ARTICLE |
Correspondence to: Patricia SimonAssmann, Unité NSERM 381, 3 avenue Molière, 67200 Strasbourg, France. E-mail: Patricia.Simon-Assmann@inserm.u-strasbg.fr
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Summary |
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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 -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 47:13571367, 1999)
Key Words: talin, cytoskeletal protein, mitosis, cleavage furrow, chromosomes, centromeres, midbodies, colon cancer cells
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Introduction |
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Intestinal morphogenesis and cell differentiation are dependent on heterotypic cell interactions (for a review see
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 -actinin, varies considerably (
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 cellcell or at the cellsubstrate junctions, such as radixin (-actinin (
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.
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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 (
The A1F1 intestinal fibroblast cell line derived from postnatal rat intestinal mucosa was provided by
The 8d4 monoclonal antibody (Sigma) and the B11 polyclonal antibody (kindly provided by Dr. Beckerle (University of Utah, Salt Lake City) (-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 (
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), lissaminerhodamine-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 35 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 (
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
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 (
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.
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Results |
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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 cellcell contacts as a thin staining (Figure 1C). Given this unusual location of talin in cellcell 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 cellcell contact.
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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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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 (
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 (-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 rhodaminephalloidin 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 (
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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
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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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Discussion |
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Talin is a high molecular weight phosphoprotein that is localized mainly in adhesion plaques (
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 (
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 (
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
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 (
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 metaphaseanaphase 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 (
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.
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Acknowledgments |
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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.
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