1433
Research Article
Structural protein 4.1R is integrally involved in nuclear
envelope protein localization, centrosome–nucleus
association and transcriptional signaling
Adam J. Meyer1,*, Donna K. Almendrala1, Minjoung M. Go1,‡ and Sharon Wald Krauss1,§
1
Department of Genome Dynamics, University of California-Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
*Present Address: Institute for Cell and Molecular Biology, University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USA
‡
Present Address: University of Illinois College of Medicine at Rockford, 1601 Parkview Avenue, Rockford, IL 61107, USA
§
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 15 December 2010
Journal of Cell Science 124, 1433-1444
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.077883
Summary
The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are
unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope
protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells
by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments.
Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and
displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic
proteins NuMA and LAP2 also have aberrant distributions, but lamin B and LAP2 have normal localizations. 4.1R-deficient mouse
embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the
association of 4.1R with emerin and A-type lamin: increased nucleus–centrosome distances, increased -catenin signaling, and
relocalization of -catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased
nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact
upon nuclear architecture, centrosome–nuclear envelope association and the regulation of -catenin transcriptional co-activator activity
that is dependent on -catenin nuclear export.
Key words: 4.1R, Nuclear envelope, Actin, Emerin, Lamin A/C, Centrosome, RNA interference, -Catenin
Introduction
The nuclear envelope comprises a double lipid membrane,
embedded nuclear pores and a filamentous lamin meshwork lying
beneath the inner nuclear membrane. Multi-protein complexes
traverse the perinuclear space between the two nuclear membranes,
linking cytoplasmic skeletal networks to inner nuclear structural
networks housing chromatin, nucleoplasmic RNA splicing and
DNA replication assemblies, and regulatory complexes (Crisp et
al., 2006; Razafsky and Hodzic, 2009; Stewart et al., 2007). Thus,
although visually distinct, the nucleus is biochemically and
physically integrated with cytoplasmic structures, the plasma
membrane and the extracellular matrix. In this current view, nuclear
structural proteins are no longer regarded as largely passive
architectural elements but are now recognized to also mediate
dynamic regulatory processes, such as macromolecular trafficking
through nuclear pores, gene expression and signaling vital to
cellular remodeling during proliferation and differentiation. The
striking discovery that defects in nuclear architectural proteins are
linked to pathobiology of diverse human diseases, including ~30
nuclear envelope diseases (for reviews, see Burke and Stewart,
2002; Worman and Courvalin, 2005), is concordant with a revised
perspective of the nuclear functions extending well beyond
safeguarding the genome.
Protein 4.1R has for decades been regarded mainly as an ~80
kDa cytoskeletal erythrocyte protein crucial for stabilizing
interactions within spectrin–actin lattices in the erythrocyte
membrane skeleton and linking them to transmembrane proteins in
the overlying plasma membrane. Defects in 4.1R linkages alter
erythrocyte shape and mechanical stability, producing hereditary
elliptocyosis characterized by erythrocyte membrane fragmentation
(Benz, 1994; Conboy, 1993; Mohandas and Chasis, 1993). It was
subsequently discovered, however, that nucleated cells express
multiple 4.1R isoforms at several subcellular locations (De Carcer
et al., 1995; Delhommeau et al., 2002; Krauss et al., 1997b; Krauss
et al., 1997a). In nucleated cells, protein 4.1R not only organizes
plasma-membrane–cytoskeletal microstructure, but, as we have
previously shown, it is also integral to mitotic spindle and
centrosome assembly and structure (Krauss et al., 2004; Krauss et
al., 2008). Furthermore, we found that 4.1R epitopes in mammalian
and Xenopus laevis nuclei are partially coincident with RNA splicing
and/or transcription factories, DNA replication assemblies, nuclear
pores and the nuclear periphery, and are largely coincident with
nuclear anti-actin signals in nuclear matrix (Krauss et al., 2003;
Krauss et al., 1997a). At the resolution of electron microscopy, we
detected 4.1R and actin closely associated on intranuclear filaments
in mammalian cells (Krauss et al., 2003). Protein 4.1 and actin
epitopes were also identified on filaments linked with nuclear pores
in Xenopus oocytes (Kiseleva et al., 2004). Independently, 4.1R
was identified by mass spectroscopy analysis of mammalian nuclear
envelope proteins (Schirmer et al., 2003).
We have previously shown that 4.1R is functionally significant
in the nucleus by using Xenopus egg extracts that recapitulate
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nuclear assembly in vitro. By 4.1 depletion and add-back
experiments, we established that 4.1 is essential for proper assembly
of nuclei capable of DNA synthesis and for transitioning into
mitosis following nuclear envelope breakdown. Furthermore, we
have documented by several different means, including use of
4.1R peptides mutant for actin binding, that the capacity of 4.1R
to bind actin is required for nuclear assembly and function (Krauss
et al., 2003; Krauss et al., 2002). Interestingly, a 4.1R nuclear
localization signal has been identified that partially overlaps with
4.1R spectrin-actin-binding domain sequences (Gascard et al.,
1999). Although 4.1R clearly is important for nuclear structure and
function, underlying mechanisms are not well understood.
One key strategy for defining mechanisms of 4.1R functions,
used successfully in the case of erythrocytes, has been to determine
its binding partners (Baines et al., 2009; Calinisan et al., 2006;
Conboy, 1999; Salomao et al., 2008; Takakuwa, 2000). The four
major domains of prototypical 4.1R in nucleated cells have diverse
interactions, indicative of its roles as a multifunctional linker and/or
adaptor protein (Krauss et al., 2002; see figure 1 within). In brief,
the 4.1R 30 kDa FERM domain contains a microtubule-binding
site, as does the C-terminal domain, and also interacts with
phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], calmodulin
and membrane proteins (An et al., 2006; Krauss et al., 2004;
Nunomura et al., 2000; Perez-Ferreiro et al., 2001). The spectrinactin-binding domain (Cohen and Foley, 1984; Discher et al., 1993;
Gimm et al., 2002; Schischmanoff et al., 1995) additionally binds
myosin, an actin-dependent ATPase (Kontrogianni-Konstantopoulos
et al., 2000; Pasternack and Racusen, 1989). The C-terminus binds
the structural protein ‘nuclear mitotic apparatus protein’ (NuMA),
present in the nucleoplasm during interphase, as well as ZO2 and
FKB13 (Mattagajasingh et al., 2000; Mattagajasingh et al., 1999;
Walensky et al., 1998).
To further delineate the nuclear roles of 4.1R, we are focusing
on our previous findings that 4.1R–actin interactions are crucial
for assembly and maintenance of nuclear structure and function.
We reasoned that 4.1R might act as a linker and/or adaptor in the
nucleus to form functionally important actin-containing complexes
with other nuclear actin-binding proteins. Actin is involved in
variety of important nuclear processes, such as transcription,
chromatin remodeling, regulation of architecture, chromatin
movement and RNA export (Chuang et al., 2006; Dundr et al.,
2007; Hofmann, 2009; Hofmann et al., 2004; Olave et al., 2002),
and several major actin-binding nuclear proteins have been
identified.
One very attractive candidate for a potential 4.1R-interacting
protein is emerin, a 34 kDa protein encoded by the EMD gene
located on the X-chromosome (Bione et al., 1994), which is
expressed in nearly all tissues and contributes to many vital cellular
functions, including nuclear assembly, chromatin segregation and
cell cycle progression (Bengtsson and Wilson, 2004; Holaska and
Wilson, 2006). Emerin resides at the inner and outer regions of the
nuclear envelope and the Wilson laboratory has extensively
characterized a number of nuclear architectural and regulatory
emerin complexes (Holaska and Wilson, 2007). Notably, emerin
caps and stabilizes the pointed end of F-actin in vitro (Holaska et
al., 2004) and has important interactions with a chromosomeassociated barrier to autointegration factor, with gene regulators
and with nesprins (Lee et al., 2001). Members of the nesprin
family also bind actin and span the nuclear envelope, as components
of LINC complexes, to connect with the cytoskeleton (Haque et
al., 2006; Libotte et al., 2005; Mislow et al., 2002; Ostlund et al.,
2009; Wheeler et al., 2007). As with 4.1R, emerin binds to
microtubules and to myosin (Holaska and Wilson, 2007). More
recently other intriguing functions of emerin have been discovered:
emerin was shown to couple centrosomes to the nuclear envelope
and to regulate -catenin activity (Markiewicz et al., 2006;
Salpingidou et al., 2007).
Another attractive actin-binding nuclear candidate is lamin A,
which has two actin-binding sites. Importantly, emerin binds Atype lamins, and the localization of emerin at the nuclear envelope
is dependent on lamin A (Vaughan et al., 2001). Mutations in the
emerin- and lamin-A-encoding genes can cause Emery–Dreifuss
muscular dystrophy (EDMD), a disease characterized by slowly
progressive skeletal muscle wasting, joint contractures and
cardiomyopathy leading to increased risk of cardiac arrest (Bione
et al., 1994; Emery, 2000; Morris, 2001). Defective A-type lamin
is also associated with a large group of diseases collectively
called laminopathies (Mounkes et al., 2003; Muchir and Worman,
2004).
Here we show that emerin and lamin A/C are coimmunoprecipitated with 4.1R, and that human cells depleted of
4.1R by RNA interference (RNAi) treatment have dysmorphic
nuclei, emerin mislocalization, disrupted A-type lamin, increased
centrosome–nuclear distances, increased -catenin transcriptional
activity and increased nuclear accumulation of -catenin. We also
studied mouse embryonic fibroblasts (MEFs) from a mouse model
in which the first two 4.1R translation initiation sites were deleted
(4.1R AUG1,2–/–) (Shi et al., 1999). Although this is not a knockout
of the entire 4.1R gene, and expression of residual 4.1R has been
reported (Stagg et al., 2008), the mice have significant phenotypes.
In addition to a predicted hemolytic anemia, the mice have cardiac
and kidney defects, neurobehavioral deficits and smaller litter sizes
with low survival (Kang et al., 2009; Rivera et al., 2006; Stagg et
al., 2008). Examining MEFs from these mice, we observed a
number of nuclear deficits resembling those in human cells with
depleted 4.1R, including emerin and A-type lamin malfunctions.
Taken together, our results from these two independent systems
indicate functional associations of 4.1R with emerin and A-type
lamins that impact upon nuclear architecture, association of
centrosomes with the nuclear envelope and -catenin transcriptional
regulation.
Results
Co-immunoprecipitation and partial colocalization of 4.1R
with emerin
To initially test for any evidence of a 4.1R association with emerin,
we probed 4.1R immunoprecipitates of HeLa whole-cell sonicates
using several different detergents to increase stringency. We readily
detected a protein migrating at ~34 kDa that reacted with
specifically with anti-emerin antibody in immune, but not nonimmune, complexes separated by SDS-PAGE (Fig. 1A). We
also confirmed that both actin and II-spectrin were coimmunoprecipitated, as would be predicted. As emerin binds lamin
A/C, but lamins can be difficult to solubilize, we checked that the
whole-cell sonicates contained lamin A/C and then probed the
4.1R immunoprecipitates and detected a lamin A/C band (Fig. 1A).
To test further association of 4.1R and emerin in a less-complex
cell fraction, we prepared HeLa nuclear extracts and
immunoprecipitated 4.1R-associated proteins (Fig. 1B). We again
detected robust emerin co-immunoprecipitation with 4.1R.
As positive controls, we probed and detected the 4.1R-binding
partners actin, II-spectrin and NuMA (Krauss et al., 2002;
Journal of Cell Science
Nuclear roles of protein 4.1R
1435
Fig. 1. 4.1R and emerin co-immunoprecipitate and partially colocalize in human and murine fibroblasts. (A)Immunoprecipitation (IP) from HeLa whole cell
sonicates. Clarified whole cell sonicates were prepared, immunoprecipitated with anti-4.1R antibody, and eluted proteins separated by SDS-PAGE were subjected
to western blotting as described in the Materials and Methods. Emerin, actin, lamin A/C and II-spectrin were detected using the antibodies indicated in lysate and
immune duplicate samples (4.1R IgG) but not in a non-immune sample (con IgG). (B)Analysis of immunoprecipitates from HeLa nuclear extract. HeLa nuclear
extract was incubated with antibodies, immunoprecipitated with anti-4.1R antibody, and eluted proteins separated by SDS-PAGE were subjected to western blotting
using the antibodies indicated on the right. In 4.1R IgG immunoprecipitates (right-hand lane) a strong 34 kDa emerin band was detected. 4.1R immunoprecipitates
also contained actin, lamin A/C, II-spectrin and NuMA. These proteins were not detected in control IgG precipitates (center lane). (C)Western blot probed with
anti-4.1R antibody showing 4.1R bands migrating at ~135, 105, 80 and 62 kDa detected specifically in 4.1R IgG immunoprecipitates but not in the pre-immune
(PI) or control (con) lanes. The right-hand panel shows a western blot demonstrating that 4.1R bands at ~135, 80, and 62 kDa were present in anti-emerin antibody
immunoprecipitates but not in those with control IgG. (D)Partial colocalization of 4.1R epitopes (red) with emerin epitopes (green) in human and murine
fibroblasts. Fixed WI38 fibroblasts and wild-type MEFs were probed by double-label indirect immunofluorescence. The nuclear envelope protein emerin was
detected at the periphery of the nucleus stained by DAPI. 4.1R epitopes were detected in the nucleus, cytoplasm and in the region of the nuclear envelope, partially
coincident with emerin signals (yellow coloration in the ‘merge’ image). Signals for an equal amount of control anti-rabbit IgG antibody staining (bottom left
panel) were not above background levels of other controls in which primary rabbit antibody was omitted but murine anti-emerin and both fluorescent secondaries
were included. Scale bar: 6m.
Mattagajasingh et al., 1999). Lamin A/C was also present in 4.1R
immunoprecipitates in HeLa nuclear extract under these conditions.
To confirm that 4.1R itself was co-precipitated, immunoprecipitated
proteins were probed with anti-4.1R antibody. Bands at ~135 and
80 kDa, consistent with full-length products generated from the
4.1R AUG1 and AUG2 translation initiation sites, were present
(Fig. 1C). Other bands detected at ~105 and ~64 kDa are likely to
be alternatively spliced 4.1R isoforms (Anderson et al., 1988;
Conboy et al., 1991; Conboy et al., 1988), although the lower band
could also be the product generated from 4.1R AUG3 (Gascard et
al., 1998; Luque and Correas, 2000). Parallel samples with control
IgG or pre-immune serum did not contain anti-4.1R-antibodyreactive bands. As further evidence of a 4.1R–emerin complex,
reciprocal emerin immunoprecipitates were probed with anti-4.1R
antibody and found to have 4.1R bands at ~135, 80 and 64 kDa
(Fig. 1C).
To additionally document a potential 4.1R–emerin association
in situ, we examined human and murine fibroblasts by doublelabel indirect immunofluorescence. As has been reported by several
investigators, in nucleated mammalian cells 4.1R epitopes are
distributed in the cytoplasm, at intranuclear sites, and at
centrosomes. At a cytoplasmic focal plane in cells double-labeled
with emerin, 4.1R staining shows an enrichment at the nuclear
periphery coincident with emerin in overlaid images (Fig. 1D).
Although this nuclear peripheral localization was apparent
previously (Krauss et al., 1997a) it was not sufficiently noted or
appreciated in light of efforts to establish 4.1 at nucleoplasmic
‘speckles’ (Correas, 1991; Krauss et al., 1997a).
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4.1R depletion induces nuclear dysmorphology with
altered emerin and A-type lamin distribution
Journal of Cell Science
Emerin-deficient MEFs, as well as cells from EDMD patients,
often have irregularly shaped nuclei and blebbing (Lammerding et
al., 2005). If 4.1R has a functional association with emerin, 4.1R
depletion might also alter nuclear morphology. To analyze the
effects of 4.1R depletion on nuclear morphology in intact
mammalian cells, we examined DAPI-stained cells after 4.1R
downregulation by RNAi (Krauss et al., 2008) and MEFs from
4.1R AUG1,2–/– mice relative to matched MEFs from wild-type
mice. We found that 4.1R-deficient cells have an ~sixfold increase
in perturbed nuclei (those that are markedly irregular with blebbing
Fig. 2. Nuclear morphology is perturbed and selective nuclear proteins are redistributed in 4.1R-deficient human cells. (A)The left-hand panels show
examples of nuclear morphology of control (noncoding RNAi) and aberrant CaSki nuclei (4.1R RNAi) stained with DAPI. The right-hand graph shows a
quantification of the number of perturbed nuclei in control compared with 4.1R-RNAi-treated cells. Numbers of nuclei examined from four independent
experiments are shown below. Nuclei were scored as normal if spheroidal or ellipsoid, and abnormal if blebbed, lobulated or at least twice as large or small as those
in controls. (B)Indirect immunofluorescent staining of nucleoplasmic protein NuMA in control and 4.1R-RNAi-treated cells. In cells with normal 4.1R, NuMA is
entirely within the nuclear area stained by DAPI, whereas in 4.1R-deficient cells, NuMA is detected in the cytoplasm, sometimes in apparent aggregates (arrow).
(C)Localization of nuclear envelope proteins by indirect immunofluorescence. The top row on the right-hand side shows that emerin is partially redistributed from
the nuclear envelope into the cytoplasm of cells when 4.1R is depleted (arrow, top row on the right-hand side). However, the nuclear envelope protein LAP2
remains in the nuclear envelope. The LEM domain protein MAN1 redistributes from the nuclear envelope/perinuclear region to further out in the cytoplasm when
4.1R is deficient (arrow, second row on the right-hand side). Localizations of two nuclear pore proteins are also affected when 4.1R is deficient. In the examples
shown in the two bottom rows, there is significant redistribution of the nuclear pore inner basket protein Tpr in a 4.1R-deficient cell although the localization of
LAP2 in the nuclear envelope remains unchanged in the same cell. Distribution of the central nuclear pore channel protein Nup62 is also altered in 4.1R-deficient
cells. A cell is shown in which emerin, as well as Nup62, is partially redistributed from the nuclear envelope but does not overlap with Nup62 in cytoplasmic
regions. (D)Localization of lamin B. Lamin B distribution, normally at the nuclear periphery (control) is not perturbed when 4.1R is deficient, even at the large
bleb in the nucleus shown. (E)Localization of lamin A/C and LAP2. Control cell nuclei have continuous lamin A/C rims with multiple nucleoplasmic lamin A/C
foci. However, 4.1R-RNAi-treated cells have nuclei without lamin A/C staining surrounding nuclear blebs (upper arrows in the top and bottom panel examples)
and can be discontinuous, as shown in bottom panels (lower arrows in bottom panel). In 4.1R-depleted cells internal lamin A/C foci are absent or severely reduced
in number. The graph shows a quantification of cells with more than six lamin A/C foci in control and RNAi-treated cells (n250 each in two independent
experiments). In the images below, the lamin-A-associated protein LAP2 localizes strictly within the boundaries of nuclei (DAPI staining) in control cells, but is
also detected in the cytoplasm in 4.1R-RNAi-treated cells, often in aggregates. Scale bars: 7m (B,C); 5m (A,D,E).
Nuclear roles of protein 4.1R
et al., 2002; Mattagajasingh et al., 1999) and both are components
of the nuclear matrix of interphase nuclei. We imaged 4.1R-RNAitreated cells and 4.1R AUG1,2–/– MEFs and detected NuMA in
cytoplasmic aggregates rather than being localized exclusively in
the nucleoplasm, as in control RNAi-treated cells or wild-type
MEFs (Fig. 2B; Fig. 3B). NuMA mislocalization could reflect a
failure of proper anchorage of NuMA by 4.1R at intranuclear sites
due to 4.1R downregulation and might also contribute to nuclear
dysmorphology (Krauss et al., 2002; Krauss et al., 1997a; Merdes
and Cleveland, 1998).
Journal of Cell Science
or are at least twofold larger or smaller relative to smooth ellipsoid
control nuclei) (Fig. 2A; Fig. 3A). Telomerase-immortalized human
diploid cells (RPE1) and HeLa cells also have increased nuclear
dysmorphology after 4.1R RNAi treatment (S.W.K., data not
shown).
To investigate whether the absence of 4.1R affects the
distribution of major nuclear proteins, notably emerin and A-type
lamin, we first analyzed whether 4.1R downregulation affected the
nuclear localization of a known binding partner of 4.1R in the
nucleus. NuMA binds directly to the C-terminus of 4.1R (Krauss
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Fig. 3. Nuclear morphology is perturbed and selective nuclear proteins are redistributed in 4.1R AUG1,2–/– MEFs. (A)The left-hand panels show examples
of nuclear morphology of wild-type and 4.1R AUG1,2–/– MEFs (4.1R–/–) stained with DAPI. The right-hand graph shows a quantification of the number of
perturbed nuclei in wild-type (4.1R+/+) compared with 4.1R–/– MEFs at equal passage in three independent experiments in which 500 nuclei of each type were
scored as described in Fig. 2. (B)Indirect immunofluorescent staining of nucleoplasmic protein NuMA in 4.1R+/+ and 4.1–/– MEFs. In cells with normal 4.1R,
NuMA is within the nuclear area stained by DAPI, whereas in 4.1R–/– cells, NuMA is detected in the cytoplasm, sometimes in apparent aggregates (arrow).
(C)Localization of nuclear envelope proteins by indirect immunofluorescence. The top row on the right-hand side shows that emerin is partially redistributed from
the nuclear envelope into the cytoplasm in 4.1R–/–MEFs (arrow). MAN1 also redistributes from the nuclear envelope/perinuclear region to further out in the
cytoplasm in 4.1R–/– MEFs (arrow). Additionally, localization of two nuclear pore proteins is affected when 4.1R is depleted. In the examples shown in the two
bottom panels, there is a major redistribution of the nuclear pore inner basket protein Tpr in 4.1R–/– MEFs, as well as the central nuclear pore protein Nup62
(arrows). However, LAP2 remains in the nuclear envelope of 4.1R–/– MEFs. (D)Localization of lamin B, lamin A/C and LAP2. The upper left-hand panels
show that wild-type cell nuclei have lamin B rims at the nuclear periphery, as do 4.1R–/– MEFs, where lamin B is continuous even at blebs and irregular contours.
The upper right-hand panels show that wild-type cell nuclei have lamin A/C rims with multiple nucleoplasmic lamin A/C foci. However, a 4.1R–/– nucleus has
discontinuous lamin A/C staining at nuclear blebs (arrows). The lower panels show that the lamin-A-associated protein LAP2 localizes within the boundaries of
the nuclei (DAPI staining) in 4.1R+/+ MEFs but is detected in the cytoplasm of 4.1–/– MEFs. Scale bars: 5m.
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We then examined the distribution of emerin in 4.1R-depleted
human and murine cells. There was a substantial mislocalization
of a portion of emerin into the cytoplasmic area relative to its
normal position at the nuclear envelope, as demarcated by another
nuclear envelope protein LAP2 (Fig. 2C; Fig. 3C). This altered
localization was confirmed by western blotting for emerin in HeLa
nuclear and cytoplasmic fractions, which showed that there was a
threefold increase in the amount of emerin present in the cytoplasm
of 4.1R-RNAi-treated cells (average 37.7% in 4.1R-deficient
cells compared with 12.9% in controls in three experiments;
supplementary material Fig. S1). Both emerin and LAP2 contain
an ~40 amino acid LEM domain (for Lamina-associated
polypeptide-emerin-MAN1), in common with the inner nuclear
envelope protein MAN1 and a group of lamin-associated proteins
(LAPs) (Foisner and Gerace, 1993; Holmer and Worman, 2001;
Lin et al., 2000; Wagner and Krohne, 2007). As with emerin,
MAN1 distribution is altered in cells with depleted 4.1R (Fig. 2C,
Fig. 3C). However, the fact that LAP2 is retained at the nuclear
envelope when 4.1R is deficient probably reflects the fact that
although these proteins each have LEM domains, their repertoire
of interactions differs. For example, LAP2 binds to B-type lamins
(Foisner and Gerace, 1993), whereas LAP2 preferentially binds
A-type lamins (Dechat et al., 2000). Indeed, when we tested lamin
B distribution in 4.1R-deficient cells, we found that lamin B forms
a continuous rim, including around nuclear blebs (Fig. 2D, Fig.
3D), consistent with our finding that LAP2 is not perturbed.
Nuclear pores are embedded in the nuclear envelope, traversing
both the inner and outer nuclear membranes and the perinuclear
space. Because several major nuclear envelope proteins are
displaced when 4.1R is deficient, we were curious as to whether
nuclear pore proteins were also affected and, hence, examined
proteins from two different regions of the pore structure. We found
that both a nuclear pore inner nuclear basket protein (Tpr) and a
nuclear pore central channel protein (Nup62) were detected in the
perinuclear region of 4.1R-deficient cells in addition to their nuclear
envelope distribution (Fig. 2C, Fig. 3C). In the examples shown,
Tpr distribution was massively altered, although LAP2 was not,
as previously noted. When 4.1R was depleted by RNAi, Nup62
and emerin are localized in the cytoplasmic region independently,
again emphasizing that it is probable that 4.1R is a pivotal linker
or adaptor anchoring components of independent, as well as
interdependent, networks (Fig. 2C).
As emerin is anchored at the nuclear membrane through its
interactions with lamin A (Vaughan et al., 2001), and emerin
localization is affected by 4.1R downregulation, it would be
predicted that lamin A/C might be perturbed. When we examined
lamin A/C staining in 4.1R-deficient mammalian cells, we found
that blebbed or multi-lobulated mammalian nuclei had
discontinuous peripheral lamin A/C at the nuclear envelope (Fig.
2E; Fig. 3D). Furthermore, we observed that 4.1R-RNAi-treated
cells, with normal or abnormal nuclear morphology, had an absence
of nucleoplasmic lamin A/C foci: fewer than 5% of the cells had
six or more internal lamin A/C foci (Fig. 2E, graph; Fig. 3D).
Additionally, a substantial amount of LAP2, a nucleoplasmic
lamin-A/C-binding partner, was detected beyond the nuclear
periphery (Fig. 2E; Fig. 3D). These data suggest that 4.1R is
required for A-type lamin integrity and not for B-type lamin
integrity. A- and B-type lamins form separate networks of
intermediate filaments beneath the nuclear envelope. The A-type
lamin-related findings are potentially significant because nuclear
dysmorphology is a hallmark of cells from laminopathic patients
and mouse models of laminopathies (Capell and Collins, 2006;
Mounkes et al., 2003; Muchir et al., 2004; Worman and Bonne,
2007) and internal lamin A/C foci are associated with the tumor
suppressor retinoblastoma protein (pRb) (Markiewicz et al., 2002).
Taken together, our data show that downregulation of 4.1R
expression causes nuclear dysmorphology with selective, but not
global, changes within multiple nuclear subcompartments:
mislocalization of a nuclear skeletal protein (NuMA), as well as
another nucleoplasmic protein (LAP2); altered distribution of
several but not all inner nuclear membrane and pore proteins
(emerin, MAN1 and two nuclear pore proteins but not LAP2);
and major disruption of one member of the nuclear lamina (lamin
A/C) but not lamin B. Interestingly, 4.1R AUG1,2–/– MEFs,
although not entirely devoid of 4.1R (Stagg et al., 2008) (S.W.K.,
unpublished), have a similar cellular phenotype.
4.1R distribution is altered in cells lacking emerin or lamin
A/C
In view of the data presented above, we reasoned that at least some
fraction of emerin and A-type lamin is associated with 4.1R such
that their subcellular localization is 4.1R-dependent. We next
decided to test whether this was also the case for 4.1R or whether
its localization was entirely independent of emerin or lamin A.
Therefore, we first analyzed patterns of 4.1R immunostaining in
emerin–/Y MEFs which do not contain any emerin (Lammerding et
al., 2005). In wild-type (emerin+/Y) MEFs, the preponderance of
4.1R epitopes localized in the nuclear area, visualized by DAPI
staining, with lesser amounts in the cytoplasm. However, in
emerin–/Y MEFs, an increased proportion of 4.1R epitopes are
cytoplasmic (the ratio of nuclear to cytoplasmic 4.1R signals was
1.97±0.28 in wild-type and 0.54±0.15 in emerin-null cells; n10
each) (Fig. 4, top left). Next we investigated the effects on 4.1R
when emerin was not absent but mislocalized. Emerin
mislocalization has been reported in fibroblasts from lamin-Aknockout mice (Lmna–/– MEFs) (Hale et al., 2008; Sullivan et al.,
1999). Immunostaining of Lmna–/– MEFs also showed increased
4.1R cytoplasmic staining relative to that in wild-type Lmna+/+
cells (the ratio of nuclear to cytoplasmic 4.1R signals was 1.97±0.17
in Lmna+/+ and 0.86±0.19 in Lmna–/–; n10 each) (Fig. 4, top
right). As lamin A organization is not perturbed in emerin–/Y MEFs
(Hale et al., 2008; Lammerding et al., 2005), these results are
consistent with the notion that emerin is a determinant for a
substantial portion of 4.1R nuclear anchorage. To see whether
4.1R-deficient cells closely resembled cells lacking emerin or
lamin A, we stained these cells for NuMA, LAP2, LAP2,
MAN1, Tpr and Nup62 (Fig. 4). Our results, summarized in Table
1, show that 4.1R-deficient human and murine cells have a unique
phenotype.
Increased centrosome–nuclear envelope distance after
4.1R depletion
The Hutchinson laboratory recently discovered a new emerin
function: emerin is essential for centrosome linkage to the outer
nuclear membrane, which, in addition to the inner nuclear
membrane, has a significant fraction of emerin (Salpingidou et al.,
2007). They demonstrated that emerin interacts with microtubules
to anchor centrosomes to the outer nuclear membrane. In addition
to emerin-null fibroblasts and emerin-RNAi-treated cells, laminA/C-null fibroblasts with mislocalized emerin also have
centrosomes detached from the nuclear envelope (Hale et al., 2008;
Salpingidou et al., 2007). Recently, it was shown that nesprin-2-
Nuclear roles of protein 4.1R
1439
Journal of Cell Science
Fig. 4. Distribution of 4.1R and nuclear proteins in MEFs
lacking either emerin or lamin A/C relative to that in cognate
wild-type MEFs. Fixed cells were stained with antibodies against
the proteins listed at the left for immunofluorescent microscopy
(green) and nuclei were stained by DAPI (blue in ‘merge’
images). Whereas in wild-type MEFs, 4.1R epitopes are
predominant in the nuclear area, emerin–/Y and Lmna–/– MEFs
show increased cytoplasmic 4.1R staining relative to that in wildtype cells. Of the other proteins tested, emerin–/Y MEFs have
increased cytoplasmic MAN1 epitopes and Lmna–/– MEFs have
mislocalized LAP2 and MAN1 (Ostlund et al., 2006). NuMA,
LAP2, Tpr and Nup62 localizations are normal in the null
MEFs. See Table 1 for a summary of these results and from 4.1Rdeficient human and murine cells (Figs 2, 3). Scale bar: 4m.
deficient cells have an increased centrosome–nuclear distance
(Schneider et al., 2010; Zhang et al., 2009) and it has been suggested
that LINC complex disruption also has this effect (Razafsky and
Hodzic, 2009).
We previously reported that 4.1R depletion causes microtubule
disorganization in vitro and in intact cells (Krauss et al., 2002;
Krauss et al., 2004; Krauss et al., 2008). We reasoned that 4.1R
could have significant interactions at the nucleo-cytoskeletal
interface affecting centrosome tethering and microtubules,
particularly if 4.1R were functionally associated with emerin and
lamin A. Therefore, we used antibodies against several centrosomal
markers and DAPI staining to determine centrosome position in
cells treated with 4.1R RNAi. In control cells, centrosomes are
located juxtaposed or within 0.8 m of the nucleus. By contrast, in
cells depleted of 4.1R, with mislocalized emerin, centrosomes are
an average of 1.8 m from the nucleus (Fig. 5A,A⬘). This is similar
to the fold increase in centrosome distance reported for emerinnull and lamin-A/C-null human diploid fibroblast lines relative to
normal human diploid fibroblasts (Salpingidou et al., 2007). To
independently test whether 4.1R depletion affects centrosome
attachment, we also measured centrosome distances in 4.1R
AUG1,2–/– MEFs stained with centrosome markers and DAPI
because these cells also have disorganized microtubules (S.W.K.,
unpublished). However, we noted increased levels of centrosome
amplification in the 4.1R AUG1,2–/– MEFs relative to matched
wild-type MEFs (supplementary material Fig. S2). Therefore, we
selected only those 4.1R AUG1,2–/– MEFs with normal centrosome
numbers for our measurements. The results show that, again, there
was an approximate twofold increase in the nucleus–centrosome
distance in murine cells with depleted 4.1R (Fig. 5A). Emerin-null
MEFs were reported to show an ~2.5-fold increase in distance
(Hale et al., 2008). Thus, decreased expression of 4.1R in MEFs
phenocopied the detached centrosomes characteristic of emerinnull MEFs. Our new data demonstrate that the relative absence of
4.1R in both human and murine cells interferes with the linkage of
centrosomes to the outer nuclear membrane that depends at least
in part on a microtubule–emerin pathway.
Increased -catenin transcriptional activity and -catenin
nuclear accumulation when 4.1R is deficient
To substantiate that a potential 4.1R association with emerin has
further biological significance, we tested the prediction that 4.1R
downregulation, with concomitant displacement of emerin from
the nuclear envelope in intact cells, compromises a second emerin
function. Markiewicz and co-workers have shown that emerin
binds to -catenin through an adenomatous polyposis coli homology
domain (Markiewicz et al., 2006). -Catenin is a transcriptional
coactivator and the downstream target of the canonical Wnt
signaling pathway, participating in many developmental processes.
Emerin was shown to regulate the flux of -catenin out of the
nucleus in a manner that was dependent on its correct localization
at the inner nuclear envelope. When an emerin mutant lacking -
Table 1. Distribution of nuclear proteins in MEFs deficient for 4.1R, emerin or lamin A/C
Protein
4.1R RNAi
4.1R AUG 1,2–/– MEFs
emerin–/Y MEFs
Lmna–/– MEFs
NuMA
Emerin
LAP2a
LAP2b
MANI
Tpr
Nup62
Lamin A/C
mislocalized
mislocalized
mislocalized
normal
mislocalized
mislocalized
mislocalized
irregular
mislocalized
mislocalized
mislocalized
normal
mislocalized
mislocalized
mislocalized
irregular
normal
absent
normal
normal
mislocalized
normal
normal
normal
normal
mislocalized
mislocalized
normal
mislocalized
normal
normal
absent
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Journal of Cell Science 124 (9)
Journal of Cell Science
Fig. 5. Functional evidence implicating a 4.1R–emerin
association. (A,A⬘) Nuclear–centrosome distances are increased
in 4.1R-deficient human and murine cells. Cells were stained with
DAPI to image nuclei (blue) and anti--tubulin antibody (red) to
localize centrosomes. Distances were measured in overlaid
images using Image J. The graph shows data from duplicate
experiments using the cells indicated (n200 cells; mean±s.e.).
An example of centrosome position in a control and a 4.1RRNAi-treated cell is shown in A⬘. The white line measures the
nuclear–centrosome distance indicated. (B)Downregulation of
4.1R expression in HEK-293 cells. Western blot of 4.1R protein
in HEK-293 cell extracts untransfected or transfected as indicated
below the image. In cells exposed to control RNAi, strong 4.1R
bands were detected at ~135, 80, and 60 kDa in extracts
regardless of whether or not they had been co-transfected with
Topflash and Renilla luciferase (left side). In 4.1R-RNAi-treated
HEK-293 cells, 4.1R bands were absent or barely detectable
(right side). The actin blot below shows that similar amounts of
extracts were probed. (C)Emerin mislocalization in 4.1Rdepleted HEK-293 cells. Indirect immunofluorescence of HEK293 cells treated with control RNAi (top) or with 4.1R RNAi
(bottom) showing that in 4.1R-depleted HEK-293 cells, as in
other cells tested (Figs 2, 3), emerin is partially mislocalized to
the cytoplasm. (D)Increased -catenin activation in 4.1Rdepleted cells. HEK-293 cells were transfected with either
TOPFLASH (a TCF-activated reporter) or FOPFLASH
(a reporter with mutated TCF-binding sites; control) plasmids and
Renilla luciferase. Luciferase activity was corrected to Renilla
luciferase signals in the same sample to normalize transfection
efficiency (four experiments, mean±s.e.). (E,E⬘) Nuclear catenin accumulation in 4.1R-RNAi-treated HEK-293 cells and in
4.1R AUG1,2–/– (4.1R–/–) MEFs. Cells were fixed and stained
with DAPI and with anti--catenin for immunofluorescent
imaging. HEK-293 cells with control RNAi (E) and 4.1R wildtype (4.1R+/+) MEFs (E⬘) show predominant staining at
peripheral cell adhesion plaques, whereas 4.1R-RNAi-treated
cells (E) and 4.1R AUG1,2–/– MEFs (E⬘) have a nuclear
accumulation of -catenin with loss of -catenin at the cell
periphery. Scale bar: 5m (C); 15m (E⬘).
catenin-binding capacity is expressed, and in emerin-null
fibroblasts, there is enhanced nuclear accumulation and activation
of -catenin (Markiewicz et al., 2006). On that basis, we
hypothesized that 4.1R depletion might lead to increased -catenin
activity and nuclear accumulation due to mislocalization of a
portion of emerin into the cytoplasm (Fig. 2C, Fig. 3C), thereby
compromising the capacity of emerin to export -catenin from the
nucleus.
To directly test whether 4.1R depletion affects emerin-dependent
-catenin activity, we measured -catenin-induced TCF-activated
transcription in human embryonic kidney (HEK)-293 cells. HEK293 cells exposed for 72 hours to 4.1R RNAi had a 93% depletion
of ~135 kDa, ~80 kDa, and ~60 kDa 4.1R bands, relative to the
levels in controls, when normalized to actin (Fig. 5B). We
confirmed that emerin is mislocalized in these cells (Fig. 5C), as
in other 4.1R-deficient cells (Fig. 2C, Fig. 3C). The HEK-293 cells
were simultaneously transfected with a luciferase reporter plasmid
in which luciferase expression is controlled by a promoter
containing multiple -catenin-TCF-binding sites (TOPGLOW). A
plasmid with a mutant promoter (FOPGLOW) was used as a
control and co-transfection with Renilla luciferase was used to
monitor transfection efficiency. Using this assay, cells with depleted
4.1R showed an ~3.5-fold increase in -catenin activity relative to
control cells expressing 4.1R (Fig. 5D). As -catenin activity is
regulated by its nuclear accumulation, we investigated expression
of -catenin in the nucleus. In control cells, the predominant catenin staining was at the cell adhesion junctions with very little
nuclear staining (Fig. 5E,E⬘). By contrast, 4.1R-RNAi-treated HEK293 cells and 4.1R AUG1,2–/– MEFs had significantly higher
levels of nuclear -catenin epitopes, with decreased staining at the
cell periphery (Fig. 5E,E⬘). Thus 4.1R depletion alters emerin
function in exporting -catenin from the nucleus, resulting in both
increased transcriptional activity and nuclear accumulation of catenin.
Discussion
Here we report for the first time that 4.1R, emerin and lamin A/C
co-immunoprecipitate, along with actin and other known 4.1Rbinding partners, and that 4.1R loss impacts upon the organization
and functions of emerin and the A-type lamin network. Depletion
of 4.1R, either by 4.1R RNAi or genetic deletion, affects nuclear
structure and function in multiple ways: emerin is mislocalized
and peripheral and nucleoplasmic lamin A is perturbed; a emerinand lamin-binding partner MAN1 (Mansharamani and Wilson,
Journal of Cell Science
Nuclear roles of protein 4.1R
2005) and the lamin-A/C-binding partner LAP2 (Dechat et al.,
2000; Naetar and Foisner, 2009) distribute aberrantly; distances of
centrosomes from nuclei increase; the emerin-binding partner catenin relocalizes from the plasma membrane to the nucleus; and
-catenin signaling activity increases. Another effect of 4.1R,
emerin and A-type lamin disruption is that in emerin- and laminA-null MEFs the amount of nuclear 4.1R is decreased. Because the
nuclear proteins that we found to be affected by 4.1R depletion are
linked to human disease, it is important and timely to examine
potential contributions of 4.1R. On the basis of our data, we
hypothesize that 4.1R acts as a linker or adaptor at nodes vital for
interconnections between nucleoplasmic substructures, nuclear
envelope components and the nucleo-cytoskeletal interface.
Our discovery of the 4.1R cellular phenotype is particularly
relevant to several previous findings. We previously reported partial
coincidence of 4.1R with nuclear pore epitopes (Krauss et al.,
1997a), and 4.1R epitopes were detected by electron microscopy
in Xenopus oocytes on nuclear pore-linked filaments thought to be
related to Tpr (Kiseleva et al., 2004). Tpr is proposed to be a
component of nuclear basket filaments projecting into the nuclear
interior that specifically function in mRNA transport (Cordes et al.,
1998; Frosst et al., 2002; Huve et al., 2008; Krull et al., 2004). As
Tpr is substantially mislocalized into the cytoplasm when 4.1R is
depleted, as is emerin, this suggests specific mechanisms to account
for how 4.1R depletion compromises emerin-dependent -catenin
nuclear export through nuclear pores.
We also previously reported that, in confocal optical sections of
nuclei, 4.1R is detected at the periphery of areas stained by the
SC35 splicing factor and is partially coincident with PCNA, a
DNA polymerase accessory protein (Krauss et al., 1997a).
Independently the Correas laboratory reported interrelationships
between 4.1R and pre-mRNA splicing factors (Lallena et al., 1998).
Intranuclear lamin A/C foci are also associated with splicing
assemblies (Jagatheesan et al., 1999) and DNA replication centers
(Kennedy et al., 2000). As 4.1R depletion decreases intranuclear
lamin A/C speckles, 4.1R–lamin-A/C complexes might have
important functions at these sites involving SC35 and PCNA,
which binds lamin C (Shumaker et al., 2008). Moreover, it has
been proposed that there is a nucleoplasmic lamin A fibrillar
network with molecular links to various proteins, including NuMA
(Barboro et al., 2003; Bridger et al., 2007; Broers et al., 1999;
Dechat et al., 2008; Naetar and Foisner, 2009; Vlcek et al., 2001).
However, NuMA was not displaced in Lmna–/– cells, although a
substantial portion was mislocalized outside the nucleus in 4.1Rdeficient cells. As Lmna–/– cells are not devoid of nuclear 4.1R, this
suggests a predominant requirement for 4.1R in NuMA nuclear
anchorage. Defective mitotic spindles with mislocalized NuMA
were also observed in 4.1R-deficient cells (Krauss et al., 2008). As
4.1R depletion affects the distribution of MAN1, it could have
downstream affects on transforming growth factor (TGF)- and
bone morphogenic protein (BMP) signaling (Cohen et al., 2007;
Lin et al., 2005; Pan et al., 2005). Clearly there remain many
tantalizing questions to resolve regarding specific roles of 4.1R in
nuclear pore structural integrity and function, DNA replication,
mRNA processing and signaling.
Several possible molecular models are consistent with our results:
4.1R could associate with emerin-specific, A-type lamin-specific
and/or emerin–lamin-A/C complexes. Formation of these
complexes could be mediated through interactions with actin, IIspectrin or microtubules that bind 4.1R; II-spectrin has been
detected in several emerin complexes purified from HeLa cells
1441
(Holaska and Wilson, 2007) and emerin has been identified in IIspectrin complexes (Sridharan et al., 2006). These are particularly
attractive models, with precedent in erythrocytes, where 4.1R forms
multiprotein complexes ultimately crucial for mature erythrocyte
morphology and mechanical properties. Future experiments could
test binary or multimeric interactions in vitro by comparing
recombinant 4.1R isoforms (particularly those detected in
immunoprecipitates) with 4.1R recombinants lacking spectrinactin-binding domains or microtubule-binding domains, or with
mutations in those domains (Gimm et al., 2002; Krauss et al.,
2004). Additional multimeric interactions might even be more
likely if nuclear networks with actin–spectrin or actin–nesprin (a
spectrin-repeat protein) were to be proven. In fact, in addition to
emerin, we find that the spectrin-repeat protein nesprin, and the
actin-binding chromatin remodeling protein BRG1 also coimmunoprecipitate with 4.1R (S.W.K., unpublished), suggesting
future avenues of investigation into nucleoplasmic and nucleocytoplasmic networks and micromechanics. Within these networks
that are beginning to be deciphered, 4.1R may link components in
series or may modulate interactions between proteins that bind
directly to each other.
Characterizing functional interactions between 4.1R, emerin and
lamin A/C has important implications for deciphering molecular
etiologies for a wide spectrum of human diseases. Multiple diseases
are associated with aberrant emerin and A-type lamins
(laminopathies) (Capell and Collins, 2006; Worman and Bonne,
2007). Analysis of 4.1R interactions with emerin and lamin A/C
mutants linked to human diseases such as EDMD and
cardiomyopathy might contribute to a more detailed understanding
of their biogenesis. However, when emerin or lamin A genes are
not mutated (~60% of EDMD cases for example), mutant 4.1R or
dysregulated 4.1R expression could alter emerin and A-type lamin
distribution in architectural or regulatory complexes with specific
pathologic consequences. Thus 4.1R should be considered as a
candidate disease gene for laminopathy. Although there are no
known human 4.1R ‘null’ patients, 4.1R deficiency is linked to
progressive heart failure in humans and 4.1R AUG1,2–/– mice
have disturbances in cardiac electrophysiology (Birks et al., 2005;
Stagg et al., 2008). In these instances accompanying pathologic
emerin and A-type lamin alterations in cardiac tissue merit
examination. Furthermore, 4.1R deficiencies are associated with
meningiomas and myeloid malignancies (Alanio-Brechot et al.,
2008), ependymomas (Rajaram et al., 2005) and
neuroancanthocytosis (Orlacchio et al., 2007). During disease
progression, formation of crucial protein complexes could be
dynamic and dependent on 4.1R, emerin and A-type lamin
partnering that may fluctuate under different physiological
conditions. An additional overlay is that 4.1R has tissue and
developmental-specific isoform expression, for example, it is highly
expressed in cardiac muscle and detected in specific subregions of
kidney, lung and brain (Ramez et al., 2003; Shi et al., 1999).
Therefore, altered 4.1R expression or mutation might impact upon
tissues differently and be an unappreciated contributor to tissue
degeneration and aging-related functional decrements, as well as
to diverse human diseases.
Materials and Methods
Cells and media
HeLa and CaSki cells were obtained from the American Type Culture Collection,
and HEK-293 cells and MDCKdN90-A cells were the gifts from Angela I. Barth,
Stanford University, Palo Alto, CA. MEFs were harvested from embryos from 12–
14 day wild-type and 4.1R-null pregnant mice (Shi et al., 1999), by standard
1442
Journal of Cell Science 124 (9)
procedures (Hogan et al., 1994), and used at early passages before spontaneous
transformation. All mouse experiments were conducted in accordance with IACUC
approved protocols at Lawrence Berkeley National Laboratory. Lamin A/C and
emerin-null MEFs were the kind gift of Colin Stewart and Teresa Sullivan (NCI,
Frederick, MD). HeLa, HEK-293 cells and MEFs were cultured in Dulbecco’s
modified Eagle’s medium H21 (Gibco BRL) and CaSki cell were cultured in RPMI
1640 (Cell Gro), both supplemented with 10% fetal calf serum and penicillinstreptomycin at 37°C and under 5% CO2, as described previously (Krauss et al.,
1997a).
Journal of Cell Science
Immunofluorescence
Cells were grown on coverslips and fixed for 10 minutes at –20°C in methanol or
with 2% paraformaldehyde and 0.2% Triton X-100, with further 1% Triton X-100
permeabilization, as described previously (Krauss et al., 2008). Alternatively,
paraformaldehyde or Triton fixation was followed addition of cold methanol.
Commercial antibodies were the monoclonal GTU-88 against -tubulin (Sigma),
rabbit anti-pericentrin antibody (Covance), mAb 414 against nuclear pore protein
p62 (Davis and Blobel, 1986) (Babco), mouse anti-NuMA antibody (Oncogene),
mouse anti--catenin antibody (BD Transduction Labs), goat anti-(lamin B) antibody
(Santa Cruz Biotechnology), anti-(spectrin II) monoclonal antibody 1622
(Chemicon) and mouse NCL anti-emerin antibody (Leica). The following antibodies
were gifts from colleagues: anti-centrin antibody 20H5 (Jeffery Salisbury, Mayo
Clinic Foundation, Rochester, MN), anti-Tpr antibody (Volker Cordes, Max Planck
Institute, Gottingen, Germany), anti-(lamin A/C) monoclonal antibody XB10 (Kyle
Roux and Brian Burke, University of Florida, Gainesville, FL), anti-MANI antibody
(Kunxin Luo, University of California-Berkeley, Berkeley, CA), and anti-LAP2
and anti-LAP2 antibodies (Roland Foisner, University of Vienna, Vienna, Austria).
Anti-4.1R Berkeley was generated in rabbits immunized with a peptide derived from
exon 19 of 4.1R (AAQTDDNSGDLDPGVC) that had no cross-reactivity with 4.1B,
4.1G, or 4.1N. Secondary antibodies with minimal species cross-reactivity were
from Molecular Probes or Jackson ImmunoResearch. Parallel samples probed with
equal amounts of control non-immune IgG or without primary antibody showed no
fluorescent patterns under conditions used for experimental samples. DNA was
stained with 4,6-diamidino-2-phenylindole (Vectorshield, VectoLabs). Images were
acquired with a Nikon Eclipse 2000 microscope using a 60⫻ 1.4 NA objective
equipped with a Retiga Ex camera and ImagePro software. Images were processed
using Adobe Photoshop. Measurements of distances between fluorescence signals
were made using Image J. Signals, within a linear range, were quantified in cell areas
using Image Pro. Nuclear signals were measured as the pixels within the area stained
by DAPI in overlay images. Cytoplasmic measurements were determined by
subtracting the nuclear pixels from the total pixels in the cell. Nuclear and cell
boundaries were visually confirmed by phase-contrast microscopy.
Immunoprecipitation
For total cell sonicates, HeLa cells were trypsinized, rinsed twice with PBS at 37°C,
(2.7 mM KCl, 1.5 mM KH2PO4, 1 mM MgCl2, 1 mM EGTA, 137 mM NaCl and
8.1 mM NaHPO4, pH 7.4) containing protease inhibitors (Mini-Complete Cocktail;
Roche), and collected by centrifugation. Pelleted cells were suspended in buffer and
sonicated four times at 15 seconds at 50–60% output using a Branson Sonifier 250
with cooling on ice between sonications. Nuclear extracts were prepared from HeLa
nuclei isolated following hypotonic shock, douncing, and pelleting through a sucrose
cushion, as described previously (Krauss et al., 1997a). Nuclei were extracted with
0.2–0.25 M KCl, centrifuged and the supernatant dialyzed (Mayeda and Krainer,
1999). Nuclear extract was diluted with immunoprecipitation buffer (IP buffer),
incubated with 10 g of IgG (immune or control), rotated overnight at 4°C, then
incubated at 4°C for 2 hours with 50 l of protein-G-coupled magnetic beads
(Dynal). The beads were collected magnetically, washed four times with IP buffer,
and the immunoprecipitated proteins were eluted and analyzed by western blotting,
as described previously (Krauss et al., 1997a). Recovery of proteins was determined
by densitometry analysis of western blots using an Alpha Imager 2200 and software.
Several IP buffers were compared: RIPA (50 mM Tris-HCl pH 7.5, 150 mM NaCl,
1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS and protease inhibitors) and buffers
containing 25 mM HEPES, 150 mM NaCl, 1 mM MnCl2 or MgCl2, protease
inhibitors and either 0.5% CHAPS or 1% Brij97. The highest amounts of 4.1R were
recovered in immunoprecipitates using 25 mM HEPES, 150 mM NaCl, 1 mM
MgCl2, protease inhibitors and 0.5% CHAPS for both whole-cell sonicates (88%)
and nuclear extracts (92%) (Fig. 1A,B).
4.1 RNAi
Small interfering RNA duplexes (Dharmacon) were used under conditions described
previously (Krauss et al., 2008) including controls for non-specific targeting.
We also used RNAi duplexes containing DNA residues (Integrated DNA
Technology) with the following sequence: 5⬘-rGrArArArGrUrCrUrGrUrGrUrArGrArArCrArUrCrArCrACG-3⬘;
5⬘-rCrGrUrGrUrGrArUrGrUrUrCrUrArCrArCrArGrArCrUrUrUrCrCrA-3⬘. This duplex was tested for 4.1R downregulation
over a concentration range of 0–100 nM during a timecourse of 24–96 hours. For
experiments reported here it was used at 30 nM complexed with Lipofectamine 2000
for transfection of 2⫻105 cells. Downregulation was assessed by western blotting
normalized to actin.
-Catenin activity measurements
HEK-293 cells (1.5⫻105) were seeded in 35-mm culture dishes and the next day
transfected with 4.R RNAi, control RNAi, or Lipofectamine alone. Samples were
co-transfected with 20 ng of Renilla luciferase (to estimate transfection efficiency)
and 1 g of either pFOPFLASH or pTOPFLASH. After 72 hours, the levels of
luciferase and Renilla luciferase were determined on a Turner Designs Model TD20/20 luminometer. Luciferase activity was corrected for differences in transfection
efficiencies determined by the Renilla luciferase in the same sample. As a positive
control, MDCK cells expressing stabilized -catenin under doxycycline repression
were grown with or without 20 ng/ml doxycycline and tested for -catenin activity.
Samples without doxycycline had 30–50-fold increased -catenin activity relative to
Renilla luciferase activity.
We would like to thank A. I. Barth, J. G. Conboy, W. J. Holaska, J.
Lammerding, W. J. Nelson, K. Roux, C. Shanahan, V. Cordes, K.
Wilson and Q. Zhang for valuable discussions. We are particularly
grateful to J. Salisbury, R. Foisner, K. Roux, B. Burke, J. Lammerding,
T. Sullivan, V. Cordes, K. Luo and A. I. Barth for sharing valuable
reagents, methods, and cells. We thank M. Parra and S. Gee for
technical advice. This work was supported by National Institutes of
Health (grant number DK059079 to S.W.K.). The authors declare no
conflicts of interest. Deposited in PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/9/1433/DC1
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