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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. Vincent’s 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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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/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
67–94, 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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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. 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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). 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).
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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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, 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. 4–7 clearly imply a role for the cytoskeleton and, in particular, the
microtubule network in transporting PTHrP toward the nucleus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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(1–141) 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 10–30% 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 1–34) 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(1–108) 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 1–108 (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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1ß and 2ß heterodimers: 1 or 2 subunit
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peptide repeat-containing nucleoporins. Proc Natl Acad Sci USA 92:6532–6536. Mol Cell Biol 19:1218–1225. J Biol Chem 274:7391–7398 associates with the cytoskeleton. Plant Cell 10:1791–1799 mediates steroid receptor interaction with microtubules
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 G. W. Moseley, D. M. Roth, M. A. DeJesus, D. L. Leyton, R. P. Filmer, C. W. Pouton, and D. A. Jans Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import Mol. Biol. Cell, August 1, 2007; 18(8): 3204 - 3213. [Abstract] [Full Text] [PDF]
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 T. Tsuji, N. Sheehy, V. W. Gautier, H. Hayakawa, H. Sawa, and W. W. Hall The Nuclear Import of the Human T Lymphotropic Virus Type I (HTLV-1) Tax Protein Is Carrier- and Energy-independent J. Biol. Chem., May 4, 2007; 282(18): 13875 - 13883. [Abstract] [Full Text] [PDF]
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 J. C. Pache, D. W. Burton, L. J. Deftos, and R. H. Hastings A Carboxyl Leucine-Rich Region of Parathyroid Hormone-Related Protein Is Critical for Nuclear Export Endocrinology, February 1, 2006; 147(2): 990 - 998. [Abstract] [Full Text] [PDF]
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 S. Mani, H. Huang, S. Sundarababu, W. Liu, G. Kalpana, A. B. Smith, and S. B. Horwitz Activation of the Steroid and Xenobiotic Receptor (Human Pregnane X Receptor) by Nontaxane Microtubule-Stabilizing Agents Clin. Cancer Res., September 1, 2005; 11(17): 6359 - 6369. [Abstract] [Full Text] [PDF]
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A. Mange, C. Crozet, S. Lehmann, and F. Beranger Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin J. Cell Sci., May 1, 2004; 117(11): 2411 - 2416. [Abstract] [Full Text] [PDF]
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N. M. Fiaschi-Taesch and A. F. Stewart Minireview: Parathyroid Hormone-Related Protein as an Intracrine Factor--Trafficking Mechanisms and Functional Consequences Endocrinology, February 1, 2003; 144(2): 407 - 411. [Abstract] [Full Text] [PDF]
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