Sohn Seongsoo, Hur Wonhee, Joe Myung Kuk, Kim Ji-Hyun, Lee Zee-Won, Ha Kwon-Soo, Kee Changwon
Department of Ophthalmology, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2002 Dec;43(12):3680-5.
To investigate the subcellular localizations and potential cytotoxicities of wild-type and truncated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells.
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.
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.
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.
研究野生型和截短型（Q368X）肌纤蛋白在培养的人小梁网（TM）细胞中的亚细胞定位及潜在细胞毒性。
利用腺病毒载体在TM细胞中将全长野生型肌纤蛋白、截短型肌纤蛋白和基质溶解素表达为绿色荧光（GFP）或DsRed融合蛋白，分别通过蛋白质免疫印迹分析和细胞增殖试验评估它们的分泌特性和细胞病变效应。为确定肌纤蛋白的亚细胞定位，用细胞器特异性抗体或荧光指示剂对感染的TM细胞的细胞器进行染色，并在共聚焦显微镜下检查。
野生型肌纤蛋白主要在TM细胞的核周区域表达，优先定位于内质网（ER），而不在肌动蛋白、微管或线粒体中。截短型肌纤蛋白也定位于内质网，发现其表达对TM细胞有潜在毒性，导致细胞形态变形和细胞增殖减少，但对基质溶解素的分泌无影响。当共表达这两种蛋白时，还发现截短型肌纤蛋白与野生型肌纤蛋白共定位并似乎聚集在一起。
参与小梁细胞外基质（ECM）成分更新的TM细胞在调节房水流出中起重要作用。截短型肌纤蛋白与野生型肌纤蛋白共定位并共聚集，被认为会导致细胞功能障碍，从而引起小梁ECM结构分区改变和房水流出受阻。
