title: Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul, Korea.
authors.forename: Seongsoo
authors.initials: S
authors.lastname: Sohn
authors.forename: Wonhee
authors.initials: W
authors.lastname: Hur
authors.forename: Myung Kuk
authors.initials: MK
authors.lastname: Joe
authors.forename: Ji-Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Zee-Won
authors.initials: ZW
authors.lastname: Lee
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha
authors.forename: Changwon
authors.initials: C
authors.lastname: Kee
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D015870:Gene Expression; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019278:Matrix Metalloproteinase 3; D011993:Recombinant Fusion Proteins; D000099179:Myocilin; C413663:fluorescent protein 583