Stress fiber formation is required for matrix reorganization in a corneal myofibroblast cell line.
Corneal wound healing fibroblasts (myofibroblasts) develop a muscle-like contractile apparatus composed of prominent microfilament bundles (stress fibers) and express alpha-smooth muscle actin (alpha-SMA). In this study, gelsolin, an actin filament-severing protein, was overexpressed in a alpha-SMA-expressing corneal myofibroblast cell line (TRK43) to assess whether intact stress fibers are required for in vitro matrix organization and wound contraction. 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 while the remaining half appeared epithelioid. One fibroblastic clone, GS56, and one epithelioid clone, GS44, were selected for detailed characterization. The GS56 cells appeared highly elongated and spindle-shaped and had prominent stress fibers and focal adhesions. GS44 cells showed disruption of stress fibers and a cortical f-actin organization as well as the down regulation of alpha-SMA expression by immunocytochemistry and Western blotting. Both phenotypes showed enhanced gelsolin expression; however, fractionation of cell extracts demonstrated differences in the subcellular distribution of gelsolin with GS44 cells having markedly reduced and GS56 cells having markedly increased cytoskeletal gelsolin. In an in vitro wound contraction assay, epithelioid GS44 cells showed a significantly impaired ability to contract a collagen matrix compared to that of TRK43 cells, CT9 or GS56 transfectants. 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 organization 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 localization of gelsolin and its effect on stress fiber formation in the two cell lines.
Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75235-9057, USA.
P K
PK
Mar
P
P
Roy
H L
HL
Yin
H D
HD
Cavanagh
J V
JV
Jester
D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism; D015536:Down-Regulation; D018274:Electroporation; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D018260:Gelsolin / Q000378:metabolism; D011817:Rabbits; D022502:Stress Fibers / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology*
D018260:Gelsolin