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Seongsoo Sohn, Wonhee Hur, Myung Kuk Joe, Ji-Hyun Kim, Zee-Won Lee, Kwon-Soo Ha, Changwon Kee; Expression of Wild-Type and Truncated Myocilins in Trabecular Meshwork Cells: Their Subcellular Localizations and Cytotoxicities. Invest. Ophthalmol. Vis. Sci. 2002;43(12):3680-3685.
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purpose. To investigate the subcellular localizations and potential cytotoxicities of wild-type and truncated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells.
methods. Full-length wild-type myocilin, truncated myocilin, and stromelysin were expressed as green fluorescence (GFP) or DsRed fusion proteins in TM cells by using adenoviral vectors, and their secretory properties and cytopathic effects were evaluated by Western blot analysis and cell proliferation assay, respectively. To determine the subcellular localizations of myocilins, the cellular organelles of the infected TM cells were stained with organelle-specific antibodies or fluorescent indicators and examined under a confocal microscope.
results. Wild-type myocilin was expressed mainly in the perinuclear region of TM cells and was localized preferentially in endoplasmic reticulum (ER), but not in actin, microtubules, or mitochondria. Truncated myocilin was also localized in ER, and its expression was found to be potentially toxic to TM cells, leading to deformed cellular morphology and diminished cell proliferation, but it had no effect on the secretion of stromelysin. The truncated myocilin was also found to be colocalized with and appeared to aggregate with wild-type myocilin when the proteins were coexpressed.
conclusions. TM cells participating in the turnover of trabecular extracellular matrix (ECM) components are important in regulating aqueous outflow. The truncated myocilin, colocalized and coaggregated with wild-type myocilin, is believed to cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.
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