Journal of Histochemistry and Cytochemistry, Vol. 47, 1357-1368, November 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Talin Concentrates to the Midbody Region During Mammalian Cell Cytokinesis

Anne Bellissent–Waydelicha, Marie-Thérèse Vaniera, Corinne Albigès–Rizob, and Patricia Simon–Assmanna
a INSERM U381, Strasbourg, France
b UMR 5538 CNRS, Institut Albert Bonniot, Grenoble, France

Correspondence to: Patricia Simon–Assmann, Unité NSERM 381, 3 avenue Molière, 67200 Strasbourg, France. E-mail: Patricia.Simon-Assmann@inserm.u-strasbg.fr


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

In this study we investigated the cellular distribution of talin, a cytoskeletal protein, during mammalian cell cytokinesis. Immunohistochemical experiments on various carcinoma cell lines and mesenchyme-derived cells reveal that talin displays a cell cycle-dependent cellular localization. During metaphase, talin is located in the centromeric region of the chromosome, like the TD-60 protein and intrinsic centromere components detected by a CREST serum. From anaphase to telophase, talin is present in the cleavage furrow. As the cells progress to cytokinesis, when the furrow is complete, talin is concentrated in the midbody structures, as assessed by immunofluorescence and confirmed by Western blot experiments on purified midbodies. Double staining experiments reveal that {alpha}-tubulin, TD-60 protein, and talin co-localize in the midbodies. These results suggest that talin, in addition to its implication in focal adhesion organization and signaling, may play a critical role in cytokinesis. (J Histochem Cytochem 47:1357–1367, 1999)

Key Words: talin, cytoskeletal protein, mitosis, cleavage furrow, chromosomes, centromeres, midbodies, colon cancer cells


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Intestinal morphogenesis and cell differentiation are dependent on heterotypic cell interactions (for a review see Kedinger et al. 1997 ). These reciprocal interactions between endodermal and mesenchymal cells first lead to the formation of a specialized extracellular matrix structure, the basement membrane (Simon-Assmann et al. 1995 , Simon-Assmann et al. 1998 ). Later, tissue differentiation results in changing and reciprocal dynamics between the cells and the extracellular matrix elements (Roskelley et al. 1995 ; Hagios et al. 1998 ). It has been proposed that cell–matrix interactions regulate gene expression via integrins (cell surface receptors), which can influence cytoskeletal organization. In vitro, integrin engagement and clustering lead to the formation of focal adhesions, also called focal contacts. The constituent cell adhesion molecules, first known to have a structural role in the attachment of cells to the substrate, are also considered to be signal transducers (Burridge and Chrzanowska-Wodnicka 1996 ).

The molecular architecture of focal adhesions is complex, and the numbers of proteins identified in the cytoplasmic face have greatly expanded. The relative abundance of the individual constituents, including talin, vinculin, paxillin, tensin, and {alpha}-actinin, varies considerably (Jockusch et al. 1995 ). Talin is particularly interesting because of its direct link with the integrin ß1-subunit (Horwitz et al. 1986 ). The human platelet talin has been proposed to be a dumbbell-shaped homodimer (Goldmann et al. 1994 ), whereas talin purified from chicken smooth muscle appeared as a majority of flexible monomers consisting of a series of globular domains (Winkler et al. 1997 ). The apparent molecular mass of the peptide is approximately 225 kD (Molony et al. 1987 ; Isenberg and Goldmann 1992 ; Goldmann et al. 1996 ). Talin can be cleaved by calpain II, a calcium-dependent protease also found in focal contacts, into an N-terminal and a C-terminal fragment with apparent molecular masses of 47 kD and 190–200 kD, respectively (Beckerle et al. 1987 ; Muguruma et al. 1995 ).

During mitosis, cells are rounding up and most focal adhesion plaques disassemble. This step is characterized by dramatic morphological changes, which occur in a strictly sequential order and include cytoskeletal disassembly, breakdown of the nuclear envelope, chromatin condensation, chromosome segregation and, finally, daughter cell separation. In the last stage of mitosis in animal cells, a cleavage furrow forms to separate the two daughter cells. It is interesting that an increasing number of cytoskeletal proteins are located in the furrowing region. Among these are proteins usually located at the cell–cell or at the cell–substrate junctions, such as radixin (Sato et al. 1991 ) and {alpha}-actinin (Fujiwara et al. 1978 ), respectively. The timing and the site of accumulation of the proteins located or concentrated at the cleavage furrow during cytokinesis vary from one molecule to the other. Despite intensive morphological and biochemical characterization of the cleavage formation, our knowledge about the molecular architecture of this structure is still not complete. Concomitantly, invagination of the plasma membrane occurs in the cleavage furrow as the result of actin–myosin interactions (for review see Fishkind and Wang 1995 ). Finally, cytokinesis is most probably accomplished by contraction of this actin ring and leads to separation of two daughter cells at the midbody. The regulation of this step is not yet clearly understood.

Here we report for the first time that talin displays a cell cycle-dependent localization. During mitosis, talin is first localized to the centromeric region of metaphase chromosomes; then it accumulates in the cleavage furrow region during anaphase and telophase, and finally becomes concentrated in the midbody region.


  Materials and Methods
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Materials and Methods
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Cell Lines and Antibodies
HT29 and Caco-2 cells are derived from human colon adenocarcinomas (Fogh and Trempe 1975 ). The HT29 cells were propagated in DMEM (Life Technologies–Gibco BRL; Cergy–Pontpoise, France) containing 10% heat-inactivated fetal calf serum (FCS; Life Technologies–Gibco BRL) and the Caco-2 cells in DMEM containing 20% heat-inactivated FCS and 1% nonessential amino acids (Life Technologies–Gibco BRL). The MCF7 human breast adenocarcinoma cells (kindly provided by Dr. M. C. Rio; INSERM U184, Illkirch, France) and the AR42J cells from rat pancreatic tumor (obtained from Dr. N. Vaysse; INSERM U.151, Toulouse, France) were cultured in DMEM supplemented with 10% heat-inactivated FCS.

The A1F1 intestinal fibroblast cell line derived from postnatal rat intestinal mucosa was provided by Fritsch et al. 1997 . Cells were propagated in DMEM with 5% heat-inactivated FCS, 0.25 IU/ml insulin (Sigma: Saint–Quentin Fallavier, France) and 10 ng/ml epidermal growth factor (EGF; Chemicon, Temecula, CA). Primary cultures of skin fibroblast cells were obtained by enzymatic dissociation of 20-day-fetal rat skin dermis as described previously (Kedinger et al. 1987 ). The culture medium is composed of a mixture (1:1) of DMEM and Ham F12 medium supplemented with 15% heat-inactivated FCS.

The 8d4 monoclonal antibody (Sigma) and the B11 polyclonal antibody (kindly provided by Dr. Beckerle (University of Utah, Salt Lake City) (Beckerle et al. 1989 ) were used for immunofluorescence and immunoblotting experiments. Both antibodies recognize the native and the cleaved form of talin (Beckerle et al. 1989 ; Otey et al. 1990 ). The following mouse MAbs were used in this study: N357 specific for ß-tubulin (Amersham, Orsay, France); h-Vin1 recognizing vinculin (Sigma), and antibody to desmoplakin I-II (Boehringer–Mannheim; Meylan, France). The YL1/2 rat MAb against tyrosinated {alpha}-tubulin was purchased from Harlan Sera-Lab (Loughborough, UK). Actin was visualized by immunofluorescence with phalloidin coupled to rhodamine (Molecular Probes; Eugene, OR). The anti-TD60 antibody (JH human autoimmune serum), recognizing a mitosis-specific human autoantigen (a kind gift of Dr. R. L. Margolis and Dr. P. R. Andreassen; Institut de Biologie Structurale J. P. Ebel, Grenoble, France) was also used (Andreassen et al. 1991 ; Martineau et al. 1995 ). An anti-centromere autoimmune serum coming from a patient displaying CREST syndrome was a generous gift from Dr. Goetz (CHU; Strasbourg, France). The HBB 2/614/88 antibody recognizing sucrase, a digestive brush-border membrane enzyme, was kindly provided by Dr. H. P. Hauri (Basel, Switzerland) (Hauri et al. 1985 ).

FITC-conjugated sheep anti-mouse (Institut Pasteur; Paris, France), FITC-conjugated goat anti-human (Jackson Immunoresearch; West Chester, PA), rhodamine-conjugated goat anti-rabbit (Nordic; Tilburg, The Netherlands), lissamine–rhodamine-conjugated goat anti-rat (Jackson), and Texas Red-conjugated sheep anti-mouse (Amersham) secondary labeled antibodies were used for immunofluorescence studies. For Western blot experiments, sheep anti-mouse secondary antibody coupled with horseradish peroxidase (Amersham) was used.

Immunofluorescence on Cultured Cell Monolayers
A total of 5 x 104 cells were cultured on glass coverslips for 3–5 days and were used before they reached confluency. For the AR42J cell line, the cells were cultured on coverslips precoated with 400 µg/ml of polylysine. Cells were washed briefly with PBS buffer, fixed for 10 min with 1% paraformaldehyde in PBS at room temperature (RT) and then permeabilized with 1% Triton X-100 in PBS for 10 min. Cells were incubated for 1 hr with the first antibody and then for 30 min with the appropriate secondary antibody. For simultaneous detection of talin and actin, fixed cells were first incubated with the primary 8d4 anti-talin MAb and then with a mixture of the secondary antibody and rhodamine-conjugated phalloidin. For double immunostaining experiments to detect talin and microtubules, we used the two-step procedure of crosslinking with DSP (dithiobis succinidylpropionate) and extracting in Triton X-100 in a microtubule-stabilizing buffer (Safiejko-Mroczka and Bell 1996 ). After that, cells were fixed in methanol for 5 min at -20C and double immunofluorescence staining was done by simultaneous incubation with the two primary antibodies, followed by simultaneous incubation with the two indicated secondary antibodies. When necessary, the nucleus was stained for 10 min with 50 ng/ml Hoechst (Sigma) in Hanks' medium. Controls consisted of substitution of the primary antibodies with an irrelevant antibody or nonimmune serum at the same dilution. The preparations were mounted in an anti-fading PBS–glycerol solution, observed under a fluorescence microscope (Axiophot; Zeiss, Oberkochen, Germany), and photographed with HP5 film (Asa 400; Ilford; Champs–sur–Marne, France). Color pictures were obtained using a Color Video Printer (SONY). Image processing was performed with Imaging C (image analySIS application) and Adobe Photoshop 5.0.

For confocal study, the Caco-2 cells were doubly stained with the B11 polyclonal anti-talin antibody and with the human CREST serum. The coverslips were fixed with nailpolish and observed under a confocal microscope (Zeiss). The software used to determine co-localization indicates by light-blue dots that there is co-localization when the distance between two points is less than 0.2 µm.

Isolation of Metaphase Chromosomes
Metaphase chromosomes were isolated from mitotic Caco-2 cells obtained by blockade with nocodazole (1 µg/ml) for 16 hr, according to the technique of Saitoh and Laemmli 1994 . The selectively detached mitotic cells were washed three times with PBS at 4C; this causes chromosomes to cluster and increases the yield significantly (Andreassen et al. 1997 ). Chromosomes were purified through a discontinuous glycerol gradient by use of a polyamine buffer system as outlined by Blumenthal et al. 1979 . The purified chromosomes migrating as a flocculent band were harvested and the cytoskeletal material remaining at the top was collected.

The purity of the chromosome preparation was checked with Hoechst staining, and both chromosome and cytoskeletal preparations were used for immunoblotting experiments. Proteins were resuspended in Laemmli buffer containing 2% SDS and 100 mM DTT and were boiled for 5 min. Samples were analyzed on 5% SDS-PAGE and electrophoretically transferred overnight onto nitrocellulose in transfer buffer (25 mM Tris HCl, 192 mM glycine, pH 8.3, 20% methanol). After transfer, the nitrocellulose was first saturated with 1% BSA, 0.5% gelatin from porcine skin (Sigma), 0.1% Tween-20 in 25 mM PBS, pH 7.4, 1 M NaCl for 1 hr at 37C and then incubated for 2 hr at RT with MAb 8d4 diluted 1:500 in 25 mM PBS, 1 M NaCl, pH 7.4, containing 0.3% BSA and 0.3% Tween-20. After washings, the nitrocellulose sheets were incubated for 1 hr with the sheep anti-mouse secondary antibody coupled to horseradish peroxidase (Amersham) and treated with the enhanced chemiluminescence (ECL) reagent according to the supplier (Amersham). Prestained molecular mass markers (Biorad; Ivry sur Seine, France) were included in each gel.

Midbody Isolation
Caco-2 cells were seeded at 1.5 x 106 cells per flask (75 cm2) and were taken on Day 5 during the phase of active growth (Pinto et al. 1983 ). Midbody isolation was performed according to Mullins and McIntosh 1982 on cells that had been detached from the culture flask after EDTA treatment (0.5 mM EDTA final in PBS). At that time, cells were pelleted at 200 x g for 3 min and gently resuspended in 25 volumes of a hypotonic swelling solution consisting of 1 M hexylene glycol (2-methyl-2,4-pentanediol), 20 µM MgCl2, and 2 mM piperazine-N-N'-bis(2-ethane sulfonic acid) (PIPES), pH 7.2, at RT. Cells were immediately pelleted at 200 x g for 3 min and vigorously resuspended in 50 volumes of a lysing solution consisting of 1 M hexylene glycol, 1 mM EGTA, 1% Nonidet P-40, 2 mM PIPES, pH 7.2, at 37C. Disruption of the cells and release of midbodies were completed by vigorous vortexing for 1 min in this solution. Midbodies were then stabilized by chilling on ice and by adding to the lysate 0.3 volume of cold 1 M hexylene glycol, 50 mM 2-(N-morpholino) ethane sulfonic acid (MES), pH 6.3, to lower the pH. After centrifugation at 250 x g for 10 min to remove large debris, the supernatant was layered over a cushion of 40% glycerol (w/v) in 50 mM MES, pH 6.3, and centrifuged for 45 min at 2800 x g. This pellet was resuspended in 50 mM MES, pH 6.3, and centrifuged again through 40% glycerol. The pellet containing the midbodies was finally resuspended in MES buffer and used for immunoblotting or immunofluorescence experiments.

For electrophoresis, the midbody samples were resuspended in Laemmli buffer under reducing conditions, run on 5% or 7.5% SDS-PAGE, and electrophoretically transferred to nitrocellulose as described above and processed for immunoblotting using the ECL technique. For immunofluorescence studies, the midbody sample was centrifuged using cytobuckets at 200 x g for 10 min onto adhesive slides (Starfrost; Microm, Francheville, France). Immunofluorescence experiments were performed as described above, the midbodies being incubated directly with the first antibodies or with the secondary antibodies as controls.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Conventional and Unconventional Localization of Talin in the Human Caco-2 Cells
The distribution of talin was studied in human colon cancer Caco-2 cells by indirect immunofluorescence experiments after several days in culture. The two talin-specific antisera yielded identical results. Talin was clustered in thin patches over the entire basal pole of the cells in a pattern typical of focal contacts (Figure 1A). The distribution of vinculin, another focal adhesion molecule, was similar to that of talin, as confirmed by double immunostaining experiments showing co-distribution of talin and vinculin in the basal patches (Figure 1B vs 1A). Talin was also detected, although faintly, at sites of cell–cell contacts as a thin staining (Figure 1C). Given this unusual location of talin in cell–cell contacts, double immunostaining experiments were performed using talin and desmoplakin (a marker of desmosomal plaques) antibodies. As shown in Figure 1C and Figure 1D, both antibodies delineated the lateral membrane in areas of cell–cell contact.



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Figure 1. Immunolocalization of talin in the human Caco-2 colon cancer cell line. Indirect immunofluorescence experiments were performed using monoclonal anti-talin (A,C,E), anti-vinculin (B), and anti-desmoplakin (D) antibodies. Talin is mainly expressed in basal patches typical of focal adhesions (A) and, at an upper focus, is also localized at the lateral cell contacts (C). Double staining experiments show that talin (A) and vinculin (B) are co-distributed in patches at the ventral part of the cells and that talin (C) and desmoplakin (D) are both localized laterally at cell–cell contacts. Arrows in A and B point to the same areas. In some cells scattered within the monolayer, talin staining is located in two bright rods (E) separated by a nonfluorescent spot (arrow) corresponding to a dark dot in phase-contrast microscopy (F, arrow). Bars: AD = 15 µm; E, F = 5 µm.

Talin was also found clustered in very bright structures, generally perpendicular to the plasma membrane between two adjacent dividing cells and at a focus higher than that of the basal patches (Figure 1E). These structures were composed of two line segments on the inner side of both plasma membranes; the central space, devoid of talin immunostaining (Figure 1E, arrow), corresponds to a thickening of the plasma membrane, as shown by phase-contrast microscopy on the same cell (Figure 1F, arrow). The various unrelated or secondary antibodies used never yielded such a typical staining.

Distribution of Talin Varies as a Function of the Cell Cycle
Because such structures look like the midbodies formed by the microtubules at the end of cytokinesis in animal cells, we analyzed the distribution of talin during the different phases of mitosis. For this purpose, Caco-2 cells were double stained with anti-talin antibodies and Hoechst, allowing visualization of the nucleus; similar data were obtained whatever antibody was used. During interphase, when chromatin was decondensed, talin was localized in basal patches and, to a lesser extent, laterally along the plasma membrane (as shown in Figure 1A and Figure 1C). During prophase, when chromatin began to condense in chromosomes, there was a decrease in the number of basal patches in the rounded mitotic cells (not shown). At metaphase, when chromosomes were becoming placed on the equatorial plate (Figure 2A), talin staining was visible as small dots in the central region where the chromosomes were located (Figure 2A'). This staining was more obvious when the polyclonal antibody was used. At early anaphase, talin began to be detected, although faintly, at the equatorial cell surface (not shown). At late anaphase, when chromosomes have separated (Figure 3A), talin immunostaining was clearly detected in the cleavage furrow within several strokes lying almost perpendicular to the equator (Figure 3A'). Later in telophase, chromosomes were less distinguishable and formed crescent daughter nuclei (Figure 3B), and talin strokes got closer to each other, staying perpendicular to the equator in concert with the decreasing diameter of the furrow (Figure 3B'). When chromosomes were no longer distinguishable and formed grossly smooth nuclei (Figure 3C), talin was present as two bright short rods, always almost perpendicular to the equatorial plane, separated by an unstained region corresponding to the dense matrix material of the midbody (Figure 3C'). In the late stage of cytokinesis, the nuclei were indistinguishable from typical interphase nuclei (Figure 3D), basal patches of talin had reappeared at the ventral part of the cell (not shown), and talin staining in the furrowing region was reduced as two thin strokes (Figure 3D').



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Figure 2. Distribution of talin in the centromeric region and Western blot analysis. Double staining experiments were performed to visualize DNA (A,B) and talin (A') or TD-60 (B'). On an upper view of a cell in metaphase (A), one can note that generally one green dot of talin (A', arrow) corresponds to one chromosome (A, arrow). Similar immunostaining was obtained with the TD-60 antibodies known to stain the centromeric region (B,B'). (C) Confocal analysis showing a cell doubly immunostained with polyclonal anti-talin antibody in red and with an anti-centromere autoimmune serum (CREST) in green. The presence of light-blue dots indicates that talin co-localized with centromeres. Bars = 5 µm. (D) Western blot analysis of a metaphase chromosome preparation with anti-talin antibodies (Lane a) shows the presence of talin in chromosomes. Note the rather low amount of talin in the metaphase chromosomes (Lane a) compared to the corresponding cytoplasmic fraction (Lane b) because lanes were loaded with proteins purified from original equivalent cell numbers. Molecular weight markers are indicated at left.

Figure 3. Distribution of talin during mitosis in Caco-2 cells. Cells were doubly stained with Hoechst and monoclonal anti-talin antibodies to visualize respectively DNA (A–D) and talin (A'D'). Cells are depicted at various mitotic stages: late anaphase (A,A'), early to late telophase (B,C'), and ending telophase (D,D'). Bars = 5 µm.

Talin Is Located in the Centromeric Region
During metaphase, as described above, polyclonal anti-talin antibodies labeled small dots in the central region where chromosomes are located. A higher view of a cell in metaphase, allowing visualization of well-separated chromosomes, suggested that one spot of talin staining might correspond to the primary constriction found between chromosome arms (Figure 2A' vs 2A, arrows). The localization of fluorescence to discrete foci in metaphase cells indicated a possible association of talin with mitotic centromeres. Such staining was also observed with the anti-TD 60 antibody (Figure 2B and Figure 2B'). This antigen, localized in centromeres and midbodies, is a marker of the telophase disc shown to bisect telophase cells (Andreassen et al. 1991 ). We also performed double immunostaining experiments on Caco-2 cells with an anti-centromere autoimmune serum from a patient with CREST syndrome. In metaphase cells, the talin-positive dots were found in the region where the autoimmune serum decorated the chromosomes in a speckled pattern. Confocal microscopic study of such double stained cells was performed in which visualization of talin was achieved by using the B11 polyclonal antibody (rhodamine labeling) and the human CREST anti-centromere antiserum (FITC labeling). Superimposition of the pictures (Figure 2C) showed that most of the anti-centromere immunoreactive dots were stained by the anti-talin antibodies.

Biochemical analysis of metaphase chromosomes from mitotic Caco-2 cells and of the corresponding cytoplasmic fraction was performed by SDS-PAGE and immunoblotting with anti-talin antibodies (Figure 2D). The results show that the antibody recognizes a 230-kD protein in the purified chromosome fraction (Figure 2D, Lane a) that corresponds to the native talin. Comparative analysis performed with the cytoplasmic fraction from equivalent cell numbers (Figure 2D, Lane b) revealed, however, that the amount of talin on chromosomes is rather low.

Talin Is Located in the Midbody Structure
At the conclusion of telophase, when the furrow is complete, talin staining is restricted to structures resembling midbodies. Because tubulin has been shown to be the major component of the midbody (Sellitto and Kuriyama 1988 ; Falconer et al. 1989 ), we compared the location of talin and tubulin by immunolabeling using a rat MAb against the {alpha}-tubulin-subunit. Double labeling experiments clearly revealed that the staining pattern obtained with the anti-tubulin antibody in the midbody region was strikingly similar to that of talin (Figure 4A vs 4A'). However, the more peripheral portions of the intercellular bridges were often devoid of talin staining, especially at the conclusion of telophase. In contrast, anti-vinculin antibodies, even if they often outlined the membrane in between the daughter cells, never co-localized with the talin or tubulin staining of the midbodies (not shown). Double staining experiments with rhodamine–phalloidin and anti-talin antibodies showed that talin labeling at the ventral face of cells co-localizes with actin at the end of stress fibers (not shown). In the dividing cells, an accumulation of actin staining was occasionally obvious at the cell cortex in the region of furrowing (Figure 4B), which corresponds to the described contractile ring of actin but is not superimposable to that of talin (Figure 4B'). In addition, the staining pattern obtained with anti-talin antibodies was superimposable to that obtained with the anti-TD-60 antibody (Figure 4C'') located in the midbody at the position of maximal furrow constriction (Margolis and Andreassen 1993 ).



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Figure 4. Talin is located in the midbody structure. Double label immunofluorescence staining of Caco-2 cells for {alpha}-tubulin (A), actin (B), TD-60 protein (C), and talin (A'C') in the midbody region. (A''C'') Corresponding images of DNA in the same cells visualized with Hoechst staining. {alpha}-Tubulin antibodies labeled the microtubules in the cytoplasm as well as the midbody (A). Comparison of the talin and {alpha}-tubulin staining reveals that talin co-distributes with {alpha}-tubulin in the more central part of the midbody region (A'). Actin visualized by rhodamine–phalloidin occurs in organized aggregates at the cell cortex (B). The TD60 (C) and talin (C') proteins are co-localized in the midbody region. Bars = 5 µm.

To confirm biochemically the presence of talin in the midbody structures, Western blotting and immunofluorescence analysis of purified midbodies were performed. Midbodies were purified from Caco-2 cells according to the technique of Mullins and McIntosh 1982 . Phase-contrast microscopy and immunodetection experiments on the midbody preparation showed that the midbodies were intact (Figure 5A and Figure 5B'). Both anti-talin (Figure 5A) and anti-TD60 (Figure 5B) antibodies revealed bright structures characteristic of those found in cultured cells, indicating the enrichment of midbodies in the preparation. It is worth noting that there was no labeling of isolated midbodies with anti-vinculin or with the secondary antibodies alone (not shown).



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Figure 5. Immunofluorescence and Western blot analysis of a midbody preparation. Purified midbodies were centrifuged, using cytobuckets, onto adhesive slides and immunorevealed with monoclonal anti-talin (A) or anti-TD60 (B) antibodies. A' and B' represent the corresponding phase-contrast pictures. Bars = 5 µm. (C) Western blot analysis: 12.5 µg (Lanes a,b) or 25 µg (Lane c) of proteins from purified midbody samples or 25 µg of proteins from total cell homogenates (Lane d) were run on 7.5% (Lane a) or 5% (Lanes b–d) SDS-PAGE. Anti-ß tubulin antibody (Lane a) revealed a major band at 55 kD and monoclonal anti-talin antibody (Lane b) a single band in the 230-kD region. Sucrase, migrating around ~210 kD, was present in high amounts in the crude homogenates of Caco-2 cells (Lane d) and was not detected in the midbody preparation (Lane c). Molecular weight markers are indicated.

Samples of isolated midbodies were analyzed by SDS-PAGE. After transfer, nitrocellulose sheets were immunorevealed with anti-ß-tubulin (Figure 5C, Lane a) and anti-talin (Figure 5C, Lane b) antibodies. As expected, the anti-ß-tubulin MAb detected a major band at 55 kD (Figure 5C, Lane a). On the same midbody preparation, the anti-talin MAb detected a band at 230 kD corresponding to the native form of talin (Figure 5C, Lane b). As negative control for the preparation, Western blotting using HBB/2/614/88 antibodies recognizing an intestine-specific marker, sucrase, was performed. This antigen, associated with intercellular organelles and the cytoplasmic membranes, was found in the whole cell homogenate (Figure 5C, Lane d) but not in the midbody preparation (Figure 5C, Lane c).

Finally, immunocytochemical analysis of various mammalian cell types differing in tissue origin and animal species revealed that the presence of talin in midbodies is a general phenomenon. Indeed, anti-talin antibodies also stained the furrowing region of the undifferentiated HT29 cells derived from another human colon adenocarcinoma (not shown), of MCF-7 cells derived from a pleural effusion of human breast adenocarcinoma (Figure 6A), and of AR42J cells established from a rat pancreatic tumor (Figure 6B). Moreover, anti-talin antibodies also labeled midbodies of mesenchymal cell types such as A1F1 cells derived from 6-day-old rat intestinal connective tissue (Figure 6C) or rat skin fibroblastic cells (Figure 6D).



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Figure 6. Midbody staining with anti-talin antibodies exists in divergent cell lines. Indirect immunofluorescence experiments were performed using the monoclonal antibody on mammary MCF7 (A) and pancreatic AR42J (B) cancer cells, on intestinal A1F1 lamina propria cells (C), and on rat skin fibroblastic cells (D). In all cell types, talin was located within the midbodies. Bars = 10 µm.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Talin is a high molecular weight phosphoprotein that is localized mainly in adhesion plaques (Burridge and Connell 1983 ; Jockusch et al. 1995 ; Burridge and Chrzanowska-Wodnicka 1996 ). In this article we provide immunocytochemical and biochemical evidence that talin distribution varies during the course of the cell cycle. In particular, talin localizes to the centromeric region during metaphase, to the cleavage furrow from anaphase to telophase, and then becomes restricted to the midbody structure. Its distribution at the cleavage furrow is different from that of the other microfilament-associated proteins or actin binding proteins that have been thus far described in this region.

Talin is one of a large number of cytoskeletal proteins that are localized in focal contacts, specialized junctions between the cells and the extracellular matrix (Jockusch et al. 1995 ). In this region, among other molecular interactions talin links the cytoplasmic domain of ß1 integrins to filamentous actin. Because of this location, talin is assumed to play, in addition to a structural role, important functions in signaling to the nucleus. Downregulation of talin using an antisense RNA strategy alters cell adhesion and impairs the folding and processing of ß1 integrins (Albiges-Rizo et al. 1995 ). Similarly, Priddle et al. 1998 showed that ES cell mutants that contain no intact talin, although expressing a truncated talin polypeptide, revealed much lower levels of ß1 integrin. In these talin -/- ES cell mutants, differentiation is also compromised.

The present finding that talin is associated with the cleavage furrow suggests that this molecule may play a role in cytokinesis in mammalian cells and can be related to previous data from the literature (Sanger et al. 1994 ; Niewohner et al. 1997 ). By microinjection in exceptionally large cells of fluorescently labeled probes for actin, myosin, filamin, or talin, Sanger et al. 1994 showed for the first time that rows of fluorescent talin are apparent as cleavage formation occurs. More recently, evidence was provided for interaction of talin with the mitotic apparatus in the Dictyostelium microorganism, because impairment of cytokinesis was observed in talin-null cells (Niewohner et al. 1997 ). However, analysis of partially talin-deprived HeLa cells (Albiges-Rizo et al. 1995 ) by cytofluorometry did not reveal any significant modification in the DNA content of the cells (unpublished work). Among several focal adhesion proteins tested, talin is a good in vitro substrate for calpain II, a calcium-dependent protease also localized in focal adhesions. Interestingly, calpain II was located in the midbody region and has been proposed to be involved in mitosis (Schollmeyer 1988 ; Yamaguchi et al. 1994 ); microinjection of calpain II at late metaphase promoted premature disassembly of the mitotic spindle (Schollmeyer 1988 ).

Despite many investigations, the mechanisms of cytokinesis and its molecular control remain poorly understood. At present, in addition to the commonly accepted contractile ring mechanism that is involved in cytokinesis, another mechanism has been proposed for mammalian cell cleavage in which a new organelle, the telophase disc, positions myosin and actin (for review see Margolis and Andreassen 1993 ). The cleavage furrow location of talin raises the possibility that this molecule is involved in cytokinesis by one or the other mechanism. During cell division, as cells round up, the stress fibers and adhesion plaques disassemble and their constituent proteins disperse throughout the cytoplasm. Some of these proteins are then relocated and become concentrated in the cleavage furrow region. Among these, myosin and actin are localized with a rather similar pattern of expression (see in Fujiwara et al. 1978 ; Cao and Wang 1990 ). The preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring (Cao and Wang 1990 ). In addition, various actin binding proteins are found concentrated at the cleavage furrow region. Cofilin, a widely distributed actin binding protein, appeared to accumulate rapidly at the cleavage furrow as cytokinesis proceeded (Nagaoka et al. 1995 ). Similarly, radixin, an end-capping actin-modulating protein first identified in cell-to-cell adherent junctions, concentrated at the equatorial cell surface of dividing cells (Sato et al. 1991 ). However, the timing and the precise site of accumulation are different among the various molecules. Like talin, zyxin, which accumulates with integrins at sites of cell–substratum contact, is able to shuttle between the focal contacts and the nucleus (Nix and Beckerle 1997 ).

During furrowing, the plasma membrane has to be linked to the contractile machinery. On the basis of previous work (a) showing the direct binding of talin to actin (Muguruma et al. 1990 ; Kaufmann et al. 1991 ) and (b) indicating that talin is an important resilient link in microfilament–plasma membrane attachment (Winkler et al. 1997 ), one can hypothesize that talin is a good candidate for attachment of the contractile ring to the plasma membrane. Furthermore, by virtue of its property of actin nucleation (Kaufmann et al. 1991 , Kaufmann et al. 1992 ; Goldmann et al. 1992 ), and by its crosslinking activity between actin and myosin (Lin et al. 1998 ), talin may promote the formation of this contractile ring.

The formation of the contractile ring is a rather late phenomenon during mitosis. However, the presence of talin in the centromeric region of metaphase chromosomes and its relocation at the equatorial plate at the metaphase–anaphase transition indicate that talin may be a member of the class of chromosomal passenger proteins. Members of this class of proteins share several traits: they are all associated during metaphase with chromosomes and become located at anaphase with the microtubules of the overlap zone of the central spindle (Earnshaw and Bernat 1991 ). It is tempting to speculate that a part of talin, like these passenger proteins, uses the chromosomes to be correctly positioned at the metaphase plate. At the metaphase–anaphase transition, talin is also released from the chromosomes and is then localized to the central spindle region. Good representatives of this family of proteins are the INCENPs antigens (INner CENtromere Proteins; Cooke et al. 1987 ). Their exact role during mitosis is still not clearly defined, although several hypotheses have emerged, such as regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle pole in anaphase (Cooke et al. 1987 ). By creating INCENP mutants, Eckley et al. 1997 recently provided clear evidence for the involvement of INCENP in cytokinesis, although no explanation for this failure was provided. In 1991, Andreassen and collaborators described an organelle, the telophase disc, that may be involved in cytokinesis. Their model recognizes the existence of the cortical ring of actin but argues that it is organized by the telophase disc rather than constituting a self-integral structure that is independently responsible for cytokinesis. This telophase disc was discovered as a result of studies on the distribution of a mitosis-specific 60-kD antigen recognized by a human autoimmune serum. Like other chromosomal passenger proteins, this antigen, designated TD60, is first apparent on centromeres and is relocated to the midzone position of mitotic spindle (Andreassen et al. 1991 ). However, in contrast to the other passenger proteins, TD60 spreads laterally, forming a disc that encompasses the entire midzone diameter. Although our present data show an almost identical expression pattern for talin and TD60, we cannot definitively conclude whether or not talin can be considered a true component of the telophase disc.

Our demonstration that talin is associated successively with centromeric and midbody regions during the cell cycle suggests a functional cooperation between chromosomes and cytoskeletal components to complete cytokinesis.


  Acknowledgments

Supported by INSERM, the Ligue Nationale Contre le Cancer, and the Association pour la Recherche sur le Cancer.

We are grateful to Drs M. Beckerle (University of Utah; Salt Lake City, UT), J. Goetz (CHU; Strasbourg, France), P. Andreassen and R.L. Margolis (Institut de Biologie Structurale J-P. Ebel; Grenoble, France), and H.P. Hauri (Biozentrum; Basel, Switzerland) for gifts of antisera and to Drs M.C. Rio (IGBMC; Illkirch, France) and N. Vaysse (INSERM U 151; Toulouse, France) for providing cell lines. We thank C. Arnold and C. Leberquier for skillful technical assistance, our colleagues in the laboratory for help and advice, and L. Mathern for photographic assistance. We are very grateful to S. Martineau (Institut de Biologie Structurale J-P. Ebel; Grenoble, France) and V. Holl (Institut d'Hématologie; Strasbourg, France) for their cooperation. Dr M. Kedinger is thanked for helpful discussion and support in this work. We also thank Dr M. Block (UMR CNRS 5538; Grenoble, France) for helpful comments and discussions and Dr D. Job (INSERM U.366, Grenoble, France) for critical reading of the manuscript.

Received for publication December 11, 1998; accepted May 17, 1999.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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