ARTICLE |
Correspondence to: Takeshi Baba, Dept. of Anatomy, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. E-mail: tbaba@res.yamanashi- med.ac.jp
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Summary |
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Interconnection between surface microdomains and the actin cytoskeleton is vital to various cellular activities. We studied the responses of okadaic acid (OKA)-treated K562 leukemia cells to crosslinking of membrane microdomains. Although OKA alone induced clustering of surface-bound F-actin, addition of a biotinylated poly(ethylene glycol) derivative of cholesterol (bPEG-Chol) and subsequent binding of streptavidin (SA) further induced accumulation of the clusters, resulting in the formation of a spherical cell extrusion. This extrusion was also induced by direct crosslinking of a raft marker, CD59, and ganglioside GM1. In addition, we found that knockout of the gene encoding Fyn kinase inhibited formation of the spherical extrusion in murine T-cells. In bPEG-Chol/SA-treated cells, CD59, ganglioside GM1, and clathrin/AP-2 were all accumulated on the surface of the actin-rich extrusion, whereas dynamin and transferrin receptors were unaffected. Intermediate filaments, mitochondria, and other vesicles also accumulated. These results suggest that crosslinking of membrane domains exaggerates the linkage between actin and a defined set of membrane proteins in OKA-treated cells.
(J Histochem Cytochem 51:245252, 2003)
Key Words: lipid rafts, clathrin-coated pits, actin, okadaic acid
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Introduction |
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LIPID RAFTS and clathrin-coated pits consist of a defined set of proteins that are functionally connected to the cortical actin cytoskeleton. In particular, the lipid raft-associated non-receptor tyrosine kinases Fyn, Lyn, and Lck are involved in stimulation of actin polymerization in lymphocytic cells (
Distinct sets of proteins that regulate endocytic pits also control cytoskeleton-dependent cell activities (for review see
In addition to these cytoskeletal systems, protein phosphorylation is crucially involved in the regulation of endocytosis and cell motility. In particular, okadaic acid (OKA), an inhibitor of ser/thr protein phosphatases 1 and 2A, induces cell rounding and inhibits many endocytic steps (
Here we describe the linkage of actin to lipid rafts and clathrin-coated pits in OKA-treated K562 leukemia cells. We addressed different strategies that are expected to aggregate surface domains or to deform the plasma membrane and cortex actin. One of the methods involved distributing a biotinylated poly(ethylene glycol) derivative of cholesterol (bPEG-Chol; see Fig 1), which was subsequently crosslinked by streptavidin (SA). The major advantage of this amphiphilic molecule is that an inserted cholesteryl moiety does not interfere with the lateral interactions between adjacent lipids. This is supported by previous results that PEG-Chol-containing liposomes effectively encapsulated water-soluble drugs without disturbing membrane structures (
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Materials and Methods |
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Cells
K562 cells were maintained in DMEM/10% FCS. The cells were incubated in serum-free medium (SFM, DMEM supplemented with 0.2% BSA and 20 mM Hepes, pH 7.4) for 30 min before the experiment. The cells were pelleted and suspended in SFM at a concentration of 5 x 106 cells/ml.
Reagents
Poly(ethylene glycol)50-cholesteryl ether (PEG-Chol) and biotinylated PEG-Chol (bPEG-Chol) were prepared as previously reported (
Transfection of pEGFPActin in K562 Cells
A plasmid containing a gene encoding pEGFPhuman-ß-actin was purchased from Clontech (Palo Alto, CA). For transfection, K562 cells were incubated overnight with an iron-depleted medium. The cells were transfected with a Transferrinfection kit (Takara; Kyoto, Japan) according to the manufacturer's protocol. The cells were used for experiments 2 days after transfection.
OKA/b-PEG-Chol/Streptavidin Treatment
K562 cells in SFM were incubated with 500 nM OKA for 30 min at 37C and with 2 µM bPEG-Chol (2 nmol/5 x 106 cells) for another 30 min at 37C without washing out OKA. The cells were chilled on ice and incubated with 10 µg/ml AlexaFluor594streptavidin (SA) for 30 min at 4C. These cells were then incubated for 530 min at 37C and fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer for 15 min at 4C. The cells were washed with PBS and mounted in PBS/BSA/Glycerol.
Immunocytochemistry
For immunolocalization of cell surface antigens, OKA/bPEG-Chol/SA-treated cells were fixed as above, attached to coverslips by a cytofuge (Statspin; Norwood, MA), and incubated with mouse MAbs for 30 min at RT. The cells were washed in PBS and reacted with AlexaFluor 488goat anti-mouse IgG for 30 min. For immunolocalization of cytoplasmic antigens, fixed cells were attached to coverslips by the cytofuge and treated with 0.1% saponin/PBS for 10 min at RT. The cells were washed in PBS, blocked with 1% BSA/PBS, and incubated with primary antibodies or with AlexaFluor 488phalloidin for F-actin. These samples were observed in a confocal laser-scanning microscope (Leica TCS4D; Heidelberg, Germany).
Fyn(-/-) Murine T-cells
Murine fyn(+/+) and fyn(-/-) T-cell lines were established from a 2C TCR transgenic mouse (
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Results |
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K562 cells were treated with okadaic acid (OKA, 500 nM) at 37C for 30 min. The cells were then incubated with bPEG-Chol at 2 nmol to 5 x 106 cells at 37C for 30 min. The structure of bPEG-Chol is shown in Fig 1. Because 2 nmol of PEG-Chol is known to occupy approximately 1% of total plasma membrane area, incorporated bPEG-Chol is equivalent to several percent of total plasma membrane cholesterol (
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The distribution of F-actin was visualized using fluorescent phalloidin in comparison with the accumulation of bPEG-Chol/SA (Fig 4). At 4C, actin was already clustered on the surface (Fig 4A). When the cells were incubated at 37C, co-migration of both molecules to the spherical extrusion was clearly seen (Fig 4D4F). A similar change in the pattern of fluorescence was observed in living K562 cells expressing EGFPactin (Fig 4G and Fig 4H). EGFPactin also distributed as patches on the surface at 4C (Fig 4G, arrows). At 37C, these patches migrated to form a spherical extrusion (Fig 4H). We hereafter refer to this spherical extrusion as "actin-rich extrusion." The total amount of F-actin/cell, determined spectrofluorometrically using fluorescent phalloidin, did not change before and after treatment with bPEG-Chol/SA (data not shown). Furthermore, the actin-rich extrusion was completely abolished by latrunculin A or jasplakinolide, suggesting that polymerization/depolymerization of actin was required for the extrusion.
Although the effect of SA was marked, we noted that bPEG-Chol alone induced an actin-rich extrusion in a few OKA-treated cells similar to those in Fig 2E and Fig 4D. This change appeared to be induced by the shear stress that was applied during pipetting or mounting on a glass slide. Indeed, gently pressing the OKA/bPEG-Chol/SA-treated cells via coverslips induced the actin-rich extrusion even at 4C (data not shown). These results suggest that membrane stresses that deform cortical actin are similarly effective for bPEG-Chol/SA.
When OKA/bPEG-Chol/SA-treated cells were observed in an electron microscope, we observed a thick actin bundle (Fig 8B, small arrows, and Fig 9A). Near this structure, intermediate filaments also bundled (Fig 8B, asterisk). Moreover, mitochondria and some electron-lucent vesicles also accumulated near the actin-rich extrusion (Fig 8C and Fig 8D). Surprisingly, the electron micrographs at higher magnification revealed accumulation of many clathrin-coated pits over the surface of the actin-rich extrusion (Fig 9B). They were homogeneously sized and open. By immunofluorescence, an AP-2 component, -adaptin, had also accumulated (Fig 5A). In marked contrast, dynamin and the transferrin receptor (TfR) homogeneously distributed over the whole surface (Fig 5D and Fig 5G). When TfR molecules were crosslinked by a combination of primary/secondary antibodies, only small patches were induced (data not shown). Moreover, distribution of an early endosomal protein, EEA1, also did not change (Fig 5J). These results suggested that TfR in OKA-treated cells are not linked to the cytoskeleton or to the early endosomal membrane.
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We examined the distribution of markers for lipid rafts, CD59, a typical GPI-anchored protein, and ganglioside GM1 (Fig 6). The molecules, visualized by an MAb and by cholera toxin B, respectively, distributed evenly over the cell surface at 4C (data not shown). In contrast, when the cells were incubated at 37C, both molecules were accumulated on the actin-rich extrusion (Fig 6D and Fig 6G). Another protein, CD45, a putative protein tyrosine phosphatase segregated from an immunological synapse (
A non-receptor tyrosine kinase, fyn, regulates raft dynamics in T-cells (
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Discussion |
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Treatment of cells with okadaic acid (OKA) is often described to mimic the cytoplasmic condition of mitotic cells. In particular, arrest of membrane traffic and cell motility in the cells has given the impression that cell activities are suppressed. However, we found that marked accumulation of actin was induced when cells were treated with bPEG-Chol and SA at 37C (Fig 4 and Fig 8). Actin was similarly accumulated when a raft component, CD59, was crosslinked. Moreover, an experiment using MAb TS2/16, which activates the linkage between integrin ß1 and actin, induced a similar response (Fig 7). Actin was also accumulated when the cells were directly compressed via a coverslip. In contrast, this response was not induced by the non-activating binding of anti-integrin MAb DF5 (Fig 7G7I) or by anti-TfR MAb (data not shown). In a previous study on normal K562 cells, we found that even fourfold more PEG-Chol (20 nmol per 5 x 106 cells) did not induce accumulation of actin (
By summarizing the microscopic results on bPEG-Chol/SA-treated cells along a time sequence, the following scenario can be drawn. (a) OKA induces actin to underline certain plasma membrane domains (Fig 4G). (b) When the surface is stimulated, these actin-bound patches move into a single pole (Fig 4D4F and Fig 4H). (c) Mobilization to the cortex is propagated to the augmentation of perinuclear intermediate filament-containing actin (Fig 8A and Fig 8B). (d) Lipid raft or clathrin-coated pit components are mobilized with the accumulated actin during steps b and c. Mitochondria and some other membrane compartments, but not EEA1-associated early endosomes, are also mobilized (Fig 5, Fig 8, and Fig 9). (e) Concentration of these elements in a single extrusion occurs, followed by separation from the enucleated part (Fig 2G and Fig 2H). We believe that each of these reaction steps is important for evaluating the molecular linkage between actin and specific sets of membrane domain elements. Analysis of T-cell lines using gene-knockout mice indicated involvement of non-receptor kinase fyn in OKA/bPEG/SA-induced cell extrusion (Fig 10). In the cells, not only actin but also a raft marker, Thy-1, failed to accumulate. In the presence of active fyn, aggregation of rafts induces association of actin filaments together with many tyrosine-phosphorylated proteins (
Formation of the actin-rich extrusion involved mobilization of the perinuclear cytoskeleton. Our observations indicated that vimentin, which initially scattered as clusters around the nucleus, assembled in a bundle due to bPEG-Chol/SA (Fig 8B and Fig 9B). Disorganization of the perinuclear cytoskeleton by phosphorylation of vimentin has been suggested for adherent cells by a number of studies. In particular, inhibitors of protein dephosphatases, OKA (
In this study we used PEG-Chol to address the coupling of cortical actin with membrane microdomains. In comparison to the commonly used reagent methyl ß-cyclodextrin, which had effects not only on lipid rafts but on clathrin-coated pits and integrity of the plasma membrane (
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Acknowledgments |
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SBS and TB contributed equally to the research. Supported by grants to SBS and TB from the Japanese Ministry of Education, Culture, Sports and Science.
We thank Drs Koichiro Miyajima (Osaka University of Pharmaceutical Sciences) and Yoshio Hamashima (Kyoto Pharmaceutical University) for PEG-Chol and bPEG-Chol, and Dr Yoshinori Fujiyoshi (Kyoto University) for support and discussion.
Received for publication June 24, 2002; accepted October 2, 2002.
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