title: Extracellular trafficking of myocilin in human trabecular meshwork cells.
abstract: Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.
authors.affiliation: Department of Cell Biology, the University of Arizona, Tucson, AZ 85724, USA.
authors.forename: Katharine M
authors.initials: KM
authors.lastname: Hardy
authors.forename: Emely A
authors.initials: EA
authors.lastname: Hoffman
authors.forename: Pedro
authors.initials: P
authors.lastname: Gonzalez
authors.forename: Brian S
authors.initials: BS
authors.lastname: McKay
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001692:Biological Transport; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D005136:Eye Proteins / Q000737:chemistry* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006684:HLA-DR Antigens / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D007371:Interferon-gamma / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D021381:Protein Transport; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry; D016622:Silver Staining; D013347:Subcellular Fractions; D013395:Sucrose / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000378:metabolism*; D000099179:Myocilin
chemical_list: D020126:Brefeldin A; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins; D019286:Endopeptidase K; D005136:Eye Proteins; D006023:Glycoproteins; D006684:HLA-DR Antigens; D007371:Interferon-gamma; D011994:Recombinant Proteins; D013395:Sucrose; D000099179:Myocilin