From the Department of Basic Sciences, University of
Crete School of Medicine, 71 110 Heraklion, Crete, Greece, the
¶ Laboratory of Pathology, University of Thessaloniki School of
Medicine, 54006 Thessaloniki, Greece, and the
Division of
Gene Expression and Development, Roslin Institute (Edinburgh),
Midlothian EH25 9PS, United Kingdom
Received for publication, August 7, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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We have previously shown that the mouse
heterochromatin protein 1 homologue M31 interacts dynamically
with the nuclear envelope. Using quantitative in vitro
assays, we now demonstrate that this interaction is potently inhibited
by soluble factors present in mitotic and interphase cytosol. As
indicated by depletion and order-of-addition experiments, the
inhibitory activity co-isolates with a 55-kDa protein, which binds
avidly to the nuclear envelope and presumably blocks M31-binding sites.
Purification of this protein and microsequencing of tryptic peptides
identify it as Heterochromatin protein 1 (HP1)1 represents the
founding member of a large protein family, which includes the
Polycomb group and other gene regulators (for recent reviews
see Refs. 1 and 2). This molecule possesses a dimeric,
quasi-symmetrical structure and contains two sequence-related and
similarly folded domains: the N-terminal chromodomain (3, 4), and the
C-terminal chromo shadow domain (5). These two domains consist of
anti-parallel, three-stranded A single HP1 species was originally identified in Drosophila
melanogaster (8). However, subsequent studies have revealed multiple variants of this protein in higher eukaryotes. Mammalian HP1
includes three distinct isotypes termed hHP1 HP1 binds to several chromatin-remodeling factors and transcriptional
regulators. Among these are CAF-1, BRG1/SNF2 The interactions between HP1 proteins and elements of the nuclear
envelope are particularly intriguing. First, peripheral heterochromatin is in physical contact with the inner nuclear membrane
during interphase and could be directly linked to the nuclear envelope;
second, transient associations involving components of condensed
chromatin and inner nuclear membrane proteins are thought to provide
the basis for nuclear envelope reassembly at the end of mitosis. In
previous studies (24) we have found that all three variants of mouse
HP1 target the nuclear periphery when injected into living cells and
bind to isolated nuclear envelopes under in vitro conditions.
To further explore these interactions, we focused on M31. In this work,
we demonstrate that in vitro binding of M31 to isolated nuclear envelopes is potently inhibited by soluble factors present in
mitotic and interphase cytosol. Using biochemical methods, we have
characterized this inhibitory activity as a soluble form of
Antibodies and Plasmids--
A previously characterized
monoclonal antibody directed to M31 (MAC 353; Refs. 13 and 14) was used
throughout this study. Fusion proteins were detected by polyclonal,
affinity-purified antibodies against recombinant glutathione
S-transferase (GST). Anti-tubulin antibodies were obtained
from Sigma. The characterization of anti-lamin B and anti-LAP2B
antibodies has been reported previously (25, 26). M31 (full length) was
expressed as a fusion protein with GST using pGEX1 and as a
His6-tagged protein employing pET-25b.
Cell Culture--
Human endometrial carcinoma cells (Ishikawa)
were maintained in minimum Eagle's medium, whereas HeLa cells and
Chinese hamster ovary cells were grown in Dulbecco's modified Eagle's
medium. All media contained 10% fetal bovine serum and antibiotics.
Synchronization in mitosis was achieved by treating the cells with
40-120 ng/ml nocodazole for 18 h.
Indirect Immunofluorescence and Immunoblotting--
Conventional
and confocal immunofluorescence, as well as Western blotting, were
performed as described previously (25, 27, 28). Staining of the cells
with propidium iodide was accomplished after a 30-min incubation with
200 units/ml RNase A and subsequent incubation with 1 µg/ml of this
dye for 5 min.
Expression, Purification, and Metabolic Labeling of Recombinant
Proteins--
GST fusion proteins and His6-tagged
polypeptides were expressed in BL21 (DE3) cells and purified from
bacterial lysates according to standard procedures (29). For metabolic
labeling, the cells were grown in methionine-free medium (M9-based) to
an OD of 0.9. Isopropyl-1-thio- Preparation of Tubulin--
Tubulin was isolated from rat brain
tissue according to published methods (30).
Partial Purification of the 55-kDa Protein--
To
isolate the 55-kDa protein, 500 µl of a thick suspension of
urea-extracted nuclear envelopes (concentration of 5-10 mg/ml in
KHM buffer (78 mM KCl, 50 mM Hepes-KOH,
pH 7.0, 4 mM MgCl2, 8 mM
CaCl2, 10 mM EGTA, 1 mM DTT, 1 mM PMSF), 1% gelatin, and protease inhibitors) was
combined with 700 µl of freshly prepared Ishikawa cytosol (5 mg/ml).
After a 45-min incubation at room temperature, the membranes were
pelleted (12,000 × g, 30 min, 4 °C) and resuspended
in 800 µl of the same buffer. Following another centrifugation, the
nuclear envelopes were eluted with 600 µl of buffer E (250 mM NaCl, 30 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, and 0.2 mM
PMSF). The eluate collected was concentrated by ethanol precipitation
or kept in its original state at Isolation of Nuclear Envelopes--
Turkey erythrocyte nuclear
envelopes were isolated as specified in Ref. 31. The membranes were
washed sequentially with 2 M KCl, 50 mM
Tris-HCl, pH 7.5, 1 mM DTT, 1 mM PMSF, and
protease inhibitors (2 µg/ml each leupeptin, pepstatin,
aprotinin, and antipain); double distilled water; and 8 M urea, 10 mM Tris-HCl, pH 8.0, 4 mM EDTA, and 1 mM PMSF (when specified). Before
use, nuclear envelopes were washed in assay buffer (see below) and thoroughly resuspended in the same buffer by mild sonication. To
prepare proteolyzed membranes, 0.8-2.0 mg/ml of nuclear envelopes were
incubated with a mixture of trypsin and chymotrypsin (0.16 mg/ml) for
30 min at room temperature. Digestion was stopped by adding PMSF (1.3 mM), protease inhibitors (see above), and 1% fish skin
gelatin (scavenger). The membranes were washed with assay buffer
containing PMSF/protease inhibitors/gelatin and resuspended in the same buffer.
Preparation of Cytosol--
Cytosol was prepared from interphase
or nocodazole-arrested Ishikawa cells according to the following
method. The cells were detached by trypsinization, pelleted at 300 × g, resuspended in culture medium containing 20 µM cytochalasin B, and incubated at 37 °C for 45 min.
After washing twice with phosphate-buffered saline and KHM buffer, the
pellet was resuspended in one volume of KHM/cytochalasin B and
Dounce-homogenized (300 strokes). The homogenate was ultracentrifuged
at 100,000 × g for 1 h at 4 °C, and the
supernatant (cytosol) was collected. When mitotic homogenates were
prepared, 1 mM MgATP, 20 mM creatine phosphate,
400 µg/ml creatine kinase, 80 mM In Situ Assays--
Unsynchronized cells grown on coverslips
were washed three times with phosphate-buffered saline and
permeabilized with 0.2% Triton X-100 in KHM buffer/protease inhibitors
(5 min, room temperature). The cells were rinsed two times with KHM,
blocked with KHM, 1% gelatin, 1 mM DTT for 10 min, and
incubated with 10 µg of recombinant proteins for 30 min at room
temperature. After rinsing with KHM, 0.02% Triton X-100, washing with
KHM, 1% gelatin, 1 mM DTT (two times, 2 min),
and rinsing again with plain KHM, the cells were fixed with 4%
formaldehyde (5 min, room temperature) and processed for indirect immunofluorescence.
Binding Assays--
All reactions were carried out in Eppendorf
tubes coated with 1% boiled/filtered fish skin gelatin. 10-50 µg of
nuclear envelopes were combined with increasing amounts of labeled or
unlabeled M31-GST and M31-His6 reconstituted in assay
buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, 0.2 mM PMSF, 10% sucrose, and 0.1%
gelatin) and adjusted in volume to 100 µl. After a 45-min
incubation at room temperature (mixing by rotation), the samples were
spun in a Microfuge (12,000 × g, 30 min), and the
pellets were washed with 300 µl of assay buffer. Following another
centrifugation (15 min, in the same fashion), the supernatants were
carefully aspirated, and the walls of the tubes were wiped with cotton
swabs. The final pellets, representing nuclear membranes and associated
material, were either solubilized in Laemmli buffer or dissolved in
scintillation fluid. Binding was detected by SDS-PAGE/Western blotting
(unlabeled proteins) or by In Vitro Reassembly Assays--
Chinese hamster ovary cells were
synchronized in mitosis with 120 ng/ml nocodazole (18 h, 37 °C).
After shake-off, the cells were washed three times with cold Pipes
buffer (50 mM Pipes-KOH, pH 7.4, 50 mM KCl, 5 mM MgCl2, 2 mM EGTA, and 1 mM PMSF) and resuspended at a density of 106
cells/ml in the same medium plus 1 mM DTT and
protease inhibitors (2 µg/ml each leupeptin, pepstatin,
aprotinin, and antipain). Digitonin was added from a 10 mg/ml stock to
a final concentration of 50 µg/ml, and the suspension was left on ice
for 5 min. The lysate was divided in equal aliquots (~2 × 105 cells). One sample (control) was diluted to 300 µl
with cold Pipes buffer and processed immediately after addition of 2 mM MgATP, 20 mM creatine phosphate, 400 µg/ml
creatine kinase, 80 mM Cytosolic Factors Inhibit M31 Binding to the Nuclear
Envelope--
To measure binding of HP1 proteins to the nuclear
envelope, we used a simple cosedimentation assay. Recombinant M31 (Fig. 1a, GST-M31 or
His-M31) was co-incubated with nuclear envelope vesicles
(NEs) that had been stripped of peripheral proteins by urea extraction
(Fig. 1a, NE; for more information see Refs.
31-33). As shown in Fig. 1, b and c, GST-M31 and
His6-M31 co-pelleted with intact NEs but did not bind to
protease-digested membranes. Furthermore, as indicated by quantitative
immunoblotting, the binding of M31-GST was
concentration-dependent and saturable (Fig. 1c).
To identify cellular factors involved in M31-nuclear envelope
interactions, we performed binding experiments in the presence and
absence of cytosol. For higher sensitivity, in these experiments we
used a metabolically labeled probe ([35S]GST-M31) and
worked under nonsaturating conditions (for details see "Experimental
Procedures"). As shown in Fig.
2a, mitotic and interphase
cytosol from Ishikawa cells inhibited M31 binding in a
dose-dependent fashion. This inhibition was specific and
did not occur when cytosol was substituted by concentrated solutions of
bovine serum albumin or gelatin (see legends to Figs. 2 and 4c). Furthermore, the decrease of M31 binding did not
reflect competition by endogenous M31 contaminating the cytosol
preparations; as could be confirmed by Western blotting, the endogenous
protein partitioned exclusively with a 100,000 × g
pellet and was undetectable in the cytosolic fraction (Fig.
2a, inset).
The Inhibitory Activity Co-isolates with a 55-kDa Nuclear
Envelope-binding Protein--
We reasoned that the factors responsible
for the inhibitory effect should act either by "neutralizing" M31
or by blocking M31-binding sites at the nuclear envelope. To
differentiate between these two possibilities, we proceeded to
depletion and order-of-addition experiments. Cytosol depleted from
M31-binding proteins after a pre-incubation with immobilized GST-M31
contained as much inhibitory activity as mock-depleted cytosol (Fig.
2b, compare columns No Cy,
+Whole Cy, and +Depl Cy). In line with this,
[35S]GST-M31 that was pre-incubated with cytosol before
incubation with the NEs did not show any significant difference from
the corresponding control (Fig.
2b, compare columns
CTL M31 and Cy-M31). However, when the NEs
were pre-incubated with cytosol before incubation with
[35S]GST-M31, a dramatic decrease in the binding was
detected (Fig. 2b, compare columns CTL
NE and Cy-NE). From these observations it can be
inferred that the inhibitory factors associate primarily with the
NEs.
To confirm this idea, we mixed NEs with metabolically labeled cytosol,
centrifuged the samples at 12,000 × g, and analyzed the pellet fraction by SDS-PAGE and autoradiography. As shown in Fig.
3a, a major 55-kDa protein and several minor species
co-sedimented with the membranes. This did not reflect aggregation or
nonspecific "sticking," because the 55-kDa protein floated up to
the 30-40% sucrose interface and co-migrated exactly with the NEs
upon analysis in sucrose density gradients (Fig. 3b, compare
panels Cy+NE and Cy+Buff.).
The 55-kDa Protein Represents a Soluble Form of Tubulin--
The
55-kDa polypeptide did not dissociate from the NEs after washing with
assay buffer but was partially extracted by distilled water and high
salt (Fig. 4a). Exploiting
this, we isolated mg amounts of the 55-kDa protein from lysates of
interphase and mitotic cells (Fig. 4b; for details see
"Experimental Procedures"). The partially purified polypeptide
abolished binding of [35S]GST-M31 to the NEs (Fig.
4c), suggesting a close relationship with the inhibitory
factor we were seeking.
To establish the identity of this protein, we excised the 55-kDa band
from SDS gels, digested the material with trypsin, and determined the
amino acid sequence of nine internal peptides. Analysis by mass
spectrometry revealed that the 55-kDa band includes two different
protein chains, corresponding to Purified Rat Brain Tubulin Associates with the Nuclear Envelope and
Inhibits Binding of M31--
Proceeding further, we tested highly
purified tubulin preparations isolated from rat brain (SDS-PAGE profile
shown in Fig. 6a).
Nonpolymeric tubulin, maintained in a soluble form by nocodazole/low salt, bound specifically to intact NEs but did not co-sediment with
proteolyzed membranes (Fig. 6b). Quantitative assays showed that binding was concentration-dependent, exhibited
saturable features, and was characterized by a relatively high affinity (apparent Kd = 10
In line with the previous results, nonpolymeric tubulin potently
inhibited the binding of [35S]GST-M31 to the NEs, whereas
a control protein (bovine serum albumin) tested in parallel had no
effect (Fig. 7a, compare
columns 3 and 4). When the experiment was
performed under conditions allowing tubulin polymerization (isotonic
salt/Mg and absence of nocodazole), inhibition of
[35S]GST-M31 binding was less pronounced (Fig.
7a, compare columns 2 and 4),
indicating that at the same nominal concentrations nonpolymeric tubulin
is a much more effective inhibitor than polymeric tubulin.
From a variety of previous studies it is known that certain isotypes of
mammalian tubulin possess a C-terminal tail consisting of 10 tandemly
arranged glutamic acids. Furthermore, tubulin is extensively
glutamoylated and phosphorylated after biosynthesis, acquiring an even
higher negative charge. Because of this peculiarity and considering
that M31-GST is also negatively charged at neutral pH (estimated
isoelectric point of 5.5), we considered the possibility that tubulin
may inhibit M31 binding by mere electrostatic repulsion. Although
M31-GST binding to the NEs was not affected significantly by negatively
charged ligands (e.g. heparin; data not shown), to confront
this problem in a more definitive fashion, we repeated the
binding/inhibition experiments at a pH of 5.7 at which both tubulin and
M31-GST carry minimal charge. At this pH, tubulin inhibited
[35S]GST-M31 binding to the same extent as at pH 7.4, ruling out any electrostatic effects (Fig. 7b).
Finally, to confirm the biochemical results by an independent method,
we employed a previously established morphological assay, which detects
in situ binding of M31 to elements of the nuclear periphery
(for details see "Experimental Procedures"). As shown in Fig.
8 (panel labeled GST-M31),
when Triton X-100-permeabilized cells were incubated with GST-M31, the
periphery of the nucleus was intensely stained. However, when the same
experiment was repeated with a mixture of M31-GST and rat brain
tubulin, the decoration of the nuclear periphery was markedly reduced
(panel labeled GST-M31+tb). This effect was specific,
because binding of M31-GST to internal nuclear structures
(e.g. perinucleolar heterochromatin) was not affected by the
presence of tubulin.
Soluble Tubulin Inhibits Nuclear Envelope Reassembly in
Vitro--
In recent studies, we have shown that M31 mediates
recruitment of nuclear envelope precursors around segregated
chromosomes and facilitates nuclear envelope reassembly at the end of
mitosis (24). Knowing that, we wanted to examine whether soluble
tubulin regulates these associations, inhibiting binding of nuclear
envelope precursors to chromosome-associated M31 at late phases of
mitosis. This interpretation makes physiological sense, because, upon
nuclear envelope breakdown, soluble tubulin is expected to have access to the nuclear interior.
To test this hypothesis, we utilized a novel in vitro
reassembly assay developed in our laboratory. In this assay,
nocodazole-synchronized (prometaphase) cells are first incubated with
low concentrations of digitonin, to gently open the plasma membrane and
allow addition of exogenous elements. Subsequently, the permeabilized
cells are incubated for 2 h at 33 °C to induce destruction of
mitotic cyclins and inactivation of the cdc2 kinase. Elimination of
this kinase allows dephosphorylation of mitotically modified nuclear
envelope proteins and initiation of nuclear envelope reassembly around condensed chromosomes. The progress of this reaction can be easily monitored by spinning the "digitonin ghosts" on glass coverslips and performing indirect immunofluorescence microscopy with anti-lamin B
and anti-LAP2B antibodies. These two proteins represent, respectively, peripheral and integral components of the nuclear envelope that disperse during prometaphase and are quantitatively recruited to the
surfaces of chromosomes at late anaphase/telophase.
As shown in Fig. 9, addition of soluble
tubulin completely abolished recruitment of LAP2B and lamin B to the
surfaces of chromosomes, whereas addition of bovine serum albumin
(control) had no effect.
Recent studies have shown that HP1 proteins interact dynamically
with the nuclear envelope. The mouse HP1 homologues mHP1 Nuclear envelope association may involve a specific interaction with
the integral membrane protein LBR, which has been shown to bind the
human HP1 homologues hHP1 The biochemical and morphological data presented here make it clear
that the inhibitory effect of tubulin is not due to a chaotropic or
purely electrostatic effect. First, nonpolymeric tubulin effectively
competes with M31 under pH conditions at which the total charge of the
molecule is nearly zero. Second, at the same nominal concentration
nonpolymeric tubulin is a much better inhibitor than polymeric tubulin,
although the charge of the protein is similar in both aggregation states.
Soluble, nonpolymeric tubulin is an abundant constituent of the
cytoplasm. Earlier studies indicate that the average hepatocyte contains 3.1 × 107 Be it as it may, tubulin would not be the only example of a protein
that participates in more than one function inside eukaryotic cells. As
it turns out, a wide variety of cellular proteins, from oligomeric
enzymes to crystallins and ion channels, switch partners and become
involved in different functions when their aggregation state or
localization is changed. These are the so-called "moonlighting proteins," a class of molecules that supposedly evolved when
"unused," surface-exposed regions of pre-existing molecules became
useful for the global physiology of the cell (for a review see Ref.
39).
2/6:
2-tubulin. Consistent with this observation,
bona fide tubulin, isolated from rat brain and maintained
in a nonpolymerized state, abolishes binding of M31 to the nuclear
envelope and aborts M31-mediated nuclear envelope reassembly in an
in vitro system. These observations provide a new example
of "moonlighting," a process whereby multimeric proteins switch
function when their aggregation state or localization is altered.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets packed against one or two
-helices and are separated from one another by a flexible hinge
region. Dimerization of HP1 involves intermolecular interactions
between chromo shadow domains that tether two polypeptide chains at
their C-terminal ends but leave the chromodomains unconstrained (6,
7).
,
, and
in humans
and mHP1
, M31, and M32 in mice (3, 9-11). Although these proteins
are structurally similar, they are distributed in different territories
of the cell nucleus (12-15).
, and the transcriptional intermediary factors
and
(7, 10, 15, 16).
Physical or spatial associations between HP1 and elements of the origin
recognition complex, actin-related proteins (Arp4), and SET or
chromodomain-containing proteins, such as Su(var)3-9 and Su(var)3-7
have also been described (14, 17-19). Finally, interactions with the
centromeric protein inner centromere protein, the nuclear
autoantigen SP100, and the inner nuclear membrane protein LBR
have been reported recently (10, 11, 20-23).
2/6:
2-tubulin. The functional implications of these findings are
discussed below.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (0.1 mM) and [35S]methionine (200-300 µCi) were
added, and incubation ensued for 3 h at 37 °C. After that, the
bacteria were collected, and the recombinant proteins were purified as usual.
70 °C.
-glycerophosphate, 50 mM NaF, and 1 µM microcystin LR were
added to the media to preserve the mitotic state.
-counting (35S-labeled
probes). Quantitative measurements were always done in duplicate or triplicate.
-glycerophosphate, 50 mM NaF, and 3 µM microcystin LR. The rest were combined with various peptides or exogenous proteins (12-120
µg), adjusted in volume to 300 µl, and incubated at 33 °C for
2 h. Half of each reaction mixture was loaded onto a cushion of
20% sucrose and spun (4 °C) for 10 min at 1,000 × g on glass coverslips. Material adhering to glass was washed
two times with cold Pipes buffer and fixed with 4% formaldehyde for 10 min. Replicas were stained with anti-LAP2B and anti-lamin B antibodies
as specified above. Morphometric analysis involved detailed examination
of at least 50 cells in the confocal microscope.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Assay system. a, SDS-PAGE
profiles of urea-stripped turkey erythrocyte nuclear envelopes (25 µg) and recombinant M31 (GST-M31, His-M31; 5 µg) used in in vitro binding assays. The arrow
points to LBR, the major integral protein of the inner nuclear
membrane. Bars denote molecular weight values of
97,000, 68,000, 45,000, and 31,000. M,
molecular weight markers. b, binding of recombinant M31 to
the nuclear envelopes, as detected by SDS-PAGE and Coomassie Blue
staining (only the relevant area of the gels is shown). The positions
of LBR, GST-M31, and His-M31 are indicated by arrows. The
input of M31 and nuclear envelopes was as in a. For more
details see "Experimental Procedures." pNE, proteolyzed
NE. c, binding of increasing amounts of GST-M31 to intact
(NE) and proteolyzed nuclear envelopes, as detected by Western
blotting. Lane 1, 0 µ g; lane 2, 0.5 µ g;
lane 3, 1.0 µ g; lane 4, 2 µ g; lane
5, 4 µ g; lane 6, 8 µg. The blots were developed by
alkaline phosphatase-conjugated secondary antibodies.
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Fig. 2.
Cytosolic factors inhibit M31 binding to the
nuclear envelopes. a, relative binding of
[35S]M31-GST to nuclear envelopes in the presence of
increasing quantities of interphase (squares) or mitotic
(diamonds) cytosol. The protein concentration of the cytosol
was 5 mg/ml; each assay mixture contained 40 µg of nuclear envelopes
and 5 µg of [35S]M31-GST in a reaction volume of 120 µl. When cytosol was omitted, the samples were supplemented with
gelatin (5 mg/ml) to compensate for the absence of soluble protein.
"100%" binding corresponds to a value of 30 µg of
[35S]M31-GST/mg of nuclear envelopes, i.e.
about one-half of the maximal binding capacity under our standard assay
conditions (65 µg of [35S]M31-GST/mg of membranes).
Each point represents averages of four independent observations, with
variation not exceeding 10%. Inset, Western blot indicating
the partitioning of endogenous M31 in mitotic and interphase cells.
Int.P, insoluble material (100,000 × g
pellet) from interphase cells; Int.Cy, soluble material
(cytosol, 100,000 × g supernatant) from interphase
cells; Mit.P, insoluble material from mitotic cells;
Mit.Cy, soluble material from mitotic cells. b,
binding of [35S]M31-GST to the nuclear envelopes under
various assay conditions. No Cy, no cytosol added; + Whole Cy, addition of whole cytosol (80 µl); + Depl
Cy, addition of cytosol (80 µl; ~400 µg of protein) that had
been pre-incubated with 2 mg of immobilized M31-GST; CTL
M31, assay with [35S]M31-GST, pre-incubated with
gelatin/KHM and reisolated by affinity chromatography; Cy-M31, assay
with [35S]M31-GST pre-incubated with 80 µl of cytosol
and reisolated by affinity chromatography; CTL NE, assay
with nuclear envelopes pre-incubated with gelatin/KHM and washed with
buffer; Cy-NE, assay with nuclear envelopes pre-incubated
with cytosol and washed with buffer. The experiments were done in
triplicate under the conditions specified in a.
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Fig. 3.
Binding of the 55-kDa protein to nuclear
envelopes as detected by flotation in sucrose gradients and direct
sedimentation assays. a, direct sedimentation assays.
One hundred µl of [35S]methionine-labeled cytosol was
co-incubated for 45 min with 100 µg of purified nuclear envelopes and
centrifuged at 12,000 × g. One-quarter of the
solubilized pellet (Cy+NE) and a sample of whole cytosol
(Cy, 10 µl) were analyzed by SDS-PAGE and
autoradiographed. The arrow indicates the position of the
55-kDa protein. b, flotation assays. One hundred µl of
[35S]methionine-labeled cytosol was mixed with an equal
volume of KHM/1% gelatin or 100 µg of purified nuclear envelopes (1 mg/ml). Following a 45-min incubation, the samples were combined with
600 µl of 80% sucrose in KHM, loaded at the bottom of gelatin-coated
tubes, and overlaid with sucrose (4 ml of 40%, 4 ml of 30%, and 3.2 ml of 20% in KHM). After ultracentrifugation (100,000 × g, 18 h, 4 °C) and collection of fractions, aliquots
were analyzed by SDS-PAGE and autoradiography. Autorad,
autoradiogram; Cy+Buff., cytosol and buffer;
Cy+NE, cytosol and nuclear envelopes; Coomassie
B.B., Coomassie Blue-stained gel. Arrows denote the
position of the 55-kDa protein. The position of the major nuclear envelope
protein LBR is also indicated. The bottom
(Bott.) of the gradients is to the left,
and the top is to the right. Note that all
cytosolic proteins remain at the loading zone when the membranes are
omitted. However, the 55-kDa protein migrates to the 30-40% sucrose
interface when nuclear envelopes are included in the reaction. The
membranes also float to the 30-40% sucrose zone, as documented in the
Coomassie Blue-stained gel.
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Fig. 4.
Isolation and functional testing of the
55-kDa protein. a, partial extraction of the 55-kDa
protein from the nuclear envelopes. Samples containing 40 µl of
[35S]methionine-labeled cytosol and 50 µg of nuclear
envelopes were incubated for 45 min and spun at 12,000 × g for 30 min. After resuspension of the pellets, the
membranes were extracted with KHM buffer, distilled water, and KHM
containing 0.5 M or 1.0 M NaCl and
recentrifuged. The panel shows an SDS-PAGE/autoradiographic profile of
the material that remains bound to nuclear envelopes (Pel.)
or is released into the supernatant (Sup.) after the various
treatments. Only the relevant area of the gel is depicted here.
b, material highly enriched in the 55-kDa protein
(arrow) that has been isolated by incubating nuclear
envelopes with interphase (ext-I) or mitotic
(ext-M) cytosol, and subsequent extraction with salt (for
details see "Experimental Procedures"). An SDS gel is shown after
staining with Coomassie Blue. The lines on the
left indicate molecular weight values (97,000, 68,000, 45,000, and 31,000 from top to bottom).
c, binding of [35S]M31-GST in the absence of
cytosol (CTL), in the presence of whole cytosol (+ whole Cy; 45 µl of 8 mg/ml), and in the presence of the
partially purified 55-kDa protein (+ ext - I; 11 µg of
protein). The assay was performed as specified in the legend to Fig.
2.
2- and
2/6-tubulin (Fig.
5a). This result was fully
confirmed by Western blotting; when whole cytosol was co-incubated with
NEs, soluble tubulin bound to the membranes and partitioned with the
pellet fraction (Fig. 5b).
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Fig. 5.
Identification of the 55-kDa protein as
2/6:
2-tubulin.
a, internal sequences obtained from the 55-kDa band. In all
but two positions, the peptides match exactly human
2- and human or
mouse
2/6-tubulin. Single amino acid differences in two of the
2
peptides (Cys instead of Met and Asp instead of His) are
underlined. b, binding of soluble tubulin to
nuclear envelopes. Nuclear envelopes and interphase cytosol were
assayed as specified in the legend to Fig. 2. Samples were analyzed by
SDS-PAGE and immunoblotted with anti-tubulin antibodies. b.
tb, rat brain tubulin; NE, turkey erythrocyte nuclear
envelopes; Cy, whole cytosol; NE+Cy, nuclear
envelopes after co-incubation with cytosol; depl.Cy,
material remaining in the supernatant fraction after co-incubating
cytosol with the nuclear envelopes.
7
M; Fig. 6c).
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Fig. 6.
Purified rat brain tubulin associates
specifically with the nuclear envelope. a, SDS-PAGE
profile of purified rat brain tubulin (b.tb) and molecular
weight markers (M) (97,000, 68,000, 45,000, and 31,000 from
top to bottom). The gel was overloaded on
purpose. b, binding of purified tubulin to nuclear envelopes
as detected by a co-sedimentation and SDS-PAGE.
NE+b.tb, brain tubulin and nuclear envelopes;
pNE, proteolyzed nuclear envelopes; pNE+b.tb,
brain tubulin and proteolyzed nuclear envelopes. c,
quantification of tubulin binding to the nuclear envelopes as assayed
by co-sedimentation and quantitative immunoblotting (ECL). The curve
shows the amount of nuclear envelope-bound tubulin as a function of its
concentration. Inset, typical ECL blot from which
quantitative data were obtained. Lane 1 (no signal)
corresponds to a sample that received no tubulin, whereas lanes
2-7 correspond to samples that received 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 µg, respectively, of tubulin. The assay volume was 100 µl,
and all samples in b and c contained 1 µM nocodazole in 10 mM Pipes-KOH to maintain
the tubulin in a nonpolymeric form.
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Fig. 7.
Purified tubulin inhibits M31 binding to the
nuclear envelope. a, binding of
[35S]M31-GST in the presence of bovine serum albumin
(columns 1 and 3) or purified rat brain tubulin
(tb) (columns 2 and 4). In one
set of samples the assay was done in medium (150 mM)
salt/Mg at 23 °C (columns 1 and 2), whereas in
the other case the experiment was done in 10 mM salt at
0 °C and in the presence of 1 µM nocodazole
(columns 3 and 4). All assays were performed as
described in the legend to Fig. 2. b, experiments similar to
that shown in a at two different pH values (black
bars, pH 7.4; gray bars, pH 5.7). In this case 1 mg of
exogenous tubulin was added to the assay mixture, and all samples
contained 1 µM nocodazole.
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Fig. 8.
Purified tubulin prevents in
situ binding of GST-M31 with the nuclear envelope.
Confocal sections of permeabilized HeLa cells incubated with M31-GST or
M31-GST and soluble rat brain tubulin (tb). In the
first case the absence of tubulin was compensated by adding an equal
amount of bovine serum albumin. All samples contained 1 µM nocodazole. Exogenous M31 was localized using
affinity-purified anti-GST antibodies (green).
Panels labeled PI (red) are the
corresponding propidium iodide profiles. The insets show
larger magnifications of the cells indicated by the
arrowheads. Note the prominent rim-like pattern in the
control and the relatively attenuated staining in the sample containing
tubulin.
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Fig. 9.
Soluble tubulin inhibits nuclear
envelope reassembly in vitro. In vitro reassembly
assays were performed as described under "Experimental Procedures."
Panels labeled Control show permeabilized mitotic
cells incubated for 2 h at 33 °C in the presence of bovine
serum albumin. Panels labeled +Sol.tb correspond
to analogous samples incubated in the presence of soluble tubulin.
Assembly of nuclear envelope proteins was assessed by staining the
particles with anti-LAP2B or anti-lamin B antibodies (green)
and propidium iodide (PI; red).
FITC, fluorescein isothiocyanate
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, M31, and
M32 accumulate at the nuclear periphery when injected into interphase
cells and decorate the nuclei of detergent-permeabilized cells in a
characteristic, rim-like fashion. Furthermore, all HP1 variants exhibit
saturable and site-specific binding to isolated nuclear envelopes
(24).
and hHP1
in two-hybrid assays (11, 20),
or constituents of peripheral heterochromatin (34). Acetylation and
other posttranslational modifications undoubtedly play a role in HP1
dynamics, because targeting of M31 and M32 to the nuclear envelope is
abolished upon treatment with deacetylase inhibitors (24). Nonetheless,
because HP1 proteins oligomerize and are capable of interacting with a
wide variety of cellular components (see the Introduction), it would be
reasonable to assume that their molecular associations are regulated by
auxiliary factors and molecular chaperones. In this study, we have
attempted to identify and characterize such factors using quantitative
in vitro assays in combination with subcellular
fractionation. Our results show that M31 binding to the nuclear
envelope is potently inhibited by nonpolymeric tubulin. This is a
surprising finding, because soluble tubulin has always been considered
to be a mere "reservoir" of microtubule subunits.
dimers, of which only 15%
are incorporated into microtubules (35). Although the proportion of
polymeric tubulin could be much higher in cultured cells (up to 62% in
porcine kidney epithelial cells, according to Zhai and Borisy (36)),
the intracellular concentration of unassembled tubulin is on the order
of 1 mg/ml (37), i.e. well above the critical concentration
for microtubule assembly. A transient increase in the concentration of
soluble tubulin should be expected at prophase-prometaphase, when the network of interphase microtubules has been destroyed, and the mitotic
spindle has not been fully assembled. At that point, soluble tubulin
may gain access to internal nuclear structures, because the nuclear
envelope is disrupted by a combination of mitotic hyperphosphorylation
and mechanical force (for a review see Ref. 38). Tubulin binding to the
disassembling nuclear envelope might prevent premature interactions
between fragments of the nuclear membrane and M31 at early phases of
cell division. Reversal of this process, i.e. dissociation
of tubulin from nuclear envelope-derived membranes, could occur at
subsequent stages of mitosis, when the spindle fully develops, the mass
of polymerized tubulin increases to interphase levels (36), and the
concentration of soluble tubulin presumably drops.
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ACKNOWLEDGEMENTS |
---|
We thank K. Weber and co-workers (Max Planck Institute for Biophysical Chemistry, Goettingen, Germany) for sequencing peptides of the 55-kDa protein. H. Polioudaki and O. Kostaki contributed excellent technical support, and F. Xekardaki contributed valuable assistance with artwork.
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FOOTNOTES |
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* This work was supported by PENED-`99 and EPET II grants (Greek Secretariat of Research and Technology) and by a core strategic grant (to P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A recipient of a predoctoral fellowship from the Graduate Program in Molecular Biology/Biomedicine of the University of Crete.
** To whom correspondence should be addressed: University of Crete, School of Medicine, Stavrakia, 71 110 Heraklion, Crete, Greece. Tel.: 0030-81-39-45-46; E-mail: takis@med.uoc.gr.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M007135200
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ABBREVIATIONS |
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The abbreviations used are: HP1, heterochromatin protein 1; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; NE, nuclear envelope vesicle; LBR, lamin B receptor.
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