ARTICLE |
Correspondence to: Takashi Kojima, Dept. of Pathology, Cancer Research Inst., Sapporo Medical Univ. School of Medicine, S1, W17, Sapporo 060-8556, Japan. E-mail: ktakashi@sapmed.ac.jp
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Summary |
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It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis. (J Histochem Cytochem 49:333339, 2001)
Key Words: connexin 32, occludin, claudin-1, ZO-1, ZO-2, midbody, mitosis
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Introduction |
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Tight junctions, the most apical component of intercellular junctional complexes, separate the apical from the basolateral cell surface domains to establish cell polarity (performing the function of a fence). They also provide a barrier function, inhibiting solute and water flow through the paracellular space (
In this study, to elucidate the mechanisms involved in maintenance of the epithelial barrier during cell division, we immunocytochemically examined changes in localization of occludin, claudin-1, ZO-1, and ZO-2 during cell division of immortalized mouse hepatocytes transfected with human Cx32 cDNA. To frequently observe cells at the M-phase of the cell cycle, the cells were treated with Taxol (Taxus brevifolia), which reduces the rate of cytokinesis at low concentrations by kinetically stabilizing spindle microtubules (
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Materials and Methods |
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Cell Culture, cDNA Construction, and Transfection
CHST8 is a clonal cell line derived from female C3H/HeJ mouse hepatocytes conditionally immortalized with the temperature-sensitive SV40 large T-antigen gene as described previously (
A fragment containing nucleotides 11558 of human connexin32 (Cx32) cDNA (
It is known that Taxol at a low concentration (10 nM) reduces the rate of cytokinesis (
Immunofluorescence Microscopy
Cells grown on glass coverslips were fixed with cold absolute acetone for 10 min. Double immunocytochemistry with monoclonal anti--tubulin (Amersham; Poole, UK) and with polyclonal anti-occludin (Zymed Laboratories; San Francisco, CA), polyclonal anti-claudin-1, polyclonal anti-ZO-1 (Zymed), polyclonal anti-ZO-2 (Zymed) antibodies, and rhodaminephalloidin (Molecular Probes; Eugene, OR) was performed. Rabbit polyclonal anti-claudin-1 antibody was raised against the synthetic polypeptide SYPTPRPYPKPTPSSGKD, which corresponds to the COOH-terminal cytoplasmic domains of mouse claudin-1 (amino acids 192209) (Immuno-Biological Laboratories; Fuzioka, Japan).
-Tubulin was visualized using Alexia 594 (red)-conjugated anti-mouse IgG (Molecular Probes) and occludin, ZO-1, ZO-2 by Alexia 488 (green)-conjugated anti-rabbit IgG (Molecular Probes).
-Tubulin was visualized using Alexia 488 (green)-conjugated anti-mouse IgG (Molecular Probes) and actin by rhodaminephalloidin (red). The specimens were examined with an epifluorescence microscope (Nikon; Tokyo, Japan) and a laser scanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA).
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Results |
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In immortalized mouse hepatocytes transfected with human Cx32 cDNA, occludin, claudin-1, ZO-1, and ZO-2 were clearly localized at cell borders (Fig 1). When the transfectants were treated with 10 nM Taxol for 24 hr, many mitotic cells (20%) at various stages of cell division were observed. However, no conspicuous changes of polymerized microtubules in cytoplasm were observed. To examine changes in localization of tight junction proteins during cell division, double immunocytochemistry with
-tubulin and either occludin, claudin-1, ZO-1, or ZO-2 was performed. Localization of
-tubulin indicated the phases of mitotic cells: the metaphase, anaphase, telophase, and cytokinesis (Fig 1a, Fig 1c, Fig 1e, and Fig 1g). All tight junction proteins, occludin, claudin-1, ZO-1, and ZO-2, were present between mitotic cells and neighboring cells throughout all phases of mitosis (Fig 1b, Fig 1d, Fig 1f, and Fig 1g).
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Next, attempting to elucidate changes in tight junctions during cell division, we further focused on changes in localization of tight junction proteins between the two daughter cells of mitosis. As shown in Fig 2a2f, staining of -tubulin and actin clearly demonstrated a tiny area not stained by either one, between two thick bundles of tubulin and actin in the middle of the midbody. Double immunostaining with
-tubulin and occludin demonstrated that occludin was concentrated in the midbody (Fig 2g2r). In Z-sections, occludin was clearly observed in the midbody on the most apical site between the two daughter cells (Fig 2j2l). Furthermore, occludin was observed not only in the midbody but also at cell borders between daughter cells and neighboring cells (Fig 2m2o). Occludin was also observed at cell borders beneath the midbody between the daughter cells (Fig 2p2r). Double immunostaining with
-tubulin and claudin-1 demonstrated that claudin-1 was also present in the midbody and between daughter cells and neighboring cells, like occludin (Fig 3a3f). Double immunostaining with
-tubulin and tight junction-associated cytoplasmic proteins ZO-1 or ZO-2 demonstrated that both ZO-1 and ZO-2 were localized just beneath the midbody, not in it (Fig 3g3r). Fig 3s and Fig 3t show schematic figures of localization of tight junction proteins during epithelial cell division. All tight junction proteins, occludin, claudin-1, ZO-1, and ZO-2, were present between mitotic cells and neighboring cells, regardless of the phase of mitosis, but only occludin and claudin-1 were localized in the midbody between the daughter cells. Line 1 of Fig 3t corresponds to Fig 2j2l and Fig 3d3f (occludin, claudin-1), and Line 2 corresponds to Fig 3j3l, and Fig 3p3r (ZO-1, ZO-2). Between the daughter cells at late telophase, the most apical part formed the midbody, in which only occludin and claudin-1 were localized, while all tight junction proteins were detected at the cell borders beneath the midbody.
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Discussion |
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In immortalized mouse hepatocytes transfected with human Cx32 cDNA, increases in the number of tight junction strands and in the amount of integral tight junction proteins occludin and claudin-1 and -2 were observed, as we previously reported (
It is well known that tight junction structures in epithelial cells are maintained during mitosis (
In ultrastructural studies, intestinal epithelial cells at late telophase still show tight junction structures between the midbody and neighboring cells (
It is known that many cytoskeletal proteins, including ERM, are located in the midbody (
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Acknowledgments |
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Supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science, and the Ministry of Welfare of Japan.
Received for publication June 13, 2000; accepted October 17, 2000.
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