It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca2+. However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca2+. More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca2+-dependent MLCK and the Rho-kinase systems. We propose that Ca2+ is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.

Insulin receptor tyrosine kinase substrate p53 (IRSp53) has been identified as an SH3 domain-containing adaptor that links Rac1 with a Wiskott-Aldrich syndrome family verprolin-homologous protein 2 (WAVE2) to induce lamellipodia or Cdc42 with Mena to induce filopodia. The recruitment of these SH3-binding partners by IRSp53 is thought to be crucial for F-actin rearrangements. Here, we show that the N-terminal predicted helical stretch of 250 amino acids of IRSp53 is an evolutionarily conserved F-actin bundling domain involved in filopodium formation. Five proteins including IRSp53 and missing in metastasis (MIM) protein share this unique domain and are highly conserved in vertebrates. We named the conserved domain IRSp53/MIM homology domain (IMD). The IMD has domain relatives in invertebrates but does not show obvious homology to any known actin interacting proteins. The IMD alone, derived from either IRSp53 or MIM, induced filopodia in HeLa cells and the formation of tightly packed parallel F-actin bundles in vitro. These results suggest that IRSp53 and MIM belong to a novel actin bundling protein family. Furthermore, we found that filopodium-inducing IMD activity in the full-length IRSp53 was regulated by active Cdc42 and Rac1. The SH3 domain was not necessary for IMD-induced filopodium formation. Our results indicate that IRSp53, when activated by small GTPases, participates in F-actin reorganization not only in an SH3-dependent manner but also in a manner dependent on the activity of the IMD.Insulin receptor tyrosine kinase substrate p53 (IRSp53), 1 also known as brain-specific angiogenesis inhibitor 1-associated protein 2, is a multifunctional adaptor protein enriched in the central nervous system (1-3). The protein contains a unique N-terminal 250-amino acid stretch, a half-Cdc42/Rac interactive binding (CRIB) motif, a proline-rich domain, a Src homology 3 (SH3) domain, and a WW domain-binding motif (WWB). IRSp53 is directly regulated by Rho family small GTPases Rac1 and Cdc42 and provides a molecular link between these GTPases and the actin cytoskeleton regulators WiskottAldrich syndrome protein (WASP) family verprolin homologous protein 2 (WAVE2) and mammalian enabled (Mena), which are involved in the formation of lamellipodia (4, 5) and filopodia (6, 7). Active Cdc42 binds to the half-CRIB motif (6, 7), whereas Rac1 binds to the unique N-terminal domain (8). The association of Rac1 or Cdc42 is proposed to liberate the C-terminal SH3 domain masked intramolecularly by its N terminus, thereby allowing the SH3 domain to interact with its binding partners (4, 7, 9). Thus, the SH3 domain is thought to be essential for IRSp53-mediated actin reorganization. However, the N-terminal half of IRSp53 lacking the SH3 domain was reported to induce neurite outgrowth in a neuroblastoma cell line (6) and filopodia in B16 melanoma cells (10), suggesting that IRSp53 promotes actin reorganization independently of SH3 domain-mediated intermolecular interactions.Recently, a novel monomeric actin-binding protein, missing in met...

High hepatocyte-selective enhancement is induced by expression patterns of transporters, which may result in accumulation of gadoxetic acid in cytoplasm of tumor cells or in lumina of pseudoglands. An HCC with gadoxetic acid enhancement is characterized by bile accumulation in tumors.

Reduced Gd-EOB-DTPA uptake might be an early event of hepatocarcinogenesis, preceding portal blood flow reduction. The hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI may help estimate histological grading, although difficulties exist in differentiating HCCs from DNs.

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.