Abstract
Abstract: :
Purpose: To determine if the membrane–associated mucin MUC16 is concentrated along membrane folds termed microplicae at the ocular surface epithelial cell–tear film interface and to determine if the cytoplasmic tail of MUC16 has the potential to be linked to the actin cytoskeleton in microplicae, through members of the ezrin, radixin, moesin (ERM) family, proteins that link the actin cytoskeleton to membrane–spanning molecules. Methods: Immunoelectron microscopy using antibody OC125, or antibody H185 which recognizes a carbohydrate epitope on MUC16, was done to localize MUC16 on sections of human corneal epithelium and on cultures of human corneal epithelium. The cytoplasmic tail sequence of the MUC16 mucin was assessed for polybasic regions that are potential ERM binding sites using GenBank Accession #AF361486. Expression of ERMs was determined by PCR, immunoblot analysis and immunofluorescence microscopy using human conjunctival epithelium or immortalized human corneal limbal epithelial cells (HCLE). Commercially available antibodies that recognize a common domain in all three ERMs or are ezrin specific were employed. Results: Electron microscopic immunolocalization of MUC16 and the H185 carbohydrate epitope carried by MUC16 demonstrated that the mucin is concentrated at the tips of the microplicae present on the human ocular surface and cultured human corneal epithelial cells. MUC16 has a 5 AA polybasic sequence in the cytoplasmic tail just inside the membrane, making it a candidate partner for ERM binding. MRNA for ezrin, radixin and moesin were found in human conjunctival epithelium and in HCLE cells. ERM proteins were detected by immunoblot analysis in the same tissue preparations and immunofluorescence microscopy demonstrated that they are localized in apical cells of the ocular surface epithelium. Conclusions: The membrane–associated mucin MUC16 is prominent at the tips of the microplicae of the ocular surface epithelium where it is a component of the glycocalyx. The mucin has the potential to be linked through the microplicae membrane to the actin cytoskeleton through members of the ERM family. Supported by NEI EY03306 to IKG.
Keywords: cornea: surface mucins • cornea: epithelium • cell membrane/membrane specializations