Stress fiber formation is required for matrix reorganization in a corneal myofibroblast cell line

Corneal wound healing fibroblasts (myofibroblasts) develop a muscle-like co ntractile apparatus composed of prominent microfilament bundles (stress fib ers) and express alpha -smooth muscle actin (alpha -SMA). In this study, ge lsolin, an actin filament-severing protein, was overexpressed in a alpha -S MA-expressing corneal myofibroblast cell line (TRK43) to assess whether int act stress fibers are required for in vitro matrix organization and wound c ontraction. Stably integrated gelsolin was introduced by electroporation of an expression construct (pREPCG8) into cultured cells. Thirty-seven clones were isolated with half of the clones showing a fibroblastic phenotype whi le the remaining half appeal ed epithelioid. One fibroblastic clone, GS56, and one epithelioid clone, GS44, were selected for detailed characterizatio n. The GS56 cells appeared highly elongated and spindle-shaped and had prom inent stress fibers and focal adhesions. GS44 cells showed disruption of st ress fibers and a cortical f-actin organization as well as the down regulat ion of alpha -SMA expression by immunocytochemistry and Western blotting. B oth phenotypes showed enhanced gelsolin expression; however, fractionation of cell extracts demonstrated differences in the subcellular distribution o f gelsolin with GS44 cells having markedly reduced and GSSG cells having ma rkedly increased cytoskeletal gelsolin. In an in vitro wound contraction as say, epithelioid GS44 cells showed a significantly impaired ability to cont ract a collagen matrix compared to that of TRK43 cells, CT9 or GS56 transfe ctants. Loss of stress fibers in GS44 cells also correlated. with enhanced cell motility. Together, these results demonstrate that the ability to form microfilament bundles or stress fibers is required for matrix; organizatio n and contraction by corneal myofibroblasts. Although no clear explanation is available, we suspect that differences in gene insertion of the gelsolin overexpression vector may have led to differential intercellular localizat ion of gelsolin and its effect on stress fiber formation in the two cell li nes. (C) 2001 Academic Press.