Isolation and Contraction of the Stress Fiber

Abstract

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-alpha-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion-associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

“…Isometrically stressed lattices are a more appropriate model of myofibroblast-populated granulation tissue during wound contraction compared with mechanically unloaded floating lattices (Grinnell, 1994). Isometric tension has been shown to be important for the development of myofibroblastic contractile features such as stress fibers (Burridge, 1981;Tomasek et al, 1992;Katoh et al, 1998;Grinnell et al, 1999a) and ␣-SMA expression in collagen lattices (Arora et al, 1999b). To test whether individual ␣-SMA-positive fibroblasts contract more efficiently than ␣-SMA-negative cells under identical conditions, we used a modification of the wrinkling silicone elastomer assay, originally developed by Harris et al (1980), which is particularly useful in order to visualize overall cell contractile forces.…”

“…Because wound contraction takes place when de novo expressed ␣-SMA is incorporated in stress fibers (Darby et al, 1990), it has been suggested that this actin isoform plays an important role in granulation tissue contraction (for review see Serini and Gabbiani, 1999). Although stress fibers have been proposed to function as contractile organelles (Burridge, 1981;Katoh et al, 1998) and their presence has been correlated with the production of isometric tension (Harris et al, 1981), at present direct evidence of a functional role of ␣-SMA in fibroblast contraction is lacking. Here, we have used several in vitro models and approaches in order to test the possible correlation between the expression of ␣-SMA and the contractile activity of fibroblastic cells.…”

On the role of the actin cytoskeleton and nucleus in the biomechanical response of spread cells

“…Previous efforts to characterise the tensile behaviour of SFs have relied on isolation/extraction of individual SFs from the cell, followed by tensile testing [29]. It is important to note that isolated/extracted SFs behave as a passive material [30], neglecting the critically important features of active myosin induced cross-bridge cycling and remodelling.…”

Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle

“…Nuclei were isolated from the cultured ECs with a chemical treatment previously reported (Katoh et al, 1998). Briefly, the confluent cells were washed with PBS (Dulbecco's phosphate-buffered saline without Ca 2+ or Mg 2+ , Wako, Osaka, Japan) and treated with an ice-cold low-ionic-strength extraction solution consisting of 2.5 mM triethanolamine (Wako), 1 µg/ml leupeptin (Wako), and 1 µg/ml pepstatin (Wako) in distilled water for 10 min.…”