Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Federal Republic of Germany
Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of Mr ~270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515-1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/ Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC- attached filaments and discuss its possible functions.
Nuclear pore complexes (NPCs)1 are constitutive
structures of the nuclear envelope in all eukaryotic cells and represent gateways between the cytoplasm and the nucleus through which the exchange and
bidirectional transport of molecules and particles takes place (for reviews see, e.g., Rout and Wente, 1994 Attached to the cytoplasmic annular rim of the NPC are
short (25-50-nm) filaments that contain not only the initial
binding sites for transport factors involved in nuclear protein import (Richardson et al., 1988 At the other, nucleoplasmic side, bundles of filaments of
~5 nm diam appear to be anchored at the NPC and often
extend into the nuclear interior for various lengths, in amphibian oocytes for >300 nm. These NPC-attached nuclear filament bundles are symmetrically arranged and appear to form a cylinder (Franke, 1970 In the course of our attempts to elucidate the composition and assembly of the NPC-attached intranuclear filament bundles, we have identified in amphibians and mammals a protein component of ~270 kD molecular mass
that turned out to be protein Tpr, localized exclusively and
constitutively to these intranuclear filaments, much to our
surprise and at variance with previous reports (Byrd et al.,
1994 Cell Cultures
Culture conditions for cells of human lines HeLa, glioma U333/CG/343
MG, PLC, A-431, CaCo2, HF-SV80, MCF-7, and HaCaT; for bovine lines
BMGE+H clone 5 and MDBK; for canine MDCK, porcine PK(15), African green monkey RC37, marsupial PtK2, and rodent RV; and for primary
cultures of human umbilical cord endothelial cells have been described in
detail (Cordes et al., 1996 Tissues
Tissues were frozen in isopentane cooled with liquid nitrogen to Antibodies
mAb 203-37 (Matritech, Cambridge, MA) against the nuclear matrix fraction from cultured cells of breast cancer line T-47D (see, however, Bangs
et al., 1996 To raise antibodies against synthetic peptides of human and Xenopus
Tpr, putative antigenic sites of this protein with high degrees of surface
probability were determined by the algorithms of Jameson and Wolf
(1988) Cell Fractionation
During the entire fractionation procedure, cultured cells grown to near
confluency (70-80%) remained in the petri dishes (10 cm diam). After
briefly washing the cells twice with hand-warm PBS containing 2 mM
MgCl2 and once with MOPS buffer (50 mM MOPS-NaOH, pH 7.0, 1 mM
EGTA, 5 mM MgCl2, and 0.75% vol/vol of saturated PMSF in ethanol) at
room temperature, all subsequent steps were performed at ~4°C using
ice-cold solutions. Solutions pipetted onto the cells were recollected after
each fractionation and washing step and were immediately centrifuged at
4°C for 4 min at 13,000 g. The resulting supernatants were combined with
4 vol of ice-cold methanol, and proteins were allowed to precipitate overnight at The manual isolation of stage VI nuclei from Xenopus laevis oocytes
has been described elsewhere (Cordes et al., 1995 Gel Electrophoresis and Immunoblotting
SDS-PAGE of proteins was by the method of Thomas and Kornberg
(1975) Isolation of cDNA Clones and PCR Products
The monoclonal IgG1 antibody 203-37, the antigen of which has not been
known by the producer (Matritech) at the beginning of this study, was
used to screen 1.2 × 106 plaques of a human fetal brain UNI-ZAPTM XR
cDNA library (Stratagene, La Jolla, CA). After plaque transfer onto isopropylthio- PCR was performed using a Xenopus laevis kidney UNI-ZAPTM XR
cDNA library (Stratagene) as template. PCR was carried out in 50 µl total
vol containing 320 ng DNA template, 10 pmol of each sense and antisense
primers, and 2 U Taq polymerase per reaction. Cycling conditions were as
follows: (1) cycle: 5 min at 95°C; 1 min at 55°C; 2 min at 72°C. (2-34) cycle:
2 min at 92°C; 1 min at 55°C; 2 min at 72°C. The end of cycle 34 was followed by 10 min at 72°C. Sense primers were initially deduced from a
GenBank sequence (accession number U12646) that had been entered as
a noncoding region of an unpublished Xenopus laevis integrin variant. Sequence comparison with human protein Tpr, however, indicated that this
DNA segment encoded partial aa sequences of the Xenopus Tpr protein;
all three forward frames of the entered DNA sequence encoded short aa
sequence stretches with apparent homology to human protein Tpr. As antisense primer for the first set of PCRs performed, the T7 primer (Stratagene) was used; its binding site on the Uni-ZAPTM XR vector flanks the
unidirectionally cloned cDNA inserts at their 3 In Vitro Translation and Immunoprecipitation
In vitro transcription-translation of the human Tpr protein cDNA clone
T1 was performed at 30°C for 90 min in the presence of [35S]methionine,
using the coupled reticulocyte lysate system and T3 RNA polymerase
(Promega, Heidelberg, FRG).
For immunoprecipitation, 25 µl of PBS-washed, preswollen protein
G-Sepharose was suspended in 200 µl PBS containing 10 µl mAb 203-37, then mixed for 1 h by rotation at 4°C, and subsequently washed four
times with 1.5 ml PBS. In parallel, 25 µl reticulocyte lysate containing the
[35S]labeled in vitro translation products was diluted with 200 µl PBS
and mixed for 1 h by rotation at 4°C with another 25-µl aliquot of PBSwashed, preswollen protein G-Sepharose; the beads were then pelleted by
centrifugation. The resulting reticulocyte lysate supernatant, containing
the [35S]labeled Tpr polypeptides, was transferred to the aliquot of protein
G-Sepharose with mAb 203-37, whereas proteins unspecifically bound to
the beads after two washings with PBS were released by boiling in SDS
sample buffer and later analyzed by SDS-PAGE for control. Protein
G-Sepharose with mAb 203-37 and reticulocyte lysate supernatant were
mixed by rotation for 1 h at 4°C; the beads were then pelleted and washed
five times in PBS. Bound proteins were released by boiling in SDS sample
buffer and analyzed by SDS-PAGE.
DNA Transfection
The entire cDNA fragment of clone T1 representing mRNA encoding
full-length human protein Tpr was isolated by NotI/ApaI or NotI/Bsp102I
digestion and subcloned into eukaryotic expression vector pRC/CMV containing a cytomegalovirus (CMV) promoter (Invitrogen, San Diego, CA)
and expression vector p163/7, containing a major histocompatibility complex (MHC) class I H-2 promoter. p163/7 is identical to p164/7 (Niehrs et al.,
1992 Immunofluorescence Microscopy
Tissue cryostat sections were fixed either in acetone or with formaldehyde
before incubation with antibodies (Cordes et al., 1995 Immunolocalization by EM
Tissue cryostat sections (5 or 10 µm in thickness) were either fixed with
2% formaldehyde, followed by washes in PBS containing 50 mM NH4Cl, or
by treatment with Manually isolated nuclei of Xenopus oocytes isolated in 5:1 buffer (Cordes
et al., 1995 Identification of a 270-kD Mammalian
Protein as a Novel Component of NPC-attached
Intranuclear Filaments
mAb 203-37 showed in immunofluorescence microscopy
of cultured cells, permeabilized either by methanol/acetone
treatment or by use of Triton X-100 after initial formaldehyde fixation, a punctate staining in the nuclear periphery,
similar to that obtained by immunostainings of NPCs. Positive cells included diverse human cell types differing in
differentiation such as cervix adenocarcinoma cells of line
HeLa (Fig. 1, a and a
When cells were permeabilized with 0.004% digitonin,
striking differences of nuclear labeling were noticed when
comparing mAb 203-37 with antibodies against RanBP2/
Nup358, a nucleoporin located at the cytoplasmic side of
the NPC. While RanBP2 antibodies showed nuclear labeling in all cells, indicating full accessibility of this protein,
mAb 203-37 stained only nuclei of some cells while others
were negative (not shown). Moreover, double-label immunofluorescence microscopy using antibodies against nucleolar and lamin proteins further showed that the antigen
recognized by mAb 203-37 is accessible only in a specific
proportion of nuclei in which intranuclear structures such
as the lamina and the nucleolus were also labeled. This indicated that the mAb 203-37 antigen is also intranuclear.
Immunofluorescence microscopy performed on cryostat
sections of human and bovine tissues confirmed immunolabeling in the nuclear periphery with mAb 203-37 in all
tissues examined, including human esophagus (Fig. 1 d),
testis (Fig. 1 d, inset), ovary, liver, skin, smooth muscles,
cerebrum, fetal cerebellum, erythroblastoma, and colon
carcinoma (not shown), as well as bovine liver (Fig. 1 e),
testis, and thymus (not shown). In most tissues, we observed occasional intranuclear dot reactions in addition to
the nuclear rim staining (an example is shown in Fig. 1 d,
inset).
To identify the antigen recognized by mAb 203-37, we
performed immunoblotting on total cell proteins of HeLa
cells and identified a protein of Mr ~270 kD (Fig. 2, a and
a
To localize p270 at the EM level, we examined its distribution by preembedding immunogold localization using
mAb 203-37 and 5-nm gold-coupled secondary antibodies
on cryostat sections of human and bovine tissues and on
monolayers of detergent-permeabilized human PLC cells
(Fig. 3, a-f). Specific labeling was found on intranuclear
filamentous structures that appeared to be attached to
NPCs. In some sections, such gold particle-decorated filament bundles projected into the nuclear interior for up to
200 nm (see, e.g., Fig. 3, b and c). Double immunogold labeling with antibodies against protein RanBP2/Nup358
(10-nm gold-coupled secondary antibodies) and mAb 20337 (5-nm gold) showed that both margins of the NPC were
accessible, and thus confirmed the specificity of the intranuclear localization of p270 (Fig. 3, g-j). Besides this labeling of NPC-attached intranuclear filaments, we occasionally also noticed specific mAb 203-37 labeling of unknown
intranuclear spheroidal structures ~40-200 nm in diam
that were located at greater distances from the nuclear envelope (see below).
cDNA Clones Encoding p270 Reveal Sequence Identity
to Protein Tpr
Using mAb 203-37, we screened a human fetal brain expression library and isolated a total of six immunoreactive
clones (T1-T6) that, on DNA sequencing, were recognized to encode protein Tpr (Fig. 4 a) recently localized
exclusively to the cytoplasmic margin of NPCs (Byrd et al.,
1994
In vitro transcription-translation of cDNA clone T1 using the reticulocyte lysate system yielded a product of
~270 kD (Fig. 4 c) that was indistinguishable in mobility
from p270 identified by immunoblotting of HeLa cell proteins and was specifically immunoprecipitated by mAb
203-37 (Fig. 4 d), indicating that p270 and at least the human fetal brain variant of Tpr represent the same antigen.
Expression of the human fetal brain tpr gene under control of a CMV promoter in African green monkey cells
(Fig. 4, e-g Antibodies against Different Epitopes of Protein Tpr
React with p270
To exclude that mAb 203-37 immunoreacts only with a
distinct Tpr variant located at the nucleoplasmic side of
the NPC but not with the Tpr protein that had been localized to the cytoplasmic side of the NPC by Byrd et al.
(1994)
Antibodies against mammalian p270/Tpr either did not
react with the Xenopus laevis Tpr orthologue or exhibited
additional unspecific cross-reactions with other Xenopus
proteins (not shown). We therefore also raised rabbit antibodies against the Xenopus Tpr protein. To this end, we
first isolated a series of overlapping PCR products using a
Xenopus kidney cDNA library as template and from
which assembled a partial DNA sequence of 1,811 bp that
encoded the 558 carboxy-terminal aa of the Xenopus Tpr
protein (Fig. 6 a). This sequence exhibited a high degree of
homology (70% identity) to the human sequence, including uninterrupted stretches of 27-45 identical aa. For immunization, a peptide located near the carboxy terminus
(Fig. 6 b) was chosen, yielding antibodies that specifically reacted with the Xenopus p270/Tpr protein on total proteins of Xenopus kidney epithelial cells of line A6 and of
manually isolated oocyte nuclei (Fig. 6, c and c
Antibodies against Human and Xenopus p270/Tpr
Label NPC-attached Intranuclear Filaments
Immunofluorescence microscopy using the human p270/
Tpr peptide antibodies on cultured mammalian cells and
on cryostat sections of mammalian tissues (liver, esophagus, epidermis, and colon) yielded results (some are shown
in Fig. 7, a-d) that were essentially indistinguishable from
those obtained with mAb 203-37 (compare with Fig. 1) but
revealed broader cross-reactivity between the various mammalian species, including rodents and African green monkey cells (not shown). The antibodies against the Xenopus
p270/Tpr sequence equally resulted in punctate staining of
the nuclear periphery of cultured Xenopus cells with some
occasional intranuclear speckles (Fig. 7, e-g In contrast with several other NPC proteins, p270/Tpr
was not detected in the pore complex-containing cytoplasmic annulate lamellae as revealed by double immunofluorescence microscopy on different mammalian and amphibian cells (Fig. 8), using the above described antibodies
against p270/Tpr and such against NPC proteins that have
been demonstrated to represent marker molecules for annulate lamellae (Cordes et al., 1995
Immunoelectron microscopy using preembedding immunogold labeling (secondary antibodies coupled to 5-nm
colloidal gold) revealed identical results on human liver
sections with antibodies against human Tpr peptides Nos.
1 and 2. Both antibodies specifically labeled the NPC-
attached intranuclear filaments, whereas the cytoplasmic
side of the NPC was devoid of gold particles (Fig. 9, a-d).
Essentially identical results were obtained on cryostat sections of human epidermis (not shown). As already noticed
by immunoelectron microscopy using mAb 203-37 (see
above), we occasionally also noticed specific immunolabeling of yet unidentified small intranuclear spheroidal
structures (Fig. 9 e). To control for the accessibility of both
sides of the NPC, double immunogold label localizations
with antibodies against RanBP2/Nup358 (10-nm gold-
coupled secondary antibodies) and p270/Tpr (5-nm gold)
were performed. The larger RanBP2 immunogold particles were seen almost exclusively at the outer NPC annulus (Fig. 9, f-h), thus confirming the localizations of others
(Wu et al., 1995
Immunogold labeling on manually isolated stage VI oocytes of Xenopus laevis, using rabbit antibodies against
peptide No. 3 of the Xenopus p270/Tpr protein and secondary antibodies coupled to 5-nm gold particles (Fig. 10,
a-d), resulted in a very dense and specific decoration of
the NPC-attached intranuclear filament bundles, whereas
both the outer aspect of the NPC and the pore complexes
of annulate lamellae were free of immunogold label. In
some micrographs with structurally well-preserved filament bundles, the gold-decorated filaments projected into
the nuclear interior for up to 350 nm. Double label immunogold localization performed as control on manually isolated oocytes (Fig. 10, e-j) and on cryostat sections through
Xenopus ovaries (Fig. 10 k), using antibodies against
RanBP2/Nup358 (10-nm gold) and p270/Tpr (5-nm gold),
showed the localization of RanBP2 at both the cytoplasmic margin of the NPCs and the cytoplasmic pore complexes of annulate lamellae (Fig. 10 j; see also Cordes et
al., 1996
We have identified a ~270-kD protein (p270) as a novel
and constitutive component of the NPC-attached intranuclear filaments in diverse kinds of mammalian and amphibian cells. Analysis of cDNAs encoding human p270
and immunobiochemistry have then revealed that p270
corresponds to the previously described human Tpr protein (Mitchell and Cooper, 1992a The amphibian oocyte (germinal vesicle) represents a
particularly suitable "classic" system to study structural aspects of the NPC and its attached filaments. Therefore, we
have isolated PCR products encoding the Xenopus p270/
Tpr protein, raised antibodies to it, and performed immunoelectron microscopy on mature oocytes of Xenopus laevis. The results have essentially confirmed those obtained
with mammalian cells: immunogold decorates intensely and
specifically the NPC-attached intranuclear filament bundles, whereas the cytoplasmic side of the NPC does not react. In these cells, the NPC-attached intranuclear bundles
are particularly well developed (see also Franke and
Scheer, 1970a In addition to the immunoreactive NPC-attached filaments, we have also noticed, in a minor proportion of cells,
immunolabeling of unknown small spheroidal intranuclear
structures that sometimes surround nucleoli. Antibodies
reactive with other spheroidal intranuclear marker structures all have been negative on these "Tpr-positive speckles" (data not shown), so that these structures could not be
related to any of the known kinds of "nuclear bodies" (for examples and references see, e.g., Bohmann et al., 1995 From the intense decoration of mammalian and amphibian NPC-attached intranuclear filaments with antibodies against near carboxy-terminal epitopes of protein
p270/Tpr, we conclude that in situ, i.e., in the actual filaments, all these epitopes are exposed and readily accessible, and thus probably located on the outer aspect of the filamentous cylinders. Sequence comparison between human and amphibian p270/Tpr shows that the carboxy-terminal domain is highly conserved and rich in acidic aa, and
therefore a reasonable candidate for interactions with basic proteins known to occur in chromatin as well as in ribonucleoprotein particles. Experiments to identify complexes
of p270/Tpr with other nuclear proteins and to examine its
possible involvement in filament-guided nucleocytoplasmic transport processes (see also the "tracks" of Blobel,
1985 The amino-terminal domain of the p270/Tpr protein contains numerous leucine zipper motifs and regions of heptad repeats typical of ; Davis, 1995
;
Pante and Aebi, 1995b
). These structures are large assemblies (110-145 MDa in vertebrates) of globular and fibrillar substructures arranged in an eightfold rotational symmetry (Franke, 1966
, 1970
; Gall, 1967
; Reichelt et al., 1990
;
Hinshaw et al., 1992
; Akey and Rademacher, 1993
; for review see Pante and Aebi, 1995a
) and estimated to be composed of ~60-100 different proteins. In vertebrates, NPC
proteins molecularly characterized include the transmembrane proteins gp210 (Gerace et al., 1982
; Wozniak et al.,
1989
) and Pom 121 (Hallberg et al., 1993
) and at least ten
nonmembranous "nucleoporins," some of which represent
components of NPC-associated filamentous structures (for
recent review see Bastos et al., 1995
).
; Newmeyer and
Forbes, 1988
; Melchior et al., 1995
; Wu et al., 1995
; for reviews see, e.g., Sweet and Gerace, 1995
; Melchior and
Gerace, 1995
; Simos and Hurt, 1995
; Pante and Aebi,
1996
) but also intracellular "docking" sites for certain DNA
viruses (e.g., Summers, 1971
; Chardonnet and Dales, 1972
). Structural components of these NPC-attached cytoplasmic filaments include the putative oncoprotein CAN/
Nup 214 (Kraemer et al., 1994
) and the recently identified
Ran protein-binding nucleoporin RanBP2/Nup358 (Yokoyama et al., 1995
; Wu et al., 1995
; Wilken et al., 1995
). Protein Tpr (translocated promotor region), whose gene has
been implicated in the activation of the protooncogenes met, raf, and trk (Park et al., 1986
; Soman et al., 1991
;
Greco et al., 1992
), has also been reported to be exclusively located in the NPC-attached cytoplasmic fibrils
(Byrd et al., 1994
) and is considered to contribute to the
formation of these filaments.
; Franke and Scheer,
1970a
,b; Kartenbeck et al., 1971
; Richardson et al., 1988
; Ris and Malecki, 1993
) built on a specific NPC-proximal
filamentous array only ~30-70 nm in length that has been
described as a "fish trap," "cage," or "basket" (Ris, 1989
,
1991
; Jarnik and Aebi, 1991
; Goldberg and Allen, 1992
).
The functions of both the long and the short (fish trap) intranuclear filament arrays are unknown. Proteins localized
to the nucleoplasmic side of the NPC at distances either
more proximal or more distal to the inner annulus include p97/Nup98 (Radu et al., 1995
; Powers et al., 1995
) and
Nup153 (Sukegawa and Blobel, 1993
; see also Cordes et al.,
1993
; Pante et al., 1994
). Of these, protein Nup153 has
been reported to occur in both the long and the short intranuclear filaments (Cordes et al., 1993
; Pante et al., 1994
).
; Bangs et al., 1996
).
Materials and Methods
). Murine fibroblasts of lines L-M(TK
) (American Type Culture Collection code ATCC CCL 13) and 3T3-L1 (ATCC
CCL 92.1) were grown in DME (ICN Biomedicals, Costa Mesa, CA) supplemented with 10% FCS, and rat hepatoma cells of line Faza 967-D2
(Venetianer et al., 1983
) were grown in Ham's F12 medium (Boehringer
Mannheim GmbH, Mannheim, FRG). Xenopus laevis kidney epithelial
cells of line A6 (ATCC CCL 102) were grown at 28°C in DME with 10%
FCS and diluted with 15% H2O.
140°C,
and then stored at
70°C. Human tissues (cerebrum, fetal cerebellum, colon and colon carcinoma, epidermis, erythroblastoma, liver, esophagus,
ovary, smooth muscle, and testis) were kindly provided by Dr. Roland
Moll (Department of Pathology, Martin Luther-University, Halle-Wittenberg, FRG). Bovine tissues (liver, testis, calf thymus) were obtained from
the slaughter house. South African clawed frogs (Xenopus laevis) were
bred in this institute, and pieces of skin, ovary, and liver were obtained by
surgery as described (Cordes et al., 1995
).
for a similar mAb) and mAb 414 (BAbCO, Richmond, CA)
against the XFXFG motif-containing domain common to some nucleoporins
(Davis and Blobel, 1986
; Sukegawa and Blobel, 1993
) have been obtained
commercially. mAb X167 against lamins A/C (Höger et al., 1991
), rabbit
and guinea pig antibodies to RanBP2/Nup358 (rb-
RBP2-2, gp-
RBP2-2;
Cordes et al., 1996
), and guinea pig antibodies against nucleolar protein
NO38 were from this institute (kindly provided by Dr. Marion SchmidtZachmann, German Cancer Research Center, Heidelberg). Guinea pig
antibodies to lamin A were a gift of Dr. Georg Krohne (University of
Würzburg, FRG).
and Emini et al. (1985)
and controlled for low homology with other
proteins by sequence database searches. Peptides were synthesized by
t-Boc chemistry (Schnölzer et al., 1992
) and coupled via a carboxy-terminal cysteine residue to maleimide-activated keyhole limpet hemocyanin
(KLH; 1 mg peptide/1 mg KLH), using the Imject Activated Immunogen
Conjugation Kit (Pierce Europe, Oud-Beijerland, The Netherlands). Human Tpr peptides correspond to amino acids (aa) 1,622-1,640 (hTpr-Pep1:
ERHLEQRDEPQEPSNKVPE) and aa 2,063-2,084 (hTpr-Pep2:
SERQAPRAPQSPRRPPHPLPPR) of the 2,363-aa Tpr sequence presented below. The Xenopus Tpr peptide (QHFFDEEDRTVPSTPT), deduced from the partial DNA sequence (see below), is homologous to aa
2,124-2,139 of human Tpr. For the first injection, the KLH-coupled peptides were emulsified with an equal volume of complete Freund's adjuvant
and, for the booster injections, with incomplete adjuvant. Guinea pigs
(hTpr-Pep1 and 2; xlTpr-Pep3) and rabbits (hTpr-Pep2; xlTpr-Pep3) were
immunized four times subcutaneously with 70-100 µg protein per injection. Booster injections were at days 14 (21), 35 (42), and 70. Guinea pigs
were killed at ~day 82. Rabbits were held for longer periods and occasionally reboostered. Blood was collected in vacutainer SST-tubes (Becton-Dickinson, Heidelberg, FRG). For affinity chromatography, peptides
containing the antigenic determinants were coupled to Ultra Link Iodoacetyl on 3 M Emphaze Biosupport Medium AB1 (Pierce Europe). The
peptide-resin was first mixed with 1% BSA in PBS by rotation for 30 min,
washed with 20 vol of PBS, and then mixed for 90 min at 4°C with 10 vol of
sera, diluted 1:3 in PBS. After washings with 20 vol each of PBS and 10 mM sodium phosphate (pH 6.8), antibodies were eluted in fractions of 400 µl with 100 mM glycine-HCl (pH 2.3) and immediately neutralized with
~0.04 vol of 1 M Tris-HCl (pH 9.5). Purified antibodies, supplemented
with an equal volume of glycerol or 0.25 vol PBS with 20% BSA, and 0.03% NaN3, were stored at
70°C.
20°C. Cells were first extracted in 2 ml MOPS buffer containing 0.2% Triton X-100 for 5 min by mildly and briefly shaking the petri dishes
every 30-60 s, briefly washed with 2 ml MOPS buffer, and then extracted
for 5 min in 2 ml MOPS containing 0.5 M NaCl, again followed by a brief
washing with 2 ml MOPS. The residual cyto- and karyoskeletons that remained attached to the dishes were immediately scraped off in 0.5 ml of
90°C SDS sample buffer and boiled. For digitonin permeabilization, cells
on dishes were incubated for 5-10 min in 2 ml ice-cold PBS containing
0.005% digitonin without protease inhibitors, and then washed with 2 ml
PBS. Soluble proteins in the digitonin and washing solutions were combined, cleared by centrifugation, and precipitated from the supernatant
with methanol. The permeabilized cells were boiled in SDS sample buffer.
).
. For two-dimensional gel electrophoresis, IEF (O'Farrell, 1975
)
was used in the first dimension, and SDS-PAGE in the second (Laemmli,
1970
). After electroblotting of proteins onto nitrocellulose, the filters
were preincubated for at least 2 h in Tris-buffered saline containing 0.05%
Tween-20 (TBST) and 5% milk powder (TBST-milk). Incubation with
primary antibodies was for 2 h. Filter washings were usually performed
with TBST. In some experiments, more stringent washing conditions were
applied, using PBS containing 0.1% Triton X-100 and PBS with additional
0.4 M NaCl, i.e., the same stringency conditions as later applied for cDNA
library screening (see below). Bound antibodies were detected by HRPcoupled secondary antibodies (Dianova, Hamburg, FRG) applying the
enhanced chemiluminescence (ECL) method (Amersham, Braunschweig,
FRG). To remove bound antibodies, filters were shaken for 3 × 30 min in
0.1 M glycine-HCl (pH 2.6) containing 20 mM magnesium acetate and 50 mM
KCl. Filters were then washed in TBST and shaken in TBST-milk for 1 h
before reincubation with antibodies.
-d-galactosidase-treated nitrocellulose, filters were washed
in TBST and saturated in TBST-milk. Incubation with mAb 203-37 in
TBST-milk was followed by washings in TBST, PBS with 0.1% Triton
X-100, and PBS with additional 0.4 M NaCl. Filters were then incubated
with HRP-coupled secondary antibodies, again followed by stringent
washings. Immunoreactive clones were identified by ECL. Subcloning of
pBluescript phagemids by in vivo excision was performed as described in
the Stratagene protocol.
-end. Other primers were
also deduced from PCR product sequences (see below). PCR assays were
generally performed without using PCR-amplified DNA from other reactions as intermediate template from which to proceed. PCR products corresponding to a given DNA segment were obtained in at least two independent reactions and sequenced. Several overlapping DNA fragments
were assembled into a DNA sequence of 1,811 bp that encoded 558 aa of
the Xenopus Tpr protein. Corresponding to this nucleotide numbering,
primer positions were as follows: 1-18, 937-957, 1137-1117, 1164-1146. Dideoxy sequencing of cDNAs and PCR products was according to Sanger
et al. (1977)
.
) with the exception of an inverted multiple cloning site. Using plasmid DNA purified on Quiagen Matrix Columns (Diagen, Hilden, FRG),
cells were transfected by the calcium phosphate precipitation method as
described (Leube et al., 1994
).
). Cultured cells
were fixed with methanol/acetone (Cordes et al., 1993
) or in 2% formaldehyde in PBS (20 min). After aldehyde fixation, cells were washed with
PBS containing 50 mM NH4Cl, followed by two washes in PBS, permeabilized by 0.1% Triton X-100 in PBS for 3 min, and again washed twice in
PBS. In some experiments, cells were only permeabilized with 0.004%
digitonin (Sigma, Deisenhofen, FRG) in PBS at 4°C for 4 min, washed in
ice-cold PBS, and fixed in 2% formaldehyde in PBS (20 min). Incubations
with primary antibodies were for 30 min; incubations with secondary antibodies coupled to Texas red sulfonyl chloride, FITC, cyanine 2-OSu bisfunctional or cyanine 3.29-OSu (Biotrend, Cologne, FRG; or Dianova)
were between 15 and 30 min. Washings were usually in PBS, but in some experiments, PBS containing 0.1% Triton X-100 and PBS with additional 0.4 M NaCl were used. For double-labeling experiments, cells were first
incubated with a mixture of antibodies from two different species, and then
incubated with the corresponding secondary antibodies. Affinity-purified
secondary antibodies specifically used for double labelings (Biotrend)
were controlled to exhibit no cross-reactivity to the primary antibodies of
the respective other species. Samples were analyzed in a Zeiss Axiophot
(Zeiss, Oberkochen, FRG) and photographed on TMY films (Eastman
Kodak Co., Rochester, NY).
20°C acetone (see above). For immunoelectron microscopy on formaldehyde-fixed cultured cells, these were permeabilized
with 0.005% digitonin in PBS for 10 min. Before the addition of antibodies, samples were kept for 15 min in blocking solution (PBS with 5% goat
serum, 0.1% cold water fish gelatine, and 5% BSA), and then washed
twice for 5 min each in PBS with 1% BSA or 0.1% acetylated BSA (BSA-c;
Biotrend). All subsequent incubations with antibodies and washings in
PBS were in the presence of 1% BSA or 0.1% BSA-c. Incubations with
primary antibodies were for 2 h, and with secondary antibodies coupled to
5- or 10-nm colloidal gold grains (Biotrend or Amersham) either for 2 h or
overnight. Control experiments, using gold-coupled secondary antibodies
alone or together with preimmune sera from Tpr-immunized animals,
were carried out in parallel under identical conditions and statistically
evaluated. Nonspecific binding of gold grains to NPCs (see Feldherr, 1974
)
was greatly reduced by the blocking agents applied and usually ranged below 1-2%. Gold-labeled secondary antibodies used for double labelings
were controlled to exhibit only minor cross-reactivity to the primary antibodies of the respective other species. After washes in PBS, sections were
fixed with 2.5% glutaraldehyde, postfixed with 2% aqueous OsO4, dehydrated, and embedded in Epon 812 as described (Cordes et al., 1995
).
) were immediately transferred into ice-cold PBS containing
2% formaldehyde. After 25 min of fixation, the nuclei were washed twice
with PBS containing 50 mM NH4Cl, and then incubated in PBS with 0.1%
BSA-c and 5% goat serum. In this solution each nucleus was ruptured
with a pair of tweezers, and the perforated nuclei were then gently pelleted by a 5-min centrifugation at 2,000 g, resulting in a very loose pellet.
Approximately 90% of the blocking solution was drawn off and replaced
by PBS with 0.1% BSA-c, thereby resuspending the nuclei. All subsequent incubations and washings were in PBS with 0.1% BSA-c. Nuclei
were first incubated with primary antibodies for 2-3 h, washed four times,
with intermediate 3-min centrifugations at 2,000 g, and then incubated
with gold-coupled secondary antibodies for 12 h. All subsequent steps
leading to embedding of nuclei in Epon 812 were as described (Cordes
et al., 1995
).
Results
), glioblastoma cells U333/CG/343 MG, primary cultures of endothelial cells, hepatocellular
carcinoma PLC cells, carcinoma cells of line A-431, colon
adenocarcinoma CaCo2 cells, SV-40-transformed fibroblasts
("SV-80 cells"), and keratinocytes of line HaCaT (not
shown), demonstrating that the antigen was widespread if
not ubiquitous. Bovine mammary gland epithelial BMGE
cells (Fig. 1 b) and kidney cells of line MDBK (not shown),
porcine kidney cells of line PK(15) (Fig. 1 c), and canine
MDCK cells (not shown) were also immunoreactive with
this mAb. In some cell cultures, occasional intranuclear
speckles were noticeable in addition to the peripheral
staining (see also below). Nuclear immunostaining was not
observed in rat cells of lines RV and 967-D2, murine cells
of lines L-M(TK
) and 3T3, marsupial PtK2 cells, and African green monkey cells of line RC37. Xenopus laevis kidney epithelial cells of line A6 were also negative.
Fig. 1.
Immunofluorescence microscopy of cultured mammalian cells and tissue cryostat sections after reaction with mAb 203-37. (a) Phase contrast and (a) epifluorescence optics of human adenocarcinoma cells of line HeLa. (b-e) Epifluorescence optics of cultured
bovine mammary gland epithelial cells of line BMGE (b), porcine kidney cells of line PK(15) (c), and cryostat sections of human esophagus (d), human testis (inset), and bovine liver (e). The finely punctate nuclear labeling in cultured cells is reminiscent of NPC staining. In a minor proportion of cells additional intranuclear dot-like structures are observed (a
, b, and c). Tissue cryostat sections (d and e) reveal staining of the nuclear periphery and also occasional intranuclear dots (see inset in d). Cells were fixed either with methanol/acetone (a, a
, and c) or with formaldehyde followed by detergent treatment (b) before incubation with antibodies. Cryostat sections were
fixed with formaldehyde without subsequent detergent treatment. Bars: (a, inset in d and e) 20 µm; same magnification in a-c; (d) 50 µm.
[View Larger Version of this Image (113K GIF file)]
), henceforth called p270. Cell fractionation revealed
that p270 remained structure associated after cell extractions with detergent and intermediate salt concentrations
(Fig. 2, b and b
). Immunoreactive polypeptides of lower
molecular weight, detected in fractions representing saltextracted soluble proteins (Fig. 2 b
, lane 3), were considered to be degradation products of p270. Indeed, fractionation of cells induced proteolytic degradation of p270 and
omission of protease inhibitors, and prolonged fractionation procedures resulted in an increase of lower molecular weight polypeptides reactive with mAb 203-37. Protein
p270 was also identified by immunoblottings with mAb
203-37 on total cell proteins of porcine PK(15) and bovine BMGE cells (Fig. 2, c and c
), as well as canine MDCK
cells (not shown), whereas rodent and marsupial p270 did
not cross-react with this antibody. Immunoblottings on
African green monkey proteins yielded a weak reaction
signal after prolonged exposure (not shown). In essence,
our immunoblotting results confirm and extend the study
of Bangs et al. (1996)
performed largely in parallel.
Fig. 2.
Immunoblot detection of a ~270-kD mammalian protein reactive with mAb 203-37. Proteins were separated by SDS-PAGE
and then, in parallel, either stained by Coomassie blue (a, b, and c) or transferred to nitrocellulose filters for immunodetection of proteins (a, b
, and c
) by enhanced chemiluminescence reaction (ECL). Same amounts of protein were loaded in corresponding lanes of gels for Coomassie staining and immunoblotting. (a) Separation of total proteins from HeLa cells and (a
) immunoblot detection of a
~270-kD protein, p270, immunoreactive with mAb 203-37. (b and b
) Fractionation of HeLa cells and immunoblot detection of p270 in
a residual detergent and salt-resistant fraction: Hela cells were first extracted with 0.2% Triton X-100 (lanes 1), washed with detergent-free buffer (lanes 2), extracted with 500 mM NaCl (lanes 3), and washed with nearly salt-free buffer (lanes 4). The residual cyto-
and karyoskeletal proteins were separated in lanes 5. Gels were loaded with the same percentage of proteins from each fraction. Note
that the major proportion of p270 remains structure-associated after detergent and intermediate ionic strength-salt treatment. A certain
amount of intact p270 has been released into solution by salt extraction as well as putative degradation products (marked by a bracket in
b
). In contrast to protein p270, a weakly immunoreactive polypeptide of ~75 kD, visible after prolonged film exposure (not shown) in
the residual cyto- and karyoskeletal fraction (lane 5), was not detected after application of more stringent filter washing conditions (see
Materials and Methods). (c and c
) Separation of total proteins from porcine kidney cells of line PK(15) (lanes 1) and bovine mammary
gland epithelial cells of line BMGE (lanes 2) and immunoblot detection of protein p270 with mAb 203-37 also in these species. Relative
molecular masses of marker proteins (M) in a-c (denoted by dots at the left margin in a
-c
) are, from top to bottom, as follows: 205, 116, 97, 66, 45, and 29 kD.
[View Larger Version of this Image (41K GIF file)]
Fig. 3.
Immunoelectron microscopy of mammalian cells and tissues using mAb 203-37 in a preembedding technique, showing intense
decoration of intranuclear sites near NPCs, often directly seen on the NPC-associated intranuclear filaments. Immunogold localization
(5-nm gold-coupled secondary antibodies) of protein p270 on cryostat sections of human liver (a-c), bovine testis (d and e), and in detergent-permeabilized human PLC cells (f). Cross-sections presented in a-e reveal exclusive labeling at the nucleoplasmic side of the
NPC and on the NPC-attached filaments extending into the nuclear interior. The grazing section presented in f allows insight into the
nuclear interior (N), and reveals nucleoplasmic labeling of two NPCs or its associated filament bundles (short thick arrows) whereas no
labeling is observed at the level of the outer, i.e., cytoplasmic annulus or in the mid-plane of the NPCs as demonstrable by the presence
of a dense central granule (long thin arrows). (g-j) Double label immunogold EM on cryostat sections of human (g) and bovine (h-j)
liver using mAb 203-37 (5-nm gold-coupled secondary antibodies) and affinity-purified guinea pig (g, gp-RBP2-2) and rabbit antibodies (h-j, rb-
RBP2-2) against RanBP2/Nup358 (10-nm gold-coupled secondary antibodies); RanBP2 representing a marker protein for
the cytoplasmic side of the NPC. Cryostat sections were fixed with acetone (a, c, and h-j) or formaldehyde (b, d, e, and g). PLC cells
were fixed with formaldehyde and then permeabilized by digitonin treatment (f). N, nuclear interior. C, cytoplasmic side. The outer nuclear membrane (denoted o in some figures) is oriented to the top in a-e and g-j. Thin long arrows in some figures mark several NPCs at
their cytoplasmic side; thicker and shorter arrows denote immunoreactive intranuclear filamentous material at or near NPCs. Bars:
(a-d, f-h) 0.1 µm; same magnification in d and e, and in h and i; (g) 0.05 µm; (j) 0.2 µm.
[View Larger Version of this Image (121K GIF file)]
; Bangs et al., 1996
). One cDNA clone, T1, contained
the entire open reading frame, encoding a 2,363-aa polypeptide with a predicted molecular mass of 267,333 daltons. Sequence comparison between human fetal brain
Tpr and the previously published, slightly shorter aa sequence (265,559 daltons) of human Tpr that had been deduced from various cDNAs isolated from fibroblast and fibrosarcoma cDNA libraries (Mitchell, 1992a,b; Byrd et al., 1994
) showed three aa exchanges in the amino-terminal
domain (positions 779, 906, and 1239) and an additional insertion of 14 mainly acidic aa in the carboxy-terminal domain of fetal brain Tpr (at position 1951) as the only differences between the two sequences (Fig. 4 b).
Fig. 4.
Characterization of p270 cDNA clones isolated from a
human expression library using mAb 203-37. (a) Schematic presentation of human p270 cDNA clones isolated from a fetal brain
Uni-ZapTMXR cDNA library. Screening of this library with mAb
203-37 yielded one clone (T1: bp 1-7321) containing the entire
coding region (bp 89-7177) for human Tpr, and five partial Tpr
cDNA clones (T2: bp 2374-7321; T3: 2723-7321; T4: 2825-7321;
T5: 3505-7321; T6: 3880-7321; lengths of polyA-tails varied between different clones and were omitted in the context of this bp
numbering). The position of the AUG start codon in clone T1 is
indicated by a circle, the stop codon present in all clones by
squares. The ORF of T1 encodes a 2,363-residue protein with a
predicted molecular mass of 267,333 D. Clones T3 and T4 represent fusion clones that also contain unrelated nt sequences at
their 3 end (not shown). Arrows denote the relative position of a
sequence insertion, present in all clones, that occurs in addition
to the previously published amino acid (aa) sequence of human
Tpr and results in a prolonged acidic aa cluster (see b). The scale
corresponds to the nt sequence of T1, each calibration line representing 1 kb. (b) An additional acidic aa sequence segment in human fetal brain Tpr. The top line shows the previously published
aa sequence of human Tpr deduced from cDNA clones isolated
from fibroblast and fibrosarcoma cDNA libraries (Mitchell and
Cooper, 1992a
,b). The bottom line shows the sequence of human
fetal brain Tpr, containing 14 additional, mainly acidic aa. (c) Radiolabeled products obtained by in vitro transcription-translation
of human Tpr cDNA clone T1 in reticulocyte lysate. The product
with the lowest mobility (arrowhead) corresponds to a relative
molecular mass of 270 kD. Radiolabeled polypeptides of lower
relative masses may represent proteolytic products of Tpr or
translation products resulting from misuse of sequence-internal AUG codons as translation initiation sites. Relative masses of reference proteins (positions denoted by dots on the left) are, from top to bottom, as follows: 205, 116, 97, 66, 45, and 29 kD. (d)
Immunoprecipitation of radiolabeled human Tpr protein. In vitro
translation products of cDNA clone T1 were incubated with protein G-Sepharose alone (lane 1) or together with mAb 203-37 (lane 2). Note that immunoabsorption of human Tpr from the
reticulocyte lysate occurs only in the presence of mAb 203-37. Relative masses of reference proteins (dots on the left) are as
in (c). (e-g
) Immunofluorescence microscopy on African green
monkey kidney cells transfected with an expression vector encoding human fetal brain Tpr. Cells were stained with mAb 203-37 reactive with human Tpr, in transfected cells, but not with the endogenous monkey orthologue of Tpr. The same fields are shown
in phase contrast (e-g) and epifluorescence optics (e
-g
). Besides a finely punctate nuclear labeling, reminiscent of NPC staining, intranuclear reaction sites are also noticeable. In a few transfected cells immunoreactive dots are located near the nucleoli
(arrows in f-g
; compare also with Fig. 7, f-g
). Cells were fixed
with methanol/acetone. Bars, 10 µm.
[View Larger Version of this Image (57K GIF file)]
Fig. 7.
Immunofluorescence microscopy of cultured cells and tissue cryostat sections after reaction with affinity-purified guinea pig
and rabbit antibodies raised against different epitopes of human and Xenopus Tpr. (a) HeLa cells stained with guinea pig antibodies
against hTpr-Pep1 (gp-hTpr-Pep1). (b) Bovine mammary gland epithelial cells of line BMGE stained with rabbit antibodies against
hTpr-Pep2 (rb-
hTpr-Pep2). (c and d) Cryostat section of human epidermis (c) stained with gp-
hTpr-Pep1 and human esophagus (d)
stained with rb-
hTpr-Pep2. Note that the nuclear staining obtained with these antibodies is essentially indistinguishable from that obtained with mAb 203-37 (see Fig. 1). In addition to the finely punctate nuclear staining resembling immunolabeling of NPCs, immunoreactive intranuclear dots are also observed occasionally (a and b). (e-g
) Xenopus laevis kidney epithelial cells stained with rabbit antibodies against xlTpr-Pep3 (rb-
xlTpr-Pep3). In addition to the finely punctate nuclear labeling, intranuclear dots are noticed in a minor
proportion of cells in which they sometimes appear to surround the nucleoli (f-g
). (h and h
) Cryostat sections through a Xenopus oocyte
stained with rb-
xlTpr-Pep3. Epifluorescence (a-e; f
, g
, and h
) and corresponding phase contrast optics (f, g, and h) are shown. Cultured cells were fixed with methanol/acetone (a, e-g
) or formaldehyde followed by detergent treatment (b). Cryostat sections were
fixed with formaldehyde (c) or acetone (d and h). N, nucleus. Bars: (a and e) 20 µm; same magnification in b; (c) 50 µm; same magnification in d; (g) 10 µm; same magnification in f; (h
) 100 µm.
[View Larger Version of this Image (159K GIF file)]
) and in rodent cells of lines Faza-967-D2 and
L-M(TK
) (not shown) confirmed that this variant of the
human Tpr protein is targeted to, and stably associates
with, nuclear structures. Immunofluorescence microscopy
on transfected cells using mAb 203-37 revealed both the
finely punctate staining in the nuclear periphery reminiscent of NPC labeling and the occurrence of intranuclear speckles, which occasionally appeared close to the nucleoli
(Fig. 4, f-g
). A similar nuclear localization of human fetal
brain Tpr was also observed when an expression vector
containing a major histocompatibility complex class I H-2
promoter was used (not shown). In some transfected cells,
with a seemingly higher level of human Tpr protein synthesis, some punctate immunoreaction sites that remain to
be characterized were also noticeable in the cytoplasm (not
shown).
, we raised antibodies against two peptides representing different regions of human Tpr that were identical
to both brain and fibroblast Tpr (Fig. 5 a). Immunoblotting using the affinity-purified antibodies revealed specific
immunoreaction with p270 (Fig. 5, b-b
). Moreover, both
mAb 203-37 and the antibodies against human Tpr peptide no. 1 (Fig. 5 a) similarly reacted also with the same
pattern of lower molecular weight polypeptides considered to represent p270 degradation products (Fig. 5, b
and b
). Two-dimensional gel electrophoresis followed by
immunoblotting definitively proved that the same mAb
203-37 reactive protein p270 was indeed also detected with
the Tpr peptide antibodies (Fig. 5, c-c
).
Fig. 5.
Immunoblot detection of p270 with affinity-purified guinea pig and rabbit antibodies raised against human Tpr protein. (a) Schematic presentation of human protein Tpr and the relative positions (arrows) of the two Tpr peptides, No. 1 (hTpr-Pep1, 1.) and No. 2 (hTpr-Pep2, 2.), against which antibodies were raised. The hatched box designates the amino-terminal domain of Tpr predicted to
form helices capable of forming coiled-coil structures. The carboxy-terminal domain (black line) is characterized by several acidic regions. The major cluster of acidic aa (corresponding to the cluster shown in Fig. 4 b) is designated by an open circle. Basic sequence elements (not denoted) separate the acidic regions and also characterize the ~50-carboxy-terminal aa. (b-b
) SDS-PAGE of total HeLa
cell proteins separated into a fraction of soluble proteins released by digitonin permeabilization of cells (lanes 1) and of residual, nonextracted proteins (lanes 2). Proteins were, in parallel, either stained by Coomassie blue (b) or transferred to nitrocellulose for immunodetection of proteins by ECL (b
-b
). Same amounts of proteins were loaded in corresponding lanes of gels for Coomassie staining and
immunoblotting. Digitonin treatment of HeLa cells already triggered proteolytic breakdown of protein Tpr and was applied to analyze
immunoreaction of degradation products with different Tpr peptide antibodies. (b
) Immunoblot using mAb 203-37 as reference, showing the detection of protein p270/Tpr and a major putative degradation product of ~210-kD. (b
) Immunoblot using guinea pig antibodies against hTpr-Pep1 showing essentially the same result as obtained with mAb 203-37: besides predominant labeling of p270/Tpr, immunoreaction is also noticed with the ~210-kD polypeptide. (b
) Immunoblot using rabbit antibodies against hTpr-Pep2 showing
labeling of p270/Tpr whereas the shorter degradation product of ~210 kD is not immunoreactive. Note that hTpr-Pep2 is located nearer
towards the protein's carboxy terminus than hTpr-Pep1. Relative masses of marker proteins (M) (denoted by dots in b
-b
) are as in Fig.
2. (c-c
) Identification of p270 as protein Tpr by double immunoblotting with mAb 203-37 and rabbit antibodies raised against hTprPep2. Residual HeLa proteins obtained after digitonin extraction were separated by IEF and SDS-PAGE and then transferred to a nitrocellulose filter (2). As reference, the same protein fraction was also separated on the left side of the same gel by SDS-PAGE alone
(1), together with marker proteins (denoted by dots in lanes M) with relative masses, from top to bottom, of 205, 116, 97, and 66 kD. The
filter (only the upper half is shown) was first incubated with mAb 203-37 and HRP-coupled secondary antibodies to murine Ig (c): the
immunoblot reaction (ECL procedure, exposure time <1 min) identifies p270 (arrow) and putative degradation products (brackets in 1 and 2) of ~210 kD. Bound antibodies were then released under acidic conditions and the filter incubated solely with HRP-coupled secondary antibodies to rabbit Ig (c
): ECL (exposure time >12 h) reveals no unspecific immunoreaction of p270 with these secondary antibodies. The filter then again was washed in acidic buffer and incubated with affinity-purified rabbit antibodies raised against hTpr-Pep2 (rb
hTpr-Pep2) and HRP-coupled
-rabbit secondary antibodies: ECL (exposure time <1 min) shows specific labeling of protein Tpr (arrow). Note that proteins identified as p270 in c and as Tpr in c
are indistinguishable by electrophoresis. As expected, the 210-kD putative degradation product is not immunoreactive with rb-
hTpr-Pep2.
[View Larger Version of this Image (39K GIF file)]
).
Fig. 6.
Generation of antibodies immunoreactive with
amphibian Tpr. (a) Isolation
of PCR products encoding
the carboxy-terminal domain of the Xenopus laevis
Tpr protein and alignment of
human and Xenopus Tpr aa
sequences. A Xenopus kidney cDNA library was used
as template for PCR assays.
Several overlapping DNA
fragments were assembled into a partial DNA sequence
of 1811 bp that encodes the
558-carboxy-terminal aa of
Xenopus Tpr (p-xlTPR)
shown in the bottom lane of
the alignment; the corresponding human sequence is
shown in the upper lane
(hTPR). Numbers at the
right margin stand for aa positions in accordance to full-length human fetal brain Tpr. Identical aa (bold letters) and homologous aa are boxed. Groups of homologous aa as relevant for this alignment were laid down as follows: R+K; D+E; S+T; V+A+P; V+I+L+M. (b) Schematic presentation
of the partial Xenopus Tpr aa sequence (p-xlTPR) and the relative position (arrow) of a Xenopus Tpr peptide, xlTpr-Pep3 (3.), against
which antibodies were raised. The human Tpr scheme (hTPR) with its relative peptide positions is shown as a reference. (c) Immunodetection of Xenopus protein p270/Tpr with affinity-purified rabbit antibodies raised against xlTpr-Pep3. Total proteins from Xenopus laevis
kidney epithelial cells (lanes 1) and from manually isolated Xenopus oocyte nuclei (lanes 2) were separated by SDS-PAGE and, in parallel, either stained by Coomassie blue (c) or transferred to nitrocellulose for immunodetection of proteins by ECL (c). Same amounts of
protein were loaded in corresponding lanes of c and c
. Relative masses of marker proteins (M) in c (denoted by dots in c
) are as in Fig. 2.
[View Larger Version of this Image (59K GIF file)]
). Punctate
nuclear periphery staining was further noticed in Xenopus
erythrocytes (not shown). On cryostat sections of Xenopus
skin, liver, and oocytes (Fig. 7, h and h
), immunolabeling
was seen at the nuclear envelope and occasionally, in somatic cells, also in additional intranuclear dots (not shown).
, 1996
).
Fig. 8.
Double immunofluorescence microscopy of mammalian and amphibian cells using antibodies against p270/Tpr (a-c),
and against other NPC proteins (a-c
) that represent marker proteins for pore complexes both of the nuclear envelope and the
cytoplasmic annulate lamellae. (a and a
) HeLa cells immunolabeled with mAb 203-37 against human Tpr (a) and affinity-purified
guinea pig antibodies (gp-
RBP2-2) against RanBP2/Nup358
(a
). (b and b
) Bovine mammary gland epithelial cells of line
BMGE immunolabeled with (b) affinity-purified rabbit antibodies against hTpr-Pep2 (rb-
hTpr-Pep2) and (b
) mAb 414 reactive with the XFXFG-family of O-glycosylated nucleoporins. (c
and c
) Cryostat section through a Xenopus laevis stage VI oocyte
immunolabeled with (c) affinity-purified rabbit antibodies against
xlTpr-Pep3 (rb-
xlTpr-Pep3) and (c
) gp-
RBP2-2. Note that no
cytoplasmic staining is detectable with Tpr-antibodies whereas
distinctive cytoplasmic structures, representing annulate lamellae, are immunoreactive with antibodies against other NPC proteins. Secondary antibodies were coupled to cyanine 2-OSu bisfunctional (a and c
) or fluorescein isothiocyanate (b
) and
cyanine 3.29-OSu (a
and c) or Texas red sulfonyl chloride (b).
Cultured cells were fixed with methanol/acetone (a and a
) or
formaldehyde followed by detergent treatment (b and b
). The oocyte cryostat section was fixed with acetone (c and c
). Bars: (a
and b) 15 µm; (c) 100 µm.
[View Larger Version of this Image (74K GIF file)]
; Yokoyama et al., 1995
; Wilken et al.,
1995
). This differential double label therefore underscores
the significance of the intranuclear location of protein p270/Tpr.
Fig. 9.
Immunoelectron microscopy of human tissues after reaction with affinity-purified antibodies raised against different epitopes of
human Tpr revealing exclusive intranuclear localization of protein p270/Tpr. (a-e) Immunogold localization (5-nm-gold) of p270/Tpr on
cryostat sections of human liver using rabbit antibodies (rb-hTpr-Pep2) against hTpr-Pep2 (a-c, and e) and guinea pig antibodies (gp
hTpr-Pep1) against hTpr-Pep1 (d). Thin long arrows in some figures mark several NPCs at their cytoplasmic side; thicker and shorter arrows denote immunoreactive intranuclear filaments. In addition to the immunolocalization of p270/Tpr in NPC-attached filamentous
material, labelings of electron-dense spheroidal intranuclear structures (arrowheads in e) are occasionally observed. (f-h) Double label immunogold EM on cryostat sections of human liver confirming the exclusively intranuclear localization of protein p270/Tpr. (f and
g) rb-
hTpr-Pep2 (5-nm-gold) and guinea pig antibodies (gp-
RBP2-2) against RanBP2/Nup358 (10-nm-gold). (h) gp-
hTpr-Pep1 (5nm-gold) and rabbit antibodies (rb-
RBP2-2) against RanBP2/Nup358 (10-nm-gold). Cryostat sections were fixed with acetone (a-g) or
formaldehyde (h). N, nuclear interior. C, cytoplasmic side. The outer nuclear membrane (denoted o in some figures) is oriented to the
top in all figures. Bars: (a-e) 0.2 µm, same magnification in a-d; (f-h) 0.1 µm; same magnification in g and h.
[View Larger Version of this Image (113K GIF file)]
), whereas the p270/Tpr protein again was localized to the intranuclear filaments.
Fig. 10.
Immunoelectron microscopy of Xenopus oocytes after reaction with affinity-purified antibodies raised against Xenopus
p270/Tpr showing dense labeling of NPC-attached intranuclear filament bundles. (a-d) Immunogold localization (5-nm-gold) of Xenopus p270/Tpr on manually isolated nuclei using rabbit antibodies raised against xlTpr-Pep3 (rb-xlTpr-Pep3). Note the exclusive localization of p270/Tpr in the intranuclear NPC-attached filament bundles that often appear laterally aggregated and partly entangled. An
individual, nuclear envelope-attached annulate lamellae cisterna (AL, marked by a bracket in c) is devoid of gold grains. (e-k) Double
label immunogold EM on manually isolated nuclei (e-j) and on a formaldehyde-fixed cryostat section through an oocyte (k) using rb
xlTpr-Pep3 (5-nm-gold) and guinea pig antibodies (gp-
RBP2-2) against RanBP2/Nup358 (10-nm-gold). Note that RanBP2 antibodies
almost exclusively label the cytoplasmic side of the NPCs, whereas Tpr antibodies label intranuclear filaments. The grazing section
through part of the nuclear envelope presented in i also shows the location of RanBP2 at the cytoplasmic side (C) of an NPC (long thin
arrow), and Tpr at its nucleoplasmic (N) side. Note that protuberances extending from the corners of the NPCs eightfold symmetrical
inner cylinder or annulus are labeled by Tpr antibodies (short thick arrows). Also, in h, intranuclear filaments extend far into the nuclear
interior and occasionally interconnect nucleoli (No) and NPCs. Also note, in j, that cytoplasmic pore complexes of annulate lamellae,
immunonegative for Tpr, are labeled by RanBP2 antibodies. Numbers of occasionally observed intranuclear 10-nm-gold grains and cytoplasmic 5-nm-gold were in the range of unspecific background labeling and secondary antibody cross-reactivities. The outer nuclear
membrane (o) is oriented to the top in a-h and j-k. Thin long arrows in b mark several NPCs at their cytoplasmic side; thick short arrows denote intranuclear sites of Tpr labeling. M, mitochondria. Bars: (a-c, f-h, j, and k) 0.2 µm; (d and i) 0.1 µm, same magnification in
d and e.
[View Larger Version of this Image (111K GIF file)]
Discussion
,b), which, however, has
been reported to be exclusively located on the cytoplasmic
side of the NPC (Byrd et al., 1994
; Bangs et al., 1996
; for
review see Pante and Aebi 1995a
,b, 1996; Bastos et al.,
1995
; Goldberg and Allen, 1995
). Because of this discrepancy and because the sequence of 2,363 aa in our p270
cDNA isolated from human fetal brain contained several
additional aa and a few minor changes compared with the aa sequence (2,349 aa) assembled from different partial
human fibroblast and fibrosarcoma cDNAs (Mitchell and
Cooper, 1992a
,b; Byrd et al., 1994
), we took special care to
prove the identity of p270 and Tpr by raising antibodies
against peptides representing different epitopes common
to both human Tpr aa sequences. All antibodies obtained
specifically react with the same p270 polypeptide and immunolabel the same NPC-attached intranuclear filaments
that are also positive with the mAb against p270 initially
used. By contrast, the cytoplasmic annulus of the NPC has
been devoid of immunolabel although it is perfectly accessible, as confirmed by double immunoelectron microscopy
using RanBP2/Nup358, a bona fide marker of the cytoplasmic NPC aspect (Yokoyama et al., 1995
; Wu et al., 1995
;
Wilken et al., 1995
) as control antigen. These immunolocalization results are also consistent with our observations made in digitonin-permeabilized cells, following the procedure recently used by others to prove exclusively cytoplasmic side labeling of NPCs (Bangs et al., 1996
). We noticed that under these conditions only some of the nuclei
were immunolabeled with p270/Tpr antibodies, whereas
others were totally negative, indicating that the antigen
was not accessible. Using RanBP2 antibodies, all nuclei, in
contrast, were immunolabeled.
,b, 1974; Richardson et al., 1988
; Cordes et al.,
1993
; Ris and Malecki, 1993
) and project into the nuclear
interior, clearly exceeding the ~50 nm of the NPC-attached
intranuclear fish trap or basket structures (Ris, 1989
, 1991
;
Jarnik and Aebi, 1991
; Goldberg and Allen, 1992
). It is
also obvious from our study that these filament bundles projecting from a given NPC into the nucleoplasm represent a distinct structural entity, in agreement with the observations of Ris and Malecki (1993)
with glutaraldehydefixed cylindrical bundles of NPC-attached filaments (see,
e.g., Fig. 3, b and c, in Ris and Malecki, 1993
). Our tracings
of p270/Tpr-containing filament bundles for up to 200 nm
in mammalian somatic cells and for up to 350 nm in Xenopus oocytes and the absence of label on the interporous aspects of the nuclear envelope further indicate that these filamentous structures are not systematic artifacts originated
from ruptures of the nuclear envelope-subjacent filament
lattice (Goldberg and Allen, 1992
, 1995
).
;
Roth, 1995; Gall et al., 1995
). Experiments to correlate the
appearance of these structures with a specific cell cycle
phase have also been inconclusive, as cell populations synchronized by colcemid or thymidine blocking and released
into G1 or G2 did not contain significantly higher numbers
of such intranuclear speckles (data not shown). At the moment, we cannot exclude that these immunoreactive dots
merely represent accidental intranuclear aggregates of
protein p270/Tpr.
; Meier and Blobel, 1992
; Stroboulis and Wolffe, 1996)
are currently underway in our laboratory. The conspicuously long and dense NPC-attached filament bundles in
amphibian oocytes might further indicate that in these
particular cells, p270/Tpr and the filaments containing it
belong to those elements that are also maternally stored to serve important roles in early embryogenesis.
helices organized in coiled-coils
(Mitchell and Cooper, 1992b
). Such a predicted conformation makes this protein a very likely candidate for assemblies of stable homo- or heterodimers (see also Byrd
et al., 1994
) and for participation in the formation of the
backbone of the intranuclear filaments described. This hypothesis should now be directly testable by self-assembly
experiments in vitro, using the pure protein made by recombinant technology.
Received for publication 30 September 1996 and in revised form 27 November 1996.
The human fetal brain p270/Tpr sequence and the partial Xenopus kidney sequence have been submitted to the GenBank and are available under accession numbers U69668 and U69669, respectively.We thank Andreas Hunziker for competent help in DNA sequencing, Horst Baron for technical assistance in immunizing animals, and Jutta Müller-Osterholt for photography. We also thank Rudi Zirwes for providing the Xenopus laevis cDNA library and Drs. Harald Herrmann, Karsten Weis, Ralf Bischoff, and Georg Krohne for valuable discussions.
aa, amino acid; CMV, cytomegalovirus; ECL, enhanced chemiluminescence; KLH, keyhole limpet hemocyanin; NPC, nuclear pore complex; TBST, TBS containing 0.05% Tween-20.