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

Purpose. Recent studies of corneal wound healing suggest that activated corneal keratocytes develop myofibroblast-like characteristics including a putative contractile apparatus comprised, in part, of intracellular microfilament bundles (i.e., stress fibers) containing f-actin, myosin, and α-actinin; extracellular fibronectin fibrils; and fibronectin surface membrane receptors (α₅β₁ integrin). The purpose of this study was to determine the expression and organization of specific components of the contractile apparatus in normal, quiescent (in situ) corneal keratocytes, and to compare the in situ organization with that of activated, tissue culture (in vitro) corneal keratocytes that potentially mimic wound healing fibroblasts. Methods. Cat corneal tissue was obtained immediately after sacrifice and was either fixed for in situ studies or cultured with MEM supplemented with 10% fetal calf serum for in vitro studies. Keratocytes (in situ and in vitro) were stained with the following probes: phalloidin, a mushroom toxin that specifically binds to f-actin; rabbit anti-bovine aortic myosin; monoclonal anti-human α-actinin; monoclonal anti-human vimentin; rabbit anti-human α₅β₁ integrin; monoclonal anti-human α₅ integrin; monoclonal anti-human connexin 43; and goat anti-human fibronectin. The cytoskeletal organization and co-localization were evaluated using epifluorescent and confocal microscopy. Results. Normal, quiescent corneal keratocytes were distributed within the cornea as a lattice network, interconnected by broad, cellular processes extending from a flattened cell body. The f-actin distribution of in situ keratocytes was predominantly cortical and appeared to be closely associated with the plasma membrane. In addition, punctate areas that appeared to correlate with the localization of adhesion sites were identified. These punctate regions appeared to stain with antibodies to α5β1 but to not α5. These data suggest that the fibronectin receptor, α5β1 integrin, is not present on normal corneal keratocytes. Based on co-localization studies, rabbit anti-bovine aortic myosin and monoclonal anti-α-actinin staining had similar distributions to FITC- phalloidin. Interconnections between keratocytes also showed staining for connexin 43, indicating the presence of gap junctions. By contrast, activated, cultured (in vitro) keratocytes showed an FITC-phalloidin staining pattern localized predominantly along intracellular stress fibers not detected in normal, quiescent keratocytes. Myosin and α-actinin staining had a similar stress fiber distribution, arranged in alternating bands and suggesting a sarcomeric distribution. Associated with stress fibers there was both anti-α5β1 and anti-α5 staining, indicating the presence of focal adhesions. Conclusions. This study demonstrates that there are major structural differences in the organization of contractile cytoskeletal proteins between normal, quiescent (in situ), and activated (in vitro) keratocytes. In situ, contractile proteins appear to be associated with the cortical f-actin network, probably related to maintenance of cell shape and interconnectivity. Alternatively, activated keratocytes were characterized by the presence of a putative contractile apparatus comprised of f-actin, myosin, and α-actinin organized into sarcomeric, muscle-like bundles (stress fibers) associated with focal contacts containing α₅β₁ integrin. These data suggest that activation of keratocytes, i.e. myofibroblast transformation, must involve the reorganization of cytoplasmic contractile proteins as well as the expression of α₅β₁ integrin and the formation of focal contacts.