ARTICLE |
Correspondence to: Richard Bennett, Jr., Dept. of Life Sciences, Winston-Salem State Univ., 601 M.L. King, Jr. Drive, WinstonSalem, NC 27110. E-mail: bennettr@ols.net
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Summary |
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MUC1, a transmembrane member of the mucin family, is believed to have anti-adhesive properties because of its highly sialylated, extended, and rigid rod-like conformation. The ERM proteins (ezrin, radixin, and moesin) function as membranecytoskeletal linkers. MUC1 and ezrin are enriched in microvilli in MCF-7az breast carcinoma cells. Similar localization was also found in peripheral membrane areas and in filopodium-like protrusions. Whereas ezrin was consistently detected in the cellcell contact region, MUC1 was less frequently found there. MUC1 was distinctly expressed in long filopodial protrusions and was highly concentrated at their tips, which also contained ezrin, whereas F-actin was found along the stalk. This localization of MUC1 suggests a role for MUC1 in transient cell structures of migrating cells and transient cell adhesion. No direct association has yet been found between MUC1 and ezrin. However, both MUC1 and ezrin had a similar overall distribution pattern in microvilli and filopodium-like protrusions in immunoelectron tomography. In addition, MUC1 and ezrin showed spatial association, because several 10-nm gold particles used to decorate ezrin were seen in the vicinity close to the clusters of 5-nm gold particles decorating MUC1. Therefore, MUC1 appears to be associated with ezrin, but the nature of this association requires further study. (J Histochem Cytochem 49:6777, 2001)
Key Words: mucin, MUC1, ezrin, cytoskeleton
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Introduction |
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Mucins are highly glycosylated proteins that make up a large part of the mucus on luminal surfaces of body epithelia (
The interactions between cell membrane structures and the cytoskeleton have an essential role in various cell functions, including coordination of cell morphology, migration, and adhesion. Several protein complexes exist that mediate interactions between cell adhesion molecules and the cytoskeleton. These include protein complexes associated with integrins in focal adhesions (e.g., -actinin, vinculin, talin, paxillin, and focal adhesion kinase) and cadherins in cellcell adherence junctions (ß-catenin/plakoglobin
-catenin complex) (
Other membrane-associated molecules, including MUC1, share ezrin's predilection for microvilli (-Catenin links ß-catenin and plakoglobin to actin filaments. Catenins may not be the only cytoskeletal proteins regulating the localization of MUC1. There also appear to be two discrete motifs in MUC1 that determine its apical localization. The first is located in the extracellular domain and the second is in the juxta-membrane region of the cytoplasmic domain (
We now show that ezrin and MUC1 have a similar localization in MCF-7 cells and are enriched in microvilli and filopodium-like protrusions, but differ in peripheral membrane areas. We have not been able to show direct molecular interaction between MUC1 and ezrin. However, the similar distribution of MUC1 and ezrin in immunoelectron tomography suggests that MUC1 and ezrin are associated, perhaps indirectly, with each other in certain cell surface protrusions. A distinct localization of MUC1 was observed in the tips of filopodial protrusions, suggesting a role in transient adhesion to the substratum.
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Materials and Methods |
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Cells and Antibodies
MCF-7az breast carcinoma cells were chosen for this study because this cell line expresses high amounts of MUC1 in immunofluorescence microscopy. The majority of breast carcinoma cell lines express MUC1, but MCF-7az cells were chosen because of their intensity of staining and the presence of structures hereafter referred to as filopodial protrusions, which contain large amounts of MUC1 at their tips. The U251-MG glioma cell line, which does not show endogenous MUC1 expression, was used to express exogenous MUC1 cDNA. MCF-7az and U251-MG cells were cultured in MEM supplemented with 10% fetal calf serum (FCS) medium. Antibodies used in this study were a polyclonal rabbit antibody to human ezrin (Ez-Bl-III) (
Immunocytochemistry/Confocal Microscopy
Cells were grown to 5070% confluence as described above, fixed for 15 min with 3.7% paraformaldehyde in PBS at room temperature (RT), and permeabilized for 10 min with 0.05% Triton X-100 in PBS. The Ez-Bl-III antibody was used at 1:501:100 dilutions, 3C12 at 1:100 dilution, and MUSE11 at 1:25 dilution in PBS. In some experiments, during secondary antibody incubation, cells were stained with rhodamine-conjugated phalloidin (Molecular Probes; Eugene, OR) or Oregon Green-conjugated DNase I (Molecular Probes) to reveal F-actin and G-actin, respectively. After incubation with the secondary antibodies, the coverslips were mounted on glass slides using Syva Microtrak mounting fluid (Behring Diagnostics; Cupertino, CA). Cells were examined by conventional fluorescence microscopy using an Olympus BH-2 microscope (Olympus America; Melville, NY) or by confocal microscopy with a confocal 410 Invert Laser Scan Microscope (Carl Zeiss; Oberkochen, Germany). The printed images from the confocal data were processed using the Apple Macintosh Adobe Photoshop program.
MUC1 cDNA (kindly provided by Dr. Sandra J. Gendler; Mayo Clinic, Scottsdale, AZ) was expressed in the pCDNA3 vector (Invitrogen; San Diego, CA) and transfected by the lipofectin method (GIBCO BRL Life Technologies; Gaithersburg, MD) to U-251 MG cells. After 2 days of growth, the transfected cells were fixed with paraformaldehyde for study by immunofluorescence.
Immunoelectron Microscopy
MCF-7az cells were grown on poly-L-lysine-coated carbonformvarNi grids, mounted on coverslips, washed with Dulbecco's salt solution (DSS), pre-fixed (1530 min) in 0.125% glutaraldehyde (GA) in HEPES, 20 mM, pH 7.4, in DSS, washed in DSS, and incubated overnight in TXHS buffer (0.05% Triton X-100, 30 mM HEPES, pH 7.4, 0.1 M NaCl, 20 mM KCl, 5 mM MgCl2, with or without 1% fish gelatin and 20 mM glycine). The grids were incubated for 12 hr at RT and overnight at 4C with rabbit polyclonal anti-ezrin antibody (Ez-Bl-III; 1:50 dilution) and mouse monoclonal anti-MUC1 antibody MUSE11 (1:25 dilution) or mouse monoclonal anti-actin (1:50 dilution) in TXHS. After washing in TXHS, the specimens were incubated for 12 hr at RT and then overnight at 4C with gold-conjugated antibody mixtures in TXHS: (a) goat anti-rabbit10-nm gold (Sigma Chemical; 1:50 or 1:25 dilution), goat anti-rabbit1.4-nm gold (NanoProbes), and goat anti-mouse5-nm gold (Sigma; 1:25 dilution), or (b) goat anti-rabbit5-nm gold (Sigma; 1:25 dilution), goat anti-mouse10-nm gold (Sigma; 1:25 dilution), and goat anti-mouse1.4-nm gold (NanoProbes). After washing with TXHS and cacodylate buffer (C) (0.1 M, pH 7.4), the specimens were postfixed in 1.25% glutaraldehyde (for 1 hr to overnight) in C, followed by brief additional fixation (15 min) in a mixture of OsO4 (final concentration 0.3%) with 1.25% glutaraldehyde in C. The specimens were then washed in C, followed by incubation in C with or without 1% tannic acid (1 hr to overnight) (
Immunoelectron Tomography
Electron micrographs of tilt series were collected with 3° increments from +60 to -60 degrees. The resulting negatives were scanned with a Umax PowerLook 2000 scanner. Images were stored in JPEG format and aligned using our Jpeganim software (
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Results |
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Localization of MUC1 and Ezrin to Similar Structures in MCF-7 Cells
A number of different cell lines were screened for endogenous expression of MUC1 and ezrin. We chose to study the localization of MUC1 and ezrin in MCF-7az cells because of the intense expression of MUC1. In double staining of cultured MCF-7az cells, both MUC1 and ezrin were seen in microvilli and in peripheral membrane areas, as earlier reported for MUC1 in mammary epithelial cells (
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Localization of MUC1 in the Tips of Filopodial Structures
Filopodium-like protrusions that were dominated by MUC1 staining over ezrin staining were seen (Fig 4). The filopodial protrusions, which projected from the cells in the plane of the glass coverslip, resided mainly between the cells in cell clusters (Fig 4). In most cases the location of filopodial protrusions was polarized; they were pointing in the same direction as the leading lamella. The filopodial protrusions were seen only in subconfluent cells, suggesting that they are involved in cell locomotion. These filopodial protrusions were drumstick-like in appearance, containing a narrow stalk and a broader headpiece staining intensely with MUSE11 antibody to MUC1. In double staining experiments, the tip regions of the filopodial protrusions contained small amounts of ezrin (Fig 4B), whereas F-actin was found mostly along the stalk (Fig 5B). The presence of F-actin was monitored by staining with rhodamine-labeled phalloidin. The tips of the filopodial protrusions were located on or close to the basal cell surface, probably attached to the bottom of the growth plate (not shown). The stalk was not always found on the basal surface. The results were controlled by omitting the primary antibodies.
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Immunoelectron Tomography
The localization of MUC1 was studied in whole-mounted microvilli and filopodial structures of MCF-7az cells by immunoelectron tomography. Immunoelectron tomography was used because it allows an unparalleled spatial resolution and provides ultrastructural detail about the specimen under inspection. 3D reconstruction enabled us to distinguish between true co-localization and apparent co-localization caused by superimposing structures. The depth of focus in an electron microscope is quite large, and 3D immunoelectron tomography enabled us to view the microvilli from all possible angles. The microvilli, which mostly projected towards the electron beam, were best visualized in specimen grids in which the Formvar membrane had been intentionally shattered, producing an array of cells in different spatial orientations and, in some cases, leading to an unobstructed view of a microvillus with no superimposing structures or Formvar membranes. Ezrin expression was monitored by 10-nm gold particles coupled to the secondary antibody detecting the polyclonal Ez-Bl-III antibody to ezrin. MUC1 expression was monitored by 5-nm gold particle labeling coupled to the secondary antibody detecting the monoclonal MUSE11 antibody to MUC1. The results were also tested by switching the secondary antibodies between MUC1 and ezrin. Omitting the primary antibody tested the specificity of binding. In these control experiments, binding of gold particles to the specimens was not seen. In addition, 1.4-nm gold particles were used to control for differences in permeability between 10-nm and 5-nm gold particles, but the visualization of 1.4-nm gold particles proved to be very difficult and is not discussed here.
As can be seen in Fig 6 and Fig 8, the similar localization of MUC1 and ezrin is also evident in the immunoelectron tomography pictures. In Fig 6, ezrin, depicted by 5-nm gold particles, is localized to the same patches as MUC1, which is labeled by 10-nm gold particles. In contrast when staining for ezrin and actin was performed a different pattern emerged (Fig 7). As before, ezrin was seen at certain patches along the length of the depicted microvillus. In contrast, actin was seen more widely dispersed along the entire length of the structure. This is congruent with the model proposed for the general structure of microvilli. Furthermore, in 3D immunoelectron tomography, 10-nm gold particles staining for ezrin were seen close to the cell membrane, whereas 5-nm gold particles representing actin were seen closer to the microvillar core. This supports the proposed model for the role and function of ezrin and actin in microvilli.
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In a more detailed view of a filopodial structure, the similar localization of ezrin and MUC1 seen in immunofluorescence pictures was also confirmed. Ezrin, labeled by 10-nm gold particles (Fig 8), was distributed along the length of the filopodial structure, accompanied by a cluster of gold-conjugated antibodies representing MUC1 (5-nm gold particles in Fig 8).
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Discussion |
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We have shown that MUC1 and ezrin are largely localized together in microvilli and, to some degree, to the same cortical cell structures and to filopodia. The localization of ezrin and MUC1 to microvilli, in the entire apical surface, is seen clearly in flattened cells. However, there are also marked differences between MUC1 and ezrin in the distribution patterns. In subconfluent cells, the localization of MUC1 is more restricted than the localization of ezrin, which is seen over the entire apical cell surface. Often, MUC1 showed a stronger concentration than ezrin at the topmost apical surface.
In immunofluorescence staining, ezrin is found at the cellcell contact region. However, the presence of ezrin at the cellcell contact region is controversial (
The typical localization of MUC1 is its presence in long filopodial protrusions, and a strong MUC1 concentration at the tips of these filopodial structures was observed. The tips formed enlarged structures, which also contained ezrin, whereas F-actin was seen in the stalk. The amount of ezrin did not correspond to the amount of MUC1. This may be due to the intense signal produced by the binding of MUC1 antibody to the extracellular repeats of the MUC1 glycoprotein. The MUC1 epitope detected by MUSE11 antibody is located in tandem repeats on the extracellular side of the membrane (
It has been shown that MUC1 contains two discrete motifs that determine its apical localization. The first is located in the extracellular domain and the second is the Cys-Gln-Cys motif at the junction of the cytoplasmic and transmembrane domains (
In general, mucins are believed to have anti-adhesion properties (
There is no evidence, as yet, for a direct association of MUC1 and ezrin in low or physiological salt concentrations, as studied in affinity chromatography, immunoprecipitation experiments, or by the Biacore surface plasmon resonance system with GST fusion protein of the cytoplasmic tail of MUC1 and purified ezrin, or ezrin in cell lysates (not shown). However, the possibility that MUC1 would not associate directly or indirectly with ezrin in certain circumstances is not fully ruled out, e.g., after certain induction signals followed by modification or conformational change in the cytoplasmic domain of MUC1. EBP50 is a protein that appears to link ezrin to anion exchangers in epithelial cells (
In vivo, MUC1 and ezrin have similar localizations in the epithelial tissues. Both proteins are located on the apical surface of the epithelial cells (
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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Supported by RIMI/NCRR grant #P20 RR0 11-583 from the National Institutes of Health.
We thank Dr Sandra J. Gendler for the MUC1 cDNA, Dr Yuri Hinoda for the MUSE11 antibody, and Ms Xeuying Wang, Ms Lynn Sydnor, Ms Johnellia Jordan, and Ms Nikki Miller for technical assistance.
Received for publication April 6, 2000; accepted August 5, 2000.
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