Department of Biology, Ochanomizu University, 211 Otsuka, Bunkyo-ku, Tokyo, 1128610, Japan, 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, 3Center for Life Science and Technology and Department of Chemistry, Keio University, Hiyoshi 3141, Kouhoku-ku, Yokohama 2238522, Japan, and 4Peptide Institute, Inc., Protein Research Foundation, 412 Ina, Minoh-shi, Osaka, 5628686, Japan
Received on May 9, 2000; revised on July 10, 2000; accepted on July 13, 2000.
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Abstract |
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Key words: ganglioside/endocytosis/extracellular matrix
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Introduction |
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In sea urchin eggs, yolk granules have been thought to provide the developing embryos with building materials or nutrients. Recent studies show that yolk granules store the components of the embryonic ECM. A 32 kDa protein that is a component of the sea urchin embryonic ECM is stored in yolk granules of unfertilized eggs (Mayne and Robinson, 1998). Also, a 22S glycoprotein in sea urchin yolk granules, called toposome, is released to the cell surface of the blastula (Gratwohl et al., 1991
). Similarly, in Xenopus embryos, a 4345 kDa lectin, detected in association with yolk platelets (Roberson and Barondes, 1983
), is secreted into the ECM in embryos through gastrulation (Outenreath et al., 1988
). Galectins, a family of lectins from Rana catesbeiana oocytes, which are mainly distributed extracellularly in several organs, are also found in yolk platelets of growing oocytes in the ovary (Uchiyama et al., 1997
). Thus, we hypothesized that M5 in yolk granules is released to the ECM with these proteins. To examine this possibility, we performed fluorescent immunocytochemistry using an anti-M5 antibody and embryo cryosections. As expected, bright fluorescence was observed at the outer surface of embryos. Immunoelectron microscopy confirmed that M5 was localized in the embryonic ECM. These results also indicate that released M5 interacts with the ECM.
Fluorescent-labeled gangliosides are used to visualize the localization of gangliosides in living cells. In this study, we used NBD-M5 to observe M5 in the ECM during embryogenesis and found that it was incorporated into endocytotic vesicles.
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Results |
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Discussion |
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In this study, using a specific antibody against M5, we showed that M5 was distributed in the embryonic ECM. Since M5 exists in unfertilized eggs in yolk granules (Shogomori et al., 1997), we concluded that it was transported from the yolk granules to the ECM and/or the plasma membrane after fertilization.
A number of studies have shown the role of gangliosides as mediators in the interaction of various cells with the ECM. Cheresh et al. (1986) demonstrated that anti-GD2 and GD3 monoclonal antibodies inhibit cell-substratum interaction in melanoma and neuroblastoma cells. Furthermore, Cheresh and Klier (1986)
showed that GD2 is localized directly on the microprocesses that make contact with the fibronectin substrate on human melanoma M21 cells. Spiegel et al. (1985)
demonstrated that mouse fibroblasts (NCTC 2071A cells), which are deficient in gangliosides and cannot retain endogenously synthesized fibronectin at the cell surface, can recover the ability of fibronectin organization when exogenous gangliosides are added to the culture medium. Also, they reported that exogenous fluorescent gangliosides are distributed on the fibrillar network of fibronectin, indicating a high affinity of gangliosides to fibronectin. Since the sea urchin embryo is surrounded by several ECM components, such as molecules similar to vertebrate fibronectin, collagen, and laminin (Wessel et al., 1984
), M5 may be involved in the organization of these molecules, although it remains to be determined whether M5 actually interacts with these proteins. Hyalin is the major protein in the embryonic ECM and is sensitive to Ca2+ for its polymerization in sea urchin (Kane, 1973
; McClay and Fink, 1982
). To determine whether M5 interacts with hyalin, we treated embryos with Ca2+-free ASW. M5, however, was not detected significantly in the Ca2+-free ASW extract (data not shown), suggesting that M5 interacts with the ECM component(s) that are insensitive to Ca2+. It has been reported that the apical and basal ECM consist of different components (Matese et al., 1997
). The strong labeling by the antibody and fluorescent analogue is observed only in the apical area, suggesting the polarized localization of M5 in sea urchin embryos and the specific interaction with components of the apical ECM. The polarized localization of sphingolipids has also been reported in epithelial cells (reviewed in Simons and van Meer, 1988
).
Since the contents of yolk granules are released to the ECM during embryogenesis, lipoprotein is also likely to be released to the ECM. If this is the case, lipoprotein may be the component of the embryonic ECM that plays a role in a ganglioside reservoir.
In this study, localization of endogenous M5 in the ECM was mimicked by NBD-M5, which encouraged us to examine the fate of extracellular M5 using NBD-M5. After fertilization, NBD-M5 in the ECM was internalized in the cells by endocytosis, suggesting that extracellular M5 was transported from the ECM to endocytotic vesicles. Although the fate of endocytotic vesicles of sea urchin embryo is still unknown, ganglioside M5 in the vesicles is likely to be used to make the body of the embryos or adult organs. Indeed, M5 is found in the somatic cells of adult esophagus and testis (Kubo et al., 1990). It is also possible that M5 in the endocytotic vesicles may be metabolized and change their structure in the recycling pathway. Further studies are necessary to reveal the metabolic and recycling pathway of the ganglioside.
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Materials and methods |
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Monoclonal antibody
The culture supernatant of the hybridoma was used as the source of the anti-M5 antibody (SUG-44, IgM). The characterization of this antibody was performed previously (Shogomori et al., 1997).
Immunofluorescence microscopy
Embryos were fixed with 4% paraformaldehyde, 0.5% glutaraldehyde and 7% sucrose in ASW for 2 h on ice. After washing with ASW, fixed embryos were sequentially transferred to 5%, 10%, and 15% sucrose in phosphate-buffered saline (PBS) for 30 min each and 20% sucrose/PBS:O.C.T. Compound (Miles Laboratories) (2:1) overnight on ice. Embryos were quickly frozen in liquid nitrogen and stored at 20°C. The embedded embryos were cut into 6 µm sections with a Minotome (Damon/IEC Division) at 20°C. The sections were incubated with SUG-44 and then with rhodamine-conjugated anti-mouse IgM Ab (Cappel, Malvern, PA).
Immunoelectron microscopy
Fixed embryos were subjected to rapid-freezing and freeze-substitution, and then embedded in LR-White as described previously (Shogomori et al., 1997). Ultrathin sections were cut with an ultramicrotome (LKB), placed on Formvar-coated nickel grids (150- or 200-mesh), and labeled with SUG-44 and 15-nm diameter colloidal gold-conjugated anti-mouse IgG + IgM Ab (Cappel). Sections were stained with uranyl acetate and lead citrate. Labeled and stained sections were examined and photographed with a JEOL 1200EX electron microscope at 80 kV.
Confocal microscopy
For confocal microscopy, zygotes and embryos labeled with NBD-M5 were held between two coverslips separated by double-stick tape and observed with a confocal microscope (TCS-NT, Leica, Germany).
Fluorescent ganglioside M5
NBD-M5 was synthesized from Neu5Gc26Glc derivative and phytosphingosine (Yamamoto et al., unpublished observations) and its structure is shown in Figure 3.
Labeling eggs with NBD-M5
Unfertilized or fertilized eggs were incubated with ASW containing 0.1 mg/ml of NBD-M5 for 20 min. Then they were washed three times with ASW. After incubation for the indicated period, they were observed with a confocal microscope.
Endocytosis assay
NBD-M5 (0.1 mg/ml) and tetramethylrhodamine dextran (Rh-dex; MW 3000; Molecular Probes, Eugene, OR) (0.3 mg/ml) were dissolved in ASW. Immediately after fertilization in ASW, the eggs were placed in ASW containing NBD-M5 and Rh-dex. After 20 min, the eggs were observed with a confocal microscope.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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