Nuclear Transport of Parathyroid Hormone (PTH)-Related Protein Is Dependent on Microtubules
Mark H. C. Lam1,
Rachel J. Thomas1,
Kate Lakoski Loveland,
Steven Schilders,
Min Gu,
T. John Martin,
Matthew T. Gillespie and
David A. Jans
Nuclear Signaling Laboratory, Division of Biochemistry and
Molecular Biology (M.H.C.L., D.A.J.), John Curtin School of Medical
Research, Canberra, ACT 2601; Department of Biochemistry and Molecular
Biology (M.H.C.L., D.A.J.), Monash University, Clayton, Victoria 3168;
St. Vincents Institute of Medical Research (R.J.T.,
T.J.M, M.T.G.), Fitzroy, Victoria 3065; Institute of Reproduction
and Development (K.L.L.), Monash Medical Centre, Clayton, Victoria
3168; Centre for Micro-Photonics (S.S., M.G.), School of
Biophysical Sciences and Electrical Engineering, Swinburne
University of Technology, Hawthorn, Victoria 3122, Australia
Address all correspondence and requests for reprints to: Professor D. A. Jans, c/o Nuclear Signaling Laboratory, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia. E-mail: David. Jans{at}med.monash.edu.au
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ABSTRACT
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PTH-related protein (PTHrP) was first discovered as a circulating
factor secreted by certain cancers and is responsible for the syndrome
of humoral hypercalcemia of malignancy induced by various tumors. The
similarity of its N terminus to that of PTH enables PTHrP to share the
signaling properties of PTH, but the rest of the molecule possesses
distinct functions, including a role in the nucleus/nucleolus in
reducing apoptosis and enhancing cell proliferation. PTHrP nuclear
import is mediated by importin ß1. In this study we use the technique
of fluorescence recovery after photobleaching to demonstrate the
ability of PTHrP to shuttle between cytoplasm and nucleus and to
visualize directly the transport of PTHrP into the nucleus in living
cells. Endogenous and transfected PTHrP was demonstrated to
colocalize with microtubule structures in situ using
various high-resolution microscopic approaches, as well as in in
vitro binding studies, where importin ß1, but not importin
, enhanced the microtubular association of PTHrP with microtubules.
Significantly, the dependence of PTHrP nuclear import on microtubules
was shown by the inhibitory effect of pretreatment with the
microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux.
These results indicate that PTHrP nuclear/nucleolar import is dependent
on microtubule integrity and are consistent with a direct role
for the cytoskeleton in protein transport to the nucleus.
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INTRODUCTION
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CONVENTIONAL NUCLEAR LOCALIZATION sequence
(NLS)-mediated nuclear protein import initially involves the
recognition of NLS-bearing proteins by the importin
/ß1
heterodimer, followed by the docking of the complex to the nuclear pore
and energy-dependent translocation into the nucleus mediated by the
monomeric GTP-binding protein Ran and regulatory factors
(1, 2, 3). Recent progress suggests a plethora of additional
but analogous Randependent nuclear transport pathways mediated by
homologs of importin ß1 (4, 5, 6, 7, 8), with the latter itself
able to recognize particular nuclear transport substrates. These
include the T cell protein tyrosine phosphatase (9), the
yeast transcription factor GAL4 (10), the viral gene
products Rev (11, 12) and Rex (13) of human
immunodeficiency virus-1 and the human T cell leukemia virus-1,
respectively, cyclin B1 (14), and the polypeptide ligand
PTH-related protein (PTHrP) (15).
PTHrP was initially discovered as a circulating factor secreted by
certain cancers and is responsible for the syndrome of humoral
hypercalcemia of malignancy (16). In normal postnatal
mammals, PTHrP has not been shown to function as a hormone but is
expressed in many normal tissues where it exerts autocrine/paracrine or
intracrine actions (16, 17, 18, 19, 20, 21). Resemblance to PTH at the
amino terminus is sufficient to confer functions similar to those of
PTH, which are mediated by the shared PTH/PTHrP receptor and adenylate
cyclase activation, such as the promotion of bone resorption and
reduction of renal calcium excretion (16, 22). Other
roles, such as the regulation of placental calcium transport to the
fetus (16, 22, 23), osteoclast inhibition
(24), and the control of cell growth and apoptosis
(17, 25), have been ascribed to distinct regions of
PTHrP.
Apart from being expressed in a cell cycle-dependent manner (26, 27), PTHrP localizes conditionally to the nucleus/nucleolus at
G1 (28) with regulation of PTHrP
subcellular localization mediated through phosphorylation by the
cyclin-dependent kinases p33cdk2 and
p34cdc2. A key phosphorylation site in regulating
PTHrP nuclear localization appears to be T85
(28), in the vicinity of an SV40 large T antigen-like NLS
(PGKKKKGK93). Intriguingly, PTHrP amino acids
6794, comprising this NLS and amino-terminal flanking regions, are
recognized with nanomolar affinity by the nuclear transport factor
importin ß1 rather than by the conventional NLS-binding importin
subunit (15), and PTHrP nuclear import in vitro
is able to be mediated by importin ß1 and the monomeric GTP-binding
protein Ran in the absence of importin
. The importance of this
nuclear import pathway is indicated by the fact that deletion of the
basic residues of the NLS results in complete cytoplasmic localization
of PTHrP and concomitant impaired PTHrP-conferred resistance to
apoptosis on the part of transfected CFK2 chondrocytes
(17). Nuclear PTHrP correlates with an increase in
mitogenesis in vascular smooth muscle cells (20) and
enhanced IL-8 expression in prostate cancer cells (29).
Nuclear/nucleolar uptake of PTHrP is also observed subsequent to
internalization of extracellular ligand by osteogenic sarcoma cells
expressing the shared PTH/PTHrP receptor (PTH1R) (15).
In the present study, we examine nuclear transport of PTHrP in living
cells using a PTHrP-green fluorescent protein (GFP) fusion protein and
the technique of fluorescence recovery after photobleaching (FRAP). We
show that PTHrP can shuttle in both directions between cytoplasm and
nucleus and can resolve the nuclear import process temporally. We
demonstrate PTHrP association with ß-tubulin in situ using
several microscopic approaches, as well as in vitro, using
taxol-polymerized microtubules where, intriguingly, binding of PTHrP
was enhanced in the presence of importin ß1. Importantly,
cells pretreated with the microtubule-disrupting agent nocodazole
show significantly reduced fractional return of nuclear and nucleolar
fluorescence in FRAP experiments, indicating that PTHrP
nuclear/nucleolar transport is dependent on microtubule integrity.
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RESULTS
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FRAP to Study Nuclear-Cytoplasmic Flux
To characterize the intracellular localization and transport of
PTHrP in living cells, a plasmid (pSMR792) was constructed for
expression of a GFP-PTHrP (amino acids 1141) fusion protein
(GFP-PTHrP, approximately 45 kDa) lacking the prepro region
(-36 to -1) to prevent secretion of the molecule (15);
this form is comparable to the nonsecreted form of PTHrP generated
through an alternate transcriptional start site (30).
Forty-eight hours after transfection, subconfluent UMR 106.01
osteogenic sarcoma cells consistently showed nuclear/nucleolar as well
as cytoplasmic localization of GFP-PTHrP when visualized by confocal
laser scanning microscopy [CLSM (Fig. 1A
, top left panel)]. The
steady state level of accumulation within the nucleus (Fn/c) and
nucleolus (Fnu/c) relative to the cytoplasm was 5.54 ± 1.0 and
6.92 ± 1.29 (n = 20), respectively. In contrast, analysis of
a control construct of GFP alone [27 kDa (Fig. 1A
, bottom left
panel)] yielded a nuclear to cytoplasmic fluorescence ratio
(Fn/c) of 1.13 ± 0.04 (n = 5), indicative of equilibration
of the protein between nuclear and cytoplasmic compartments through
simple diffusion.

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Figure 1. Results for FRAP of UMR 106.01 Cells Expressing
GFP-PTHrP or GFP Alone
A, Visualization using CLSM of the recovery with time of fluorescence
into the nucleus after photobleaching, performed as described in
Materials and Methods, for GFP-PTHrP
(top) and GFP (bottom). A movie of the
former experiment is available at
http://jcsmr.anu.edu.au/dbmb/jans/lam/frap/ (Fig. 1 .avi).
B, Quantitation of the recovery with time of fluorescence into the
nucleus/nucleolus after photobleaching for GFP-PTHrP (top
panels) or GFP (bottom panels) expressed in
terms of specific nuclear (Fn) or nucleolar (Fnu) fluorescence
(left panels), or fractional recovery of Fn, Fnu, or Fc
(cytoplasmic fluorescence) (right panels).
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To compare intracellular movement of GFP-PTHrP to that of GFP as a
control, the nucleus of the cell was bleached using the confocal laser
at high energy, and the return of fluorescence was then monitored at
lower laser energy every 20 sec for up to about 8 min. The recovery of
fluorescence to the nucleus/nucleolus of GFP-PTHrP and GFP through
transport from the cytoplasm, after the initial pre-/postbleach images
is shown in Fig. 1A
. Representative quantitative data are shown in Fig. 1B
, indicating that the average fractional return of PTHrP to the
nucleus and nucleolus was 52.6 ± 3.1 and 61.4 ± 4.1%
(n = 19), respectively, with t1/2 values of
21.6 ± 3.1 and 23.8 ± 3.4 sec (n = 19), respectively.
When photobleaching of a volume of the cytoplasm equivalent to that in
the bleach of the nucleus was performed, fluorescence due to GFP-PTHrP
was observed to relocate to the cytoplasm with a consequent fall in
nuclear and nucleolar fluorescence (data not shown); the rate of export
from the nucleus was about 3-fold slower than that of nuclear import
(see also Ref. 42). Experiments on cells expressing GFP
alone (e.g. Fig. 1B
, bottom panels) indicated
almost 5-fold slower rates of return of nuclear fluorescence
(t1/2 of 106 ± 8.3, n = 3),
representing the difference between simple diffusion (GFP) and
NLS-facilitated transport (GFP-PTHrP). Together, these data imply that
PTHrP fluxes continuously between the nucleus and cytoplasm, with
transport in the import direction being faster (42).
Localization of PTHrP to the Microtubule Network
Although we have previously observed colocalization of PTHrP with
cytoskeletal structures, we had not been able to demonstrate
colocalization with actin filaments (28). Accordingly, we
used high-resolution fluorescence imaging using two-photon laser
excitation (31) to re-examine GFP-PTHrP subcellular
localization, results clearly indicating that, in addition to
predominant nucleolar and nuclear localization, cytoplasmic PTHrP was
observed associated with distinct filamentous structures (Fig. 2
). Because two-photon excitation is not
ideally suited to colocalization studies, we used more conventional
CLSM and coimmunofluorescence to determine whether the filamentous
structures with which PTHrP appeared to associate were microtubules.
Coimmunofluorescence of endogenous PTHrP and microtubules (Fig. 3A
), as well as colocalization of
GFP-PTHrP expressed in transiently transfected cells with microtubules
detected using an anti-ß-tubulin antibody and Texas Red-labeled
secondary antibody (Fig. 3B
), was performed; results from both
approaches supported the idea that PTHrP colocalized with microtubules.
Predominant PTHrP localization in the nucleus/nucleolus was evident, as
was the localization of cytoplasmic PTHrP to filamentous,
microtubule-like structures (Fig. 3
); colocalization with ß-tubulin
was clearly evident when green and red channel
images were merged (Fig. 3
, right panels). Studies (Fig. 3B
, middle panels) performed using cells pretreated with
nocodazole, a specific microtubule-disrupting agent that binds to
microtubule subunits and prevents heterodimers from repolymerizing,
indicated that concomitant with the disruption of microtubule
structure, filamentous localization of cytoplasmic GFP-PTHrP was absent
(Fig. 3B
; middle panels); limited colocalization in the
GFP/Texas Red-merged image (not in filaments; Fig. 3B
, middle
right panel) was observed, consistent with the association of
PTHrP with depolymerized ß-tubulin. Control experiments using cells
transfected to express GFP (Fig. 3B
, bottom panels)
indicated a lack of filamentous structure localization and lack of
colocalization with ßtubulin (Fig. 3B
, bottom right
panel); thus, these results strongly implicated the specific
nature of PTHrP association with microtubules.

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Figure 2. Visualization of Subcellular Localization of
GFP-PTHrP in UMR 106.01 Transfected Cells Using Two-Photon Excitation
(100x Objective)
Imaging was performed as described in Materials and
Methods section.
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Figure 3. Colocalization of Endogenous and Transfected PTHrP
with Microtubules
A, Immunolocalization of endogenous PTHrP in UMR 106.01 cells was
performed using a rabbit anti-PTHrP antibody (Oregon Green-labeled
secondary antibody), whereas ß-tubulin was detected by
immunolocalization using a mouse anti-ß-tubulin antibody
(rhodamine-labeled secondary antibody). PTHrP (green) is
shown in the left panel, ß-tubulin
(red) in the middle panel, and merged
images on the right with colocalization in
yellow. Magnification, 2,500x. B, UMR 106.01 cells
transfected with GFP-PTHrP (top and
middle panels) or GFP (bottom panels)
were left untreated (top and bottom
panels), or treated with 10 µg/ml nocodazole for 1 h
(middle). ß-Tubulin was detected by immunolocalization
using a mouse anti-ß-tubulin antibody (rhodamine-labeled secondary
antibody). PTHrP-GFP (green) is shown in the
center panels, ß-tubulin (red) in the
left panels, and merged images on the
right with colocalization in yellow.
Magnification, 1,200x.
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To confirm these observations, transiently transfected GFP-PTHrP
expressing UMR 106.01 cells were microinjected with rhodamine-labeled
tubulin. Forty five minutes after injection, cells that had formed
rhodamine-labeled microtubules were subjected to FRAP analysis, and
both GFP-PTHrP and rhodamine-labeled microtubules were visualized
through separate channels on a CLSM (Fig. 4A
). Quantitation of GFP-PTHrP nuclear
import in these cells showed no difference in the rate or extent
of transport between microinjected and nonmicroinjected cells,
indicating that the cells were not damaged during the microinjection
procedure (Fig. 4B
). There was no rhodamine-tubulin in the nuclei of
these cells and no movement of rhodamine-tubulin into the nucleus for
the duration of the experiment (Fig. 4B
). As expected, not all the
microinjected rhodamine-tubulin was assembled into microtubules, but
careful analysis of the cytoplasm (highlighted by the white
arrows in Fig. 4A
, i and ii) revealed that GFP-PTHrP colocalized
with rhodamine-labeled microtubules (yellow in the merged
images); in some cases, it was possible to discern the movement of
PTHrP (seen as yellow spots) along the microtubules toward
the nucleus during the course of the experiment (data not shown).
Association of PTHrP to the Microtubule Network Is
Enhanced in the Presence of Importin ß1
It has previously been shown that plant importin
is able
to interact with microtubules in an NLS-dependent fashion
(32). To test whether a similar mechanism might operate in
a mammalian system for the importin ß1-mediated import substrate
PTHrP, a microtubule association assay was used. Taxol- assisted
microtubule formation was performed in vitro, followed by
the addition of combinations of PTHrP, importins, and UMR106.01 total
cell lysate, followed by incubation for 30 min. The microtubule/protein
mixtures were then spun through a glycerol gradient, and the resulting
microtubule pellet was analyzed for the presence of PTHrP by SDS-PAGE
and Western blotting (Fig. 5
). First,
exogenously added PTHrP was found to associate with microtubules in the
presence of total cell lysate, which contained all the necessary
components for nuclear import (Fig. 5A
, lane 3), with endogenous PTHrP
in the cell lysate also found to associate with the microtubules (Fig. 5A
, lane 2). Addition of recombinant importin ß1 caused the strongest
association of PTHrP to the microtubules (Fig. 5A
, lane 7). As negative
controls, cell lysate or protein alone (not mixed with preformed
microtubules) was spun through the glycerol cushion and analyzed by
Western blotting. PTHrP alone (Fig. 5A
, lane 10) or in cell lysates
(Fig. 5A
, lane 9) did not pellet through the glycerol cushion; nor did
importin
or ß1 (Fig. 5A
, lanes 12 and 11, respectively).
Whole-cell lysate run on the same blot (Fig. 5A
, lane 13) demonstrated
functionality of the anti-PTHrP antibody.

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Figure 5. PTHrP Associates with Microtubules, and Its
Association Is Enhanced by Importin ß1
Microtubule association was determined by the ability of PTHrP to
pellet with the microtubules through a 60% glycerol cushion. The
resulting microtubule pellets were analyzed by SDS-PAGE, blotted on a
nitrocellulose membrane, and probed using an anti-PTHrP antibody and
horseradish peroxidase-coupled secondary antibody, developed using
enhanced chemiluminescence, and imaged using a cooled charge-coupled
device camera (see Materials and Methods). For control
experiments, proteins that were not incubated with taxol-stabilized
microtubules were subjected to the same protocol as above (lanes
1012). Microtubules formed in the presence of taxol were
incubated in the presence (A) or absence (B) of UMR106.01 cell lysate
with combinations of exogenously added PTHrP, importin ß1, and
importin (as indicated below each lane). As
controls, PTHrP, importin ß1, and importin were also added to
glycerol cushions and spun (panel A, lanes 912). Whole-cell lysates
from UMR 106.01 cells (no centrifugation through a glycerol cushion)
were used to test the efficacy of the antibody (panel A, lane 13).
C, Quantification of data shown in A (lanes 18; left
panel) using ImageGauge software (Fuji Photo Film Co., Ltd.). D, Quantification of data shown in panel B.
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To test whether PTHrP alone could bind to microtubules in the absence
of cell lysate, recombinant PTHrP was added (Fig. 5B
, lane 1), its
microtubular association being enhanced about 2-fold in the presence of
importin ß1 (Fig. 5B
, lane 2, and Fig. 5D
); microtubular association
of importin ß1 could also be detected (data not shown). In support of
its specific requirement for importin ß1, and not importin
, for
nuclear import (15), addition of importin
did not
enhance but rather decreased microtubular association of PTHrP more
than 3-fold (Fig. 5B
, lane 3, and Fig. 5D
). These results indicated
that PTHrP associates directly with microtubules, with importin ß1
able to enhance this process.
Microtubule Integrity Is Essential for PTHrP Nuclear Import
To test directly whether an intact microtubule network is
necessary for nuclear import of PTHrP, transiently transfected cells
expressing GFP-PTHrP were pretreated for 60 min with nocodazole, and
PTHrP subcellular localization examined by CLSM. Steady state analysis
revealed increased levels of nuclear/nucleolar GFP-PTHrP in the treated
cells compared with controls (data not shown), indicating reduced
nucleocytoplasmic flux for PTHrP. FRAP experiments indicated that
nocodazole pretreatment reduced the extent of the return of
nuclear/nucleolar fluorescence after photobleaching (29.8 ± 2.1
and 34.8 ± 3.2%, n = 20, for nucleus and nucleolus,
respectively; Fig. 6
, A and B, and Fig. 7
) significantly (P <
0.0001) compared with untreated cells (52.6 ± 3.1 and 61.4
± 4.1%, n = 19, for nucleus and nucleolus, respectively; Fig. 6
, A and B and Fig. 7
). The t1/2 of fluorescence
recovery was markedly longer (t1/2 of 37.8
± 7.2 and 42.2 ± 7.6 sec, n = 19, for nuclear and nucleolar
fluorescence, respectively) when compared with that of untreated cells
(Fig. 7
). Similar results were obtained after 24-h treatment with
nocodazole (results not shown). Taken together, the results from
Figs. 47


clearly imply a role for the cytoskeleton and, in particular, the
microtubule network in transporting PTHrP toward the nucleus.

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Figure 6. Results for FRAP Experiments in Untreated and
Nocodazole-Treated UMR106.01 Cells Expressing GFP-PTHrP
A, Visualization of the return of fluorescence after photobleaching in
control and nocodazole-treated (+ Noc) cells. A movie of
this experiment is available at
http://jcsmr.anu.edu.au/dbmb/jans/lam/frap/ (Fig. 6 .avi). B,
Quantitative analysis from panel A.
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Figure 7. Pooled Data for the Fractional (A) and Half-Maximal
(B) Return of Nuclear and Nucleolar Fluorescence of GFP-PTHrP
Subsequent to Nuclear Photobleaching
Values for the maximal fractional return of nuclear fluorescence
(Fn500 sec/Fnprebleach) and nucleolus
(Fnu500 sec/ Fnuprebleach)
(top) and time in seconds for half-maximal return
(t1/2) (bottom) are shown for untreated and
nocodazole-treated cells. * Represents a signifiant
(P < 0.0001) difference between
nocodazole-treated and untreated cells.
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DISCUSSION
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The technique of FRAP has been used extensively in biological
systems to study the lateral mobility of proteins within the plasma
membrane (33, 34, 35, 36, 37), the nuclear membrane (38),
or within organelles (39). In terms of nuclear transport,
FRAP had been used together with fluorescent dextrans to determine the
diffusion size limit across the nuclear pore complex (40)
as well as to assess the lateral mobility of nuclear import substrates
in the nuclear or cytoplasmic compartments (41). In this
study, we use FRAP in conjunction with a GFP-fusion protein for the
first time as a means of studying, in living cells, the
nucleocytoplasmic flux of a protein for which the nuclear transport
mechanism has been delineated (15). This approach has the
advantage over conventional nuclear transport assays in that 1) cells
are not physically damaged by microinjection, detergent, or mechanical
perforation, and 2) that proteins are expressed intracellularly and
hence do not have to be introduced exogenously, which allows for the
study of their nuclear transport in as physiological a context as
possible. The technique is quantitative, enables actual nuclear
transport rates to be determined, and permits temporal resolution of
the nuclear import process in intact living cells.
The present results suggest that under normal conditions, PTHrP is able
to cycle between the nucleus and cytoplasm via the nuclear pore
complex. Although nuclear import is mediated by importin ß1, based on
previous work (15), export of PTHrP is leptomycin B
sensitive, implying involvement of the nuclear export receptor CRM1
(42). PTHrP association with cytoskeletal elements has
recently been reported (30), and, as shown here for the
first time, microtubule integrity clearly plays an important role in
PTHrP nuclear import; whether the integral role of the microtubule
network in PTHrP nuclear transport relates directly to the role of
importin ß1, as opposed to that of the importin
/ß1 heterodimer
in conventional NLS-dependent nuclear protein import, is unclear. The
role of microtubules in PTHrP subcellular localization is indicated by
the demonstration of colocalization with ß-tubulin, the direct
visualization of GFP-PTHrP on rhodaminelabeled microtubules after
FRAP, and the fact that the microtubule-disrupting agent nocodazole
alters the steady state level of PTHrP nuclear/nucleolar accumulation
and its nucleocytoplasmic flux. Further, we show that PTHrP is able to
bind to in vitro polymerized microtubules and that this
association is enhanced by importin ß1. We have also detected
PTHrP in microtubule preparations from mouse brain, testis, and UMR
106.01 cells (data not shown); significantly, importin ß1 was
also detected in all of these preparations.
Interestingly, the conventional NLS-binding importin
subunit has
been shown to associate with microtubules/microfilaments in mammalian
cells (43) and tobacco protoplasts (32), as
well as with microtubules in vitro in an NLS-dependent
manner, whereas yeast importin
has also been reported to bind
directly to the actin-related protein Act2p (44).
Microtubule/microfilament association of armadillo repeat-containing
proteins, such as catenins and Vac8p (involved in vacuolar protein
targeting), has also been reported (32, 45). In the case
of several viruses, nuclear import appears to be negatively regulated
by association with the actin cytoskeleton (46) or to
involve movement along microtubule filaments (47) in
analogous fashion to our observations here for PTHrP. Our finding that
PTHrP binding to microtubules is enhanced by the presence of its NLS
receptor importin ß1 is comparable to the NLS-dependent association
of plant importin
with microtubules. The close relationship of
nuclear import pathways with cytoskeletal components (see Ref.
48) thus may be a general phenomenon of mechanistic
importance. The differences in the requirements of individual
substrate/importin complexes in terms of binding to microtubules
indicate the presence of highly selective mechanisms in transport
toward the nuclear pore complex for different NLS-bearing
substrates. Intriguingly, preliminary results for FRAP experiments (our
unpublished data) on confluent (stationary phase) cells (as opposed to
the subconfluent cells exclusively analyzed in this study) are
comparable to those for nocodazole-treated cells in terms of a reduced
rate of nuclear import and low fractional return of nuclear and
nucleolar fluorescence, implying that the importin ß1-PTHrP complex
interaction with the cytoskeleton may be modulated differentially
during cell growth and the cell cycle. The extent to which cell
cycle-related phosphorylation at T85 of PTHrP
(28) may play a role in this is currently being
examined.
Although clearly implicated in delaying apoptosis (17) and
promoting proliferation (20) in certain cell types, the
precise role of PTHrP in the nucleus/nucleolus (49, 50)
remains unclear. Poly (G) RNA binding on the part of PTHrP has recently
been reported (51), pointing to a possible role of PTHrP,
perhaps in conjunction with other proteins, as a nuclear export factor
for RNA, consistent with its ability, as shown here, to shuttle between
nuclear/nucleolar and cytoplasmic compartments (see above). We have
recently shown that treatment of GFP-PTHrP-expressing cells with the
RNA-polymerase inhibitor actinomycin D inhibits association of PTHrP
with the nucleolus (data not shown), providing further evidence for a
role of PTHrP in RNA transport and/or regulation thereof. RNA binding
on the part of PTHrP may also contribute to its cytoskeletal
association, since a large part of cytoplasmic mRNA appears associated
with cytoskeletal elements (52, 53), and nuclear proteins
such as the mRNA binding protein mrnp41 (52) and Cbf5p, a
yeast nucleolar protein that regulates rRNA synthesis
(53), have also been demonstrated to associate with the
cytoskeleton.
PTHrP was originally discovered as a factor responsible for the
syndrome of humoral hypercalcemia of malignancy, with its role as a
growth/malignancy factor being implicated by a number of observations
(16); MCF-7 and MDA-MD-231 breast cancer cell lines made
to overexpress PTHrP, for example, possess increased tumorigenic
capacity and metastatic potential (56, 57). The fact (see
above) that PTHrP nuclear localization is integral to its function
implies that strategies to block PTHrP nuclear localization in cancers
overexpressing it could lead to, at least potentially, increased
apoptosis and hence reduced tumorigenic potential. The present results,
with respect to cytoskeletal and cell cycle control over PTHrP
nuclear-cytoplasmic flux, thus may have important application in
anticancer therapies, which is the focus of future work in this
laboratory.
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MATERIALS AND METHODS
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Tissue Culture
UMR106.01 rat osteosarcoma cells (56) were
maintained in
-MEM supplemented with 10% FBS, at 37 C in 5%
C02 atmosphere.
GFP-PTHrP Fusion Protein Expression Construct
A plasmid expressing GFP fused to the amino terminus of
PTHrP(1141) was generated using oligonucleotide primers as previously
described (28). For control experiments in which GFP was
analyzed alone, the pEGFP-C1 plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA) was used.
Transfection and Photobleaching Experiments
UMR106.01 cells were passaged onto 22-mm diameter round
coverslips for 2 d and then transfected with the
GFP-PTHrP-expressing construct using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD; Ref. 57).
Forty eight hours after transfection, the cells were transferred into
an open-perfusion microincubator cell chamber (PDMI-2, Medical Systems
Corp., Greenvale, NY) and maintained at 37 C in phenol red-free
-modified Eagles medium. GFP-PTHrP expressing cells were identified
using CLSM (Bio-Rad MRC-1024; Bio-Rad Microscience, Hemel Hempstead,
UK). The CLSM was equipped with a multiline 15-mW Krypton/Argon laser,
which allowed for a maximum illumination intensity of approximately 0.3
mW at the point of focus. The cells were visualized (x1.5, zoom;
x100, oil immersion lens) by illumination with 1030% total laser
power with excitation at 488 nm. One image (five scans in Kalman mode)
was collected before photobleaching, after which an area covering at
least 50% of the nucleus was selected by zooming 20-fold. This area
was then bleached by removing all barrier filters on the laser to allow
for maximum illumination of the area selected for 10 scans (in 8 sec);
this did not result in cell death as determined by monitoring the
uptake of propidium iodide (data not shown). Images of cells were
collected 20 sec after photobleaching, and subsequent images were
acquired at 20-sec intervals for about 500 sec using detector and laser
settings identical with those used before photobleaching. Because PTHrP
binds to nuclear components (15), lateral diffusion from
the nonbleached area of the nucleus was assumed to contribute minimally
to the return of fluorescence, which was not monitored before 20 sec
after photobleaching to avoid this rapid diffusive component; similar
approaches have been used and validated with respect to FRAP
experiments and the nucleus (39, 40, 60, 61, 62). For some
experiments, cells were treated with 10 µg/ml nocodazole
(Sigma, St. Louis, MO) for 60 min before experimentation.
Image analysis was performed using the NIH Image software as described
previously (10, 15); autofluorescence was quantitated and
subtracted from all other values (Fn, nuclear fluorescence; Fnu,
nucleolar fluorescence; Fc, cytoplasmic fluorescence). Fn/c and Fnu/c
are the nuclear and nucleolar to cytoplasmic ratios, respectively. FRAP
data to calculate the fractional return of specific fluorescence and
t1/2 for the return of fluorescence was fitted
exponentially as described previously (33, 34, 39, 40).
Two-Photon Excitation Microscopy
High-resolution imaging using two-photon excitation to determine
GFP-PTHrP subcellular localization was performed using a Fluoview
Confocal Scan system (Olympus Corp., Lake Success, NY)
coupled to an Olympus Corp. IX70 Microscope with
excitation from a Tsunami/Verdi femtosecond pulsed laser
(Spectra-Physics, Mountain View, CA) at 900 nm (31) using
a x100 oil objective.
Microinjection
GFP-PTHrP-expressing UMR 106.01 cells grown on coverslips were
microinjected with rhodamine-labeled tubulin (Cytoskeleton, Denver, CO)
using a Narshige IM-200 (Narshige, Tokyo, Japan) microinjector as
previously described (63). The injected cells were
returned to tissue culture for 45 min and then assessed for microtubule
formation by CLSM followed by analysis for GFP-PTHrP movement by FRAP
as above.
Immunofluorescence
UMR106.01 cells grown on glass coverslips were transfected with
the GFP-PTHrP-expressing construct using LipofectAMINE and allowed to
express the protein for 24 h. Cells were then left untreated or
treated with 10 µg/ml nocodazole for a further 24 h and then
fixed with dithiobis succinimidyl propionate (Pierce Chemical Co., Rockford, IL) at 1 mM (in PBS) for 30 min at 37
C, incubated for 5 min with stop solution (0.5% Triton X-100, 1
mM EDTA, 4% polyethylene glycol 6000 in serum-free DMEM),
and then incubated for 15 min in 3.2% paraformaldehyde in PBS, pH 7.4,
for 15 min at 21 C. The fixed cells were permeabilized and preblocked
in 0.1% (wt/vol) BSA and 0.3% (vol/vol) Triton X-100 in PBS for
1 h before incubation with anti-ß-tubulin antibody (Roche Molecular Biochemicals, Castle Hill, New South Wales, Australia)
overnight at 4 C, followed by a 1-h incubation (21 C) with Texas
Red-X-conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR) and mounting with antifade solution
(DAKO Corp., Glostrup, Denmark). To colocalize endogenous
PTHrP with tubulin, cells were fixed and permeabilized as described
above, then coincubated with a polyclonal rabbit anti-PTHrP antibody
(R87) specific for the amino terminus (amino acids 134) of the
molecule together with the anti-ß-tubulin antibody, followed by
hybridization with Oregon Green-conjugated anti-rabbit and Texas Red-X
antimouse conjugated secondary antibodies. Imaging was performed using
CLSM. The red and green channels were collected individually to prevent
bleed-through of fluorescence, although the iris settings were set such
that uniform confocality was achieved. Images were merged using
Confocal Assistant (Bio-Rad version 4.02) and prepared for presentation
in Corel Photopaint 8 (Corel Corp.).
Protein Expression and Purification
Recombinant PTHrP(1108) was expressed and purified as
previously described (64). Glutathione
S-transferase (GST)-tagged mouse importin
1 (PTAC58) and
GST-tagged mouse importin ß1 (PTAC97) were expressed and purified as
previously described (65).
Microtubule Association Assays
To polymerize microtubules, 100 µM bovine brain
tubulin, in the form of 33% rhodamine-labeled tubulin and 66%
unlabeled tubulin made up in general tubulin buffer [80 mM
piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.8, 1
mM MgCl2, 1
mM EGTA, and 10% glycerol; Cytoskeleton, Denver,
CO], was polymerized in PMGT, which consists of general
tubulin buffer, protease inhibitors (Complete, Roche Molecular Biochemicals), 1 mM GTP, and 100
µM taxol (Sigma) at 35 C for 20
min. Polymerized microtubules were then visualized by CLSM.
For microtubule association, 100 µl of combinations of UMR106.01 cell
extract, recombinant PTHrP 1108 (150 µM), GST-tagged
mouse importin ß (200 µM), and/or GST-tagged mouse
importin
(200 µM) were mixed with the polymerized
microtubules and incubated at room temperature for 30 min. The samples
were carefully layered onto 400 µl of glycerol cushion buffer [80
mM piperazine-N,N'-bis(2-ethanesulfonic acid),
pH 6.8, 1 mM EGTA, 1 mM
MgCl2, 60% vol/vol glycerol, 0.005%
chlorohexadine, and 5 nM taxol] and centrifuged
at 100,000 x g (Beckman TL100 Ultracentrifuge with a
TLA-100.3 rotor, Beckman Coulter, Inc., Palo Alto, CA) at 25 C for 45
min. The microtubule pellet was then resuspended in 100 µl of
PMGT, and the microtubule population was monitored using CLSM. The
association of PTHrP or importin ß with microtubules was assessed by
running 20 µl of the microtubule preparations on SDS-PAGE gels
followed by transfer to a nitrocellulose membrane and detection with an
anti-PTHrP rabbit polyclonal antibody (1903) used at 1:5,000 dilution
or an antiimportin ß1 goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) used at 1:1,000 dilution
followed by a 1-h incubation in a 1:1,000 dilution of a donkey
antirabbit or rabbit antigoat horseradish peroxidase-conjugated Ig
(Amersham Pharmacia Biotech, Little Chalfont,
Buckinghamshire, UK). Blots were developed using ECL Plus
Western blotting detection system (Amersham Pharmacia Biotech), and chemiluminescence was detected using a cooled
charge-coupled device camera (Fujifilm LAS1000, Fuji Photo Film Co., Ltd., Tokyo, Japan).
 |
ACKNOWLEDGMENTS
|
---|
We thank Ginny Leopold and Elizabeth Allan for help with tissue
culture, and Damian Myers for setting up the microincubator cell
chamber.
 |
FOOTNOTES
|
---|
This work was supported by National Health and Medical Research Council
Australia Program Grant 003211 (to T.J.M. and M.T.G.), an Anti-Cancer
Council of Victoria Grant (to T.J.M. and M.T.G.), an Australian
National University Institute of Advanced Studies bilateral
collaborative scheme grant (to D.A.J.), a Rebecca Cooper Foundation
Grant (to M.H.C.L. and D.A.J.), and the Chugai Pharmaceutical Company,
Tsukuba, Japan (to T.J.M. and M.T.G.). M.L. is a National Health
and Medical Research Council of Australia Peter Doherty Fellow.
1 These authors contributed equally to this work. 
Abbreviations: CLSM, Confocal laser scanning microscopy; FRAP,
fluorescence recovery after photobleaching; GFP, green fluorescent
protein; GST, glutathione-S-transferase; NLS, nuclear
localization sequence; PTHrP, PTH-related protein.
Received for publication February 1, 2001.
Accepted for publication October 23, 2001.
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