Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore,loss of radial glia disrupts the inner and outer barriers of the neural tube,disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells,transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.

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.

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.

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-α-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.

Stress fibres and associated focal adhesions in cells constitute a contractile apparatus that regulates cell motility and contraction. Rho-kinase, an effector molecule of small GTPases, regulates non-muscle cell motility and contractility. Rho-kinase mediates the contraction of stress fibres in a Ca ²⁺ -independent manner, and is responsible for slower and more finely tuned contraction of stress fibres than that regulated by myosin light chain kinase activity in living cells. The specific inhibition of the Rho-kinase activity causes cells to not only lose their stress fibres and focal adhesions, but also to appear to lose their cytoplasmic tension. Activated Rho-kinase is also involved in the organization of newly formed stress fibres and focal adhesions in living cells.

Mechano-signal transduction is a process in which cells perceive extracellular mechanical signals, convert them into intracellular biochemical signals, and produce a response. Integrins are cell surface receptors that sense the extracellular mechanical cues and bind to the extracellular matrix (ECM). This binding induces integrin clustering and activation. Cytoplasmic tails of activated integrins interact and induce cytoskeleton tensions via several adaptor proteins. Integrins monitor extracellular stiffness via cytoskeleton tensions and modulate ECM stiffness via downstream signaling pathways regulating the expression of genes of ECM components. Integrin-mediated mechano-transduction is very crucial for the cell as it regulates the cell physiology both in normal and diseased conditions according to extracellular mechanical cues. It regulates cell proliferation, survival, and migration. Abnormal mechanical cues such as extreme and prolonged mechanical stress result in pathological conditions including fibrosis, cancers, skin, and autoimmune disorders. This paper aims to explore the role of integrins and their associated proteins in mechano-signal transduction. It highlights the integrins and their associated proteins as targets for therapy development. Furthermore, it also presents the challenges to the targeted drug development, which can be drug resistance and cytotoxicity. It is concluded in this paper that research on integrin-mediated mechano-signal transduction and its relationship with cell physiology and pathologies will be an important step towards the development of effective therapies.