Keiji Sugimoto

Oblique Alignment of Stress Fibers in Cells Reduces the Mechanical Stress in Cyclically Deforming Fields

The stress fiber (bundles of actin filaments) is one of the most prominent cytoskeletal components that contributes to the maintenance of cell architecture. It has generally been believed that upon cyclic stretching, both cells and their stress fibers become perpendicularly aligned to the direction of stretching. However, using our newly developed stretching device, we have recently found the contrary evidence that stress fibers in endothelial cells rapidly become rearranged at a specific oblique angle relative to the direction of stretching [Takemasa, T., K. Sugimoto, K. Yamashita: Exp. Cell Res. 230, 407-410 (1997)]. In light of this finding, we attempted to establish the explanation for such a phenomenon. First, we investigated the effects of possible modulators on the angle of the stress fibers; those were, modification of the stretching program, dependency of extracellular matrix types, and their reproducibility in other cell species. However, it seemed that the orientation was solely depending on the stretching amplitude applied. Next, we analyzed alterations in stress fiber length during loading tests using two kinds of deforming experiment systems. It was thus revealed that stress fibers aligned at a particular angle so as to minimize their length alterations in cyclic deforming fields. Rearrangement of the stress fibers at this angle probably occurs as a result of avoiding compressive stress and may be interpreted as a way of reducing the mechanical stress to which they are subjected during the deformation. This hypothesis well explains the reason not only for the survival of the stress fibers at a particular oblique angle, but also for the reduced numbers of stress fibers found at the other angles on cyclic deforming fields.

Comparative studies on the circulatory system of the compound ascidians, Botryllus, Botrylloides and Symplegma

The circulatory systems of four polystyelids, Botryllus schlosseri, B. primigenus, Botrylloides violaceus and Symplegma reptans, were compared. The palleal buds are connected to the parent zooid by a peduncle and to the colonial vascular system by connecting vessels. The peduncle of S. reptans disappears at an earlier stage of bud development than in B. primigenus; it survives the dissolution of the parent zooid in B. schlosseri and B. violaceus. The connecting vessel is formed by anastomosis between an epidermal outgrowth from the bud and a neighboring colonial vessel, and is characterized by the presence of a sphincter. The number of connecting vessels formed in a palleal bud is three in S. reptans, two in B. primigenus and one each in B. schlosseri and B. violaceus. In each species, the larva has eight rudiments of ampullae. In B. primigenus, the original ampullae degenerate soon after metamorphosis and new ampullae extend from the ventral epidermis of the oozooid. In the other species, the colonial vascular system is derived from the original ampullae.

Role of Heat Shock Protein 70 in Induction of Stress Fiber Formation in Rat Arterial Endothelial Cells in Response to Stretch Stress

We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress.

Expression of stress fibers in bullfrog mesothelial cells in response to tension

The relationship between stress fibers and tension in mesothelial cells of the bullfrog small intestine was examined by fluorescence cytochemistry using en face mesothelial cell preparations. In nontreated controls, actin revealed by rhodamine-phalloidin staining was localized only along the margins of the mesothelial cells. On the other hand, many stress fibers were formed in the mesothelial cells within 5-7 min after stretching of the intestinal wall in a given direction. The orientation of stress fibers within the cells was coincident with the direction of the tension applied. These cytoplasmic fibers disappeared almost completely from the mesothelial cells within 30 min after the release of tension. According to a difference in the intensity of tension necessary for stress fiber expression, the intestinal mesothelial cells were classified into two groups. Furthermore, cells containing stress fibers in each group showed a rapid increase in number once a given value of tension was applied. The present results indicate that the mesothelial cells of bullfrog small intestine may develop stress fibers to counteract tension exerted on the intestinal wall. Such stress fibers may serve to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue.

Expression of stress fibers in bullfrog mesothelial cells in situ

SummaryActin-containing cytoplasmic fibers in mesothelial cells of the abdominal wall of the bullfrog, Rana catesbeiana, were visualized by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers ran straight and were aligned parallel with each other. They also showed immunofluorescence staining with antibody against myosin or α-actinin. Electron microscopy revealed the presence of microfilament bundles in the basal cytoplasm of the cells. These cytoplasmic fibers may be comparable to the stress fibers observed in cultured cells. The mesothelial cells of tadpoles initially developed stress fibers when they underwent transformation from a polygonal to a spindle-like shape. Such fibers were also present in the polygonal cells of frogs. Expression of stress fibers in these cells seems to correspond to the expansion of the abdominal wall caused by marked growth of some intraperitoneal organs. The stress fibers in the mesothelial cells may serve to regulate cellular transformation and, further, may play a role in maintaining cellular or epithelial integrity by strengthening the cellular attachment to subepithelial tissue against probable tension load on the abdominal wall.

Role of marginal stress fibers formed in the rat vascular endothelial cells

Fluorescence cytochemistry using en face preparations of rat vascular endothelial cells (ECs) revealed the localization of actin, fibronectin (FN) and fibronectin receptor (FNR) along not only central stress fibers (SFs) but also the cell margins. Electron microscopy showed very close proximity between the topographical distribution of intracellular microfilament bundles and that of subendothelial FN in the EC margins. Therefore, these basal and marginal actin cables may be comparable to the well-established central SFs present in ECs. Formation of the central SFs was induced in ECs or mesothelial cells in response to tension, by which their cellular integrity seems to be effectively maintained. However, even when central SF formation was inhibited by cytochalasin D, the ECs with marginal SFs showed high resistance to mechanical tension, whereas mesenteric mesothelial cells having no such fibers easily lost their integrity. Thus, together with central SFs, the marginal SFs characteristic of rat vascular ECs may play an essential role in strengthening cell-matrix adhesion.

Endogenous peroxidase activity in brush cell-like cells in the large intestine of the bullfrog tadpole, Rana catesbeiana

SummaryA special cell type was identified in the mucosal epithelium of the large intestine of the tadpole of the bullfrog, Rana catesbeiana. It is a slender, columnar cell, with a dark, basally situated nucleus. By electron microscopy the cell displays prominent bundles of filaments emerging from each microvillus and extending deep into the cytoplasm without ending in the terminal web. It has longer and more crowded microvilli than the absorptive cell. The specialized cell is also characterized by the presence of many apical vesicles and numerous subapical dense bodies. These cytological features suggest that it may be a brush cell (Rhodin and Dalhamn 1956). These cells displayed endogenous peroxidase activity in smooth and rough endoplasmic reticulum, in the well-developed Golgi apparatus and in apical vesicles. Furthermore, peroxidase reaction product was frequently observed on their luminal surface membrane. These findings suggest that the brush cell in the large intestine of the bullfrog tadpole may be a secretory cell.

Simple method for the identification of oxidative fibers in skeletal muscle

Skeletal muscle is composed of several different types of myofiber: slow oxidative (SO), fast glycolytic oxidative and fast glycolytic. However, the classification is usually determined by myosin heavy chain typing rather than by metabolic index. In this study, the oxidative metabolic index was investigated as a possible method of myofiber typing. Myoglobin, which is involved in oxygen transport and storage in myofibers, and mitochondria, which are the central organelles for oxidative metabolism, were studied. High levels of myoglobin and mitochondria are believed to exist in SO fibers, but the current study showed that they are considerably richer in some fast type fibers. As myofiber typing using the oxidative metabolic index is important physiologically, an attempt was made to find a simple method for this purpose. Some mitochondrial proteins have been observed to auto-fluoresce but until now this effect was too faint to detect easily. Owing to the recent advances in cooling charge-coupled device technology, such auto-fluorescence can now be used for myofiber typing, and the simple and rapid method for doing so is reported here.

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog, Rana catesbeiana

SummaryActin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or α-actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.

The fate and role of openings formed in the mesentery of bullfrog tadpoles, with reference to the contour of mesothelial cells

Using morphological techniques, histological changes of the mesentery were observed during the development of the bullfrog, Rana catesbeiana. The tadpoles of this species had many openings all over the mesentery from the duodenum through the large intestine. Most of the openings were elliptical and less than 3 × 2 mm in size. The openings became remarkably decreased in size and number with rapid narrowing of the mesentery occurring during the period of metamorphic climax, and had almost completely disappeared by the end of metamorphosis. Appearance and disappearance of the openings were closely correlated with the changes in the dimensions of the mesentery. Furthermore, in parallel with these changes in the openings, a noticeable alteration occurred in the shape of the mesothelial cells of the mesentery. In tadpoles having no mesenteric openings, the mesothelial cells had a polygonal contour, which became transformed once the openings were formed in the mesentery. The shapes of the transformed cells were classified into two types, one having many radiating cell processes and the other a very slender and spindle‐shaped contour. Both types of cells eventually became transformed into a definitive type of cell exhibiting a roundish polygonal contour by the end of metamorphosis. From these findings it was concluded that the growing mesentery might, of necessity, give rise to the openings and transformation of the mesothelial cells to enable rapid lengthening and shortining of the intestinal tract to occur during the postembryonic development of anuran amphibians.