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Cell Cycle 11:7, 1-14; April 1, 2012; © 2012 Landes Bioscience
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The peptidyl prolyl isomerase cyclophilin A
localizes at the centrosome and the midbody
and is required for cytokinesis
John H. Bannon,† Darragh S. O’Donovan,† Susan M.E. Kennelly and Margaret M. Mc Gee*
School of Biomolecular and Biomedical Science; Conway Institute of Biomolecular and Biomedical Research; University College Dublin; Belfield, Dublin, Ireland
These authors contributed equally to this work.
†
Key words: cyclophilin A, prolyl isomerase, centrosome, cytokinesis, midbody
Abbreviations: CypA, cyclophilin A; Cep55, centrosome protein 55; PPIase, peptidyl prolyl isomerase
Failed cytokinesis leads to tetraploidy, which is an important intermediate preceding aneuploidy and the onset of
tumorigenesis. The centrosome is required for the completion of cytokinesis through the transport of important
components to the midbody; however, the identity of molecular components and the mechanism involved remains
poorly understood. In this study, we report that the peptidyl prolyl isomerase cyclophilin A (cypA) is a centrosome
protein that undergoes cell cycle-dependent relocation to the midzone and midbody during cytokinesis in Jurkat cells,
implicating a role during division. Depletion of cypA does not disrupt mitotic spindle formation or progression through
anaphase; however, it leads to cytokinesis defects through an inability to resolve intercellular bridges, culminating in
delayed or failed cytokinesis. Defective cytokinesis is also evidenced by an increased prevalence of midbody-arrested
cells. Expression of wild-type cypA reverses the cytokinesis defect in knockout cells, whereas an isomerase mutant does
not, indicating that the isomerization activity of cypA is required for cytokinesis. In contrast, wild-type cypA and the
isomerase mutant localize to the centrosome and midbody, suggesting that localization to these structures is independent
of isomerase activity. Depletion of cypA also generates tetraploid cells and supernumerary centrosomes. Finally, colony
formation in soft agar is impaired in cypA-knockout cells, suggesting that cypA confers clonogenic advantage on tumor
cells. Collectively, this data reveals a novel role for cypA isomerase activity in the completion of cytokinesis and the
maintenance of genome stability.
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Introduction
Cytokinesis is the final step in cell division and results in the
separation of two daughter cells. It involves the assembly of an
actomyosin contractile ring, which constricts the plasma membrane and compacts midzone microtubules to form the midbody,
which is located within a thin intercellular bridge that connects
two daughter cells.1-3 Membrane vesicles fuse with the ingressing
plasma membrane to form a new plasma membrane, and abscission of the intercellular bridge finally separates daughter cells.
Failure of cytokinesis leads to tetraploidy and genomic instability, which have been implicated in the onset of tumorigenesis.4-7
The centrosome is a structurally conserved organelle that
consists of a pair of centrioles surrounded by pericentriolar
material.8 The centrosome is the primary site of microtubule
nucleation within the cell, and it orchestrates the formation of
the mitotic spindle.9,10 It also anchors a number of regulatory proteins involved in cell cycle progression and DNA replication.11
Centriolin and centrosome protein 55 (cep55) migrate from the
centrosome to the midbody and regulate the abscission of the
intercellular bridge during cytokinesis through the recruitment
of vesicle trafficking and fusion machinery.12,13 While the recruitment of these components is critical for efficient abscission, the
biochemical events that regulate the final plasma membrane
fusion during abscission are only beginning to emerge.
Cyclophilins are members of a class of ubiquitously expressed
peptidyl-prolyl isomerases (PPIases), which also include the parvulins, the FK-506 binding proteins and the protein Ser/Thr
phosphatase 2A (PP2A) activator PTPA,14-16 which catalyze the
cis-trans isomerization of proline amide bonds. Cyclophilins act
as chaperone proteins and assist in protein folding;17 however,
their true physiological function remains elusive. One family
member, cyclophilin A (cypA), interacts with the receptor tyrosine kinase Itk and modulates its activity18 and with the human
immunodeficiency virus type 1 capsid protein Gag, increasing
viral infectivity.19,20 Additional functions have been attributed to
cypA in apoptosis and in chemotaxis, and cypA is the intracellular
receptor for the immunosuppressive drug cyclosporine A (CsA).21
*Correspondence to: Margaret M. Mc Gee; Email: [email protected]
Submitted: 02/14/12; Accepted: 02/14/12
http://dx.doi.org/10.4161/cc.11.7.19711
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CypA is overexpressed in hematopoietic malignancies, lung, pancreatic and breast cancer, implicating a role in tumorigenesis;22-25
however, the function of cypA during cell growth remains poorly
understood.
The parvulin pin1 specifically catalyzes the isomerization of
proline residues that are preceded by a phosphorylated serine or
threonine26 and regulates cell cycle progression, DNA synthesis,
centrosome duplication and cytokinesis, supporting a role in the
maintenance of genome stability27-29 and raising interest in prolyl
isomerization as a therapeutic target.29
The relationship between prolyl isomerization, malignant
transformation and tumor progression is poorly understood;
however, it is believed that the normal enzymatic function of
PPIases is exploited to promote the growth and survival of tumor
cells.30 In support of that, recent evidence reveals that the cistrans interconversion catalyzed by PPIases regulates important
signaling events, and isomerization catalyzed by Pin1 is critical
for JNK activation in breast cancer cells.31
In this study, we demonstrate a role for cypA in the regulation
of mammalian cell division. We show that cypA is a centrosomal
protein that forms part of the spindle poles during mitosis and
migrates to the midbody during telophase, where it regulates
abscission. The isomerase activity of CypA is not required for
its targeting to the centrosome or to the midbody; however, it
is essential for the timely completion of cytokinesis. Depletion
of cypA leads to cytokinesis defects, including the generation
of tetraploid cells with elevated centrosome number. Thus, our
findings unveil a novel role for phosphorylation-independent
isomerization in the regulation of cytokinesis and the maintenance of genome stability, which may have important implications for the initiation and progression of cancer.
and Jurkat cells. The absence of GAPDH confirms the absence of
contaminating cytosolic proteins (Fig. 1E). Finally, cypA localizes at the centrosome in the absence and presence of nocodazole
or taxol, indicating that it does not require an intact microtubule
network for localization to the centrosome (Fig. 1F).
CypA localizes to the spindle poles during metaphase and
early anaphase and migrates to the midzone and midbody during late anaphase and telophase. Endogenous cypA was monitored during the cell cycle by confocal immunofluorescence and
was found to form part of the spindle poles during metaphase
and early anaphase. During late anaphase, cypA levels are diminished at the spindle poles and become concentrated at the midzone and finally, during telophase cypA becomes concentrated
at the midbody connecting two daughter cells (Fig. 2A). The
midbody staining of cypA resembled that of the centrosome protein cep55.34 To confirm this, Jurkat cells were co-transfected
with GFP-Cep55 and mCherry-α-tubulin to highlight midbody
and intercellular bridge formation during cytokinesis (Fig. 2B,
top part). Consistent with endogenous cypA and cep55, GFPCypA concentrates at the midbody, whereas the empty vector
does not (Fig. 2B, bottom and middle parts). CypA also localizes
to the midbody in H1299 and K562 cells (Fig. 2C). Collectively,
these results illustrate the cell cycle-dependent relocalization of
cypA from the centrosome to the midzone and midbody during
mitosis, suggesting potential sites of action during cell division.
CypA-deficient cells form a bipolar spindle and proceed
through normal anaphase. We investigated a role of cypA in
bipolar spindle formation and the progression through anaphase.
Wild-type and cypA-/- Jurkat cells were probed with antibodies
to detect α-tubulin and pericentrin, which highlight the mitotic
spindle and centrosomes, respectively. DNA was highlighted by
DAPI staining. No apparent defects in mitotic spindle formation
were observed in the two cell lines by confocal immunofluorescence (Fig. 3A). Using phase contrast live cell imaging, the time
from cell elongation during anaphase onset to furrow ingression
during telophase was measured in single cells, and it was found to
be similar in the two cell lines (Fig. 3B). Cells were synchronized
in prometaphase using a low dose of nocodazole (0.14 μM) and
subsequently released. Western blot analysis of whole-cell extracts
isolated at the indicated times after release highlight that the rate
of cyclin B and phosphorylated histone H3 degradation during
mitotic progression was similar in the two cells lines (Fig. 3C).
Cell synchronization was validated by flow cytometry (data not
shown). Extracts from unsynchronized cells represent a population predominantly comprised of interphase cells, where low
levels of cyclin B and phosphorylated histone H3 were detected
(Fig. 3C, lanes 1 and 2). In addition, the spindle assembly checkpoint protein, BubR1 was phosphorylated in the wild-type and
cypA-/- cells at 0 h and underwent dephosphorylation, which correlates with cyclin B and phosphorylated histone H3 degradation, suggesting that the spindle assembly checkpoint is satisfied
in the two cell lines (Fig. 3C). Finally, BubR1 was detected at
the kinetochore in the two cell lines by confocal microscopy following treatment with nocodazole and taxol, which activate the
spindle assembly checkpoint (Fig. 3D). Collectively, this data
illustrates that the spindle assembly checkpoint is functional in
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Results
CypA is a centrosome protein. We have previously reported a
nuclear location for cypA in hematopoietic cells.22 We examined
the subcellular localization of cypA more closely and detected
a prominent single or paired dot-like structure at the nuclear
periphery by high power confocal microscopy that resembled
centrosomal structures (Fig. 1A). Cells were double stained
with antibodies to detect cypA and the core centrosome protein
γ-tubulin, and merged images show co-localization of cypA and
γ-tubulin at the centrosome in H1299 lung carcinoma cells,
chronic myeloid leukemia cells (K562, KYO.1 and Lama84) and
Jurkat T lymphoma cells (Fig. 1A). To confirm the centrosome
localization of cypA, a GFP-tagged full-length cypA construct
(GFP-CypA) was expressed in Jurkat cells that lack cypA, which
was confirmed by western blotting (Fig. 1B), and cells were analyzed by immunofluorescent microscopy. GFP-CypA co-localizes
with the centrosome protein centrin, whereas the GFP-empty
control vector (GFP-E) does not (Fig. 1C). CypA localizes to
a single centrosome in interphase cells and to two centrosomes
immediately following S-phase duplication (Fig. 1D). In contrast,
centrosomal staining of cypA was not detected in Jurkat cells that
stably lack cypA (Fig. 1D). Furthermore, cypA and γ-tubulin
co-migrate in enriched centrosome fractions isolated from K562
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Figure 1 (See previous page). CypA is a centrosome protein. (A) H1299, K562, Lama84, KYO1 and Jurkat cells were prepared on slides and stained
with anti-γ-tubulin (green), anti-cypA (red) and DAPI (blue). (B) Jurkat CypA-/- cells were transfected with GFP-CypA or GFP vector control (GFP-E). GFP
protein expression was confirmed by western blot with anti-GFP. (C) Jurkat CypA-/- cells expressing GFP-CypA and GFP-E were stained with anti-centrin
(red) to visualize centrosomes. (D) Jurkat cells were prepared on slides and stained with anti-γ-tubulin (green), anti-cypA (red) and DAPI (blue). (E)
Centrosome extracts were prepared from K562 or Jurkat cells by fractionation on a discontinuous sucrose gradient and resolved using SDS-PAGE followed by western blotting with anti-cypA, anti-γ-tubulin and anti-GAPDH. (F) Jurkat cells were incubated with vehicle (0.5% (v/v) DMSO), 0.1 μM taxol
or 0.1 μM nocodazole for 24 hr. Samples were prepared on slides and stained with anti-γ-tubulin (green), anti-cypA (red) and DAPI (blue). Arrowheads
indicate co-localization. Bar: 10 μm.
wild-type and cypA-/- cells, and the cells proceed from anaphase
to telophase with similar kinetics.
CypA is required for abscission. The mitotic index of WT
and cypA-/- cells was measured using multivariate flow cytometry
following treatment with nocodazole and taxol. A representative cell profile illustrates that 2.13% of wild-type cells are in
mitosis (contain 4N DNA), and this increased to 9.56% and
17.8% following treatment with nocodazole and taxol, respectively (Fig. 4A, top part). However, while the overall mitotic
index is not significantly different in the cypA-/- cells, there was
a dramatic shift in the mitotic cell population from 4N to an
8N state. Representative data outlined in Figure 4A shows that
1.87% of vehicle-treated cells contain 8N DNA, which increased
to 11.1% and 13.9% following treatment with nocodazole and
taxol, respectively, whereas only 2% of mitotic cells contain 4N
DNA (Fig. 4A, bottom part). The increase in DNA content of
the mitotic population was confirmed in triplicate determinations following treatment with nocodozole and taxol (Fig. 4B).
The ability of nocodazole and taxol to effectively block cells in
prometaphase confirms our earlier finding that cypA-/- cells have
a functional spindle assembly checkpoint. Thus, the polyploidization observed in cypA-/- cells is most likely due to post-anaphase defects in cell division.
A role of cypA during cytokinesis was investigated by monitoring the time from intercellular bridge formation during telophase
to complete abscission of the bridge in single cells transfected
with the midbody marker cep55 (GFP-Cep55) and mCherryα-tubulin to highlight central midbody and intercellular bridge
structures, respectively. Representative images from single Jurkat
cells are shown in Figure 4C. The time from intercellular bridge
formation to final abscission of the bridge is significantly prolonged in cells depleted of cypA. The midbody and intercellular
bridge is undetectable in wild-type cells after 60 min, and two
daughter cells with a complete plasma membrane are formed
within 90 min. However, the midbody and intercellular bridge
persist in cypA-/- cells up to 120 min after initial bridge formation, and the cell eventually divided by 150 min (Fig. 4C and
Movies S1 and S2). A similar delay was detected in K562 cypA
siRNA-transfected cells but was absent from control scramble
transfected cells (Movies S3 and S4). The mean time from bridge
formation to abscission was 84 min and 134 min for wild-type
and cypA-/- Jurkat cells, respectively (Fig. 4D), and 120 min and
280 min for scrambled control and cypA siRNA K562 cells (Fig.
4E). In some instances, the intercellular bridge was not resolved
8 h after telophase onset in the cypA siRNA-knockdown cells.
Consistently, a significant increase in Jurkat and K562 midbody-arrested cells was detected 180 min after release from the
nocodazole block (Fig. 4F–H) that was not detected at 0 min or
60 min. A similar increase in midbody-arrested cells was detected
in an unsynchronized cell population (Fig. 4G). Collectively,
this data suggests that loss of cypA expression in a number of cell
lines leads to delayed cytokinesis through an inability to resolve
the intercellular bridge during abscission.
CypA isomerase activity is not required for localization to
the centrosome or midbody. We investigated the role of cypA
isomerase activity in its localization to the centrosome and midbody. CypA-/- Jurkat cells were transfected with either GFPCep55 as a centrosome and midbody marker, GFP empty vector
control (GFP-E), GFP-CypA or the isomerase defective mutant,
GFP-R55A. Results illustrate that the GFP-R55A mutant localizes to the centrosome in a manner similar to wild-type cypA
and cep55 (Fig. 5A). Furthermore, GFP-R55A concentrated at
the midbody during cytokinesis, similar to wild-type GFP-CypA
and GFP-Cep55 (Fig. 5B). The staining intensity of GPF-R55A
at the centrosome and midbody was similar to wild-type GFPCypA and suggests that the centrosome and midbody localization of cypA in Jurkat cells is not dependent on its isomerase
activity.
CypA isomerase activity is required for abscission. Cis-trans
isomerization is an important regulator of cell cycle progression.35
A role for the isomerization activity of cypA during cytokinesis
was investigated through a series of rescue experiments, whereby
Jurkat cypA-/- cells that undergo delayed cytokinesis were cotransfected with mCherry-α-tubulin and either GFP vector control (GFP-E), GFP-CypA or the isomerase defective GFP-R55A,
and the time from telophase to abscission was measured by live
cell imaging as before. The average time to complete cell division
was approx 130 min for cypA-/- cells, which is consistent with
earlier findings (Fig. 4C). The re-expression of wild-type cypA
reduced the time to complete division to approximately 90 min
(Fig. 5C) and is similar to that measured in the wild-type Jurkat
cells (Fig. 4C). However, transfection of cypA-/- cells with the
R55A mutant failed to rescue the division delay observed, and
the time taken to complete division did not differ significantly
from cypA-/- cells (Fig. 5C). These results outline that the cistrans isomerization catalyzed by cypA in required for the timely
completion of cytokinesis.
Loss of CypA generates tetraploid cells. Failed cytokinesis
is often characterized by bi- or multi-nucleated cells or enlarged
mononuclear cells formed through the fusion of bi-nucleates.6
The loss of cypA expression in Jurkat cells led to a decrease in the
2N DNA population with a concomitant increase in 4N and 8N
polyploid populations, a phenotype consistent with failed mitosis
(Fig. 6A). This was accompanied by a pronounced increase in
mononuclear cells with large nuclei (diameter >10 nm) (Fig. 6B
and C). The number of bi- and multi-nucleated cells was elevated in
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Figure 2. CypA undergoes cell cycle dependent localization to the midzone and midbody. (A) Jurkat cells were prepared on slides and stained with
anti-γ-tubulin (green), anti-cypA (red) and DAPI (blue). (B) Jurkat CypA-/- cells were transfected with mCherry-α-tubulin and either GFP-Cep55, GFP vector control (GFP-E) or GFP-CypA. Transfected cells were subjected to videomicroscopy at 30 sec intervals for 3 hr. (C) H1299 and K562 cells stained with
anti-γ-tubulin (green) and anti-cypA (red). Arrows highlight midbody. Bar: 10 μm.
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Figure 3. Loss of CypA does not abrogate bipolar spindle formation or progression through mitosis. (A) Jurkat WT and CypA-/- cells were prepared
on slides, fixed and stained with anti-pericentrin (red), anti-α-tubulin (green) and DAPI (blue). Representative images from interphase and mitosis
are shown. (B) Jurkat cells were observed by phase contrast live cell imaging. The time from cell elongation at anaphase onset to contraction of the
cleavage furrow during telophase was recorded, n = 20. (C) Jurkat WT and CypA-/- cells were synchronized in prometaphase. Upon release whole cell
lysates were prepared at the indicated times and samples were resolved by SDS-PAGE followed by western blotting with anti-BubR1, anti-cyclin B,
anti-Phospho-Histone H3 (PH-H3) and anti-GAPDH. Unsynchronized whole cell extracts were included as a control. (D) Jurkat WT and CypA-/- cells
were incubated with vehicle (0.5% (v/v) DMSO), 0.1 μM taxol or 0.1 μM nocodazole for 24 hr. Cells were prepared on slides and stained with anti-BubR1
(green) and DAPI (blue). Error bars are SEM. Bars: 10 μm.
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Jurkat and K562 cells following loss of cypA expression (Fig. 6B
and D). Increased ploidy was confirmed by metaphase chromosome counts from WT and cypA-/- Jurkat cells, as shown in
Figure 6E and F. This data reveals that wild-type cells exhibit
a diploid chromosome number, whereas cypA-knockdown cells
display a near tetraploid chromosome number.
CypA deficiency leads to supernumerary centrosomes and
decreased proliferation. Tetraploid cells produced through failed
cytokinesis contain multiple centrosomes that lead to multipolar mitosis and eventual aneuploidy.36 Centrosome number was
examined in wild-type and cypA-/- Jurkat cells in culture. Early
passage cells contained 1 or 2 centrosomes following depletion of
cypA; however, a significant increase in centrosome number was
detected in late passage cells (P + 20) with a concomitant decrease
in the number of cells containing < 2 centrosomes (Fig. 7A). Up
to four centrosomes were detected clustered together in mononuclear cells with large nuclei (Fig. 7B). Jurkat and K562 cells
deficient in cypA also display a significant reduction in their
proliferative capacity compared with wild-type cells in culture
(Figs. 7C and S1), which is consistent with the delay in division detected. Finally, the clonogenic potential of wild-type and
cypA-/- Jurkat cells was tested in colony-forming assays. Wildtype Jurkat cells display clonogenic ability consistent with previous reports in reference 33, and depletion of cypA significantly
reduced colony formation (Fig. 7D), suggesting that overexpression of cypA confers a clonogenic advantage in tumor cells.
and progress through normal anaphase.43 In addition, we have
shown that the spindle assembly checkpoint is functional and
satisfied in the wild-type and cypA-deficient cells, thus enabling
anaphase onset, and cells progress from anaphase to telophase
with similar kinetics. Furthermore, cypA was not detected at
the kinetochore or centromere during anaphase, suggesting that
a function in chromosome assembly or segregation during early
anaphase is unlikely. Therefore, although cypA is present at the
spindle poles, it is dispensable for spindle formation, and cells
lacking cypA proceed through an apparently normal anaphase.
CypA-deficient cells display a dramatic increase in the proportion of mitotic cells that harbor 8N DNA. These cells have
a functional spindle assembly checkpoint and are efficiently
arrested in metaphase by microtubule poisons, and the polyploid phenotype observed is consistent with failed division
after anaphase.44 Consistent with this, cytokinetic defects were
observed in cypA-knockout cells. The predominant phenotype
observed is persistence of the intercellular bridge joining two
daughter cells for extended periods, and the time to complete
division was almost doubled in cypA-knockout cells. This phenotype is similar to that detected following centriolin silencing
and following overexpression of the Cdc14A phosphatase.12,45
Imaging data also revealed that the cleavage furrow and intercellular bridge forms with similar kinetics in the two cell lines.
Impaired cytokinesis was also shown by an increase in midbody-arrested cells in unsynchronized and synchronized cell
populations. Importantly, re-expression of full-length cypA in
knockout cells restored the time to complete division to that
of wild-type cells; however, in contrast, the isomerase defective mutant did not, and cells continue to display persistent
intercellular bridges and prolonged cytokinesis. Collectively,
this data reveals that cypA isomerase activity is required for
the final stage of cell division and supports the recent finding that isomerization of midbody components is an important
regulator of abscission.46 In this study, we also reveal that isomerization activity of cypA is not required for efficient targeting
of cypA to the centrosome and midbody. Further research is
required to provide insight into the structural basis of cypA at
these locations.
Failed cytokinesis results in the formation of bi- and multinucleated cells or enlarged mononuclear cells,47 which was
detected in Jurkat and K562 cells following loss of cypA expression. Enlarged nuclear volume in Jurkat cells correlates with
increased ploidy and is consistent with an increase in 4N and
8N DNA detected and a concomitant decrease in cells with 2N
DNA. Cells with > 8N DNA were not detected, suggesting that
endoreplication did not occur. Increased ploidy and genomic
instability was confirmed in metaphase chromosome spreads,
which revealed that cypA deficiency induces tetraploidization
and is consistent with that observed during cytokinesis failure
following loss of Nek7 kinase activity.47
Genomic instability and tetraploidization is associated with
amplified chromosome number.48 Up to four centrosomes were
detected in late-passage cypA-deficient Jurkat cells with a concomitant decrease in cells with less than two centrosomes. The
proportion of multicentrosomal cells correlates with the number
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Discussion
In recent years, important molecular components of the cytokinetic machinery have been identified that regulate plasma
membrane remodeling and membrane severing.37-39 While the
recruitment of these components is critical for efficient abscission, the underlying mechanism is not completely understood.
Pin1-induced isomerization of cep55 has recently been shown to
facilitate its phosphorylation by polo-like kinase 1 (Plk-l) and
function during abscission, highlighting the importance of isomerization as a mechanism to control cytokinesis.1-3 In this paper,
we report a novel role for phosphorylation-independent cis-trans
isomerization during cytokinesis. CypA is localized to the centrosome during interphase in a range of tumor cells, including
lung carcinoma, leukemia and lymphoma. Centrosome localization of cypA was confirmed by immunofluorescent imaging
of endogenous protein and following the exogenous expression
of a full-length GFP-cypA construct and by co-migration with
the core centrosome protein γ-tubulin in enriched centrosome
fractions. Centrosome localization is independent of a nucleated
microtubule array confirming that cypA is a bona fide centrosome component. CypA is present in two centrosomes following
S-phase duplication and forms part of the mitotic spindle poles
during metaphase. During late anaphase, cypA translocates to
the midzone and the midbody, and its localization resembles
that of cep5534 and other centrosome proteins, including Plk1, centriolin, PTP-BL and LAPSER-1.12,40-42 Loss of cypA does
not abrogate spindle formation in Jurkat cells, which is consistent with reports that acentrosomal cells form mitotic spindles
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Figure 4. (A–C) CypA is required for cytokinesis. Jurkat WT and CypA-/- cells were incubated with vehicle (0.5% (v/v) DMSO), 0.1 μM taxol or 0.1 μM nocodazole for 24 hr and samples were fixed and immunostained with anti-PH-H3 primary antibody followed by AlexaFluor488 secondary antibody and
analyzed by flow cytometry. Representative histograms are shown in (A). Boxes indicate mitotic populations. Mean mitotic indices for Jurkat WT and
CypA-/- are shown in (B), n = 3. Jurkat WT and CypA-/- cells were transfected with mCherry-α-tubulin and GFP-Cep55 and subjected to videomicroscopy
at 30 sec intervals for 3 hr. Representative images up to 150 min are displayed in (C) and the mean time of division is illustrated for Jurkat.
of cells with enlarged nuclei. Given that our data reveals a latestage cytokinesis defect, it is most likely that the multi-centrosomal cells arise due to the continued cycling of binucleate cells
produced from impaired cytokinesis and is not a consequence
of uncontrolled centrosome duplication. In support of this, a
large proportion of the tetraploid cells stained positive for the
phosphorylated histone H3, confirming that they entered mitosis. Tetraploid cells are inherently unstable and contribute to the
tumorigenic process.48 As expected, the centrosome and chromosome abnormalities observed increased after prolonged time in
culture. In addition, the proliferative capacity of cypA depleted
cells was dramatically impaired after 48 h in culture, which may
be a direct result of prolonged or failed cytokinesis. In addition,
while it is clear that Jurkat cells can tolerate a certain level of
ploidy, some tetraploid cells may undergo multipolar mitosis and
subsequent p53-dependent growth arrest and/or cell death which
may also contribute to the decrease in cell number detected.
8
Furthermore, the colony-forming ability of cypA-deficient cells
was significantly impaired under conditions that supported the
colony formation of wild-type cells. This data supports the finding that deregulated cypA confers a growth advantage on tumor
cells and is reminiscent of other cytokinesis regulators, including PLK-1 and Aurora B, which confer clonogenic advantage to
tumor cells.49
In summary, we have characterized the role for cypA during
cell division and have shown that the prolyl isomerization catalyzed by cypA at the midbody is essential for the timely completion of cytokinesis and the maintenance of genomic stability. The
midbody acts as a site for the recruitment of secretory vesicles
and membrane fusion machinery. Thus, cypA may regulate the
functional isomers of important midbody proteins required for
abscission. Interestingly, cypA is an important component of the
retroviral budding machinery, and it co-localizes with Rab24,
implicating a role in vesicular trafficking.50 The identification of
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Figure 4. (D–H) CypA is required for cytokinesis. (D) and K562 (E), n = 10. Jurkat WT and CypA-/- cells were unsynchronized or synchronized in prometaphase. At the indicated time after release cells were stained with α-tubulin (green) and DAPI (blue). Representative images from each time point are
shown (F) and intercellular bridges were quantified for Jurkat (G) and K562 cells (H) in a field of view of ~50 cells, n = 5. Error bars are SEM *p < 0.05, **p
< 0.01. Bars: 10 μm.
cypA substrates at the midbody will provide important insight
into the molecular mechanism of abscission, which has significant implications for tumorigenesis.
Materials and Methods
Cell culture and synchronization. Jurkat, KYO1, Lama84, K562
and H-1299 cells were grown in RPMI-1640 medium containing
10% (v/v) FCS, 2 mM L-glutamine and 100 μg/ml penicillin/
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streptomycin solution. Cells were synchronized in mitosis by
incubating 0.14 μM nocodazole for 16 hrs and released by washing twice in growth medium. Jurkat CypA-/- cells were obtained
through the AIDS Research and Reference Reagent Program
and were created as described previously in reference 22.
Plasmids and transfections. pmCherry-α-tubulin was created by excising the α-tubulin fragment from pEYFP-Tub (BD
Biosciences Clontech) and ligating into pmCherry‑C1 (BD
Biosciences Clontech). pEGFP-CypA-WT/R55A plasmids were
Cell Cycle
9
© 2012 Landes Bioscience.
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Figure 5. Cis-trans isomerization catalyzed by CypA is required for abscission. Jurkat CypA-/- cells were transfected with GFP-Cep55, GFP vector control
(GFP-E), GFP-CypA or GFP-R55A. Cells were prepared on slides and were stained with anti-centrin (red). Colocalization is indicated by a yellow color (A).
Jurkat CypA-/- cells were transfected with mCherry-α-tubulin and either GFP-Cep55, GFP vector control (GFP-E), GFP-CypA or GFP-R55A. Transfected
cells were subjected to videomicroscopy at 30 sec intervals for 3 hr. Representative images are shown (B). Arrows highlight midbody. In (C) expression
of GFP-E, GFP-CypA or GFP-R55A was confirmed by western blot using anti-GFP antibody and GAPDH as a loading control. Average time was calculated from telophase to the abscission of the intercellular bridge, n = 10. Error bars are SEM **p < 0.01. Bar: 10 μm.
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Figure 6. CypA is required for genome stability. (A) Jurkat WT and CypA-/- cells were fixed, stained with PI and DNA content was quantified by flow
cytometry. n = 3. *p < 0.05. Nuclei were evaluated by fluorescent microscopy and the number of nuclei per cell and nuclear size (>10 μm diameter) was
tabulated (B). n = 50. *p < 0.05. Jurkat (WT and CypA-/- cells) and K562 (control scrambled and cypA siRNA KD cells) were prepared on slides, stained
with DAPI and observed using a Zeiss fluorescent microscope. Representative images of Jurkat WT and CypA-/- cells are shown (C). Multinucleated K562
cells are tabulated (D). Metaphase chromosome spreads from WT and CypA-/- cells were observed using a Zeiss fluorescent microscope. Representative
images of WT and CypA-/- cells are shown in (E) and the distribution of chromosomes is shown (F). n = 20. All error bars are SEM. Bar: 10 μm.
created by excising CypA-WT/R55A from pcDNA3.1-CypAWT/R55A, a kind gift from Professor Jeremy Luban, Department
of Microbiology and Molecular Medicine, University of Geneva,
and ligating into or GFP-C1 (BD Biosciences Clontech). pEGFPC3-Cep55 was a kind gift from Professor Kerstin Kutsche, Institut
für Humangenetik, Universitätsklinikum Hamburg-Eppendorf.
All sequences were verified by automated sequencing. Plasmid
www.landesbioscience.com
transfections in Jurkat and K562 cells were performed using the
Amaxa biosystems Cell Line Nucleofector® Kit V.
Antibodies. The following primary antibodies were used:
anti-phospho-histone-H3 (Ser10) (Cell Signaling), anti-cypA
and anti-GAPDH (Millipore), anti-γ-tubulin and anti-β-actin
(Sigma), anti-BubR1 and anti-cyclin B1 (BD PharMingen),
anti-α-tubulin and anti-pericentrin (Abcam), anti-centrin and
Cell Cycle
11
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Figure 7. Loss of cypA leads to supernumerary centrosomes and decreased proliferation. Jurkat WT and CypA-/- cells were prepared on slides and
stained with anti-pericentrin. Centrosome number was counted in early passage (P + 6) and late passage (P + 20) Jurkat cells in a field of 100 cells per
experiment (A), n = 3. Representative images are shown (B). (C) WT and Jurkat CypA-/- cells were seeded (5.5 x 106/50 ml) in complete growth medium
and cell number was determined after 24, 48, 72 and 96 hr. n = 3. (D) Jurkat WT and CypA-/- cells were seeded in soft agar, incubated for 28 d at 37°C,
95% O2 and 5% CO2 and stained using crystal violet. n = 3. **p < 0.01.
anti-GFP (Santa Cruz). Secondary anti-rabbit and anti-mouse
HRP conjugated antibodies (Cell Signaling) were used for western blot, and AlexFluor488 or AlexaFluor594 conjugated antirabbit and anti-mouse secondary antibodies (Invitrogen) were
12
used for imaging and flow cytometry. Anti-FLAG antibody was
from Sigma-Aldrich.
Flow cytometry. Cells were harvested by centrifugation at
450 RCF for 3 min. The cell pellet was resuspended in PBS,
and cells were fixed in ice-cold 70% ethanol at 4°C overnight.
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Cells were centrifuged at 800 RCF for 5 min and resuspended
in PBS containing RNase A (300 μM) and propidium iodide
(PI) (200 μM), and samples were incubated in the dark at 37°C
for 30 min before analysis. Multivariate analysis was performed
using anti-phospho-histone-H3, to label mitotic cells. Cells were
fixed in 70% ethanol, blocked in 2% BSA/PBS and incubated
with primary antibody. AlexaFluor 488-conjugated secondary
antibody (Invitrogen) was used to detect the primary antibody,
and samples were co-stained with PI. PI was detected using a
488:613 band pass dichroic, and AlexaFluor488 was detected
using a 488:525 band pass dichroic on a CyAn ADP Flow
Cytometer. Analysis was performed using Summit v4.3 software.
Immunoblotting and immunofluorescence. Immunoblotting
was performed as described previously in reference 22. Proteins
were visualized by enhanced chemiluminescence solution (Pierce)
and X-ray film (Fujifilm).
For immunofluorescence, fixation and immunostaining were
performed as described previously in reference 32. Cells were
incubated with relevant primary antibodies, which were detected
with Alexa Fluor 488- or 594-conjugated secondary antibodies.
DNA was stained with DAPI (Sigma). Images were acquired
at 63x magnification on a Zeiss 510UV Meta confocal microscope at room temperature. Images were captured using the LSM
Imaging software.
Time-lapse imaging. For phase contrast live cell imaging,
Jurkat cells were transferred to multichambered microscopy
slides (IBIDI), which were coated with poly-l-lysine (Sigma).
Cells were observed using phase contrast at 60x magnification
on a Zeiss microscope with an Andor iXonEM EMCCD camera,
and images were captured every 30 sec for 2 h. Movies were made
using Andor IQ software.
For fluorescent imaging, Jurkat cells were transfected with
fluorescent plasmids as described and transferred to multichambered microscopy slides (IBIDI), which were coated with
poly-l-lysine. Fluorescent proteins were observed using 488 nm
and 594 nm laser excitation at 100x magnification on a Nikon
confocal spinning disc microscope with an Andor iXonEM
EMCCD camera. Images were captured every 30 sec for 3 h at
37°C, 95% O2 and 5% CO2. Movies were made using Andor
IQ software.
Centrosome preparations. Centrosomes were purified from
cells using a sucrose gradient as described previously in reference 33. Ten fractions were collected, and centrosomes were
recovered by centrifugation at 15,000 RCF for 10 min, boiled
in SDS sample buffer and resolved by SDA-PAGE followed by
western blotting.
Colony formation assay. A cell suspension (5 x 105 cells/ml)
was prepared in a liquid 0.7% agarose/growth media solution,
plated onto soft agar as described previously in reference 33
and incubated at 37°C, 95% O2 and 5% CO2. After 28 d, the
samples were fixed and stained with 0.2% crystal violet as outlined. Colonies were photographed using a digital camera and
counted.
Metaphase spread. Cells were incubated with 10 μM
nocodazole for 2 hrs at 37°C, 95% O2 and 5% CO2. Cells were
centrifuged at 450 RCF for 3 min. The pellet was washed in PBS
and resuspended in 900 μl KCl (0.075 μM) and incubated at
37°C for 17 mins. Cells were fixed in 100 μl of cold fixative (3:1,
methanol:acetic acid) and centrifuged at 1,500 RCF for 2 mins.
The supernatant was removed, the pellet was resuspended, and
20 μl of the sample was dropped onto the center of a glass slide
and allowed to dry. The DNA was stained using DAPI. DNA was
visualized using a Zeiss fluorescent microscope and Axiovision
LE 4.8.1 software.
© 2012 Landes Bioscience.
Disclosure of Potential Conflicts of Interest
Do not distribute.
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No potential conflicts of interest were disclosed.
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Financial Support
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