Yumiko Kano

Lateral Zone of Cell-Cell Adhesion as the Major Fluid Shear Stress–Related Signal Transduction Site

It has been proposed previously that actin filaments and cell adhesion sites are involved in mechanosignal transduction. In this study, we present certain morphological evidence that supports this hypothesis. The 3D disposition of actin filaments and phosphotyrosine-containing proteins in endothelial cells in situ was analyzed by using confocal microscopy and image reconstruction techniques. Surgical coarctations were made in guinea pig aortas, and the same 3D studies were conducted on such areas 1 week later. Stress fibers (SFs) were present at both basal and apical regions of endothelial cells regardless of coarctation, and several phosphotyrosine-containing proteins were associated with SF ends. Apical SFs had one end attached to the apical cell membrane and the other attached to either the basal membrane or the lateral cell border. Within the coarctation area, the actin filament-containing and vinculin-containing structures became prominent, especially at the apical and the lateral regions. Substantially higher levels of anti-phosphotyrosine and anti-Src staining were detected in the constricted area, particularly at the cell-cell apposition, whereas the anti-focal adhesion kinase, anti-CT10-related kinase, anti-platelet endothelial cell adhesion molecule-l, anti-vinculin, and phalloidin staining intensities increased only slightly after coarctation. We propose that apical SFs directly transmit the mechanical force of flow from the cell apex to the lateral and/or basal SF anchoring sites and that the SF ends associated with signaling molecules are sites of signal transduction. Our results support the idea that the cell apposition area is the major fluid shear stress-dependent mechanosignal transduction site in endothelial cells.

Rho-associated kinase-dependent contraction of stress fibres and the organization of focal adhesions

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 Ca2+-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.

Localization of a developmentally regulated neuron-specific protein S54 in dendrites as revealed by immunoelectron microscopy

We sought to determine the ultrastructural localization of the developmentally regulated neuron-specific protein S54 in the chicken cerebellar cortex and optic tectum. The brains were fixed by perfusion with paraformaldehyde and glutaraldehyde. Frozen sections were immunocytochemically labeled with a monoclonal antibody to S54 protein. The immunoreactivity for S54 protein was localized in dendrites. No immunoreactivity for S54 protein was detected in axons and their presynaptic terminals.

Role of stress fibers and focal adhesions as a mediator for mechano-signal transduction in endothelial cells in situ

Fluid shear stress is the mechanical force generated by the blood flow which is applied over the apical surface of endothelial cells in situ. The findings of a recent study suggest that stress fibers and its associated focal adhesions play roles in mechano-signal transduction mechanism. Stress fibers are present along the apical and the basal portion of the endothelial cells. Endothelial cells respond to fluid shear stress and change their morphological characteristics in both their cell shape and cytoskeletal organization. Atherosclerosis is a common disease of the arteries and it occurs in areas around the branching site of blood vessels where the cells are exposed to low fluid shear stress. The organization of stress fibers and focal adhesions are strongly influenced by shear stress, and therefore the generation of atherosclerotic lesions seem to be associated with the cytoskeletal components of endothelial cells. This review describes the possible role of the cytoskeleton as a mechano-transducer in endothelial cells in situ.

Isolation and in vitro contraction of stress fibers.

Publisher Summary Isolated stress fibers are useful for studying their properties and functions, but they are also useful as a nonmuscle contraction model system for investigating the regulatory mechanism for the actomyosin-based contractility in nonmuscle cells. This chapter discusses the isolation of stress fibers en masse from cultured cells. It also describes the way to observe contraction of stress fibers. The isolation procedure is simple and provides stress fibers that are pure enough for biochemical as well as structural and other studies. The contraction of stress fibers is best observed by using those still attached to the substrate surface. The contraction is an actomyosin-based, ATP-driven contraction. Rho kinase is involved in stress fiber contraction. Both RhoA and Rho kinase are present in isolated stress fibers. The chapter explains that the stress fiber model can contract in the absence of Ca 2+ and that this contraction is inhibited by a Rho kinase inhibitor. In this case, the myosin regulatory light chain is phosphorylated, not by myosin light chain kinase, but by Rho kinase. The studies presented in the chapter indicate that stress fiber contraction is regulated by two independent systems: one by the Ca 2+ - dependent myosin light chain kinase system and the other by the Ca 2+ - independent Rho kinase system.

Response of Vascular Endothelial Cells to Fluid Flow

KEIGI FUJIWARA, MICHITAKA MASUDA, MASAKI OSAWA, KAZUO KATOH, YUMIKO KANO, NOBORU HARADA, AND ROSANGELA B. LOPES Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, 56.54565 Japan Fluid flow triggers a variety of responses in vascular endothelial cells (ECs), such as initiation of signal trans- duction, modulation of gene expression, and remodeling of cytoskeletal and related structures. However, the pri- mary steps of mechanosensing are not known. Because fluid flow is a mechanical (thus vectorial) stimulus, we decided to study how ECs respond in a vectorial manner. Among the various types of responses (for review, see Davies, 1995), those involving the cytoskeleton are clearly vectorial. Using a parallel plate flow chamber mounted on a light microscope, we first analyzed morphological responses of ECs to laminar flow (Masuda and Fujiwara, 1993a, b). We found that, in addition to the already known morpho- logical responses of ECs to flow (i.e., the elongation and alignment of ECs parallel to the direction of flow and alignment of stress fibers in the flow direction), flow in- duced preferential development of lamellipodia in the di- rection of flow. This latter response caused ECs to migrate preferentially in the flow direction. Although it takes many hours for both the cell shape change and the align- ment responses to become recognizable, the motility pat- tern change was detectable in 5 - 10 min. This is the fastest morphology-related response of ECs exposed to flow. ECs exhibit little morphologically detectable responses when exposed to fluid shear stress of less than 0.4 Pa (4 dyn/cm2), although it is known that many signal-transduc-