* Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.
Structural proteins via diverse molecular interactions determine cell morphology, organize subcellular compartments, stabilize cell attachments, and
even regulate essential cellular responses to internal or external signaling. The 80-kD structural protein, protein 4.1, was initially characterized as a crucial member of the red
cell membrane skeleton where it stabilizes complexes between spectrin and actin within the skeletal network and
anchors them to the overlying plasma membrane through
interactions with integral membrane proteins. Deficiencies
in 80-kD protein 4.1 profoundly alter red cell morphology
and decrease membrane mechanical strength, leading to
membrane fragmentation and hemolytic anemia.
In subsequent studies, the 80-kD 4.1 of mature red cells
was identified as only one member of a large protein 4.1 family that is relatively abundant in nucleated erythroid and
nonerythroid cells. In fact, Western blots of many types of
mammalian and avian cells revealed 4.1 immunoreactive
protein species ranging from 30-210 kD (Anderson et al.,
1988 Several binding partners for specific 4.1 domains have
been characterized. The amino-terminal domain of erythrocyte protein 4.1 contains binding sites for glycophorin C,
calmodulin, p55 (Kelly et al., 1991 In contrast to the strictly peripheral localization of 4.1 in
mature red cells, 4.1 epitopes in nucleated cells have been observed by immunofluorescence throughout the cytoplasmic compartment (Cohen et al., 1982 The nucleus contains an internal nonchromatin scaffolding called the "nuclear matrix" or "nucleoskeleton." The
nuclear matrix is a three-dimensional structure, and when
viewed using resinless section electron microscopy, it
appears as a network of polymorphic filaments enmeshing
larger masses or "dense bodies" (Capco et al., 1982 We report here that protein 4.1 isoforms are resident
proteins of the nucleoskeleton during interphase and undergo dynamic reorganization in their association with nuclear components during mitosis. This suggests that by molecular interactions either previously identified or entirely
novel, protein 4.1 isoforms could be integral both to nuclear structure and to modulation of nuclear activities.
Materials
WI38 cells (CCL 75: diploid human fetal lung fibroblasts), CaSki cells
(CRL 1550: human cervical epidermoid carcinoma), and 3T3 cells (CCL 92:
murine fibroblasts) were obtained from American Type Culture Collection (Rockville, MD). HCA cells, from human fetal foreskin, were the gift
of Dr. J. Campisi (Lawrence Berkeley National Laboratory, Berkeley,
CA). The plasmid pSV2NeoCMV was generously provided by Dr. P. Yaswen
(Lawrence Berkeley National Laboratory). The vector contains a cytomegalovirus (CMV)1 promoter, SV-40 polyA addition and splice signals, and
an EcoRI restriction site for insertion of test sequences. B4A11 monoclonal antiserum was the gift of Dr. J.A. Nickerson (Massachusetts Institute of Technology, Cambridge, MA). FITC-conjugated goat anti-rabbit IgG and 5-(and-6)-carboxy-rhodamine succinimidyl ester were purchased from Molecular Probes (Eugene, OR). Tetramethyl-rhodamine-conjugated goat anti-mouse IgG and fluorescein 5-isothiocyanate isomer 1 were
from Sigma Chemical Co. (St. Louis, MO). Anti- Western Blot Analysis
Protein samples separated on 7% SDS-PAGE were transferred to nitrocellulose for 7 h at 100 V. After blocking, the blots were incubated first
with 0.5 µg/ml primary IgG for 1 h at room temperature, washed, and then
incubated with a secondary antibody coupled to horseradish peroxidase.
Immunoreactive proteins were visualized by chemiluminescent autoradiography.
Immunofluorescence
Subconfluent WI38 fibroblasts or CaSki cells grown on coverslips were
rinsed twice in PBS at pH 7.4, fixed for 10 min at 4°C in PBS containing
2% formaldehyde/0.2% Triton X-100, further permeabilized with 1% Triton X-100 for 10 min at room temperature, and then rinsed twice in PBS.
Alternatively, cells were fixed in Microscopy
Immunofluorescent cells were photographed using an epifluorescence microscope with a 63× objective and a 1.4 NA oil immersion lens. A microscope (model Axioplan; Carl Zeiss, Inc., Thornwood, NY) equipped with
a 63× 1.3 Neofluor oil immersion lens, a Quantitative Image Processing
System based on the MicroImager 1400 digital camera (Xillix Technologies Corp., Vancouver, BC, Canada), and the Scil-Image image analysis
software package (The University of Amsterdam, The Netherlands) were
used to acquire and analyze 0.2-µm optical sections of cells. A confocal
scanning laser microscope (model MRC 1000; BioRad Labs, Hercules,
CA) equipped with a Nikon Diaphot 200 (Melville, NY), a fluor 60× 1.4 planapo oil immersion lens, and a krypton/argon laser was also used to obtain 0.5-µm optical sections. Double-label images were collected sequentially and both signals were of similar intensity. Confocal images were constructed by means of BioRad software.
High resolution resinless section EM of immunostained cells was performed as previously described (Capco et al., 1984 Cell Culture
WI38 cells were grown in a 5% CO2 incubator at 37°C in DME and CaSki
cells in RPMI-1640 medium containing 10% FCS, 4 mM glutamine, antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) and 2.2 g/liter sodium
bicarbonate. Human fibroblasts were used at passages 20-24 where >90%
were capable of DNA synthesis.
Preparation of Nuclear Matrix Proteins
For isolation of nuclear matrix from nuclei, proliferating cells were trypsinized, washed three times in PBS, swollen in RSB (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2) for 30 min on ice, and dounced in RSB containing 0.3% NP-40 for about ten strokes until nuclei were free of cytoplasm by microscopic examination. Nuclei were then pelleted through a
0.25 M sucrose-RSB cushion for 10 min at 1500 g at 4°C. Typically, >95%
of nuclei were recovered. Sequential extraction procedures were performed as described previously (Fey et al., 1984 Antibodies
Rabbits were immunized with 80-kD protein 4.1 purified from human
RBC or with synthetic peptides comprised of sequences encoded by either
exon 16 (peptide 10-1), exon 19 (peptide 24-2), or exon 21 (peptide 24-3)
coupled to thyroglobulin (see Fig. 1). Sequences of the peptide antigens
were: 10-1, KKRERLDGENIYIRHSNLMLEC; 24-2, TDDNSGDLDPGVLLTAQTITSETPSSTTTTQITKC; or 24-3, HPDMSVTKVVVH
QETEIADEC. In addition, the sequence encoding peptide N-2, containing 209 amino acids following the AUG-1 start site of translation of protein 4.1 (Conboy et al., 1991
PCR Amplification of Protein 4.1 mRNA
WI38 mRNA, prepared from early passage, proliferating cells by the
guanidinium thiocyanate method (Chirgwin et al., 1979 Transient Transfections
A DNA construct was engineered encoding the sequence for 80-kD 4.1 fused at its COOH terminus to an epitope tag (KPPTPPPEPET) derived
from SV-40 large T antigen. The 80-kD 4.1 sequence began at AUG-2 and
included coding sequence through exon 21 except for the alternatively
spliced exons 14, 15, 17A, and 17B. The tag sequence, detected with the
monoclonal antibody KT3 (MacArthur and Walter, 1984 Subconfluent murine fibroblasts (3T3 cells) growing on coverslips were
transfected using LipofectAMINE and plasmid DNA according to the
manufacturer's instructions. A CMV- Protein 4.1 Localizes to Intranuclear Sites
To investigate nuclear localization of protein 4.1, we initially used diploid human fibroblasts (WI38 cells) since their
flattened morphology is particularly amenable by microscopy to distinguish nucleus versus cytoplasm and since human fibroblasts do not spontaneously transform in culture.
Multiple protein 4.1 isoforms have previously been observed in fibroblasts (Cohen et al., 1982 To detect 4.1 epitopes by immunofluorescence microscopy, we used a panel of affinity-purified IgGs. As depicted by the map of multiple alternative splicing pathways in Fig. 1 A, protein 4.1 isoforms contain varying
patterns of exonic inclusion and exclusion. Using exonspecific antibodies allows detection of 4.1 isoforms having
the peptide encoded by that particular exon. Our antibody
panel included antibody 10-1, recognizing sequence encoded by exon 16; antibody 24-2, specific for peptide encoded by
exon 19; antibody 24-3, recognizing epitope encoded by exon
21; antibody N-2, recognizing the NH2-terminal 209 amino
acids translated from the AUG-1 start site (Fig 1, B and C);
and anti-RBC 80-kD, against the 80-kD red cell 4.1 isoform
purified from human RBC (Fig. 1 B). We established the
specificity of our antibody panel for protein 4.1 by showing
that IgGs 10-1, 24-2, 24-3, and anti-RBC 80-kD 4.1 each
reacted with red cell 80-kD 4.1 in Western blot analysis (Fig. 2 A) and with protein 4.1 in the plasma membrane of
human red cells using immunofluorescent detection (Fig. 2
B). Furthermore, immunofluorescent signals were coincident when human red blood cells were probed with 24-2 and 24-3 directly labeled with two different fluorophores
(see Fig. 4).
Using three different classical fixation techniques (paraformaldehyde, methanol, or acetone), prominent punctate
staining in the nuclear area of WI38 fibroblasts was consistently observed by indirect immunofluorescence with each
antibody. Some antibodies also generated a more diffuse
nuclear staining pattern and several of the antibodies produced cytoplasmic staining (Fig. 3). Parallel samples incubated with control IgG did not produce nuclear immunofluorescence with any of these fixation methods (Fig. 3).
Fibroblasts were also probed with preparations of 24-2 or
24-3 IgGs directly labeled with either of two different fluorophores; these cells displayed immunofluorescent patterns similar to those obtained by indirect methods (data
not shown). Nuclear immunofluorescent staining was also
observed when another human fibroblast line (HCA), a
transformed epithelial human line (CaSki; see Fig. 9) and
a transformed murine fibroblast line (3T3; see Fig. 8) were
probed with the 4.1 antibody panel.
The nucleus is bounded by two concentric lipid bilayers
perforated by nuclear pores and an underlying ring of
lamin, a specialized intermediate filament. Nuclear-associated 4.1 immunofluorescent signals could have arisen from
cytoplasmic epitopes overlying the nucleus, from nuclear
membrane sites, or from interior nuclear locations. In fact,
since 80-kD protein 4.1 in mature erythrocytes is a component of the plasma membrane skeleton (Fig. 4), one might predict that 4.1 would localize to the nuclear membrane or
even the underlying lamin protein ring. To distinguish
among these possibilities, double-label confocal microscopy was performed to localize 4.1 foci relative to epitopes
for two well-characterized nuclear proteins, lamin B and the
nuclear pore complex. Optical horizontal sections through
the center of the nucleus revealed well-separated rhodamine (Fig. 5 A, stain for lamin; Fig. 5 B, stain for pores)
and FITC (stain for 4.1) signals, leading to the conclusion
that 4.1 epitopes are within the area bounded by nuclear
pores embedded in the nuclear membrane and by the underlying lamin protein ring. Furthermore, the smooth circumferential staining of both pores and lamin in the preparations rules out the possibility that internal 4.1 foci are
being generated by invaginations of the nuclear membrane.
To further characterize the distribution of 4.1 within the
nucleus, fibroblast nuclei stained with IgGs against 4.1 peptide domains were optically sectioned into horizontal
planes using conventional and confocal immunofluorescence microscopy, and the information was digitized. Using image analysis software, three-dimensional reconstructions were made through vertical planes of the nuclear interior. As illustrated in Fig. 5 C, 4.1 foci detected in horizontal planes are at multiple levels within the nucleus
when analyzed by vertical cuts through horizontal layers
(Fig. 5 C To determine whether 4.1 foci colocalize with nuclear
domains involved with pre-mRNA splicing factors or with
DNA replication, additional double-label immunofluorescence experiments were performed. Nuclear 4.1 epitopes
were imaged relative to SC-35 splicing protein (Fu and
Maniatis, 1992 Identification of Protein 4.1 in Nuclear Matrix
Since 4.1 is an important structural protein in red cells, we
hypothesized that isoforms in the nucleus might biochemically cofractionate with nonchromosomal nuclear scaffold
proteins. To test this prediction, purified nuclear matrix
was prepared by established techniques using sequential
permeabilization, salt extraction, nuclease digestion, and
salt reextraction from cells grown on coverslips and also
from purified fibroblast nuclei (Capco et al., 1982 Electron microscopy was used to achieve greater resolution in localizing 4.1 epitopes in the nucleus. Cells growing
on coverslips were prepared for embedment-free ("resinless") section electron microscopy, a technique developed
specifically to image three-dimensional cytoplasmic and
nuclear structures normally concealed in conventionally embedded samples (Capco et al., 1984
Western Blot Analysis of Nuclear Matrix Proteins
To analyze nuclear-associated 4.1 isoforms, nuclear matrix
proteins isolated from purified fibroblast nuclei were separated on SDS-PAGE and transferred to nitrocellulose,
and identical gel lanes were probed with each of the 4.1 antibodies. A number of protein bands in the matrix preparation were reactive with multiple 4.1 IgGs. (Fig. 7 A,
lanes 1-5). Prominent clusters of bands migrating with molecular masses in the range of ~55 and 80-100 kD were
detected. Other less abundant bands also reacted with
antibodies against several different protein 4.1 epitopes:
~65 kD (antibodies 24-2, 24-3) and ~120-150 kD (antibodies 10-1, 24-3, 4.1). No reaction with the nuclear matrix
proteins occurred when control rabbit IgG was used as a
probe (Fig. 7 A, lane 6). Thus multiple isoforms of 4.1 are
retained in the most insoluble fraction of nuclear proteins.
To verify that the blotted proteins contained characteristic nuclear matrix proteins, a lane was probed with antibody against a major nuclear matrix protein, lamin B. A
single band at the expected molecular mass of 66 kD was
detected (Fig. 7 A, lane 9). To assess cross-contamination
of nuclear matrix by cytoplasmic proteins, lanes containing
cytoplasmic and nuclear matrix fractions were probed with
antibody against the cytoplasmic protein In both immunofluorescence and Western blotting experiments with antibody N-2, the results reported above
suggest that 4.1 isoforms initiated at the upstream translation initiation site AUG-1 are expressed in fibroblasts. As
an independent means of confirming the existence of such
isoforms in fibroblasts, 4.1 mRNA was selectively amplified by reverse transcriptase-PCR techniques using primers designed to detect the AUG-1 sequence. As shown in
Fig. 7 B, WI38 fibroblast RNA encodes AUG-1, consistent
with the N-2 antibody results.
Furthermore, after Western blotting of nuclear matrix
using 24-2 and 24-3 IgGs, we detected several protein
bands with similar apparent molecular masses as well as
other bands not in common (Fig. 5, lanes 4 and 5). This result predicted nuclear 4.1 isoforms with differing combinations of the 24-2 and 24-3 epitopes. Using preparations of
24-2 and 24-3 antibody each directly labeled with a different fluorophore, double-label immunofluorescent staining of fibroblasts revealed coincident signals in the nucleus
along with some noncoincident staining for each antibody
(data not shown). This result is consistent with data from
Western blot analysis of nuclear matrix proteins. Taken
together, immunofluorescent and Western blot data suggest that there are multiple nuclear 4.1 isoforms or homologues and that protein 4.1 fractionates with nuclear matrix proteins.
Expression of Epitope-tagged 4.1 in the Nucleus
To test for protein 4.1 in the nucleus by a second experimental strategy, a construct encoding the prototypical
80-kD protein 4.1 (Conboy et al., 1991 Dynamic Rearrangement of 4.1 Epitopes during the
Cell Cycle
During mitosis, the nucleoskeleton is dismantled, a mitotic
spindle is assembled, and chromatin is condensed, packaged, and partitioned into daughter nuclei. Nuclear structural proteins change their subcellular localizations and
some vary their roles during these events. The rearrangements of protein 4.1 epitopes as cells enter mitosis were
determined in rapidly proliferating CaSki cells. Cells were
labeled with antibody probes to 4.1 peptide epitopes (Fig.
9, A, C, E, and G) and with B4A11, a monoclonal antibody against a nuclear matrix protein associated with a
subset of RNA splicing factor storage/assembly sites (Fig.
9, B, D, F, and H) (Blencowe et al., 1994 Antibodies to 4.1 epitopes exhibited a pattern of diffuse
nuclear staining with dispersed intense fluorescent foci
within interphase nuclei (Fig. 9 A, arrow I; see also C, E,
and G) and stained structures resembling centrosomes in
the perinuclear region (Fig. 9 A, below arrow I). This pattern dramatically altered during metaphase, when 4.1 antibodies intensely stained the spindle and spindle pole of the
mitotic apparatus (Fig. 9 A, arrow M and inset) and diffusely stained the outlying areas of the dividing cell. As the
spindle poles separated, 4.1 epitopes clustered around two
distinct perichromosomal regions beginning to egress toward each daughter cell (Fig. 9, C, E, and G). During telophase, 4.1 antigenic material formed an intensely staining
mass in each nascent nucleus and the midbody region in
the vestigial connection between the daughter cells showed
a distinct band of fluorescence. B4A11 epitopes, which had
completely disappeared during mitosis (Fig. 9 B), began to
form larger foci during telophase (Fig. 9 H) preceding movement into maturing interphase nuclei. The patterns
of 4.1 epitope distribution during the cell cycle were reproducibly observed in cell populations partially synchronized
by a thymidine block. Thus, throughout the cell cycle, 4.1 epitopes remain accessible to antibody binding but display
a complex pattern of rearrangements, migrating from intranuclear sites after interphase to regions associated with
the mitotic spindle, perichromosomal areas, and the midbody.
While mature red cells contain predominantly an 80-kD
4.1 isoform, multiple protein 4.1 isoforms are expressed in
nucleated erythroid and nonerythroid cells. Combinatorial
alternative splicing of 4.1 mRNAs could in principle generate many 4.1 isoforms that participate in distinct sets of
molecular interactions. This could provide mechanisms for
differential cellular localization of 4.1 isoforms as well as
isoform functional specificity. There are numerous precedents for a role of alternative splicing in isoform-specific localization. Among membrane skeletal proteins, for example, band 3 isoforms with different 5 The focus of the present investigations has been to explore in depth the 4.1-immunoreactive epitopes in the nucleus. A currently evolving view is that the nucleus contains a highly structured internal skeletal lattice that can
organize chromosomes and numerous other nuclear components into physical and functional subdomains. At a biochemical level, this internal nuclear structure could serve
as a nonsoluble integrator of nuclear metabolic reactions (Nickerson et al., 1995 An important strategy was to use a panel of affinitypurified antipeptide IgGs against distinct areas of the 4.1 polypeptide sequence as well as traditional IgG preparations generated against red cell 80-kD 4.1. The peptides
were selected in part because of their evolutionary conservation and in part because they are encoded in different
functional domains of 4.1. Each antibody preparation reacted with red cell 80-kD 4.1 by Western blotting of human red cell membranes and immunofluorescent staining
of human red cells. Members of the antibody panel consistently showed immunoreactivity with fibroblast nuclear
structures using multiple immunofluorescent protocols,
by Western blot analysis and immunoelectron microscopy.
To avoid potential immunological cross-reactivity with
known members of the 4.1 superfamily (ezrin, moesin, radixin, merlin/schwannonin, talin, and certain tyrosine phosphatases), antibodies used in this study were directed
against domains of 4.1 not shared with these related proteins. Thus, the 4.1 epitopes located in nuclear regions should
identify authentic 4.1 isoforms or very highly related homologues yet to be discovered.
However, to further verify by an independent approach
that protein 4.1 can localize to the nucleus, fibroblasts were
transiently transfected with a vector encoding the 80-kD
red cell 4.1 sequence fused to an epitope tag. This 4.1 isoform coding sequence was selected for a number of reasons: (a) It encodes a well-characterized full-length 4.1 isoform; (b) antibody against this sequence produced strong
nuclear immunofluorescent signals in human and murine
fibroblasts; (c) protein bands migrating ~80 kD were detected in nuclear matrix Western blots; and (d) this sequence contains a potential nuclear localization signal (De
Carcer et al., 1995). This transfection experiment allowed
the unequivocal demonstration that an individual 4.1 isoform of known structure can be localized to the nucleus.
Since 4.1 in red cells mechanically links the spectrin and
actin membrane skeleton to the overlying lipid bilayer, we
explored its intranuclear localization to test the idea that
nuclear 4.1 might associate with the nuclear membrane-
lamin region. By reconstructing three-dimensional optical
sections through the nucleus, we found that 4.1 epitopes
are not predominantly colocalized with nuclear membrane
pores or with the underlying fibrous lamin network but are
distributed throughout the interior of the nucleus.
Additional double-label experiments were performed to
test for colocalization of 4.1 with known intranuclear structural or functional domains. The 4.1 distribution is complex and does not precisely colocalize with PCNA. Doublelabel confocal microscopy provided evidence for a potential
association of 4.1 with SC-35-enriched splicing assemblies,
which in fibroblasts are concentrated in a plane just below
the midline, parallel to the growth surface (Carter et al.,
1993 After extensive biochemical extraction, 4.1 epitopes were
detected in nuclear matrix preparations by both immunofluorescence light microscopy and immunogold electron
microscopy. At the higher resolution of electron microscopy, gold bead-coupled antibodies did not cluster preferentially near the nuclear pore-lamina complex, consistent
with our double-label immunofluorescent confocal data. Rather, gold beads were distributed in the vicinity of dense
bodies within the internal fibrous nuclear scaffold network. This observation is also entirely consistent with the
double-label immunofluorescence experiments showing
that 4.1 foci appeared at positions within the nuclear area
stained by NuMA but were not primarily coincident. At
the level of resinless electron microscopy, NuMA antibodies have been seen to decorate subsets of filaments in nuclear matrix preparations (Zeng et al., 1994 Nuclear matrix proteins, isolated from purified fibroblast nuclei under extraction conditions identical to those
used for immunofluorescence microscopy, revealed several polypeptides that reacted with multiple anti-4.1 IgGs
in Western blots. We speculate that the complex pattern of
4.1 expression results from splicing-mediated inclusion or
exclusion of various internal peptides as well as posttranslational modifications such as phosphorylation (Horne et
al., 1990 One of the bands in the ~80-100-kD cluster may be
similar to the 4.1p75 polypeptide observed by De Carcer
et al. (1995) using an alternative method of nuclear extraction and an independently generated 4.1 antibody. More
extensive alternative splicing or usage of new unidentified
promoters may be responsible for lower molecular mass
bands. The broad band in the nuclear matrix preparation migrating at ~55 kD reacted with all the 4.1 IgGs tested.
This pattern could represent an authentic 4.1 isoform that
is the product of an as yet unrecognized 4.1 alternative
splicing pathway, or it could represent a new 4.1 homologue. For example, in the ankyrin membrane skeletal
protein family, a novel small isoform with a truncated
membrane binding domain has been identified in Golgi (Devarajan et al., 1996 This speculation is particularly intriguing because during mitosis, 4.1 epitopes display dynamic rearrangements
within the nuclear microarchitecture that are correlated
with cell division events: at interphase they reside throughout the nucleus, then accumulate at the spindle during metaphase, coalesce around condensed chromatin during telophase, and some remain at the midbody region during
cytokinesis. Many nuclear-derived components redistribute at the time of nuclear membrane dissolution to several
structural zones: the chromosome scaffold, the perichromosomal region, the centrosome/kinetochore region, midbody, and spindle poles (for review see He et al., 1995 The recent elucidation of an isoform kindred for structural protein 4.1 suggests uncharted roles in addition to its
traditionally defined function in the erythroid plasma
membrane skeleton. As a structural component of the nucleus, protein 4.1 presents a new challenge to decipher
mechanisms underlying its targeting to nuclear subdomains, to identify its localized molecular interactions, and
to understand signals orchestrating its dynamic redistribution during the cell cycle. The versatile rearrangements of
4.1 epitopes during cell division could reflect the disposition of individual isoforms with unique structural assignments and patterns of expression. Ultimately, protein 4.1 may contribute fundamentally to modulation of the cell
cycle and possibly to more specialized phenomena such as
apoptosis or mammalian erythroid enucleation.
; Granger and Lazarides, 1984
, 1985
). As in many
other structural protein families, 4.1 isoform structural and
functional diversity can be generated by a number of mechanisms including complex alternative splicing of 4.1 premRNA (Conboy et al., 1988
, 1991
; Tang et al., 1988
, 1990),
usage of at least two translation initiation sites, and posttranslational modifications of 4.1 proteins. These variations as well as regulated 4.1 mRNA expression can be both
tissue- and differentiation-specific (for review see Conboy,
1993
).
; Tanaka et al., 1991
;
Pinder et al., 1993
; Gascard and Cohen, 1994
; Hemming
et al., 1994
, 1995
; Marfatia et al., 1994
, 1995
), and band 3 (Jons and Drenckhahn, 1992
; Lombardo et al., 1992
), while
a domain towards the COOH terminus contains binding
sites for spectrin and actin complexes (Correas et al., 1986a
,b; Discher et al., 1993
; Schischmanoff et al., 1995
).
Purified red cell 4.1 also interacts specifically with tubulin
(Correas and Avila, 1988
) and myosin (Pasternack and
Racusen, 1989
). The multiplicity of 4.1 isoforms combined
with the diversity of possible protein-protein interactions
suggests that individual 4.1 isoforms may have specific and
discrete functions. However, the roles of 4.1 isoforms,
other than in red cells, have not yet been defined.
; Lue et al., 1994
),
including perinuclear regions such as apparent centrosomal and Golgi structures (Leto et al., 1986
; Marchesi, V.T.,
S. Huang, T.K. Tang, and E.J. Benz. 1990. Blood. 76:12A;
Chasis et al., 1993
; Krauss, S.W., C.A. Larabell, C. Rogers, N. Mohandas, and J. Chasis. 1995. Blood. 86:415a; Beck, K.A., and W.J. Nelson. 1996. Mol. Cell. Biol. 302:22). Immunolabeling of 4.1 epitopes has also been observed in the
nucleus (Madri et al., 1988
; Tang, T.K., C.E. Mazoucco,
T.L. Leto, E.J. Benz, and V.T. Marchesi. 1988. Clin. Res.
36:A405; Marchesi, V.T., S. Huang, T.K. Tang, and E.J.
Benz. 1990. Blood. 76:12A; Correas, 1991
; Krauss, S.W. 1994. J. Cell. Biochem. 18c:95, M208; Krauss, S.W., J.A. Chasis,
S. Lockett., R. Blaschke, and N. Mohandas. 1994. Mol.
Biol. Cell. 5:343a; De Carcer et al., 1995; Krauss, S.W., J.A.
Chasis, C.A. Larabell, S. Lockett, R. Blaschke, and N. Mohandas. 1995. J. Cell Biochem. 21B:140, JT 309). Isoforms
of protein 4.1 in the nucleus presumably could serve as
structural elements. This is particularly intriguing in light
of emerging evidence of the important relationship between
nuclear architecture and regulation of nuclear functions.
; Fey
et al., 1986
; Nickerson et al., 1995
; for review see Penman,
1995
). Matrix spatial organization appears to provide functional subcompartmentalization for nuclear metabolic processes and requisite machinery (Nakamura et al., 1986
; Spector, 1990
, 1993
; Carmo-Fonseca et al., 1991
; Saunders et
al., 1991
; Spector et al., 1991
; Wang et al., 1991
). The largest nuclear domains are the nucleoli, sites of ribosomal
RNA synthesis and partial assembly (Fischer et al., 1991
;
Scheer et al., 1993
). RNA transcription and pre-mRNA
splicing occur in smaller domains and along tracks within
the matrix reticular network (Spector, 1990
; Spector et al.,
1991
; Carter et al., 1993
; Xing et al., 1993
, 1995
). DNA is
attached to the nuclear skeleton, and factors involved in
replication and cell cycle control are clustered into discrete replication foci (Smith and Berezney, 1982
; Jackson
and Cook, 1986
; Cook, 1988
; Leonhardt et al., 1992
; Hozak
et al., 1993
). However, during each round of cell division,
the nucleus itself undergoes dramatic morphologic remodeling: its nuclear envelope dissolves, the underlying lamin
ring depolymerizes, and chromosomes condense. Thus,
the nuclear scaffold must be capable of a series of ordered
yet dynamic protein-protein interactions necessitated by
perturbations of nuclear architecture and function during
the cell cycle.
Materials and Methods
-tubulin and high molecular weight "Rainbow" markers were products of Amersham Corp.
(Arlington Heights, IL). Pefabloc (AEBSF; 4-[2 aminoethyl]benzenesulfonyl fluoride) was obtained from Boehringer Mannheim Corp. (Indianapolis, IN). Renaissance chemiluminescence reagents were purchased from DuPont/NEN (Boston, MA). DME and LipofectAMINE were the product of GIBCO BRL (Gaithersburg, MD) and FBS was from Hyclone Labs (Logan, UT). Antibodies against lamin B (Ab-1), PCNA (Ab-1), nuclear mitotic apparatus protein (NuMA) (Ab-1), and nuclear pores (Ab-3) were either purchased or provided by Oncogene Research (Cambridge, MA).
80°C acetone or in cold MeOH for 5 min. After incubation in 10% goat serum to block nonspecific protein
binding, fixed cells were incubated with 5-10 µg/ml primary antibodies for
1 h at room temperature in PBS containing 10 mg/ml BSA, washed, and
then incubated with FITC- and rhodamine-conjugated secondary antibodies. Cells were stained with 4
,6-diamidino-2-phenylindole (DAPI) at 0.5 µg/ml (Sigma Chemical Co.) to confirm nuclear location and washed again, and the coverslips were mounted on slides using Vectashield (Vector Laboratories, Burlingame, CA). Controls with equivalent amounts of nonimmune IgG or with primary antibody omitted were also included in each
experiment. When photographed under the same conditions as the experimental samples, the controls did not show fluorescence.
; Nickerson et al., 1990
,
Nickerson and Penman, 1992
). Extracted cells were fixed with glutaraldehyde, blocked with goat serum, labeled with gold bead-conjugated goat
anti-rabbit secondary antibodies (Amersham Corp.) after incubation with
primary IgG, and fixed again. The samples were embedded in diethylene
glycol distearate (Polysciences, Warrington, PA). Thin sections were cut,
and the resin was removed before critical point drying and carbon coating.
Resinless sections were examined using an electron microscope (model
1200EX; JEOL U.S.A., Inc., Peabody, MA).
, 1986; Nickerson et al., 1994
)
using buffers containing protease inhibitors (1 mM Pefabloc, 0.1 µg/ml
aprotinin, 1 µg/ml pepstatin and leupeptin). Briefly, nuclei were successively extracted in CSK buffer (10 mM Pipes, pH 6.8, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, and 0.5% Triton X-100), extraction buffer (10 mM Pipes, pH 6.8, 300 mM sucrose, 250 mM ammonium
sulfate, 3 mM MgCl2, 1 mM EGTA, and 0.5% Triton X-100), digestion buffer (10 mM Pipes, pH 6.8, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM
EGTA, and 0.5% Triton X-100) with DNase and RNase or RNase-free DNase as indicated, and reextracted with 250 mM ammonium sulfate. To
remove additional proteins from nuclear matrix, the preparation was extracted with 2 M NaCl buffer. For fractionation of nuclear matrix for in
situ microscopy, subconfluent cells growing on coverslips were rinsed in
PBS and sequentially processed as described above.
) (Fig. 1), was fused to glutathione-S-transferase (GST). The construct was overexpressed, purified by glutathione agarose affinity chromatography, and used for immunization. Immune IgGs were affinity purified against the immunizing 4.1 peptide or RBC
80-kD 4.1 and analyzed to confirm no cross-reaction with either thyroglobulin or the GST moiety. Antibodies 10-1 and 24-2 have been used by us in
other studies (Chasis et al., 1993
; Conboy et al., 1993
; Schischmanoff et al.,
1995
; Chasis et al., 1996
). For direct labeling, IgGs (0.4 µg/ml) were conjugated to rhodamine or FITC fluorophores and then extensively dialyzed
according to the instructions provided by Molecular Probes.
Fig. 1.
Protein 4.1 peptide
domains used to generate antibodies. (A) A schematic diagram of protein 4.1 mRNAs
displaying multiple combinations of splicing pathways
possible among 4.1 alternative exons. In this format, exons are coded as follows:
solid bars, constitutive;
shaded bars, alternative;
open bars, noncoding. The
arrows on top indicate the
positions of alternative translation initiation sites, AUG-1
and AUG-2. The figure is derived from Conboy et al.
(1991). (B) Examples of protein 4.1 isoforms derived from different translation initiation sites. The 80-kD prototypical red cell isoform is produced from AUG-2 and can be present in nucleated and nonnucleated cells.
Chymotryptic fragments of this isoform include a 30-kD membrane binding domain, a 16-kD domain, a 10-kD spectrin and actin binding domain, and the 22-24-kD domain (Leto and Marchesi, 1984). Higher molecular mass 4.1 isoforms, present in nucleated cells, use
AUG-1 to generate an additional 209-amino acid "NH2-terminal extension" (N-term). (C) Synthetic peptides derived from the 4.1 amino acid sequence at the positions indicated by the arrows were used to immunize rabbits. IgGs were prepared from N-2, 10-1, 24-2, and
24-3 sera by affinity purification using the homologous peptide. The sequences for the peptides are given in the Methods section.
[View Larger Version of this Image (25K GIF file)]
) and human reticulocyte mRNA prepared as described (Temple et al., 1977
) were transcribed into cDNA and amplified by PCR under the conditions previously
used by Conboy et al. (1991). For amplification of AUG-1, the oligonucleotides used were: sense strand, AACATCATGACAACAG, and antisense strand, GTGTGTTTCTGCACTGCTTA.
), does not contain the SV-40 nuclear localization signal. The 80-kD 4.1 epitope-tagged
construct was fused to GST in pGEX-KT and expressed in Escherichia
coli, and the product was isolated by glutathione chromatography (Discher et al., 1993
). It was analyzed by Western blotting to verify its apparent molecular weight and reactivity both with 4.1 and KT3 antibodies.
-gal vector was used to optimize transfection conditions. 3T3 cells were transfected for 24-48 h with pSV2NeoCMV
containing an insert encoding 80-kD red cell protein 4.1 fused to an
epitope tag at its COOH terminus or a control vector without an insert.
Transfected 3T3 cells were fixed either with paraformaldehyde or methanol, processed for indirect immunofluorescence using Texas red-conjugated secondary antibody, and finally DAPI stained to determine subcellular distribution of expressed epitope-tagged protein. Three independent
transient transfection experiments were performed.
Results
; Conboy et al., 1993
).
Fig. 2.
Characterization of antibodies directed against red cell 80-kD 4.1 and 4.1 peptide domains. (A) Human red cell membrane
fractions (Conboy et al., 1993) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with the following IgGs:
lane 1, anti-RBC 4.1; lane 2, anti-N-2; lane 3, anti-10-1; lane 4, anti-24-2; lane 5, anti-24-3; lane 6, control IgG. Protein 4.1 in red cells migrates at ~80 kD. Although this is the predominant 4.1 isoform in red cells, a faint band at ~135 kD detected by anti-N-2 (lane 2) is also
detected by other 4.1 IgGs when increased amounts of red cell membrane preparations are applied to gels (Conboy et al., 1991
). (B) Human red cells were fixed by methanol and probed by indirect immunofluorescence using primary antibodies as indicated and FITCconjugated secondary antibodies. The faint staining by anti-N-2 most likely reflects the small amount of ~135-kD 4.1 also detected by
Western blot analysis of red cell membranes. Bar, 10 µm.
[View Larger Version of this Image (56K GIF file)]
Fig. 4.
Coincidence of immunofluorescent signals on human
red cells probed with antibodies
against two 4.1 peptide domains.
Normal human red cells, which
contain 80-kD 4.1 in their
plasma membrane, were fixed in
methanol and probed with antibody 24-2 directly conjugated to
a FITC fluorophore (green) and
antibody 24-3 directly conjugated to a rhodamine fluorophore (red). The yellow image indicates that the red and green
signals coincide. Bar, 6 µm.
Fig. 5.
Distribution of protein 4.1 epitopes in the interphase nucleus of human fibroblasts visualized by immunofluorescence. Location of the nucleus was confirmed in all cases by DAPI staining (not shown). (A and B) Double-label confocal microscopy of cells
stained by protein 4.1 antibody 24-2 (green) and (A) antibody against nuclear pores (red) or (B) lamin B (red). The majority of nuclear protein 4.1 foci are interior to the periphery of the nucleus as demarcated by immunofluorescence of pores within nuclear membrane and by its subadjacent network of lamin B fibers. (Additional lamin B sites in the nuclear interior have been reported [Bridger et al.,
1993; Moir et al., 1994
]). Similar results were obtained by imaging epitopes for protein 4.1 antibodies 24-3, 10-1, and N-2 relative to antipore
and antilamin epitopes. (C) Three-dimensional image analysis of nuclear epitopes probed with anti-RBC 80-kD 4.1 showing a 0.3-µm
horizontal optical midsection of an immunofluorescent cell with a line indicating the position of the vertical reconstruction depicted in
C
. Since optical resolution in the z-axis (vertical) is less than in the x-y (horizontal) axis, the reconstructed image is less sharp; the dome
shape of the top of the cell and the flat bottom plane of the coverslip are somewhat obscured by the coincidence of out of focus light. (A
cartoon depicting the approximate shape of the vertical cross-section through the nucleus is presented in the bottom panel.) Thus, it is apparent that epitopes for protein 4.1 lie in numerous planes within the volume of the cell. Similar results were obtained in three-dimensional reconstructions using the 24-2, 24-3, and 10-1 antibodies as probes. (D and D
) Double-label confocal microscopy of a fibroblast
using anti-24-2 (green) and anti-NuMA (red). In a horizontal plane through the midsection of the cell (D), protein 4.1 foci appeared
within the NuMA-stained nuclear interior. (D
) Three-dimensional reconstructions were made of three different vertical planes from
this NuMA:24-2 double-labeled cell. The flattened plane of the coverslip appears at the top of the vertical reconstructed images. The
conclusion that 4.1 epitopes localized at multiple planes throughout the volume of the nucleus is entirely consistent with the observations
obtained in images A, B, and C. The same conclusion was reached when cells were probed with antibodies against NuMA and RBC 80-kD
protein 4.1. (E and F) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against PCNA (red).
Optical sections through the fibroblast nucleus show that 24-2 (green) and PCNA (red) epitopes coincided (yellow) in many areas except in the vicinities of the nucleoli, which either are relatively devoid of any signal or sometimes contained a clustering of 24-2 (green) signals. Both PCNA and 24-2 epitopes resided in multiple planes throughout the nucleus (not shown). (G and H) Double-label confocal microscopy of cells probed with antibody 24-2 (green) and antibody against SC-35 (red). Optical sections are in the plane of the cell containing SC-35 domains; most SC-35 domains in fibroblasts are located in a single plane (Carter et al., 1993
). It is apparent that the vast
majority of the SC-35 domains contained areas of coincident staining with 24-2 (seen as yellow coloration), most often at the periphery
of the SC-35 domains. In optical sections above and below SC-35, additional 24-2 signals are observed (not shown), consistent with the
conclusions from the localization data presented in A-F. Bars, 3.5 µm (8 µm in the vertical dimension [C
and D
]).
[View Larger Versions of these Images (42 + 69 + 84 + 69 + 30 + 106 + 77 + 155K GIF file)]
Fig. 3.
Detection of nuclear epitopes of protein 4.1 by immunofluorescent light microscopy in WI38 cells. Cultured WI38 human fibroblasts were fixed in acetone for probing with anti-RBC 80-kD 4.1 and N-2, in methanol for probing with anti-24-2, or in formaldehyde for probing with anti-10-1 and anti-24-3, followed by incubation with a FITC-labeled secondary antibody. Punctate nuclear signals, detected with all the protein 4.1 antibodies, were particularly prominent with anti-RBC 80-kD 4.1 and anti-24-2. Localization of
FITC signals within the nuclei of all cells was confirmed by comparison to DAPI fluorescence of the cells with 4.1 fluorescence (not
shown). Antibodies N-2, 10-1, and 24-3 also produced considerable cytoplasmic staining. Staining patterns were in large part independent of the fixation method. Controls showed no fluorescent patterns when imaged under the same conditions as experimental samples. Bar, 10 µm.
[View Larger Versions of these Images (107 + 115 + 134 + 50 + 114 + 134K GIF file)]
Fig. 9.
Dynamic redistribution of protein 4.1 antigens during
the cell cycle. CaSki cells were permeabilized with 0.5% Triton
X-100 in CSK buffer to remove membranes and soluble proteins
before formaldehyde fixation, incubated with DNase I, and extracted with 0.25 M ammonium sulfate. The cell preparations
were immunostained with protein 4.1 antibody 24-2 (A, C, E, and
G) and with B4A11 (B, D, F, and H), a monoclonal antibody
against a nuclear matrix protein that displays an intense nuclear
speckle pattern at interphase but is not detectable at mitosis (Blencowe et al., 1994). Micrograph pairs show the fluorescent pattern
with anti-24-2 (left) and B4A11 (right) of the same fields. Cell cycle stages were also confirmed by viewing cells using phase contrast microscopy (not shown). (A) In the center, a mitotic cell
(M) showed staining of the mitotic spindle with particularly
strong staining of the spindle poles. The mitotic cell is surrounded
by interphase cells. Note that at opposite sides of an interphase
nucleus (I), immunostained centrosomes (small spots) are visible.
The inset shows the mitotic spindle of another cell intensely immunolabeled by anti-10-1. (B-F) Epitopes for B4A11 have disappeared in mitotic cells, but a strong speckled staining pattern is
present in interphase nuclei. (C) In the center, a cell in anaphase
retained a high degree of 4.1 staining in the area of the condensed
chromosomes. (E) As the cells approached telophase and cytokinesis, the bridge between the intensely stained perichromosomal
regions became visible by antibody deposition. As the daughter
cells separated further apart (G), bright 4.1 staining appeared at
the midbody (arrow). In the companion B4A11 fields, diffuse
staining began to condense into a more focal pattern, foreshadowing the appearance of the prominent B4A11 speckles characteristic of interphase cells. Bar, 5 µm.
[View Larger Version of this Image (59K GIF file)]
Fig. 8.
Expression of epitope-tagged 4.1 in nuclei after transient transfection. A construct was engineered to encode the sequences for red cell 80-kD protein 4.1 fused to an epitope tag derived from SV-40 large T antigen. (A) The construct was bacterially expressed,
isolated, and then analyzed by Western blotting to confirm the presence of both 4.1 and SV-40 tag epitopes. Both 24-2 IgG (lane 1) and
KT3 antibody (against the epitope tag; lane 2) recognized a protein with the same apparent molecular mass. The KT3 antibody did not
recognize epitopes in a whole cell lysate of 3T3 cells (lane 3). (B) Murine fibroblast 3T3 cells, probed with anti-RBC 80-kD 4.1, displayed punctate nuclear immunofluorescent signals. (C and C) 3T3 cells, transiently transfected with pSV40NeoCMV containing sequences encoding epitope-tagged RBC 80-kD 4.1, strongly expressed epitope-tagged protein localized in nuclei, which was detected by
indirect immunofluorescence using KT3 antibody. (D and D
) After parallel transient transfection of 3T3 cells with pSV40NeoCMV
without a construct inserted, there was no immunofluorescent staining with KT3 antibody. Bar: (B and B
) 12 µm; (C-D
) 20 µm.
[View Larger Versions of these Images (41 + 59 + 25K GIF file)]
). This observation was confirmed by a doublelabel experiment in which nucleoplasm, highlighted by
rhodamine with an antibody probe against a generally distributed nuclear matrix protein NuMA (nuclear mitotic
apparatus protein; Lydersen and Pettijohn, 1980
; Compton et al., 1992
; Yang et al., 1992
), was imaged relative to
4.1 epitopes detected by FITC labeling (Fig. 5 D). The
confocal optical vertical sections show 4.1 epitopes to be
scattered throughout the nucleoplasm (Fig. 5 D
).
) and relative to proliferating cell nuclear antigen (PCNA), an accessory protein for the replicative DNA
polymerase delta (Tan et al., 1986
; Bravo et al., 1987
; Prelich et al., 1987
) (Fig. 5, E-H). Although some coincident
fluorescent signals (denoted by pseudo yellow coloration)
were apparent in confocal horizontal planes through fibroblast nuclei stained for 4.1 (probed with FITC) relative to
either SC-35 or to PCNA (each probed with rhodaminelabeled secondary antibody), each protein was also detected alone. This is consistent with previous observations
that splicing and replication centers do not coincide with
each other (Leonhardt et al., 1992
). Protein 4.1 foci were
not detected in areas occupied by nucleoli, seen as dark
round "holes" within the nucleus (Fig. 5 E). These data
show 4.1 epitopes to be generally distributed throughout nonnucleolar nuclear domains involved in RNA and DNA
metabolism. Taken together, it is clear from the data in
Fig. 5 that 4.1 foci are intranuclear but do not localize predominantly at the nuclear membrane.
; Fey et al.,
1986
). Fibroblasts and CaSki cells retained epitopes recognized by 80-kD RBC 4.1, 10-1, 24-2, and 24-3 antibodies when monitored by immunofluorescence during each stage
of nuclear matrix preparation. When analyzed by threedimensional optical reconstructions, all extraction steps
before nuclease digestion displayed nuclear foci at multiple planes. After completion of the matrix preparation,
there was no detectable staining with DAPI, attesting to
the near complete removal of chromatin. In contrast, immunofluorescent foci of 4.1 were retained in the matrix preparation. Microscopic optical sectioning of these samples showed a collapsed nucleus so that, in contrast to previous extraction steps, all nuclear 4.1 epitopes resided on a
single plane.
; Nickerson and
Penman, 1992
; Nickerson et al., 1994
). Samples were sequentially extracted, lightly fixed, and incubated with primary IgGs against 4.1 peptides or normal rabbit IgG. A
secondary antibody coupled to gold beads was added, and
the preparation was fixed again and then embedded and
sectioned. After removal of the temporary embedding material, epitopes for 4.1 were detected within the residual
nuclear matrix structures visualized by electron microscopy (Fig. 6), consistent with the results obtained with immunofluorescence assays run in parallel samples. At the
higher resolution of electron microscopy, the 4.1 epitopes
retained in nuclear matrix preparations were associated
with dense bodies and not with the supporting filamentous network. Parallel samples incubated with control IgG did
not show labeling of nuclear structures.
Fig. 6.
Immunolocalization of protein 4.1 in nuclear matrix visualized by high-resolution resinless electron microscopy. CaSki cells were extracted with 0.5% Triton X-100 in CSK buffer, chromatin was removed by HaeIII and PstI digestion, and proteins were extracted with 0.25 M ammonium sulfate followed by extraction with 2 M NaCl. After fixation, the matrix preparation was incubated with
protein 4.1 antibody 24-3 (A) and 24-2 (B) and then with colloidal gold-coupled secondary antibody. The EM micrograph of a resinless
section shows a network of nuclear matrix core filaments (CF) and dense bodies (DB). The 10-nm gold beads decorated principally the
periphery and some internal areas of the dense bodies (arrowheads). This localization pattern at dense bodies in the matrix was also observed in a parallel experiment using anti-10-1 IgG but not with control IgG. Bar, 100 nm.
[View Larger Version of this Image (141K GIF file)]
Fig. 7.
Analysis of fibroblast
nuclear matrix 4.1 protein by
Western blot and 4.1 mRNA by
reverse transcriptase-PCR. (A,
lanes 1-6) Nuclear matrix proteins isolated from 3 × 106 WI38
nuclei were separated by SDSPAGE, transferred to nitrocellulose, and incubated with the
following 4.1 antibodies: lane 1,
RBC 80-kD 4.1; lane 2, N-2; lane
3, 10-1; lane 4, 24-2; lane 5, 24-3;
lane 6, control IgG. To test the
relative separation of nuclear
matrix proteins from soluble cytoplasmic proteins, a cytoplasmic
fraction from 2 × 105 cells was
electrophoresed in parallel with
a sample of nuclear matrix from
6 × 105 cells derived from the
same fibroblast preparation and
probed after transfer with an antibody against the cytoplasmic protein -tubulin. While
-tubulin was abundant in the cytoplasmic fraction (lane 7), no protein band with a similar migration could be detected in the nuclear matrix fraction (lane 8) even after long exposure times. An antibody against lamin B used to probe fibroblast nuclear matrix proteins produced a band at the appropriate position, verifying the presence of a predicted nuclear matrix protein (lane 9). (B) PCR analysis of protein 4.1 mRNA containing AUG-1 in WI38 fibroblasts. The products of reverse transcriptase-PCR using primers encompassing AUG-1 were analyzed on a polyacrylamide gel. The
following cDNAs were amplified: lane 1, WI38 cells; lane 2, human reticulocytes; lane 3, no DNA. The sizes of molecular weight standards are: 1353, 1078, 872, 603, 310, 234, and 194 bp.
[View Larger Versions of these Images (50 + 35K GIF file)]
-tubulin (Fig.
7 A, lanes 7 and 8);
-tubulin could be detected only in the
cytoplasmic fraction (Fig. 7 A, lane 7) and not in the lane containing nuclear matrix (Fig. 7 A, lane 8).
; Tang et al., 1988
,
1990) fused to an epitope tag was transiently transfected
into murine fibroblast 3T3 cells. Before transfection, we
determined that the protein expressed by the epitopetagged construct was recognized on Western blot analysis
both by antibodies against 4.1 (24-2 IgG, Fig. 8 A, lane 1;
10-1 IgG, data not shown) and KT3 antibody directed against the epitope tag (Fig. 8 A, lane 2). As expected, the
KT3 antibody did not react with proteins in extracts of
the recipient host fibroblasts (Fig. 8 A, lane 3). To confirm
the presence of endogenous nuclear 4.1 in murine fibroblasts, 3T3 cells were probed with anti-RBC 80-kD 4.1. The 3T3 nuclei displayed punctate immunofluorescent 4.1 signals (Fig. 8 B) similar in pattern to diploid human fibroblasts (Fig. 3). After transient transfection with the vector
encoding the 4.1-tagged construct, expressed protein bearing the epitope tag produced a very strong immunofluorescent signal in the nuclei of the 3T3 cells (Fig. 8, C and
C
) when probed with KT3 antibody. Of note, in parallel
experiments, no immunofluorescent signal was detected
when cells were transfected with the vector not containing the construct (Fig. 8, D and D
) or in mock (i.e., no vector
DNA) transfected cells (data not shown).
). B4A11 epitopes
produce a strong speckled staining pattern in interphase
nuclei that almost completely disappears in detergentextracted cells undergoing mitosis (Blencowe et al., 1994
);
cells undergoing mitosis were thus identifiable by the loss
of B4A11 epitopes along with altered morphology.
Discussion
ends generated by
alternative transcription initiation localize either to peripheral membranes or to perinuclear regions (Cox et al.,
1995
). Similarly, two
-spectrin isoforms having different
carboxy termini appear to interact with different binding
partners and exhibit different cellular localization (Malchiodi-Albedi et al., 1993
). Alternative splicing of CaM kinase generates a nuclear localization signal that targets a
specific isoform of this multifunctional protein to the nucleus (Srinivasan et al., 1994
). Alternative splicing of WT1
determines if a WT1 isoform associates within the nucleus
mainly with RNA splicing factors or with DNA in transcription factor domains (Larsson et al., 1995
).
). Isoforms of protein 4.1 may contribute to nuclear structure. The nucleus houses potential
binding partners for 4.1, such as spectrin (Bachs et al.,
1990
; Beck et al., 1994
), actin (Nakayasu and Ueda, 1986
;
Milankov and DeBoni, 1993
; Chaly, N., and X. Chen. 1995. Mol. Biol. Cell. 6:423a), myosin (Berrios and Fisher, 1986
;
Hagen et al., 1986
; Milankov and DeBoni, 1993
), and calmodulin (Bachs et al., 1990
). Moreover, protein 4.1 is known
to be glycosylated and phosphorylated in vivo, posttranslational modifications used by nuclear proteins to modulate
interactions during the cell cycle.
). This potential association, however, is not exclusive: 4.1 foci are detected at lower and upper regions of the
nucleus and some 4.1 signals are noncoincident even
within the spliceosomal plane. Taken together, these data
may represent a composite of several functionally distinct
4.1 protein isoforms or could suggest a more unified role
for protein 4.1 at interfaces between the nucleoskeleton
and subnuclear assemblies such as centers for splicing or
DNA replication. However, many nuclear subdomains, including DNA replication foci, change in size and distribution during S phase (D'Andrea et al., 1983
; Goldman et al.,
1984
; Hatton et al., 1988
; O'Keefe et al., 1992
), and it is not
known whether protein 4.1 associations are static or dynamic during interphase. Therefore, further studies using
tightly synchronized cell cultures and specific inhibitors
will be required to quantitate the spatial associations of 4.1 with these key nuclear functional domains.
); we observed
4.1 epitopes near dense bodies rather than associated with
the surrounding filaments. The biochemical composition
of these dense bodies within the matrix is not entirely understood. Some contain various combinations of RNA
splicing factors corresponding to speckles seen in immunofluorescence but others appear to be related to centers
of transcription and DNA replication, coiled bodies,
RNA-protein complexes, and nucleolar remnants (for review see Nickerson et al., 1995
). It remains to be established if 4.1 localization is specific for subclasses of dense
bodies or is more generally associated with a variety of nuclear substructures.
; Subrahmanyam et al., 1991
) and/or glycosylation (Haltiwanger et al., 1990
). Some additional variation may
be generated by expression of alternative NH2 or COOH
termini not yet characterized. For example, the lack of significant reaction of protein 4.1 antibody 24-3 with the
~85-kD band suggests that the sequence of COOH termini may vary. Similarly, the N-2 IgG prepared against the
209-amino acid NH2-terminal extension of 80-kD 4.1 detected only a single band in the 80-100 kD region with the
slowest migration (~100 kD), indicating that apparently
not all matrix polypeptides have identical NH2 termini.
). In sum, our final matrix fraction
contains multiple 4.1 isoforms, some with distinct immunoreactive patterns. This suggests that 4.1 polypeptides
have differing peptide domains or modifications that may
enable unique molecular interactions in the nucleus.
).
Some nuclear structural proteins, such as lamin, even migrate into the cytoplasm to be later reimported into newly
formed nuclei. The 4.1 pattern appears to be a unique hybrid of several patterns reported for specific matrix-associated proteins (for review see He et al., 1995
).
Received for publication 24 July 1996 and in revised form 20 January 1997.
1. Abbreviations used in this paper: CMV, cytomegalovirus; DAPI, 4This work was supported by grant DK 32094 from the National Institutes of Health.