H2-calponin Gene Knockout Increases Traction Force of Mouse Fibroblasts in vitro

Abstract

Cell traction force (CTF) plays a critical role in controlling cell shape, enabling cell motility, and maintaining cellular homeostasis during various biological processes such as wound healing, angiogenesis, and cancer metastasis. It has been demonstrated that h2-calponin, an actin binding protein found in smooth muscle and non-muscle cells including epithelial cells, endothelial cells, macrophages and fibroblasts, plays a role in regulating the actin cytoskeleton activities in cell adhesion, migration and cytokinesis. We recently found that knockout (KO) of h2-calponin gene increased cell motility when compared to wild-type (WT) cells. This finding indicated a potential involvement of h2-calponin in producing CTF. The present study investigated the role of h2-calponin in mouse fibroblast traction force. Primary fibroblasts were isolated from leg muscles of h2-calponin KO and WT mice and analyzed using CTF-microscopy. CTF-microscopy is the current state-of-the-art method to determine CTF in a cell spread on a two-dimensional substrate. Using CTF-microscopy, we determined the root-mean square traction force, the total strain energy, net contractile movement produced by mouse fibroblasts cultured on a thin layer of 8-kPa polyacrylamide gel containing fluorescent beads of 0.2 µm in diameter. The results showed that h2-calponin KO fibroblasts had greater traction force than WT control. In comparison to WT cells expressing abundant tropomyosin-2, h2-calponin KO fibroblasts lost tropomyosin-2, a phenotype mimicking that of metastatic cancer cells. H2-calponin KO fibroblasts also adhered to cultural substrate slower than WT control, had smaller cell spreading area, and rounded up faster during trypsin treatment, supporting the role of h2-calponin in stabilizing the actin cytoskeleton. Our findings indicate that h2-calponin has an inhibitory role in the production of CTF, consistent with the increased motility of h2-calponin-null cells. Further studies on the mechanisms of h2-calponin-mediated CTF regulation and cell motility are underway.