ARTICLE |
Correspondence to: Beatrice Y.J.T. Yue, U. of Illinois at Chicago, Department of Opthalmology and Visual Sciences, 1855 W. Taylor Street, Chicago, IL 60612. E-mail: u24184@uic.edu
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Summary |
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We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites. (J Histochem Cytochem 48:13211329, 2000)
Key Words: cell cultures, glaucoma, human, immunoelectron microscopy, myocilin, TIGR, tissue, trabecular meshwork
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Introduction |
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The trabecular meshwork (TM), a specialized eye tissue located at the chamber angle of the eye next to the cornea, regulates the outflow of the aqueous humor and controls the intraocular pressure (IOP) (
The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified (
The myocilin transcript has been detected in the TM (
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Materials and Methods |
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Cell Culture and DEX Treatment
Normal human donor eyes with no history of glaucoma or other eye diseases were obtained from the Illinos Eye Bank at Chicago. TM tissues from three donors (ages 19, 19, and 29 years) were excised and cultured on Falcon Primaria flasks in complete medium composed of Eagle's minimal essential medium, 10% fetal bovine serum, 5% calf serum, essential and nonessential amino acids, and antibiotics (
Specimen Preparation
Cultured human TM cells without or with the DEX treatment were fixed at room temperature (RT) for 3 hr in a fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.05% Triton X-100 in 0.1 M phosphate buffer, pH 7.4. After rinsing three times in PBS, the cells were dehydrated at -20C through a graded series of N,N-dimethyl formamide (DMF). DMF was then exchanged stepwise to glycol methacrylate (GMA) embedding medium that was made up of 65 ml 2-hydroxyethyl methacrylate, 35 ml N-butyl methacrylate, 5 ml ethylene glycol dimethacrylate, and 0.5 g benzoin methyl ether (
For in vivo studies, normal human eyes from three donors (ages 48, 58, and 72 years) with no history of ocular diseases were obtained from the Illinois Eye Bank. The anterior segments that included TM tissues were excised from donor eyes. They were fixed at RT for 3 hr in 4% paraformaldhyde, 0.1% glutaradehyde, 0.1 M phosphate buffer, pH 7.4, and were further dissected into small wedge-shaped pieces. The specimens were dehydrated through a DMF series and embedded in GMA block as described above. Ultrathin sections cut transversely through the entire layers of TM were mounted on EM grids.
Polyclonal Antibody and Western Blotting Analysis
Polyclonal anti-myocilin was generated in rabbits against a synthetic polypeptide corresponding to the amino acid sequence DKSVLEEEKKRLRQ (residues 148161) of human myocilin. The peptide was coupled to keyhole limpet hemocyanin via a carboxy-terminal cysteine residue not present in myocilin. The sythetic peptide and the antibody were prepared by Alpha Diagnostic International (San Antonio, TX). The antibody was purified by an affinity column and its specificity was confirmed by ELISA. To further verify its specificity, Western blotting analysis was performed. Briefly, protein extracts from normal human TM tissues (donor ages 43, 52, and 53 years) were subjected to SDS-PAGE and were transferred to nitrocellulose membranes. The blots were immunoblotted with either anti-myocilin (1:5000) or the antibody preabsorbed with the immunogenic peptide. The immunoblots were further incubated with biotinylated goat anti-rabbit IgG (Jackson Immuno Research; West Grove, PA), horseradish peroxidase-conjugated streptavidin (Jackson Immuno Research), and Super Signal West Pico (Pierce; Rockford, IL). Using a Fluoroimager Image Station 440CF (Eastman Kodak; Rochester, NY), the typical 66- and 57/55-kD myocilin bands (
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Immunogold Labeling and EM Study
Ultrathin sections were incubated in 1% bovine serum albumin in PBS for 15 min at RT to minimize nonspecific binding. The blocking solution was removed and the grids were incubated at RT for 3 hr with anti-myocilin (1:200). After the antibody incubation, the grids were rinsed by shaking thoroughly in a mixture of 0.5 M NaCl and 0.05% Tween-20 in 0.1 M phosphate buffer. The sections were incubated at RT for 1 hr with 12-nm colloidal gold-conjugated goat anti-rabbit IgG (Jackson Immuno Research) at a dilution of 1:30 in blocking solution. They were then washed, counterstained with uranyl acetate, and examined under a transmission EM (JEM-1220; JEOL, Tokyo, Japan) at 80 kV accelerating voltage. For negative controls, anti-myocilin at the same dilution preabsorbed with the immunogenic sythetic peptide was used.
To identify the structures with which myocilin was associated, double labeling (
Quantification of Immunogold Labeling
To quantify the density of gold labeling, 10 photomicrographs at a magnification of x10,000 were taken and a total of 50 images were randomly chosen and enlarged to x50,000. The gold particles associated with intracellular structures such as mitochondria, vesicles, acting filaments, and intermediate filaments in both control and DEX-treated cultured cells were counted using a Microtek imaging system and the MetaMorph software (Universal Imaging; West Chester, PA). The labeling density was expressed as either number of particles/µm2 or number of particles/structure. The density in the surrounding cytosol in cultured cells (nonspecific labeling) was also counted. Differences in labeling between each structure and the cytosol were statistically evaluated by Student's t-tests. In normal TM tissues, gold particles associated with TM cells and the ECM were analyzed in the same manner. The labeling density was statistically compared with that in the intertrabecular space.
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Results |
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Myocilin in Cultured Human TM Cells
In both DEX-treated (Fig 2A) and control TM cells, immunogold labeling of myocilin was localized to mitochondria. This protein was also associated with cytoplasmic filaments (Fig 2A and Fig 2B) and, to a lesser degree, with intracellular vesicles (Fig 2B). When the peptide-preabsorbed antibody was used in place of anti-myocilin, the gold labeling was reduced to a minimal level (Fig 2C). Quantification of the gold labels (Table 1) confirmed that the mitochondrion was a major site of myocilin association. The cytoplasmic filaments and vesicles were also significantly (p<0.0001) more densely labeled than the surrounding cytosol.
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Double labeling experiments performed using different sizes of gold particles further revealed that myocilin co-localized with cytochrome c oxidase subunit II to mitochondria (Fig 3), with vimentin to intermediate filaments in the cell cortex (Fig 4A), and with actin to stress fibers (Fig 4B). The co-localization was observed in all three lines of cultured human TM cells, with or without the DEX treatment. Myocilin did not appear to co-localize with ß-tubulin, a component of microtubules, although an association with centrosomes was evident (Fig 4C).
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Myocilin in Normal Human TM Tissues
In normal human TM tissues, myocilin-associated colloidal gold particles were associated with TM cells and with elastin-like fibers, as well as their surrounding electron-dense matrices, in the beams of the uveal and corneoscleral meshworks (Fig 5A). In the JCT region, immunogold labeling for myocilin was observed in the JCT cells, the inner wall cells of Schlemm's canal, and in the electron-lucent and electron-dense extracellular matrices (ECMs), including elastin-like fibers (Fig 5C). The labeling density (Table 2) in each region was significantly (p<0.0001) differently from that in the intertrabecular space. In negative controls, the gold particles were mostly eliminated by replacement of anti-myocilin with the peptide-preabsorbed antibody (Fig 5B and Fig 5D).
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Co-localization of myocilin and cytochrome c oxidase subunit II to mitochondria was also seen in the cells of normal human TM tissues (Fig 6A). A giant mitochondrion was observed in a TM cell of the corneoscleral meshwork, in which myocilin was localized particularly to the mitochondrial inner membrane (Fig 6B). Furthermore, in the cells of the corneoscleral meshwork, myocilin was associated with intermediate filaments labeled by vimentin (Fig 6C).
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Discussion |
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Using a postembedding immunogold EM method, this study provides conclusive evidence that myocilin is localized at both intra- and extracelluar sites in the TM. Of special interest in the association of myocilin with mitochondria. In a previous immunofluorescence study, confocal scanning laser microscopy disclosed that myocilin co-localized with mitochondria, especially in the perinuclear regions of cultured human TM cells either with or without DEX treatment (
The mitochondrial localization was observed not only in cultured TM cells but also in cells residing in TM tissues. The biological significance of such a mitochondrial connection is presently unknown. However, because further experiments demonstrated myocilin location in the inner membrane of mitochondria, it is tempting to speculate that myocilin may participate in the energy transfer and metabolic activities that normally take place at this site (
Because of the myosin-like domain in its gene structure, myocilin has been suspected to be connected with actin and/or microtubules (
Our data, although demonstrating the association of the endogenous myocilin with actin and vimentin, indicated no myocilin connection with ß-tubulin or microtubules. This observation is in direct contrast to a recent result showing that, with green fluorescent protein as a marker, highly overexpressed myocilin in transfected cells co-localized with microtubules (
In their study of the retina, myocilin was noted by -tubulins to form ring-like complexes (
Myocilin labeling was observed to a lesser degree on vesicles. The vesicular association, also suggested recently by
The investigations using in vivo TM tissues, demonstrating the same pattern of myocilin intracellular distribution as that in cultured cells, validate the in vitro culture findings. More significantly, the tissue studies provide, for the first time, experimental evidence depicting the extracellular localization of myocilin. In the uveal and corneoscleral meshwork, myocilin labeling was detected in the central core of trabecular beams, particularly in association with elastin-like fibers. Myocilin was also seen dispersed in the ECM of JCT regions. ECM components underlying the cells are believed to be important for a properly functioning TM (
Topical and systemic treatments with glucocorticoids such as DEX affect the fluid dynamics of the eye and raise IOP in normal human eyes (
In summary, myocilin was ultrastructurally localized to multiple intracellular and extracellular sites in normal human TM. This protein, depending on the sites at which it localizes, may exert different biological functions. The current localization data help shed light into the possible functions of myocilin and provide a basis for future investigative pursuits on this novel protein.
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Acknowledgments |
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Supported in part by research grants EY 05628, EY 03890, and core grant EY 01792 from the National Institutes of Health (Bethesda, MD) and by a Senior Investigator Award from Research to Prevent Blindness, Inc., New York, NY. K.K.W.H. was supported by an Individual NSRA EY 06889 from the National Institutes of Health.
We thank Kira Lathrop for expert advice in imaging and Chan Boriboun for assistance in tissue culture experiments.
Received for publication December 27, 1999; accepted April 20, 2000.
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