Corneal keratocytes: In situ and in vitro organization of cytoskeletal contractile proteins

摘要: Purpose Recent studies of corneal wound healing suggest that activated keratocytes develop myofibroblast-like characteristics including a putative contractile apparatus comprised, in part, intracellular microfilament bundles (i.e., stress fibers) containing f-actin, myosin, and alpha-actinin; extracellular fibronectin fibrils; surface membrane receptors (alpha 5 beta 1 integrin). The purpose this study was to determine the expression organization specific components normal, quiescent (in situ) keratocytes, compare situ with activated, tissue culture vitro) potentially mimic fibroblasts. Methods Cat obtained immediately after sacrifice either fixed for or cultured MEM supplemented 10% fetal calf serum vitro studies. Keratocytes were stained following probes: phalloidin, mushroom toxin specifically binds f-actin; rabbit anti-bovine aortic myosin; monoclonal anti-human vimentin; alpha integrin; connexin 43; goat fibronectin. cytoskeletal co-localization evaluated using epifluorescent confocal microscopy. Results Normal, distributed within cornea as lattice network, interconnected by broad, cellular processes extending from flattened cell body. f-actin distribution predominantly cortical appeared be closely associated plasma membrane. In addition, punctate areas correlate localization adhesion sites identified. These regions stain antibodies but not 5. data receptor, integrin, is present on normal keratocytes. Based studies, myosin anti-alpha-actinin staining had similar distributions FITC-phalloidin. Interconnections between also showed 43, indicating presence gap junctions. By contrast, an FITC-phalloidin pattern localized along fibers detected Myosin alpha-actinin fiber distribution, arranged alternating bands suggesting sarcomeric distribution. Associated there both anti-alpha staining, focal adhesions. Conclusions This demonstrates are major structural differences proteins situ), situ, appear probably related maintenance shape interconnectivity. Alternatively, characterized comprised organized into sarcomeric, muscle-like (stress contacts integrin. activation i.e. myofibroblast transformation, must involve reorganization cytoplasmic well integrin formation contacts.