3703
Journal of Cell Science 113, 3703-3713 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1711
HA95 is a protein of the chromatin and nuclear matrix regulating nuclear
envelope dynamics
Sandra B. Martins1, Turid Eide1, Rikke L. Steen1, Tore Jahnsen1, Bjørn S. Skålhegg2 and Philippe Collas1,*
1Institute
2Institute
of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway
for Nutrition Research, PO Box 1046 Blindern, 0317 Oslo, Norway
*Author for correspondence (e-mail: [email protected])
Accepted 25 August; published on WWW 17 October 2000
SUMMARY
We report a role for HA95, a nuclear protein with high
homology to the nuclear A-kinase anchoring protein
AKAP95, in the regulation of nuclear envelope-chromatin
interactions. Biochemical and photobleaching data indicate
that HA95 is tightly associated with chromatin and
the nuclear matrix/lamina network in interphase, and
bound to chromatin at mitosis. HA95 resides in a complex
together with lamin B receptor (LBR), lamina-associated
polypeptide (LAP)2 and emerin, integral proteins of the
inner nuclear membrane. Cross-linking experiments,
however, illustrate a tight association of HA95 with LBR
and LAP2 only. Intra-nuclear blocking of HA95 with antiHA95 antibodies abolishes nuclear breakdown in a mitotic
HeLa cell extract. The antibodies inhibit nuclear
membrane breakdown and chromatin condensation – the
latter independently of nuclear membranes. However,
lamina disassembly is not affected, as judged by
immunological analyses of A/C- and B-type lamins. In
contrast, immunoblocking of HA95 bound to condensed
chromosomes does not impair chromatin decondensation,
nuclear membrane reassembly or lamina reformation. Our
results argue for a role for HA95 in anchoring nuclear
membranes and lamins to chromatin in interphase, and in
releasing membranes from chromatin at mitosis. The data
also suggest that HA95 is not involved in initial binding of
membranes to chromatin upon nuclear reassembly. We
propose that HA95 is a central platform at the chromatin/
nuclear matrix interface implicated in regulating nuclear
envelope-chromatin interactions during the cell cycle.
INTRODUCTION
Proteins of the INM, lamina and chromosomes are
extensively interconnected. The INM harbors specific integral
proteins including lamin B receptor (LBR; Worman et al.,
1990), lamina-associated polypeptides (LAP)1 and LAP2
(Foisner and Gerace, 1993), emerin (Nagano et al., 1996),
nurim (Rolls et al., 1999) and MAN1 (Lin et al., 2000). LBR,
LAP1, LAP2β and emerin bind to lamins A/C and/or B in vitro
(reviewed by Wilson, 2000). LBR and LAP2β also bind
chromatin via interactions with heterochromatin protein (HP)1
(Ye and Worman, 1996) and the small DNA-binding protein
BAF (barrier to autointegration factor; Furukawa, 1999). An
intranuclear LAP2 variant, LAP2α, also interacts with lamins
A/C (Dechat et al., 1998) while H2-type histones bind lamins
A/C and B in vitro (Goldberg et al., 1999). Thus, the chromatin
provides multiple anchoring sites for the NE.
HA95, also called HAP95 or NAKAP95, is a novel nuclear
protein recently cloned by us (Ørstavik et al., 2000) and others
(Seki et al., 2000; Westberg et al., 2000). HA95 exhibits high
homology to the human nuclear A-kinase (PKA) anchoring
protein, AKAP95 (Eide et al., 1998). Like AKAP95, HA95
harbors two zinc fingers and a putative nuclear localization
signal but does not contain the PKA-binding domain of
AKAP95 (Ørstavik et al., 2000). HA95 and AKAP95 colocalize in interphase and at mitosis, yet the two proteins do
The nuclear envelope (NE) is a highly dynamic structure
consisting of two concentric membranes underlaid by the
nuclear lamina, a network of intermediate filaments called
A/C- and B-type lamins (reviewed by Collas and Courvalin,
2000; Wilson, 2000). The outer nuclear membrane (ONM) is
in direct continuity with the endoplasmic reticulum (ER) and
shares biochemical and functional properties with the ER. The
inner nuclear membrane (INM) harbors specific integral
proteins that provide attachment sites for chromatin and the
nuclear lamina. ONM and INM are connected by sharply bend
membranes associated with nuclear pore complexes. The
dynamic nature of the NE is best illustrated at mitosis when
the NE breaks down and reassembles around daughter
chromosomes. Disassembly of the NE at prophase correlates
with phosphorylation of proteins of the chromatin, nuclear
membranes and lamina, leading to solubilization of lamin A/C
while B-type lamins remain mostly in a membrane-bound form
(Gerace and Blobel, 1980). In late anaphase, ER-derived
membranes associate with chromatin to reform nuclear
membranes (Chaudhary and Courvalin, 1993; Ellenberg et al.,
1997), while in telophase, lamins are dephosphorylated and
repolymerize (Ottaviano and Gerace, 1985).
Key words: HA95, Chromatin, Nuclear envelope, Mitosis
3704 S. B. Martins and others
not interact. Nevertheless, HA95 is found in multiprotein
complexes directly associated with one another or indirectly
associated through other nuclear proteins. To support this view,
HA95/HAP95 was recently shown to interact with RNA
helicase A and enhance expression of CTE, an RNA element
involved in nuclear export of retroviruses (Westberg et al.,
2000).
We report here that HA95 is a chromatin- and nuclear
matrix-associated protein implicated in the regulation of NEchromatin interactions. HA95 directly interacts with the lamina
and with a subset of integral proteins of the INM. Antibodymediated blocking of HA95 inside purified nuclei inhibits
chromatin condensation and disassembly of nuclear
membranes, but not of the lamina, in mitotic cytosol. However,
immunoblocking HA95 bound to mitotic chromosomes does
not affect nuclear reformation. We propose that HA95 is a
central element of the chromatin and the nuclear matrix
implicated in regulating NE-chromatin interactions during the
cell cycle.
MATERIALS AND METHODS
Reagents and antibodies
Affinity-purified rabbit polyclonal antibodies directed against the
NH2-terminal domain of human LBR, against a peptide of human
lamin B (not distinguishing between lamin B isoforms), and against
a peptide of human LAP2 (generous gifts from Drs J.-C. Courvalin
and B. Buendia, Institut Jacques Monod, Paris, France) were
described elsewhere (Buendia and Courvalin, 1997; Chaudhary and
Courvalin, 1993; Collas et al., 1996). Anti-HA95 polyclonal
antibodies (designated α-HA95) directed against amino acids 65-84
of human HA95 were produced and affinity-purified as described
earlier (Ørstavik et al., 2000). The HA95 peptide (residues 65-84) was
synthesized by Eurogentec. The anti-Myc antiserum was from
Invitrogen (cat. no. R950-25). Anti-emerin monoclonal antibodies
(mAbs) were a generous gift from Dr G. E. Morris (N. E. Wales
Institute, Wrexham, UK; Manilal et al., 1996).
Cell culture and transfection
The human fibroblast cell line 293T and the B cell line Bjab were
cultured in RPMI1640 (Gibco BRL) as described previously (Ørstavik
et al., 2000). HeLa cells were grown as monolayers in EMEM (Gibco
BRL) and synchronized in M phase with 1 µM nocodazole as reported
earlier (Eide et al., 1998).
Bjab cells were stably transfected with an EcoRI/NotI fragment of
full-length HA95 cDNA ligated to a triple c-myc epitope at the 3′ end
(HA95-Myc) and cloned into pcDNA3 (Invitrogen) as described
previously (Ørstavik et al., 2000). Fibroblasts (293T) were transiently
transfected with an EcoRI/BamHI fragment encoding residues 1-347
of human HA95 and ligated into pEGFP-N1 (Clontech) as reported
earlier (Ørstavik et al., 2000). Fibroblasts were also transfected with
a BglII-EcoRI fragment encoding amino acids 326-772 of the human
transcription factor TCF11 (Luna et al., 1995) and ligated into pECFPN1 (Clontech). This TCF11-CFP vector was a kind gift from Dr A.
B. Kolstø (Biotechnology Center, University of Oslo; A. B. Kolstø,
unpublished data). Cells were examined for HA95-GFP or TCF11CFP expression after 24 hours.
Mitotic and interphase cell extracts
Cytosolic extracts were prepared from mitotic HeLa cells as described
earlier (Collas et al., 1999). In short, mitotic cells were homogenized
by sonication in 1 volume of lysis buffer and the lysate sedimented at
10,000 g for 15 minutes. The supernatant was cleared at 200,000 g
for 3 hours in a Beckman SW55 rotor. The clear supernatant (mitotic
cytosol) was aliquoted and frozen in liquid nitrogen and stored at
−80°C. Interphase cytosol was prepared as above except that EDTA
was omitted from the lysis buffer.
Mitotic membrane vesicles were recovered from the 200,000 g
pellet following mitotic cytosol preparation, washed by resuspension
and sedimentation at 100,000 g for 30 minutes in membrane wash
buffer, frozen in liquid nitrogen and stored at −80°C (Collas et al.,
2000).
Isolation of nuclei, nuclear matrices and interphase
chromatin
Nuclei from Bjab cells used for fractionation or immunological
procedures were purified as described elsewhere (Ørstavik et al.,
2000). Briefly, cells were suspended in hypotonic buffer (10 mM TrisHCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2) and lysed by addition of
0.5% NP-40. Nuclei were sedimented, washed in buffer N (10 mM
Hepes, pH 7.5, 2 mM MgCl2, 250 mM sucrose, 25 mM KCl, 1 mM
DTT and protease inhibitors). HeLa cell nuclei were isolated by
Dounce-homogenization as described previously (Collas et al., 1999).
Bjab and HeLa nuclei were frozen at −80°C in buffer N containing
70% glycerol.
For biochemical experiments, high salt (2 M NaCl)-extracted
nuclear matrices were prepared from purified Bjab nuclei after
extractions of nuclei with 1 mg/ml DNAse I and 0.5% Triton X-100
as reported previously (Reyes et al., 1997). Nuclear matrices were
further extracted with 2 M NaCl for 30 minutes at 4°C before being
solubilized in SDS sample buffer. Interphase chromatin was prepared
from Bjab nuclei essentially as described (Collas et al., 1999). Briefly,
approx. 108 nuclei were digested with 5 U microccocal nuclease and
nuclei sedimented. The supernatant was collected and the pellet was
resuspended in 2 mM EDTA and incubated for 15 minutes at 4°C.
After sedimentation the supernatant was combined with the first
supernatant to yield an interphase chromatin fraction. Proteins were
precipitated with trichloracetic acid and dissolved in SDS sample
buffer. To solubilize mitotic chromatin, chromosomes were recovered
from a mitotic cell lysate (Collas et al., 1999), extracted with 1%
Triton X-100, sedimented and the detergent-insoluble fraction was
digested with 5 U microccocal nuclease. After sedimentation, proteins
of the supernatant were precipitated and dissolved in SDS-sample
buffer.
In situ extraction of nuclear matrices was carried out after affixing
Bjab nuclei onto poly-L-lysine-coated glass coverslips. Nuclei were
washed in PBS, extracted with 0.1% Triton X-100 in PBS for 5
minutes, incubated for 5 minutes with ice-cold cytoskeleton
stabilization buffer (CSK; Collas et al., 1999) containing 0.1% Triton
X-100 and washed twice with CSK buffer. DNA was digested for 30
minutes with 1 mg/ml DNase I made in CSK buffer. Digested nuclei
were washed twice 5 minutes with PBS and fixed with 3%
paraformaldehyde for immunofluorescence analysis.
Cross-linking of anti-HA95 antibodies to nuclear
localization signal peptides and introduction of
antibodies-NLS conjugates into nuclei
Synthetic nuclear localization signal (NLS) peptides of the SV40 large
T antigen (CGGPKKKRKVG-NH2) were conjugated to α-HA95 or
pre-immune IgGs using the cross-linker MBS (Pierce Biochemicals).
One hundred micrograms of immunoglobulins in 100 µl were added
to 10 µl MBS (at 1 mg/ml in dimethylformamide) and the mixture
stirred for 30 minutes. Antibodies derivatized with MBS were
combined with 100 µg NLS (at 1 mg/ml in sodium phosphate, pH 7.2)
for 6 hours at room temperature and unconjugated peptide was
removed by dialysis against PBS overnight at 4oC. NLS cross-linking
to antibodies was verified by nuclear import of antibodies in
interphase cytosol as described below. NLS-conjugated α-HA95 did
recognize HA95 by immunofluorescence and immunoblotting,
indicating that NLS cross-linking to the antibody did not interfere
with antigen recognition (data not shown).
Nuclear envelope anchoring to chromatin by HA95 3705
A nuclear import assay for antibody-NLS conjugates typically
consisted of 20 µl interphase cytosol, an ATP-generating system (2
mM ATP, 20 mM phosphocreatine, 50 µg/ml creatine kinase), 100 µM
GTP, 2-4×104 nuclei and 2 µl antibody-NLS import substrate (0.3
µg/µl final protein concentration). Reactions were incubated at 23°C
for 1 hour. At the end of incubation, nuclei were recovered by
sedimentation at 1,000 g through a 1 M sucrose cushion. Nuclei were
either resuspended in buffer N for immunofluorescence or in mitotic
cytosol for nuclear disassembly reactions. Nuclear import of
antibodies was monitored by immunofluorescence using TRITCconjugated anti-rabbit antibodies (data not shown).
Immunoprecipitation of the HA95 complex after crosslinking
Purified Bjab nuclei (approx. 2×107) suspended in buffer N were
cross-linked with 1 mM of the cleavable cross-linker dithiobissuccinimidylproprionate (DSP) for 15 minutes at room temperature
(Zhao et al., 1998). The reaction was stopped with 50 mM Tris (pH
7.5) from a 1 M stock and the mixture incubated for 15 minutes at
room temperature. Proteins were precipitated with 0.25 M (NH4)2SO4
and dissolved in 8 M urea. Urea was diluted to 2 M with
immunoprecipitation buffer and HA95 was immunoprecipitated as
described above.
Nuclear disassembly and chromatin condensation
A nuclear disassembly/chromatin condensation reaction typically
consisted of 20 µl mitotic cytosol and 1 µl of nuclei (2-4×104 nuclei,
intact or loaded with antibodies). The reaction was started by
addition of the ATP-generating system and allowed to proceed at
30°C for up to 2 hours (Collas et al., 1999). Condensation of
chromatin into compact chromosomes was monitored by phase
contrast microscopy or by staining DNA with 0.1 µg/ml Hoechst
33342 (Steen et al., 2000). Percent chromatin condensation was
calculated as the ratio of condensed chromatin masses per number
of nuclei examined. For biochemical analyses, the condensed
chromatin was sedimented at 1,000 g through 1 M sucrose and
recovered from the pellet.
Fluorescence recovery after photobleaching (FRAP) and
fluorescence loss in photobleaching (FLIP) experiments
FRAP was carried out at room temperature with an Argon-Krypton
laser (model 643LICA-A01, Omnichrome, Carlsbad, CA, USA) using
a confocal scanning laser DM RXA Leica microscope fitted with a
×63 water-immersion objective. Cells expressing HA95-GFP or
TCF11-CFP (used as a control for fluorescence recovery) were
illuminated at 488 nm and detection done at 495-585 nm. The
indicated box in Fig. 2B was bleached by continuous scanning and
recovery of fluorescence monitored by scanning the whole cell in 5minute intervals. We noted that the diffusion rate of TCF11-CFP was
so high that it was virtually impossible to detect photobleaching in
nuclei of TCF11-CFP-expressing cells (data not shown). Thus, FLIP
was carried out using TCF11-CFP-expressing fibroblasts by
continuously photobleaching the area shown in Fig. 2C for five
successive 50-second periods and monitoring fluorescence by
scanning the entire cell between each bleaching sequence. In neither
FRAP nor FLIP experiments did scanning laser intensity significantly
photobleach the sample.
Nuclear reconstitution assay
A chromatin substrate was prepared from interphase nuclei
disassembled in mitotic cytosol as described above. Condensed,
membrane-free chromatin masses were recovered by sedimentation
through 1 M sucrose and resuspended in interphase cytosol to their
initial concentration (approx. 2,000 chromatin units/µl extract). It was
necessary to supplement the interphase cytosol with mitotic vesicles
to provide membranes harboring proteins required for NE assembly
(see Results; Collas et al., 1996; Pyrpasopoulou et al., 1996). An ATP
generating system and 100 µM GTP were added to promote chromatin
decondensation, nuclear membrane vesicles binding to chromatin and
fusion of vesicles into nuclear membranes (Collas et al., 1996). In
some instances, cytosol and chromatin masses were pre-incubated
with antibodies for 30 minutes prior to adding the ATP-generating
system. The reaction was incubated at 30°C for up to 2 hours and
nuclear reassembly was monitored by phase contract microscopy,
DNA labeling with Hoechst 33342, membrane staining with 10 µg/ml
of the lipophilic dye DiOC6 and immunofluorescence.
Immunological procedures
Immunoblotting analysis was performed as described earlier (Collas
et al., 1999) using the following antibodies: anti-HA95 (1:250
dilution), anti-lamin B (1:1,000), anti-LBR (1:500), anti-LAP2
(1:500), anti-emerin (1:250). For immunofluorescence analysis
(Collas et al., 1996) cells, nuclei or chromatin masses were settled
onto coverslips, fixed with 3% paraformaldehyde, permeabilized with
0.1% Triton X-100 (cells and nuclei only) and proteins blocked with
2% BSA in PBS/0.01% Tween-20. Primary and secondary antibodies
were used at a 1:100 dilution and DNA was stained with 0.1 µg/ml
Hoechst 33342. Observations were made and photographs taken as
described earlier (Collas et al., 1999). For immunoprecipitations
(Collas et al., 1999), whole mitotic or interphase cells were sonicated
on ice in immunoprecipitation buffer (10 mM Hepes, pH 7.5, 10 mM
KCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitors) and the
lysate centrifuged at 10,000 g for 15 minutes. The supernatant was
pre-cleared with Protein A/G agarose. Immunoprecipitations were
carried out with relevant antibodies (diluted 1:50) from the
supernatant at room temperature for 2.5 hours, followed by incubation
with Protein A/G-agarose for 1.5 hours and centrifugation at 4,000 g
for 10 minutes. Immune complexes were washed three times and
proteins eluted in SDS sample buffer.
RESULTS
HA95 is a non-diffusible nuclear protein
The subcellular localization of HA95 was examined
throughout the cycle in the human B cell line Bjab.
Immunofluorescence analysis using affinity-purified antiHAP95 antibodies (α-HA95) indicated that HA95 was
exclusively nuclear in interphase, co-localized with DNA and
was excluded from nucleoli (Fig. 1A). During all mitotic
stages, HA95 staining was restricted to chromosomes (Fig.
1A). Cell fractionation data confirmed these observations
and showed that HA95 was nuclear in interphase and cofractionated with micrococcal nuclease-soluble chromatin
at mitosis (Fig. 1B). These results were verified by
immunofluorescence analysis of 293T fibroblasts transiently
expressing HA95-GFP, in which endogenous HA95 colocalized with HA95-GFP (Fig. 1C). Additionally, Myc-tagged
HA95 stably expressed in Bjab cells also co-localized with
endogenous HA95, as judged by double immunofluorescence
using anti-Myc and anti-HA95 antibodies (Fig. 1D). Thus,
HA95 is restricted to the nucleus in interphase and associates
with chromatin at mitosis.
To address the extent of association of HA95 with nuclear
structures, purified Bjab nuclei were extracted with
incremental concentrations (0.05-1 M) of NaCl in the presence
of 0.1% Triton X-100. Western blotting analysis of particulate
and soluble fractions indicated that HA95 remained insoluble
even at the highest salt concentration (Fig. 2A). Insolubility of
HA95 after detergent and salt extraction was verified in a
FRAP experiment to qualitatively examine the mobility of
3706 S. B. Martins and others
Fig. 1. Subcellular distribution of HA95 during the cell cycle. (A) Distribution of HA95 was examined by immunofluorescence analysis of
unsynchronized Bjab cells using an affinity-purified polyclonal anti-HA95 antibody. DNA (insets) was labeled with Hoechst 33342.
(B) Interphase and mitotic Bjab cells were fractionated into nuclei or chromatin, cytosol and cytoplasmic membranes, and proteins of each
fraction were immunoblotted using the anti-HA95 antibody. (C) Human 293T fibroblasts were transiently transfected with HA95-GFP. After 24
hours, cells were fixed, labeled with anti-HA95 antibodies and localizations of HA95-GFP and endogenous HA95 were examined by
fluorescence microscopy. (D) Bjab cells stably expressing HA95 tagged with a Myc epitope (HA95-Myc) were analyzed by
immunofluorescence using anti-Myc and anti-HA95 antibodies. DNA was stained with Hoechst 33342. Bars, 10 µm.
HA95-GFP in living 293T fibroblasts. HA95-GFP was found
to be highly immobile in interphase nuclei, as judged by lack
of fluorescence recovery up to 15 minutes after photobleaching
(Fig. 2B). In a control experiment, photobleaching fibroblast
nuclei expressing residues 326-772 of the transcription factor
TCF11 fused to CFP was followed by immediate recovery of
fluorescence into the bleached area, such that the bleach was
virtually impossible to monitor (data not shown). However,
repeated 50-second bleaches elicited a loss of TCF11-CFP
fluorescence in the entire nucleus over a 275-second period
(Fig. 2C, right nucleus, arrow; the nucleus on the left was not
bleached). This ascertained that a mobile nuclear protein was
capable of diffusing under the present conditions. Taken
together, the data of Fig. 2 argue that HA95 is a non-diffusible
protein tightly anchored to nuclear structures.
HA95 is a protein of the chromatin and of the
nuclear matrix scaffold
To address the subnuclear distribution of HA95, purified Bjab
nuclei were fractionated into salt-extracted nuclear matrices
(defined as a nuclear scaffold resistant to 1 mg/ml DNase I,
0.5% Triton X-100 and 2 M NaCl) and micrococcal nuclease-
soluble chromatin. Western blots and densitometric analyses of
each fraction revealed that approx. 30% of HA95 partitioned
with the matrix while approx. 70% co-fractionated with
chromatin (Fig. 3A).
These biochemical data were verified by in situ preparations
of Bjab nuclei and nuclear matrices as described in Materials
and Methods, followed by immunofluorescence analysis. As
expected, intranuclear HA95 labeling was detected in intact
nuclei, whereas antibodies against B-type lamins (referred to
as ‘lamin B’ in the text), markers of the nuclear matrix/lamina
network, decorated the NE (Fig. 3B, Nuclei). DNase digestion
of nuclei however, removed most intranuclear HA95 labeling
(Fig. 3B, Matrices), suggesting the association of a fraction of
HA95 with nucleic acids. Nevertheless, strong perinuclear
labeling of HA95 remained and co-localized with lamin B
throughout the lamina/matrix network, as evidenced by varying
the focal plane of examination (Fig. 3B, Matrices). Thus,
although HA95 is primarily associated with DNase-sensitive
chromatin, a significant fraction of HA95 also associates with
the nuclear matrix/lamina network.
Association of HA95 with the nuclear lamina was verified
biochemically after immunoprecipitation of HA95 or lamin B
Nuclear envelope anchoring to chromatin by HA95 3707
Fig. 2. HA95 is a non-diffusible nuclear protein. (A) Salt
extractability of HA95. Purified Bjab nuclei were extracted with
0.1% Triton X-100 and increasing NaCl concentrations as indicated.
Nuclei were sedimented and pellet (P) and supernatant (S) fractions
were immunoblotted using anti-HA95 antibodies. (B) Qualitative
FRAP experiment in interphase 293T fibroblast nuclei expressing
HA95-GFP. The sequence displays the absence of fluorescence
recovery over a 15-minute period after photobleaching the marked
area. (C) FLIP experiment in a 293T fibroblast expressing TCF11CFP (right nucleus, arrow). The sequence displays the gradual loss of
fluorescence after photobleaching the marked area continuously for
50 seconds between each observation. The nucleus on the left was
not bleached and was used as a control to show that scanning laser
intensity did not significantly photobleached the samples. Apparent
increased fluorescence in the bleach nucleus between 165 and 220
seconds is due to refocusing on nucleus. Bars, 5 µm.
from Bjab cells. HA95 and lamin B co-precipitated from
interphase cell lysates regardless of the precipitating antibody,
whereas at mitosis the proteins were segregated (Fig. 3C). We
noted however, that not all HA95 was co-precipitated with
lamin B under conditions where all detectable lamin B could
be immunoprecipitated, as judged on immunoblots of the nonprecipitated material (Fig. 3C, Sup.). Interaction of HA95 with
lamin B in interphase was verified by co-precipitation of the
lamin from Bjab cells expressing Myc-tagged HA95, using
anti-Myc antibodies (Fig. 3D). Lamin B also co-precipitated
with HA95 after treating Bjab nuclei with the cleavable,
membrane-permeable, cross-linker DSP (Zhao et al., 1998) and
denaturation in 8 M urea (Fig. 3D, DSP). These results indicate
a tight interaction between HA95 and the lamina that is cell
cycle-regulated.
HA95 interacts with integral proteins of the inner
nuclear membrane
The existence of HA95 at the NE-chromatin interface raises
the possibility that HA95 interacts with the inner nuclear
membrane (INM). To test this hypothesis, HA95 was
immunoprecipitated from interphase cells and the precipitates
were immunoblotted with antibodies against the INM markers
LBR, LAP2β and emerin. These three markers co-precipitated
with HA95, but not with pre-immune IgGs (Fig. 4A).
Interaction of HA95 with INM proteins was confirmed by the
co-precipitation of LBR, LAP2β and emerin with HA95-Myc
from HA95-Myc-expressing cells, using anti-Myc antibodies
(Fig. 4B). Moreover, anti-Myc antibodies co-precipitated
HA95-Myc as well as endogenous HA95, suggesting that
HA95 associates with itself (see also Ørstavik et al., 2000; Fig.
4B). Finally, to evaluate the strength of the interaction of HA95
with INM proteins, HA95 was immunoprecipitated from Bjab
nuclei following reversible cross-linking with DSP under low
efficiency cross-linking conditions and protein denaturation in
8 M urea (see Zhao et al., 1998). Neither LBR, LAP2β nor
emerin co-precipitated with HA95 when proteins were
denatured with urea without previous DSP cross-linking (data
not shown). In contrast, both LBR and LAP2β co-precipitated
with HA95 from DSP-treated nuclei (Fig. 4C). Interestingly
however, emerin did not co-precipitate with HA95 under these
conditions (Fig. 4C). These results argue that associations of
HA95 with LBR and LAP2β are stronger than that with
emerin.
Functional HA95 is required for disassembly of
nuclear membranes, but not of the lamina, in mitotic
cytosol
Disassembly of the NE at mitosis involves the disruption of
INM-lamina-chromatin interactions. We determined whether
HA95 played a role in NE disassembly by blocking HA95
function within purified HeLa nuclei using the affinity-purified
3708 S. B. Martins and others
Fig. 3. HA95 is associated with chromatin and the nuclear matrix. (A) Purified Bjab nuclei were fractionated into chromatin and high saltwashed nuclear matrices and proteins were immunoblotted using anti-HA95 antibodies. Relative amounts (means ± s.d.) of HA95 in each
fraction were determined by densitometric analysis of duplicate blots. (B) Bjab nuclei settled on coverslips were permeabilized with 0.1%
Triton X-100 (Nuclei) or further extracted with DNase I to produce nuclear matrices (Matrices). Nuclei and matrix preparations were fixed and
examined by immunofluorescence using anti-HA95 and anti-lamin B antibodies. DNA was labeled with Hoechst 33342. Photographs of
matrices were taken in two different focal planes. Bar, 10 µm. (C) HA95 and lamin B (LB) were immunoprecipitated (IP) from interphase and
mitotic cell lysates and immune precipitates immunoblotted with anti-HA95 and anti-lamin B antibodies. Sup., immunoblot of a reaction
supernatant following complete immunodepletion of B-type lamins from interphase cell lysate. (D) Left panel, HA95-Myc was
immunoprecipitated from Bjab cells expressing Myc-tagged HA95 using anti-Myc antibodies and precipitates were immunoblotted using antilamin B antibodies. Right panel, co-precipitation of HA95 and lamin B after cross-linking nuclear proteins with DSP and denaturing proteins
with 8 M urea. HA95 was immunoprecipitated and precipitates immunoblotted with anti-lamin B antibodies. IgG, control
immunoprecipitations using pre-immune rabbit IgGs.
Fig. 4. HA95 associates with
integral proteins of the inner
nuclear membrane. (A) HA95
was immunoprecipitated (IP)
from interphase Bjab cell lysates
and immune precipitates were
immunoblotted with anti-LBR,
anti-LAP2 and anti-emerin
antibodies. Control
immunoprecipitations were
performed using pre-immune
rabbit IgGs. (B) Co-precipitation
of HA95 and INM proteins from
Bjab cells expressing HA95-Myc. Interphase cell lysates were immunoprecipitated using anti-Myc antibodies and immune precipitates
immunoblotted using anti-HA95, anti-LBR, anti-LAP2 and anti-emerin antibodies. (C) Immunoprecipitation of HA95 after cross-linking. Bjab
nuclei were cross-linked with DSP, proteins were precipitated, denatured in 8 M urea and HA95 was immunoprecipitated. The precipitates were
immunoblotted using anti-LBR, anti-LAP2 and anti-emerin antibodies.
Nuclear envelope anchoring to chromatin by HA95 3709
Fig. 5. Intranuclear
immunoblocking of HA95
inhibits nuclear breakdown in
mitotic cytosol. (A) Anti-HA95
antibodies were introduced into
purified HeLa nuclei as
described in Materials and
Methods (Input). After
recovery by sedimentation
through sucrose, nuclei were
resuspended in mitotic cytosol
and nuclear morphology was
examined after 2 hours by
phase contrast microscopy and
membrane labeling with
DiOC6. No Ab, control nuclei
not exposed to any antibody;
IgG, control nuclei loaded with
pre-immune IgGs; α-HA95,
nuclei loaded with α-HA95; αHA95+pep., nuclei loaded with
α-HA95 bound to the
competitor HA95 peptide. Bar,
10 µm. (B) Proportions (± s.d.)
of chromosome condensation
in nuclei containing α-HA95
(䊉), preimmune IgGs (ⵧ), no
antibodies (䉱) or α-HA95 plus
competitor peptide (䉲) during
incubation in mitotic cytosol.
anti-HA95 antibody, and monitoring nuclear breakdown in a
cytosolic extract derived from mitotic HeLa cells. This mitotic
cytosol was previously shown to support NE disassembly and
chromosome condensation (Steen et al., 2000). Anti-HA95
antibodies (or pre-immune rabbit IgGs) were conjugated to
NLS peptides and imported into purified HeLa nuclei using a
cell-free nuclear import assay.
To address the effect of immunoblocking HA95 on nuclear
disassembly, antibody-loaded interphase nuclei (Fig. 5A,
Input) were exposed for 2 hours to mitotic cytosol and nuclear
disassembly was assessed by phase contrast microscopy and
nuclear membrane labeling with the lipophilic dye, DiOC6.
Control nuclei not exposed to immunoglobulins or loaded with
pre-immune IgGs condensed into chromosomes while nuclear
membranes disassembled (Fig. 5A,B, No Ab, IgG). In contrast,
α-HA95 inhibited chromatin condensation and nuclear
membrane solubilization (Fig. 5A, α-HA95) even after
prolonged exposure to the cytosol (Fig. 5B). This inhibition
was specific since it did not occur with α-HA95 introduced into
nuclei with 160 µM of the competitor HA95 peptide used to
generate the antibody (Fig. 5A,B α-HA95+pep.).
To ascertain the maintenance of the NE after
immunoblocking HA95, markers of the INM and of the lamina
were examined by immunofluorescence following nuclear
disassembly in the presence or absence of α-HA95. Clearly,
the INM persisted after blocking HA95 function as shown by
LBR labeling (Fig. 6A, α-HA95). However both A/C- and Btype lamins were eliminated (Fig. 6A, α-HA95). Solubilization
of B-type lamins, but not of LBR, was verified on western blots
of nuclei and chromatin fractions (P) and reaction supernatants
(S) at the end of a 2-hour incubation in mitotic cytosol (Fig.
6B). Thus, the absence of lamin B immunostaining resulted
from solubilization of the lamin B isoforms rather than from
antigen masking. Control nuclei not exposed to antibodies (not
shown), or loaded with pre-immune IgGs or with α-HA95
bound to the competitor HA95 peptide underwent nuclear
membrane breakdown, lamina disassembly and chromatin
condensation (Fig. 6A, IgG, α-HA95+pep.). Additional
immunoprecipitation experiments indicated that interactions of
HA95 with lamin B, LBR and LAP2β were maintained in
interphase nuclei loaded with α-HA95, indicating that the
disruption of the interaction of HA95 with B-type lamins was
not a mere consequence of introducing anti-HA95 antibodies
into nuclei (data not shown). Rather, the results argue that
intranuclear immunoblocking of HA95 prevents disassembly
of the nuclear membranes, but not of the lamina, in mitotic
cytosol.
HA95 function is required for chromatin
condensation
Intranuclear blocking of HA95 prevents nuclear membrane
breakdown and chromatin condensation. To determine whether
the lack of chromosome condensation resulted from inhibition
of membrane disassembly or from an effect of anti-HA95
antibodies on chromatin per se, we assessed the consequence
of immunoblocking intranuclear HA95 after removal of
nuclear membranes with detergent. HeLa nuclei were extracted
with 0.1% Triton X-100 to solubilize nuclear membranes
(verified by staining with DiOC6; data not shown), incubated
with α-HA95 for 1 hour in nuclear buffer and subsequently
exposed to mitotic cytosol for 2 hours. As anticipated,
demembranated nuclei not exposed to immunoglobulins or
exposed to pre-immune IgGs condensed normally, albeit at a
lower frequency than intact nuclei (Fig. 7; compare with Fig.
3710 S. B. Martins and others
Fig. 7. Anti-HA95 antibodies inhibit chromatin condensation in
mitotic extract. Purified HeLa nuclei were extracted with 0.1% Triton
X-100 to remove nuclear membranes and incubated in nuclear buffer
for 40 minutes with no antibodies, control pre-immune IgGs, or αHA95. Nuclei were sedimented through sucrose and resuspended in
mitotic cytosol. After 2 hours, chromatin condensation was assessed
by phase contrast microscopy. Proportions of chromatin
condensation were determined in a total of 138 (No Ab), 113 (IgG)
and 135 (α-HA95) nuclei per group. Bars, 10 µm.
Fig. 6. Immunoblocking of HA95 inhibits nuclear membrane
disassembly but not lamina solubilization in mitotic cytosol.
(A) Purified HeLa nuclei were loaded with either pre-immune IgGs,
α-HA95, or α-HA95 bound to the HA95 competitor peptide (αHA95+pep.) and exposed to mitotic cytosol as in Fig. 5. After 2
hours, distributions of LBR and lamin B and lamin A were examined
by immunofluorescence. Input nuclei (Input) were interphase nuclei
loaded with α-HA95 prior to exposure to mitotic cytosol. Bars, 10
µm. (B) At the end of incubation, nuclei/chromatin masses were
sedimented and distributions of lamin B and LBR were analyzed by
immunoblotting pellets (P) and reaction supernatants (S). Input
nuclei (Input, P) and input mitotic cytosol (Input, S) were also
analyzed.
5B). However, pre-incubation of demembranated nuclei with
α-HA95 abolished chromosome condensation (Fig. 7). As the
nuclear lamina is solubilized in mitotic cytosol, this implies
that inhibition of condensation after intranuclear blocking of
HA95 (see Fig. 5) does not solely result from the persistence
of nuclear membranes. Rather, immunoblocking of
intranuclear HA95 also affects chromosome condensation.
Antibody blocking of HA95 does not inhibit nuclear
reassembly in interphase cytosol
The role of HA95 in nuclear membrane disassembly in vitro,
as judged from the antibody inhibition experiments, raises the
possibility that HA95 might also be implicated in the
reassembly of the NE (membranes or lamina, or both) at the
end of mitosis. To test this hypothesis, we examined whether
antibody-mediated blocking of HA95 associated with
condensed chromosomes would affect nuclear reassembly in
vitro. To this end, we developed a cell-free system that
recapitulates nuclear reassembly at the end of mitosis. A
condensed chromatin substrate was prepared by exposing
purified HeLa nuclei to mitotic cytosol as described above.
Nuclei breakdown took place and the condensed chromatin
was purified by sedimentation through a sucrose cushion. The
condensed chromatin (see Fig. 8, Input chromatin) was
exposed to pre-immune IgGs, α-HA95 or no antibodies for 30
minutes. After sedimentation through 1 M sucrose to remove
unbound antibodies, the chromatin was resuspended into an
interphase cytosol containing an ATP-regenerating system,
GTP and membrane vesicles to promote nuclear reformation.
After 2 hours, chromatin decondensation and NE reformation
were monitored. Phase contrast examination shows that nuclei
reassembled whether the chromatin was exposed to preimmune
IgGs or α-HA95 (Fig. 8). Moreover, all nuclei reformed
membranes, as judged by DiOC6 labeling, and a nuclear
lamina, as shown by immunofluorescence analysis of B-type
lamins (Fig. 8). Based on these criteria, we concluded that
antibody-mediated blocking of HA95 associated with
condensed chromosomes does not impair nuclear
reconstitution in vitro.
DISCUSSION
Using cell and nuclear fractionation techniques and in vitro
nuclear disassembly and reconstitution assays, we report
biochemical and functional characterizations of HA95, a
ubiquitous nuclear protein recently cloned by us and others
(Ørstavik et al., 2000; Seki et al., 2000; Westberg et al., 2000).
HA95 is homologous to AKAP95, a PKA-binding protein of
the nucleus, but lacks the RII-binding motif of AKAP95 and
thus, does not bind PKA-type II. HA95 is primarily a
chromatin-associated protein, although a significant proportion
co-fractionates with the nuclear matrix/lamina network. This
positions HA95 primarily at the chromatin-NE interface.
A previous study involving human-mouse heterokaryons
suggested that HA95/HAP95 was able to shuttle in and out of
Nuclear envelope anchoring to chromatin by HA95 3711
Fig. 8. Immunoblocking chromatin-bound HA95
does not inhibit nuclear reassembly. A condensed
chromatin substrate was prepared from HeLa nuclei
disassembled in a mitotic cytosol and sedimented
through sucrose. Chromatin masses (Input chromatin)
were resuspended in interphase cytosol containing an
ATP-generating system, GTP and cytoplasmic
membrane vesicles to promote chromatin
decondensation and NE reassembly. After 1.5 hours,
nuclei were examined by phase contrast microscopy,
membrane staining with DiOC6 and
immunofluorescence using anti-lamin B antibodies.
DNA was labeled with Hoechst 33342 (insets). Bar,
10 µm.
the nucleus, although a steady state nuclear localization of
HA95 was reported (Westberg et al., 2000). Our studies,
however, do not support a shuttling property of HA95 for three
reasons: (i) HA95 is not detected on western blots of cytosolic
fractions even after overloading the gels (data not shown); (ii)
HA95 remains insoluble after extraction of nuclei with
detergent and high salt; (iii) FRAP experiments demonstrate
that HA95-GFP does not roam in the nucleus. Thus, we
concluded that HA95 is tightly anchored to insoluble nuclear
structures throughout the cell cycle.
HA95 constitutes the third example to date of a chromatin
protein interacting with integral proteins of the INM. First,
heterochromatin protein HP1 was identified as a binding
partner for LBR (Ye and Worman, 1996). Second, BAF, a small
DNA-binding protein implicated in retroviral genome insertion
into chromosomes (Cai et al., 1998; Lee and Craigie, 1998),
was also found to bind LAP2β in a yeast two-hybrid screen
(Furukawa, 1999) through a domain conserved in emerin
and MAN1 (Lin et al., 2000). Third, cross-linking and
immunoprecipitation experiments indicate that HA95 tightly
interacts with LBR and LAP2β but not with emerin, although
emerin lies in a multi-protein complex including HA95.
Selective interaction of HP1, BAF and HA95 with proteins
of the INM and of the lamina may underline implications of
these associations in intranuclear processes. Deletion analyses
have shown that LAP2β plays a role in DNA synthesis and in
expansion of in vitro reconstituted Xenopus nuclei (Gant et al.,
1999). Furthermore, lamins have been proposed to be involved
in nuclear functions such as DNA replication (Ellis et al., 1997;
Spann et al., 1997) and organization of pre-mRNA splicing
complexes (Jagatheesan et al., 1999). Interactions of HA95
with LAP2β and B-type lamins raise the possibility that HA95
acts as an effector of LAP2β and lamin functions. Other
chromatin-associated proteins interacting with the INM or the
lamina, such as LAP2α (Dechat et al., 1998) and BAF
(Furukawa, 1999), may also participate in these effector
functions (see Wilson, 2000). Additionally, a cross-talk
between HA95- and BAF-associated complexes through HA95
and BAF may not be excluded. To support this view,
preliminary data from our laboratory indicate that HA95 and
BAF co-precipitate in interphase (S. Martins, K. Wilson and P.
Collas, unpublished data), suggesting that the two proteins
biochemically and perhaps functionally interact. It emerges
from these observations that the multiplicity of protein
complexes linking chromatin to the NE not only provides
control mechanisms for mitotic NE disassembly and
reformation, but also establishes functional links between the
NE and the genome.
Attachments of heterochromatin to the NE are reversible and
cell cycle-regulated. This dynamics is best illustrated at
mitosis, when the NE breaks down in prophase and
reassembles in anaphase/telophase (reviewed by Marshall and
Wilson, 1997; Collas and Courvalin, 2000). In interphase, most
heterochromatin is subjacent to the NE. However in late S
phase, replicating heterochromatic DNA is released from the
nuclear periphery, translocated towards the replication
machinery and moves back to the nuclear periphery after
replication (Li et al., 1998). Reversible heterochromatin
anchoring to the NE may be regulated by LBR, LAP2β or
lamins, which all bind chromatin. The role of HA95 in
anchoring chromatin to the INM further suggests that HA95
provides an additional level of regulation of NEheterochromatin interactions during interphase and at mitosis.
Immunoblocking of HA95 in interphase nuclei abolishes
chromosome condensation and nuclear membrane disassembly
in mitotic cytosol; however, lamina disassembly is not affected.
Mitotic chromosome condensation requires multiple proteins,
including the multiprotein condensin complex (Hirano et al.,
3712 S. B. Martins and others
1997) and the HA95-related PKA-binding protein AKAP95,
acting as a targeting protein for the condensin complex (Collas
et al., 1999; Steen et al., 2000). The putative role of HA95 in
chromosome condensation may be independent of that of
AKAP95, since anti-HA95 antibodies do not recognize
AKAP95 (and vice-versa) and presumably maintain AKAP95
active. This suggests that the two proteins have distinct
functions in chromosome condensation. This view is supported
by our observation that anti-AKAP95 antibodies do not block
NE disassembly in vitro (Collas et al., 1999), and by the lack
of co-precipitation of HA95 and AKAP95 in interphase and
mitosis (Ørstavik et al., 2000; our unpublished data).
Anti-HA95 antibodies interfere with interactions of HA95
with LBR or LAP2β such that disruption of these interactions
in a mitotic environment is abolished. How anti-HA95
antibodies affect nuclear membrane disassembly remains
speculative at present. A first possibility is that anti-HA95
antibodies mask mitotic phosphorylation sites of LBR and/or
LAP2β that are implicated in NE disassembly (Courvalin et
al., 1992; Foisner and Gerace, 1993). Whether HA95 is
phosphorylated at mitosis is currently unknown. Alternatively,
anti-HA95 antibodies may cross-link components of the HA95
complex, preventing their segregation. A third possibility is
that, as anti-HA95 antibodies block chromatin condensation,
LBR or LAP2β association with chromatin is regulated not
only by phosphorylation but also by chromatin conformation,
and that changes in both are required for NE breakdown. Thus,
one can envisage that a role of lamin phosphorylation at mitosis
is to alter lamin-lamin interactions, and that this is all that is
required to eliminate the lamins from the NE. In contrast to
nuclear membranes, disruption of HA95 from the lamina is not
impaired by anti-HA95 antibodies as both A/C- and B-type
lamins are solubilized in the mitotic cytosol.
Implications of anti-HA95 antibody-mediated inhibition of
nuclear membrane – but not lamina – disassembly, are twofold.
First, this inhibition strongly supports previous reports that
membrane and lamina disassembly may occur independently
and involve biochemically distinct pathways (Miake-Lye and
Kirschner, 1985; Newport and Spann, 1987; Collas, 1998).
Second, it argues for a separation of the INM and lamina
domains of the NE – as judged by the segregation of LBR or
LAP2β from A/C- and B-type lamins in the cytosol, as
originally proposed (Chaudhary and Courvalin, 1993).
It is intriguing that anti-HA95 antibody-mediated blocking
of HA95 function on condensed chromosomes does not
prevent any of the nuclear reassembly steps examined, namely,
chromatin decondensation, nuclear membrane and lamina
assembly. An implication is that the NH2-terminal domain of
HA95 (in which the antigen resides) does not contribute to
membrane anchoring to chromatin upon nuclear reformation.
Alternatively, HA95 may not be involved at all in targeting or
initial binding of nuclear membranes to chromatin. The role of
LBR in this process (Collas et al., 1996; Pyrpasopoulou et al.,
1996) may be mediated by LBR interaction with HP1 (Ye and
Worman, 1996) rather than with HA95. Instead, HA95 may
conceivably anchor the INM to chromatin at a later stage of
NE assembly, possibly by the time the nuclear lamina is
reassembled.
The authors are thankful to Drs E. Kieff for HA95-Myc-transfected
cells, A. B. Kolstø for the gift of the TCF11-CFP plasmid, J.-C.
Couvalin and B. Buendia for generous gifts of antibodies and fruitful
discussions, G. E. Morris for anti-emerin antibodies, K. Taskén for
discussions and J. Helm for his expertise in confocal microscopy. S.
Eikvar is thanked for assistance with transfections. This work was
supported by grants from the Anders Jahre’s Foundation, the Novo
Nordisk Foundation, the Norwegian Research Council and the
Norwegian Cancer Society. Confocal microscopy work was supported
by Norwegian Research Council Grant 129550/310 and Prof. Letten
F. Saugstad Fund. S.M. was partly supported by a grant from
Universidade Lusófona, Lisbon, Portugal.
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