively. β-tubulin antibody was used as loading control. c: untreated cells. (C)
HOPS coimmunoprecipitation with γ-tubulin antibody in H35 cells. Total lysate was
immunoprecipitated with HOPS antibody (IP) and preimmune serum (PS). Resulting
immunocomplexes were tested by immunoblotting using γ-tubulin antibody. c: total
protein extracts. (D) Centrosome isolation from H35 cells. Immunoblot analysis on
gradient resulting fractions using HOPS, γ-tubulin, CRM-1 antibodies. Lamin B receptor
(LBR) and H3S10ph antibodies were used as centrosome purification controls. c:
total protein extracts.
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
HOPS function in centrosome assembly
1464 Cell Cycle 2008; Vol. 7 Issue 10
cell division, resulting in aberrant genome segregation that in
turn drives the cell towards mitotic arrest.
Similar results have been obtained using a different oligoduplex
for HOPS siRNA (data not shown).
HOPS in the cytosolic complex. The description of a
cytosolic complex as putative precursor of centrosome in
which γ-tubulin is associated with eEF-1A and HSP70 in
vitro16 and the interaction of HOPS with eEF-1A in vivo15
prompted us to examine the possibility that HOPS might
be a component of this complex.
Cytosolic extracts from hepatoma cells fractionated
by a 40–10% continuous sucrose
gradient, revealed a cosedimentation of HOPS
with γ-tubulin, eEF-1A and HSP70 in several
fractions (Fig. 4A, fractions 6 to 9). The previously
observed association of HOPS with CRM-1
and γ-tubulin in centrosomes led us to investigate
the presence of CRM-1 with HOPS also in
cytosolic complexes. Interestingly, we revealed
CRM-1 in the same fractions also containing
HOPS, γ-tubulin, eEF-1A and HSP70 (Fig.
4A). Furthermore, to better characterize binding
between HOPS and CRM-1, we performed
coimmunoprecipitation of HOPS with CRM-1
in the cytosolic complex (Fig. 4B).
Our results show that HOPS binds to CRM-1
and together they constitute a complex with
γ-tubulin, eEF-1A and HSP70 (Fig. 4B). The
identification of cytosolic complexes containing
HOPS suggests that essential proteins for
centrosome duplication and spindle formation
might be assembled in the cytosol where they
might become rapidly available for centrosome
arrangement and function. A cytoplasmic form
of γ-tubulin, associated in a complex, as precursor
for centrosome assembly has been described.22-24
This might result into a rapid recruitment of
γ-tubulin in the centrosome at the onset of
mitosis.25
The organization of a preassembled complex
containing γ-tubulin, HOPS, CRM-1, eEF-1A
and HSP70 in cytosolic fractions prompted us
Figure 2. HOPS depletion in NIH-3T3 cells. (A) Cells were transfected
with Hops-siRNA and scramble-siRNA and analyzed after 24,
48 and 96 hours from depletion. Western blotting was performed
using HOPS antibody. β-tubulin antibody was used as loading
control. (B) QPCR on Hops-siRNA interferred NIH-3T3 cells. Bars
indicate relative quantity of mRNA referred to control sample.
A.U. represents arbitrary unit expression referred to control. (C)
Diagram in bars showing percentage of cells containing more
than two centrosomes. (D) Immunofluorescence staining in HOPS
depleted cells with γ-tubulin and centrin antibodies. A larger inset
of centrosome is shown in box. Bar is 10 μm. (E) Double immunofluorescence
staining in HOPS depleted multinucleated cells using
γ-tubulin antibody and DAPI for nuclear staining. Bar is 10 μm. (F)
Diagram in bars showing percentage of multinucleated cells. In
diagrams: white bars indicate untreated cells, grey bars indicate
scramble-siRNA transfected cells, black bars indicate Hops-siRNA
depleted cells.
Figure 3. HOPS depletion and cell cycle modification. (A) Tri-, tetra- and n-polar spindles images
obtained by immunofluorescence staining of Hops-siRNA treated cells using γ-tubulin and α-tubulin
antibodies. (B) Percentage of abnormal spindles per mitosis at different times following HopssiRNA
treatment. (C) Percentage of mitotic figures. White and grey bars indicate controls: untreated
cells and cells transfected with scramble-siRNA. Black bars indicate depleted cells transfected
using Hops-siRNA. (D) Cell cycle profiles of HOPS depleted NIH-3T3 cells by FACS analysis. Bars
indicate the percentage of cells in each mitotic cycle phase. Grey, white and black bars indicate
G0/G1, S and G2/M phases respectively. (E) Western blot analysis on HOPS depleted cells using
p53 and p21 antibodies.
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HOPS function in centrosome assembly
www.landesbioscience.com Cell Cycle 1465
to investigate whether all of these components are assembled in
centrosomes.
Western blot analyses performed on centrosome enriched fractions
showing cosedimentation of HOPS, CRM-1 and γ-tubulin
(Fig. 1A), were found to also contain eEF-1A and HSP70, thus
generating a protein macrocomplex of high molecular weight (Fig.
4C, lanes 6 to 11; Suppl. Fig. 3 online). These results support the
presence of a previously assembled cytosolic complex that might act
as centrosome precursor.
The mechanisms by which precursor complexes of centrosomes
are recruited during cell duplication have still to be revealed. It has
been proposed that cytosolic complexes are targeted to centrosome
as precursors,24,25 alternatively the cytosolic complex can regulate
centrosome assembly by retaining precursors until centrosome
duplication.26 It has been demonstrated that centrosome proteins
are present in cytosolic pools and that treatment with cycloheximide
does not affect centrosome duplication.27,28
Here, we present the first evidence that HOPS is an essential
protein important for centrosome assembly. Knockdown of HOPS
leads to hyperamplification of centrosomes, multinucleated cells
and multipolar spindles. This in turn results to cell cycle arrest and
activation of p53-dependent cell cycle checkpoint. We demonstrate
that HOPS is present in cytosolic fractions with well characterized
centrosome proteins and that this complex might play a role in
centrosome assembly. Future studies will be aimed to identify the
mechanisms by which HOPS is recruited and assembled from the
cytosol to the centrosome and to elucidate its function during cell
division.
Materials and Methods
Cell culture and cell cycle synchronization. H35 hepatoma cells
and NIH-3T3 mouse immortalized fibroblasts were maintained and
synchronized as previously described.15
Immunofluorescence assays. H35 hepatoma cells, NIH-3T3
and HOPS-depleted-NIH-3T3 cells were fixed and stained using
anti-HOPS, anti-γ-tubulin, anti-α-tubulin (Sigma Aldrich), antipericentrin
(Santa Cruz Biotechnology), anti-centrin (kind gift of
Prof. J.L.Salisbury) antibodies. Antibodies binding was revealed by
incubation with appropriate secondary antibodies: Alexa Fluor® 488
FITC-conjugated anti-mouse IgG and Alexa Fluor® 568 Texas-Red®
-conjugated anti-rabbit IgG (Molecular Probes™). Nuclei were
stained with DAPI (Sigma Aldrich). Images were captured with a
Zeiss Axioplan fluorescence microscope controlled by Spot-2 cooled
camera (Diagnostic Instruments).
Coimmunoprecipitations. H35 hepatoma cells were lysed in
RIPA buffer with proteolytic inhibitors (Sigma-Aldrich). 1 mg of
whole cell lysate was immunoprecipitated with rabbit polyclonal
anti-HOPS antibody as previously described.15 Immunoprecipitation
products were probed by immunoblot analysis using mouse monoclonal
anti-γ-tubulin antibody.
Anti-HOPS reacting cytosolic fractions from sucrose gradients
were pooled, dialyzed and subjected to immunoprecipitation with
rabbit polyclonal anti-HOPS antibody. Immunoprecipitation products
were probed using mouse monoclonal anti-CRM-1 antibody.
Centrosome isolation. Centrosomes were isolated from H35
hepatoma cells as described.17 In brief, exponentially growing cells
were subjected to cytochalasin D and nocodazole (Sigma-Aldrich)
treatment and lysed. Centrosomes were harvested by centrifugation
onto a 60% sucrose cushion and centrosomal complexes were further
purified by centrifugation through a discontinuous (70%, 50% and
40%) sucrose gradient. Fractions were collected and analyzed.
Cytosolic fractions preparation. H35 hepatoma cells were grown
to 80% confluence and then arrested in a state resembling late
prophase/early metaphase by aphidicolin treatment (Sigma Aldrich)
for 16 hours. Cells were washed and incubated for additional 7 hours
in fresh medium and then subjected to nocodazole treatment (Sigma
Aldrich) for 16 hours. Citosolic complexes were obtained from H-35
cells as previously described16 and loaded onto a continuous sucrose
gradients from 40 to 10%. Resulting fractions were collected and
analyzed.
Immunoblot analysis. H35 hepatoma cells lysate was probed
using anti-HOPS antibody. Anti-cyclinA (Santa Cruz Biotechnology)
and anti-H3S10ph (Ser10 phosphorylated H3 histone) were used as
G2/M and S phase specific markers respectively.
HOPS-depleted-NIH-3T3 cells were lysed at different times
from transfection and probed using anti-HOPS antibody, anti-p53
(Santa Cruz Biotechnology) and anti-p21 (Dako) antibodies. Anti-
β-tubulin (Sigma Aldrich) was used as loading control.
Sucrose gradient fractions containing γ-tubulin cytosolic complex
and centrosomes were tested by immunoblot analysis using anti-
HOPS, anti-CRM-1 (Becton Dickinson), anti-γ-tubulin (Sigma
Aldrich), anti-eEF1A, anti-HSP70 antibodies (Abcam). All the hybridizations
were detected by chemiluminescence ECL™ (Amersham).
Figure 4. HOPS in the cytosol and in the centrosome. (A) Western blotting
on the cytosolic fractions using HOPS, γ-tubulin, CRM-1, HSP70 and eEF-
1A antibodies. c: total protein extract. (B) Immunoprecipitation on pooled
fractions from 20 to 15% sucrose density (lanes 6–9 Fig. 4A) with HOPS
antibody (IP) and preimmune serum (PS). Western blot analysis on immunoprecipitates
using anti-CRM-1 antibody. (C) Immunoblot analysis on the
centrosome fractions using HOPS, γ-tubulin, CRM-1, HSP70 and eEF-1A
antibodies. c: total protein extracts. LBR and H3S10ph antibodies were used
as centrosome purification controls.
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
HOPS function in centrosome assembly
1466 Cell Cycle 2008; Vol. 7 Issue 10
HOPS depletion by siRNA. Hops-siRNA Stealth™ oligoduplex
(target 5'-gctaggagacgacactcagacacta-3') and scramble-siRNA
oligoduplex
(medium GC content) (Invitrogen) as negative control
were transfected into NIH-3T3 cells according to the manufacturer’s
instructions. To efficiently deplete HOPS, cells were subjected to a
second round of transfection performed 48 hours after the first one.
Cells were harvested 24, 48 and 96 hours after the first transfection
and processed for further analyses. Data are an average of three separate
experiments, shown as mean ± s.d.
Quantitative PCR. Total RNA was isolated from controls and
HOPS-depleted-NIH-3T3 cells as previously described.29 3 μg of total
RNA were retrotranscribed using RevertAid™ H Minus M-MuLV
Reverse Transcriptase (Fermentas) and random hexamer primers.
Real-Time PCR amplifications were performed using Mx3000P™
Real Time PCR System using Brillant® SYBR® Green QPCR Master
Mix (Stratagene) and ROX as reference dye. Hops specific primers
were: Forward 5'-TGCTTGCTTGCCTTCTGG-3', Reverse
5'-TGTGCTGGTGTTGTGGTC-3' (Invitrogen). Housekeeping
control was mouse HPRT (Hypoxanthine Phosphorybosiltransferase)
gene.
All the experiments were performed in triplicate.
Acknowledgements
We thank E. Borrelli, P.G. Pelicci, S. Brancorsini, E. Ayroldi for
the fruitful suggestion and discussion of the paper. We thank J.L.
Salisbury for the kind gift of anti-centrin antibody. We also thank
S. Pagnotta for the technical assistance. This work was supported
by grants from Associazione Italiana Ricerca sul Cancro (AIRC),
Fondazione Guido Berlucchi, Ministero della Università e Ricerca
(MiUR) prot. 2006061141_003 and Fondazione Cassa di Risparmio
di Perugia.
Note
Supplementary materials can be found at:
www.landesbioscience.com/supplement/PieroniCC7-10-Sup.pdf
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