CORNEAL KERATOCYTES - IN-SITU AND IN-VITRO ORGANIZATION OF CYTOSKELETAL CONTRACTILE PROTEINS

Purpose. Recent studies of corneal wound healing suggest that activate d corneal keratocytes develop myofibroblast-like characteristics inclu ding a putative contractile apparatus comprised, in part, of intracell ular microfilament bundles (i.e., stress fibers) containing f-actin, m yosin, and alpha-actinin; extracellurar fibronectin fibrils; and fibro nectin surface membrane receptors (alpha(5) beta(1) integrin). The pur pose 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 organizatio n with that of activated, tissue culture (in vitro) corneal keratocyte s that potentially mimic wound healing fibroblasts. Methods. Cat corne al tissue was obtained immediately after sacrifice and was either fixe d for in situ studies or cultured with MEM supplemented with 10% fetal calf serum for in vitro studies. Keratocytes (in situ and in vitro) w ere stained with the following probes: phalloidin, a mushroom toxin th at specifically binds to f-actin; rabbit anti-bovine aortic myosin; mo noclonal anti-human alpha-actinin; monoclonal anti-human vimentin; rab bit anti-human alpha(5) beta(1) integrin; monoclonal anti-human alpha( 5) integrin; monoclonal anti-human connexin 43; and goat anti-human fi bronectin. The cytoskeletal organization and co-localization were eval uated using epifluorescent and confocal microscopy. Results. Normal, q uiescent corneal keratocytes were distributed within the cornea as a l attice network, interconnected by broad, cellular processes extending from a flattened cell body. The f-actin distribution of in situ kerato cytes was predominantly cortical and appeared to be closely associated with the plasma membrane. In addition, punctate areas that appeared t o correlate with the localization of adhesion sites were identified. T hese punctate regions appeared to stain with antibodies to alpha 5 bet a 1 but to not alpha 5. These data suggest that the fibronectin recept or, alpha 5 beta 1 integrin, is not present on normal corneal keratocy tes. Based on co-localization studies, rabbit anti-bovine aortic myosi n and monoclonal anti-alpha-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-p halloidin staining pattern localized predominantly along intracellular stress fibers not detected in normal, quiescent keratocytes. Myosin a nd alpha-actinin staining had a similar stress fiber distribution, arr anged in alternating bands and suggesting a sarcomeric distribution. A ssociated with stress fibers there was both anti-alpha 5 beta 1 and an ti-alpha 5 staining, indicating the presence of focal adhesions. Concl usions. This study demonstrates that there are major structural differ ences 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 int erconnectivity. Alternatively, activated keratocytes were characterize d by the presence of a putative contractile apparatus comprised of f-a ctin, myosin, and alpha-actinin organized into sarcomeric, muscle-like bundles (stress fibers) associated with focal contacts containing alp ha(5) beta(1) integrin. These data suggest that activation of keratocy tes, i.e. myofibroblast transformation, must involve the reorganizatio n of cytoplasmic contractile proteins as well as the expression of alp ha(5) beta(1), integrin and the formation of focal contacts.