Abstract
Abstract: :
Purpose: To study the normal secretory pathway of myocilin using agents that disrupt the secretory pathway and to visualize fluorescent protein (FP) tagged normal and mutant myocilin in relation to cellular organelles. Methods: CHO/C15 which constitutively secrete myocilin into the media were treated with brefelden A, tunicamycin and monensin to look at their effects on the production/secretion of myocilin. After treatment, cell extracts and/or media were run on NuPAGE gels, blotted to PVDF, and probed vs. anti- myocilin antibody (AB129). Plasmids of wild type and disease-causing mutant myocilin were constructed fusing red fluorescent protein (DsRED) at the C-terminus. Transformed trabecular meshwork cells (TM5) were co-transfected using Lipofectamine 2000 with the DsRED-tagged myocilin plasmid along with plasmids encoding Enhanced Green Fluorescent Protein (EGFP) tagged proteins directed to subcellular organelles ( ER, Golgi, peroxisome, mitochondria or cytoskeleton). Cellular localization was observed using confocal microscopy and deconvolution microscopy in cells 48 hrs after transfection. Results: Myocilin is a 54 and 57kDa doublet on SDS-PAGE immunoblots. Brefelden A leads to cessation of secretion of myocilin and an accumulation of myocilin inside the cell. Both myocilin bands are affected by treatment with brefelden A. Tunicamycin prevents secretion of the upper band of myocilin, but does not prevent secretion of the lower band. Monensin decreases the amount of both bands of myocilin into the media. Wild-type myocilin was observed in ER and Golgi. Also, small vesicle-like structures could be seen throughout the cell and even in the long processes extending out from the cell body. The myocilinY437H mutation was mainly localized in the ER with very few vesicular structures showing the DsRED label. The myocilin G364V mutation showed localization in the ER and formed large complexes in the cytoplasm, suggestive of aggresomes. Conclusions: Our data suggest that two cellular pools of myocilin are secreted into the media of cultured cells. The larger mw form, is less stable and requires N-glycosylation, while, the smaller, more stable form does not. Wild-type myocilin is synthesized via the normal secretory pathway whereas disease-causing mutations remain in the cell for degradation by the cellular machinery. Our results support a role for protein misfolding and aggregation in the mutant myocilin phenotype.
Keywords: mutations • proteins encoded by disease genes • trabecular meshwork