JOURNAL

OF STRUCTURAL

BIOLOGY

108,

129-139

(1992)

Transient Storage of a Nuclear Matrix Protein along Intermediate-Type Filaments during Mitosis: A Novel Function Cytoplasmic Intermediate Filaments R. A. L)epartment

of Anatomy,

University Received

of Ziirich-Irchel, July

29,

1991,

MARUGG’ Winterthurerstrasse

and

in revised

INTRODUCTION The chromatin-depleted nuclear substructures are operationally defined as nuclear matrix or scaffold Department 100. CH-8006

form

190, January

CH-8057

Ztirrch,

Switzerland

20, 1992

(reviewed in Nelson et al., 1986; Schroder et al., 1987; Verheijen et al., 1988; see also Fig. 5e). The nuclear matrix is structurally organized in two different parts within the nucleus: the nuclear lamina and the interior part of the nucleus (Aaronson and Blobel, 1975; Capco et al., 1982; Van Eekelen et al., 1982). The nuclear lamina consisting of assembled lamins forms a fibrillar network of lo-nm filaments at the nucleoplasmic surface, which interacts with the nuclear pores and the chromatin (Aebi et al., 1986; Fischer et al., 1986; Franke et al., 1981; Paddy et al., 1990; Glass and Gerace, 1990). Wellcharacterized nuclear fractionation procedures lead to a more detailed understanding of the structural organization of the nuclear matrix. These biochemical and differential extraction procedures of the cells in situ include the use of detergent to remove membranes; insoluble material or matrix attached material are removed by both high and low ionic strength buffers, extensive DNase treatment removes the chromatin, and RNase is used to remove the RNA (Berezney and Coffey, 1974, 1977; Berezney, 1980; Staufenbiel and Deppert, 1984). Many observations indicate a significant functional role of the nuclear matrix, aside from structural support, in higher chromatin organization (Mirkovitch et al., 19841, DNA replication (Ciejek et al., 19831, RNA synthesis, and RNA processing !Jackson et aI.. 1981). DNA attachment sites on the nuclear matrix seem to play a role of the higherorder chromatin organization (Mirkovitch et al., 19843. Active genes and replicating DNA have been suggested to be physically associated with the nuclear matrix (Ciejek et al., 1983; Jackson et al., 1981; Berezney, 1980). Over 90% of the newly synthesized heterogeneous nuclear RNA (hnRNA) is retained with the matrix and may be an integral component necessary for maintaining the nuclear structure (Van Eekelen and Van Venrooij, 1981; Fey et al., 1986). Compared to the fundamental importance of the interior matrix? only few data concerning its struc-

We recently identified a nuclear matrix protein, named NMP125 for its molecular weight (M, 125 kDa). On the basis of immunofluorescence analysis with monoclonal antLNMP125 antibodies of differentially extracted cells in situ, including detergents, DNase I, RNase A, and high/low ionic strength conditions, it is concluded that NMP125 is a component of a chromatinand histonedepleted nuclear substructure, operationally defined as nuclear matrix in interphase cells. The protein revealed evolutionary conservation in man, rat, chicken, and Xenopus, at least at the level of immunological crossreactivity. The subcellular distribution of NMP125 is cellcycle-dependent; in interphase cells NMP125 is confined to a nuclear substructure with a granular aspect, whereas after nuclear envelope breakdown, it is freed into the cytoplasm. However, most of the protein remains attached to a cytoskeletal ligand that we have identified as the intermediate-type filament vimentin. In late mitotic stages the protein forms punctuate aggregates of relatively large size, which get passively closer to the newly formed telophase nuclei together with the reorganized vimentin around the nuclei in late telophase. From the morphological point of view, although static in nature, a dynamic cell-cycle-dependent distribution of NMP125 is found, revealing dissociation and spreading throughout the cytoplasm in metaphase, binding to vimentin filaments, cytoplasmic aggregation, and transport to nuclei in telophase. The transient affinity of the nuclear protein NMP125 to vimentin filaments during mitosis together with a passive cytoplasmic dislocation of the vimentin/NMP125 conjugate toward the telophase nuclei could represent a novel and dynamic function of cytoplasmic intermediate filaments, implicating a transient repository and passive shift of nuclear proteins during mitosis. L 1992 Academic Press, Inc.

’ Present address: Hospital, Rtimstrasse

of

of Neurosurgery, University Ziirich, Switzerland. 129

Copyright t 1992 Al! rights of reproductmn

1047-8477192 53.00 by Academic Press, Inc m any form reserved.

130

R. A. MARUGG

ture and the composition exist (Capco et al., 1982; Fey et al., 1984, 1986; Hakes and Berezney, 1991). Fractionation of the nuclei revealed the existence of nuclear core filaments forming a 3D lattice within the nucleus in chromatin-depleted HeLa cell nuclei (Dacheng et al., 1990). The mechanisms that regulate the dispersal and subsequent reconcentration of nuclear components at successive stages of the cell cycle are an interesting and important problem in cell biology. Mitosis in all higher eucaryotes is characterized by a profound reorganization of the cytoarchitecture. During condensation of the chromatin into chromosomes, both nuclear membranes and the underlying nuclear lamina break down. This results in a disintegration of the nuclear and the cytoplasmic compartments (Gerace and Blobel, 1980). The nuclear lamina fraction of rat liver is composed of three major proteins of 62 to 72 kDa. Its mitotic dynamics is the best studied component (Gerace and Burke, 1988). The lamins undergo cell-cycle-dependent phosphorylation, whereby the cyclin-activated cdc2 kinase was implicated for lamin phosphorylation (Murray, 1989; Balter, 1991). The work described here investigates the dynamics of a nuclear matrix protein during mitosis. The high molecular weight protein, named NMP125 according to its apparent molecular weight, can be visualized by monoclonal antibodies. To follow the distribution of NMP125 during mitosis we used a human mesenchymal-derived cell (M617 cell line). We further characterized the protein in M617 cells by differential extraction of the cells in situ and subsequent analysis of the extracts by SDS-PAGE, immunofluorescence, and immunoelectron microscopy of chromatin-depleted cells. To investigate the affinity of NMP125 to vimentin during mitosis, we used double-immunostaining procedures, immunoelectron microscopy, and an in vitro affinity test. In addition we looked for immunologically related proteins in chicken, rat, and Xenopus. MATERIALS Cell

AND

METHODS

Culture

Human mesenchymal-derived M617 cells (kindly provided by U. Wiesmann, Department of Pediatrics, University of Berne, Switzerland) were grown in DMEM with 10% fetal calf serum (GIBCO, Grand Island, NY) supplemented with penicillin/streptomycin. For differential extraction of the nuclei in situ and immunofluorescence analyses, glass coverslips, which had been washed with ethanol and sterilized, were included in the culture. For electron microscopic analyses, cells were grown on termanox tissue culture coverslips (Miles Scientific, Naperville, IL). Protein

Analysis

and

Immunoblot

Analysis

M617 cells were extracted with 1% nonidet P40 or Triton X-100 in nuclear matrix (NM) buffer (10 mM Mes, pH 6.2, 10 mM NaCI, 1.5 mM MgCl,, 10% glycerol, 30 pg aprotinin (200 kIU: Trasylol,

Bayer, Leverkusen, Germany)). Detergent extracts and residual cytoskeletons were centrifuged at 100 OOOg for 1 hr at 4°C. The proteins in the pellet were dissolved in NM buffer containing 8 M urea. The proteins of both fractions were separated on mini-SDS PAGE (58%) and then were transferred to 0.22 pm nitrocellulose membrane (20 V/O.1 A, 12 hr). Rat liver nuclei were prepared according to Berezney and Coffey (1977). The isolated nuclei were extracted with NM buffer containing 8 M urea, centrifuged, and processed as described above. A crude cytoskeletal and nuclear protein fraction of chick neuronal tissue was obtained according to Delacourte et al. (1980) and Marugg and Baier-Kustermann (1988). The nitrocellulose blots were washed two times in TBS for 15 min each, incubated in TBS containing 1% BSA and 10% FCS, incubated with an anti-NMP125 antibody, washed 5~ 3 min in TBS, incubated with a rabbit anti-mouse IgG-peroxidase conjugate, washed 5x 3 min in TBS, and developed. The apparent molecular weight (M,) was estimated by measurement of the relative mobility (Rr) of the protein in SDSPAGE, blotted on nitrocellulose, and subsequently immunostained with anti-NMP125. Rf values versus log M, for the standards (high molecular weight standards from Bio-Radl were plotted on semi-log paper. The Rrvalue of the nuclear protein was obtained by interpolation. Differential

Extraction

of the Cells

in Situ

Cells on glass coverslips were washed three times in NM buffer. The first extraction step was performed with NM buffer containing 1% nonidet P40, 1 mM EGTA, and 5 n-&f dithiothreitol (D’lT) for 30 min at 4°C. PMSF (1 mM) was added to the extracts, which were immediately frozen for further analysis. The cell residues in situ were washed three times with NM buffer and incubated for 15 min at 37°C with 100 pg/ml DNase I (Boehringer-Mannheim GmbH, Germany). Ammonium sulfate was added from a 1 M stock solution for a final concentration of 0.25 M. The cell residues were further extracted with 2 M NaCl, 1 mM EGTA, and 5 mM DTT in NM buffer. After digestion with 100 kg/ml DNase I and 100 kg/ml RNase A (Boehringer-Mannheim GmbH) in NM buffer for 30 min at 37°C the residual cellular skeletons were dissolved in NM buffer containing 6 M urea. Each extraction step was monitored by gel electrophoresis and the cellular residues after each extraction step were fixed with 2% freshly prepared paraformaldehyde in PBS, pH 7.2, and immunostained with monoclonal anti-NMP125 antibodies. The monoclonal anti-NMP125 antibodies were obtained by immunization with a crude cytoskeletal fraction including nuclear extracts obtained from bovine spinal cords. The supernatants of hybridomas were screened in immunodot tests and by indirect immunofluorescence of M617 cells. Zmmunofluorescence In situ fractionated cells (see above) or fixed cell monolayers were washed 2x 15 min with TBS. Nonspecific binding sites were blocked with blocking buffer BB (1% BSA (w/v) + 10% FCS in TBS) for 20 min at room temperature followed by incubation overnight with anti-NMPl25 and anti-vimentin antibody for double-immunostaining. The monoclonal antibodies were diluted in BB alone or both together as an antibody cocktail in case of double-immunofluorescence staining. The cells were washed 5~ 3 min with TBS and incubated with a goat anti-mouse FITC or TRITC conjugate, diluted in BB or as an antibody cocktail in case of double-immunofluorescence staining. The double-immunofluorescence labeling with two mouse monoclonal antibodies requires Ig-subclass-specific secondary antibodies. The cells were washed 5 x 3 min with TBS and mounted with a mounting medium based on polyvinyl alcohol. Two negative controls with nonimmune serum or blocking buffer instead of anti-NMP125 antibodies were made. The cells were viewed in an ZEISS IM35

MITOTIC equipped on Kodak Affinity

with epifluorescence T-MAX film.

optics.

DYNAMICS

Photographs

were

OF A NUCLEAR recorded

Test

A crude vimentin and nuclear protein fraction of M617 cells was obtained by removing soluble proteins and membranes with NM buffer containing 1% Triton X-100. The chromatin was removed by centrifugation at 100 OOOgil hri4”C after dissolving the cytoskeleton in 8 M urea. The supernatant was dialyzed against vimentin assembly buffer (5 mM Tris-HCl, pH 7.3, 1 mhf DTT, 1 mM EGTA, 0.17 M KCl). Cocentrifugation of vimentin with NMP125 was tested in immunodot tests of the pellet and the supernatant. Protein (100 pg) was placed as dots on nitrocellulose. These dots were screened for vimentin and NMP125 by immunostaining according the immunoblot procedure described above. Electronmicroscopy,

Preembedding

Immunolabeling

Cells on Thermanox plastic coverslips were subfractionated in situ, fixed, and immunostained as outlined above, with the exception that an immunogold conjugate (GAM-G15) was used as secondary antibody. The labeled cells were dehydrated and embedded in EPON, and the ultrathin sections were contrasted with uranyl acetate and lead citrate. Each immunolabeling procedure included two negative controls as described above. The sections were viewed in a transmission electron microscope (PHILLIPS EM3001. RESULTS

Protein Analysis

Immunoblots of electrophoretically separated polypeptides from the detergent-insoluble fractions of cells of different species reveal one sharp immunoreactive protein band when incubated with the anti-NMPlZ5 antibody M2 (Fig. 1). Human M617 cells were homogenized in a buffer containing Triton X-100 and centrifuged to separate detergent insoluble (lane 1) from detergent soluble proteins (lane 2). No immunoreactivity was found in the supernatant (lane 2). Detergent insoluble proteins of a sample of 123

NMP125

II

4

- -

i”e

5

MATRIX

131

PROTEIN

chicken brain (lane 3) and rat liver tissue (lane 4) were immunostained with the same monoclonal anti-NMP125 antibody. The immunoreactivity with another anti-NMP125 antibody (M9) with detergent insoluble proteins of rat liver tissue is shown in lane 5. A strong reaction with NMP125 was found, but an additional protein of low molecular weight is also stained. In addition, we could identify immunologically related proteins in Xenopus brain tissue extracts (experiments were performed by Dr. B. Riederer, University of Lausanne, Switzerland; personal communication, results not shown). As shown in Fig. 2, lanes 1 and 2, the apparent molecular weight of the protein recognized by the monoclonal antibody was 125 kDa based on its relative mobility in SDS-PAGE. Immunoblots with anti-NMPl25 and a monoclonal anti-vimentin antibody document the monospecificity in M617 cell extracts (Fig. 2, lanes 3 and 4). Immunofluorescence

and Differential

Extraction

Since successive stages of the cell cycle can easily be followed in a single cell culture of M617 cells, all the cell cycle data were obtained from these cells. Due to the monospecificity of the anti-NMP125 antibody M2 (see protein analysis), all immunostainings were done with this antibody. Immunolabeling of all interphase cells with anti-NMP125 yielded a granular staining of the nuclei without significant immunoreactivity of the nucleoli and the cytoplasm. A representative nuclear staining is shown in Fig. 3a, marked with an arrowhead, whereas in mitotic cells the immunofluorescence was spread throughout the whole cytoplasm without any significant staining of the chromosomal area. Figure 3a shows a cell in prophase with a high concentration around

1

2

3

4

NMP125

FIG. 1. Immunoblots of cells and tissues of different species. Immunolabeling with two monoclonal anti-NMP125, M2 (lanes 14 and M9 (lane 5). Human M617 cells: the cells were homogenized in Triton X-100 and the detergent insoluble (lane 1) and the soluble proteins (lane 21 were separated by centrifugation. No staining was found in the Triton X-100 extract [lane 21. Detergent insoluble proteins of chicken brain ilane 3) and rat liver tissue (lane 4 and 5).

FIG. 2. Specificity of the monoclonal antibodies used. SDSpolycrylamide gel electrophoresis (518%) of a vimentin-enriched protein fraction of M617 cells (lane 1: v, vimentin). Molecular weight standards (lane 2: myosin, 200 kDa; B-galactosidase, 116.25 kDa; phosphorylase B, 97.4 kDa; bovine serum albumine, 66.2 kDa; ovalbumin, 45 kDa). Immunoblot stained with antivimentin (lane 3) and anti-NMP125 M2 (lane 41.

132

R. A. MARUGG

FIG. 3. Immunocytochemistry with anti-NMPl25 M2 in different mitotic stages of the cell. Bar = 5 pm for all figures. (a) Fixed tissue culture cells (M617, human). Note the granular and patchy staining of the interphase cell nucleus (arrowhead), but no staining within the cytoplasm. Note the accumulation of NMP125 around the central area of the prophase cell corresponding to a condensed DNA region (asterisk), and the cytoplasmic punctuate units. (b) Distribution pattern in a M617 metaphase and anaphase cells. NMP125 immunoreactivity is found within the whole cytoplasm, whereby the chromosomal areas are excluded. (c) Distribution pattern in a telophase cell. Note the granular aggregates within the cytoplasm and the reorganization of NMP125 within the newly forming nuclei.

the central area of the cell from which it is excluded (asterisk). In metaphase and anaphase cells the protein is spread diffusely within the cytoplasm and the chromosomes become clearly visible, due to the lack of immunoreactivity in the chromosomes (Fig. 3b). Hence NMP125 stained with mab M2 seems to be freed into the cytoplasm when the nuclear membrane is broken down. In late telophase the protein aggregates to punctuate units of relatively large size within the cytoplasm and reappears within the newly formed nuclei (Fig. 3~). Immunocytochemistry and biochemical cell fractionation have both inherent advantages and disadvantages that limit their use for subcellular and, in particular, subnuclear localization of proteins. To circumvent these problems, both approaches can be combined by differential extraction of cells in situ and analyses of the extracted proteins by SDSPAGE and immunoblotting (Staufenbiel and Deppert, 1984). The first extraction step with NP40 before fixation leaves the cytoskeleton with associated polysomes and the extracted nuclei behind. As illustrated in Fig. 4a, immunofluorescence of the residual structures still displays the same morphology as in unfractionated cells. Hence NMP125 is associated with the detergent-resistant cellular fraction both in interphase as well as in mitotic cells indicating that it is not a soluble cytoplasmic component in mitotic cells. The second extraction step with DNase I removes quantitatively over 99% of the DNA, whereas histone proteins are completely extracted by high salt treatment (Staufenbiel and Deppert, 1984). The same authors have also demonstrated that RNA extraction occurs during these treatments. About 10% of RNA is still associated tightly with the nuclear matrix after DNase I/high salt treatment. Most of this RNA is removed by subsequent RNase A treatment. After DNase I treatment the M2 immunoreactivity is still present in the nuclei (Fig. 4b). The nuclear immunofluorescence remains after the third extraction step under high salt conditions (Fig. 4~). The last extraction step with DNase URNase A removes the remaining DNA and RNA. The NMP125 immunoreactivity was still located within the nuclear residue after DNase I/RNase A digestion, and therefore, according to our operational definition, is a component of the nuclear matrix of interphase cells (Fig. 4d). The pattern of solubilized polypeptides after each extraction step is also revealed in Fig. 4. NP40 extracts the membranes and soluble proteins (lane 21, DNase I removes most of the DNA and probably solubilizes some core histones (lane 3). The DNase I extraction was also monitored by DNA-agarose gels (result not shown) and by electron microscopy (Fig. 5e). High salt extraction removes the core histones

MITOTIC

DYNAMICS

OF A NUCLEAR

MATRIX

133

PROTEIN

h DNase

DNase

I

I + RNase A

5

67

FIG. 4. Differential extraction of M617 cells in situ and SDSgel electrophoretic protein patterns of the extracts. Bar = 10 pm for figures. (a) Detergent extracted cells. MW standards (lane 1: see also Fig. 2, lane 2) and soluble proteins (lane 2). (b) DNase I-extracted cells with similar nuclear staining. DNase I (lane 3: h, core histones and histone HI). (c) Histone extraction with 2 M NaCl. Note unchanged pattern and the cytoplasmic staining of the mitotic cell. Lane 4, extracted core histones and histone HI (h) and actin (a). DNase I and RNase A digestion. Note the remaining nuclear fluorescence. Lanes 5 and 6: the protein pattern after dissolving the residues in 6 M urea, dialysis against TBS, and centrifugation. Supernatant, containing lamins A, B, and C (lane 51, pellet containing mainly cytoskeletons (lane 6: a, actin; v, vimentin; and small amounts of the lamins, la). HMW-standards (lane 7).

and histone HI (lane 4), and the residual cellular skeleton after sequential DNase IiRNase A extraction is shown in lanes 5 and 6). The major bands are vimentin (57 kDa), actin (42 kDa), and the lamins A, B, and C (72, 68, and 62 kDai. The NMP125 and vimentin were monitored with immunoblots ilanes 3 and 4 in Fig. 2). Immunofluorescence analyses of NMP125 of in situ fractionated and chromatin-depleted cells have shown that no significant amounts of NMP125 can be removed by detergent treatment. Afinity

of NMP125

for Vimentin

in Mitotic

Cells

If the cells were in situ fractionated with NP40 to remove the soluble proteins before fixation of the

all the (d) cell

cells, NMP125 remains associated with the cytoplasm of mitotic cells, indicating that NMP125 has a high affinity to the Triton-insoluble part of the cytoplasm. To identify the cytoplasmic ligand of NMP125, we first studied the cells by immunoelectron microscopy. A representative M617 cell in telophase is shown in Fig. 5a. Before fixation and immunostaining the cells were extracted with Triton X-100 and the DNA was fragmented with DNase I. The remaining cytoskeletons and nuclear matrices were labeled with anti-NMP125 and incubated with a secondary antibody conjugated with 15 nm gold particles (goat anti-mouse IgG G15). Electron micrographs of these immunolabeled, in situ fractionated cells demonstrate colocalization of NMP125 with intermediate-type filaments (Figs. 5b and 5d). The

R. A. MARUGG

FIG. 5. Electron microscopy of chromatin-depleted M617 cells. (a) DNase I-extracted telophase cell. Note the midbody region between of a the two telophase cells (arrow). The rectangular region is further enlarged in Fig. 5b. Bar = 2 pm. (b) Electronmicrograph cytoplasmic area of a telophase cell. Preembedding staining with anti-NMPl25 and goat anti-mouse/l5 nm gold conjugate. Note the association of the antigen with filaments. Bar = 200 nm. (c) Representative area of negative control cell in telophase. Bar = 200 nm. (d) Higher magnification of the rectangular area of Fig. 5b. Note the colocalization of the antigen with filaments of approx. 10 nm diameter. Gold particles = 15 nm; bar = 80 nm. (e) DNase I-extracted cell in interphase demonstrating the nuclear matrix. enm, external nuclear matrix (peripheral nuclear lamina); inm, internal nuclear matrix (nuclear core filaments) with residual nucleoli (n). Bar = 2 pm.

MITOTIC

DYNAMICS

OF A NUCLEAR

gold particles could be found as single particles along filaments or as clusters on globular supramolecular structures corresponding to the punctuate aggregates found in immunofluorescence pictures (Figs. 3c, 8a, and 8b). In negative controls that included nonimmune serum instead of the primary antibody and secondary antibody without primary antibody, only a few gold particles could be found (Fig. 5c).

Since M617 cells express the intermediate-type filament vimentin and the monoclonal antibody does not crossreact with vimentin, we decided to doublestain cytoskeletal proteins together with NMP125. Figure 6a shows interphase cells with the typical distribution of NMP125 within the nucleus without any immunoreactivity within the cytoplasm. Vimentin filaments in the same cells yield the typical cytoplasmic distribution around the nucleus (Fig. 6b). In prophase cells NMP125 is spread throughout the cytoplasm with an accumulation of the protein around the condensed chromatin (Fig. 6~). The vimentin pattern in the same cells demonstrate a compact distribution pattern around the forming chromosomes with a distribution similar to that of NMP125 (Fig. 6d). In late telophase NMP125 forms a granular pattern within the cytoplasm and the beginning reorganization within the newly forming nuclei (Fig. 6e). The granules can be colocalized with the intermediate filaments (Fig. 60. The transient character of the affinity of NMP125 to vimentin during mitosis is shown in Fig. 7. The cells were extracted with Triton X-100 before fixation and double-staining. In prophase cells NMP125 (Fig. 7a) is evidently colocalized with the vimentin basket around the forming chromosomes (Fig. 7b) indicating a rapid transfer of the nuclear matrix protein to the cytoplasmic cytoskeleton. A higher magnification of the cytoplasm of a cell in late telophase is shown in Figs. 8a and 8b. The cells were double-stained with anti-vimentin and antiNMP125, but the same secondary antibody (goat anti-mouse IgG conjugated with FITC) was used. Vimentin as filaments and NMP125 as telophase granules can be seen simultaneously within the same cell. The colocaiization of the two proteins in these immunofluorescence pictures (Figs. 7c and 7d) and in immunoelectron micrographs (Fig. 5) is evident. Since vimentin can be extracted by urea and partially purified by dialysis against a vimentin assembly buffer, we could demonstrate in immunodot tests that after dialysis vimentin together with NMP125 can be detected in the pellet fraction (Fig. 8a, inset: 2, vimentin spot; 3, NMP125) after centrifugation of the dialysate, whereas only small quantities could be detected in the supernatant (Fig. 8a, inset: 1, NMP125).

MATRIX

135

PROTEIN DISCUSSION

NMP125 Is a Nuclear Matrix Evolutionary Conservation

Protein

with

Monoclonal antibodies obtained by immunization of mice with a crude cytoskeletal and nuclear fraction of bovine spinal cord reveal a granular staining of M617 cell nuclei when stained by indirect immunofluorescence. Crossreactivity with known nuclear matrix proteins such as lamins can be excluded, because these proteins differ significantly in their molecular weights (62-72 kDa for the lamins) and, in addition, reveal a completely different nuclear distribution in immunofluorescence studies compared with the nuclear staining of NMP125. The absence of fluorescence within the chromosomes indicates that anti-NMP125 does not recognize a chromatin structure, centromeres, kinetochores, or an already known nuclear scaffold protein (Mirkovitch et al., 1984). To characterize the cellular state of NMP125, we used well-established fractionation methods combined with immunofluorescence staining and ultrastructural studies of differentially extracted cells in situ (Staufenbiel and Deppert, 1984; Mirkovitch et al., 1984; Ascoli and Maul, 1991; Fey et al., 1986). Our results revealed that NMP125 was not extracted by any of these treatments. All extracts were analyzed by SDS-PAGE. In addition, the extraction efficiency was monitored by electron microscopy and bisbenzimide staining (not shown) to visualize the DNA. The results of the differential extraction of the cells in situ allowed us to conclude that NMP125 fulfills the criteria to be a nuclear matrix protein. At least at the level of immunological crossreactivity in man, rat, and chicken, we have found that NMP125 is conserved during evolution. In addition NMP125 can be detected in immunoblots of Xenopus brain tissue (Dr. B. Riederer, University of Lausanne, Switzerland, personal communication). We therefore suggest that this conservation could reflect a functional importance at least in amphibians, birds, and mammals. The Distribution of NMP125 Is Cell-Cycle-Specific and Exhibits a Transient Affinity for Vimentin Filaments during Mitosis

There are similarities of NMP125 with lamins A and C that are also released into the cytoplasm at the onset of nuclear envelope breakdown. At that stage these two lamins become soluble proteins, whereas lamin B remains attached to membrane structures (reviewed by Gerace and Burke, 1988). In contrast to the lamin dynamics, NMP125 is a part of the insoluble, detergent-resistant pool of cytoplasmic proteins in mitotic cells. This indicates a different dynamic behavior of NMP125 compared with

136

R. A. MARUGG

FIG. 6. Double-immunostaining of M617 cells in representative mitotic stages with anti-NMPl25 (a, c, e) and anti-vimentin (b, d, D. Bar = 5 pm for all figures. (a, b) Cells in interphase. Note the absence of NMP125 in the cytoplasm in a and the vimentin basket around the nucleus in b. (c, d) Cell in an early mitotic stage. Note the NMP125 distribution in the cytoplasm in c and the colocalization of NMP125 and vimentin in d around the condensed DNA. (e, f) Cell in telophase. NMP125 aggregates within the cytoplasm and around the newly forming nuclei in e and vimentin in f.

lamins A and C at a mechanistic level. A cell-cycledependent behavior of other nuclear proteins such as centromer- or kinetochor-associated proteins has

also been reported (Compton et al., 1991; Mirkovitch et al., 1984). Most of these nuclear scaffold proteins have been localized to the chromosome scaffold by

MITOTIC

DYNAMICS

OF A NUCLEAR

FIG. 7. Double-immunostaining of NMP125 and vimentin after extraction istry with anti-NMP125 and goat anti-mouse IgG FITC (see Fig. 3a). Bar = 5 same cells. Note the colocalization of the two proteins around the chromosomal basket around the nuclear area (b), where most of NMP125 can be found (a).

monoclonal antibodies. Nevertheless, dissociation of two scaffold antigens from chromosomes after anaphase and accumulation at the midplate region has been described (Pankov et al., 1990; Yen et al., 1991). The metaphase scaffold that is a compound of a subset of nonhistone proteins is localized along the axis of the metaphase chromatid (Mirkovitch et al., 1984). Other nuclear matrix domains like mRNA processing sites, sites of tRNA, and 5s RNA synthesis disappear or redistribute with advancing mitosis (Ascoli and Maul, 1991). Our observations concerning the cell-cycle-specific dynamic behavior of NMP125 differ from the dynamics of all these scaffold proteins because during DNA condensation into chromosomes NMP125 dissociates from nuclear structures and the chromosomes remain unstained during all mitotic stages. Since vimentin can be dissolved in 8 M urea and reassembled into filaments by dialysis against an assembly buffer, we could demonstrate that NMP125 can be centrifuged together with reassembled vimentin filaments. In addition, we could colocalize NMP125 and vimentin in mitotic cells with double-immunostainings, and we have shown the association of NMP125 to intermediate-type filaments at an ultrastructural level. These results together with the insolubility of NMP125 in mitotic cells could indicate a novel mechanism of dissociation and redistribution of nuclear matrix proteins during mitosis. This mechanism implies the transient aspect of NMP125/vimentin interaction. Since we could never detect a significant soluble pool of NMP125 in detergent extracts, we concluded that a very rapid binding of the

MATRIX

PROTEIN

of soluble proteins with detergents. (a) Immunocytochemkm. (b) Anti-vimentin and goat anti-mouse TRITC in the area of the prophase cell. The vimentin filaments form a Bar = 5 pm.

protein to vimentin filaments occurred. This is supported by double-immunostainings of cells in prophase, in which most of NMP125 could be colocalized with the vimentin basket around the nuclear area. In subsequent mitotic stages the vimentin/NMP125 conjugate is divided into two parts and redistributed in approximately equal amounts to the two daughter cells. Preliminary semiquantitative analysis of the amount of NMP125 allowed us to speculate that the synthesis of NMP125 in the two daughter cells starts after telophase (results not shown). NMP125 reappears in the newly forming nuclei in late telophase cells, whereas the cytoplasmic NMP125 forms punctuate units of relatively large size thus suggesting an aggregation of the antigen or an association with globular supramolecular structures. These granules gradually disappear in late telophase cells and can be detected no more in interphase cells. These findings could implicate an aggregation of the protein and subsequent transport to the nucleus, or it could probably reflect protein synthesis to double the protein amount within the two daughter cells. Another possible explanation of the disappearance of these granules could be that of the degradation of the remaining cytoplasmic pool of the protein. The reappearance of the protein in the newly formed nuclei let us speculate that the mechanism of redistribution of NMP125 could be important for the reconstruction of the nuclear matrix architecture. The affinity of NMP125 to vimentin filaments during mitosis supports a passive intracellular transport mechanism of a nuclear protein. The functional role

138

R. A. MP LRUGG REFERENCES Aaronson, R. P., and Blobel, G. (1975) Isolation of nuclear pore complexes in association with a lamina, Proc. N&l. Acad. Sci. USA 72, 1007-1011. Aebi, U., Cohn, J., Buhle, L., and Gerace, L. (1986) The nuclear lamina is a meshwork of intermediate type filaments, Nature 323, 560-564. Ascoli, C. A., and Maul, G. G. (1991) Identification of a novel nuclear protein, J. Cell Biol. 112, 785-795. Balter, M. (1991) Cell cycle Science 252, 1253-1254. Berezney, R. (1980) Biol. 85, 641-646.

Fractionation

research:

Down of the

to the

nuclear

nitty

matrix,

gritty, J. Cell

Berezney, R., and Coffey, D. S. (1974) Identification of a nuclear protein matrix, Biochem. Biophys. Res. Commun. 60, 14101417. Berezney, R., and Coffey, D. S. (1975) Nuclear protein matrix: Association with rapidly labeled DNA, Science 189, 291-292. Berezney, R., and Coffey, D. S. (1977) Nuclear matrix, J. Cell Biol. 73, 616-637. Capco, D. G., Wan, K. M., and Penman, S. (1982) The nuclear matrix: Three-dimensional architecture and protein composition, Cell 29, 847-858. Ciejek, E. M., Tsai, M. J., and C’Malley, B. W. (1983) Actively transcribed genes are associated with the nuclear matrix, Nuture 306, 607-609.

FIG. 8. Simultaneous labeling of NMP125 aggregates and vimentin in telophase cells. Immunodot test (inset). (a and b) Detail micrographs of the cytoplasmic area of telophase cells which are labeled with anti-NMP125 and anti-vimentin. Both antibodies were labeled with the same secondary antibody (goat anti-mouse IgG FITC) to visualize simultaneously both antigens. Most of the NMP125 aggregates can be colocalized with vimentin filaments (see also Fig. 5). Bars = 1 pm. (Inset) Immunodot test of urea extracts of detergent insoluble proteins after dialysis against vimentin assembly buffer. 1, Supernatant dot stained with antiNMP125; 2, pellet dot stained with anti-vimentin; 3, pellet dot stained with anti-NMPl25. NMP125 can be cocentrifuged with assembled vimentin filaments after dialysis against vimentin assembly buffer.

of intermediate filaments is essentially structural and mechanical, but different dynamic aspects of intermediate filaments have been reported (Steinert and Liem, 1990). Vimentin attachment sites occurring at the nuclear envelope in vitro, namely an attachment to lamin B has been described (Georgatos and Blobel, 19871, and an association between peripherin (a neuronal, type III IF subunit) and lamin B has been reported (Djiabali et al., 1991). Mediated by intermediate filaments, the nuclear matrix protein gets closer to the newly formed nuclei in telophase cells. A regulatory control of this mechanism could be the cyclin-dependent phosphorylation (Murray, 1989).

Compton, D. A., Yen, T. J., and Cleveland, D. W. (1991) Identification of novel centromer/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds, J. Cell Biol. 112, 1083-1097. Dacheng, H., Nickerson, J. A., and Penman, S. (1990) Core filaments of the nuclear matrix, J. Cell Biol. 110, 569-580. Delacourte, A., Filliatreau, G., Boutteau, F., Biserte, G., and Schrevel, J. (1980) Study of the lo-nm-filament fraction isolated during the standard microtubule preparation, Biochem. J. 191, 543-546. Djabali, K., Portier, M.-M., Gros, F., Blobel, G. (1991) Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments, Cell 64, 109121. Fey, E. G., Krochmalnic, G., and Penman, S. (1986) The nonchromatin substructures of the nucleus: The ribonucleoprotein (RNP)-containing and RNP-depleted matrices analysed by sequential fractionation and resinless section electron microscopy, J. Cell Biol. 102, 1654-1665. Fey, E. G., Wan, K., and Penman, S. (1984) Epithelial cytoskeleta1 framework and nuclear matrix-intermediate filament scaffold: Three-dimensional organization and protein composition, J. Cell Biol. 98, 1973-1984. Fisher, D. Z., Chaudhary, N., and Blobel, G. (1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filaments, Proc. Natl. Acad. Sci. USA 83, 6450-6454. Franke, W., Scheer, U., Krohne, G., and Jarasch, E. (1981) The nuclear envelope and the architecture of the nuclear periphery, J. Cell Biol. 91, 399-50s. Georgatos, S. D., and Blobel, G. (1987) Lamin B constitutes an intermediate filament attachment site at the nuclear envelope, J. Cell Biol. 105, 117-125. Gerace, L., and Blobel, G. (1980) The nuclear envelope lamina reversibly depolymerized during mitosis, Cell 19, 277. Gerace, L., and Burke, B. (1988) Functional organization nuclear envelope, Annu. Rev. Cell Biol. 4, 336-376.

is

of the

MITOTIC Glass, J. R., and Gerace, assemble at the surface 111, 1047-1057.

L. (1990) of mitotic

DYNAMICS

OF A NUCLEAR

Lamins A and C bind and chromosomes, J. Cell Biol.

Hakes, D. J., and Berezney, R. (1991) DNA binding properties of the nuclear matrix and individual nuclear matrix proteins: Evidence for salt-resistant DNA binding sites, J. Biol. Chem. 266, 11131-11140. Jackson, D. A., McCready, S. J., and Cook, P. R. (1981) RNA is synthesized at the nuclear cage, Nature 292, 552-555. Marugg, R. A., and Baier-Kustermann, W. (1988) Electroelution of high yield purified neurofilament constituent proteins: Monoclonal antibodies directed against structurally defined NF binding sites, in Rousset, B. A. F., (Ed.), Structure and Function of the Cytoskeleton, Vol. 171, pp. 19-24, Colloque INSERMJohn Libbey Eurotext Ltd. Mirkovitch, J., Mirault, M.-E., and Laemmli, U. K. (1984) Organization of the higher-order chromatin loop: Specific DNA attachment sites on nuclear scaffold, Cell 39, 223-232. Murray, A. W. (1989) The cell cycle as a cdc2 cycle, Nature 342, 14-15. Nelson, W. G., Pienta, K. J., Barrack, E. R., and Coffey, D. S. (1986) The role of the nuclear matrix in the organization and function of DNA, Annu. Rev. Biophys. Chem. 15, 457-475. Paddy, M. R., Belmont, A. S., Saumweber, H., Agard, D. A., and Sedat, J. W. (1990) Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery, Cell 62, 89-106.

MATRIX

PROTEIN

139

Pankov, R., Lemieux, M., and Hancock, R. (1990) An antigen located in the kinetochore region in metaphase and on polar microtubule ends in the midbody region in anaphase, characterized by a monoclonal antibody, Chromosoma (Berlin) 99, 95101. Schroder, H. C., Bachmann, M., Diel-Seifert, B., and Muller, W. E. (1987) Transport of mRNA from nucleus to cytoplasm, Prog. Nucleic Acid Res. Mol. Biol. 34, 89-141. Staufenbiel, M., and Deppert, W. (1984) Preparation of nuclear matrices from cultured cells: Subfractionation of nuclei in situ, J. Cell Biol. 98, 1886-1894. Steinert, P. M., and Liem, R. K. H. (1990) Intermediate filament dynamics, Cell 60, 521-52.3. Van Eekelen, C. A. G., Salden, M. H. L., Habets, W. J. A., van de Putte, L. B. A., and van Venrooij, W. J. (1982) On the existence of an internal nuclear protein structure in HeLa cells, Exp. Cell Res. 141, 181-190. Van Eekelen, C. A. G., and Van Venrooij, W. J. (1981) hnRNA and its attachment to a nuclear protein matrix, J. Cell Biol. 88, 554-563. Verheijen, R., Van Venrooij, W., and Ramaekers, F. (1988) J. Cell Sci. 90, 11-36. Yen, T. J., Compton, D. A., Wise, D., Zinkowski, R. P., Brinkley, B. R., Earnshaw, W. C., and Cleveland, D. W. (1991) CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase, EMBO J., 10, 12451254.