Keigi Fujiwara

Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells

Abstract Both DNA and the centriole pairs are replicated once in each cell generation. The cyclic changes in both must be coordinated so that the two centriole pairs can participate in mitosis when the genetic material is to be partitioned to the two daughter cells. One of the centriole pairs also forms a primary (9 + 0) cilium sometime during the cell cycle. In this study, we asked whether some aspects of the coordination of the DNA and centriole cycles occur in G 1 , a part of the cell cycle when non-neoplastic cells become irreversibly committed to DNA synthesis. We used indirect immunofluorescence with antitubulin antibody to reveal the centriole pairs as a microtubule organizing center with or without a cilium. Quiescent Balb/c and Swiss 3T3 cells in low serum or at high cell density stopped in G 1 with ciliated, probably unduplicated centrioles. When these quiescent 3T3 cells were stimulated to enter DNA synthesis, the centrioles ciliation changed in three phases: first, an initial but transient deciliation within 1–2 hr; second, a return of the cilium by 6–8 hr; and third, a subsequent final deciliation of the centriole coincident with the initiation of DNA synthesis at 12–24 hr. The deciliated and duplicated centrioles subsequently separated in preparation for mitosis. Together with other information, these results imply that centrioles in growing mammalian cells are primarily ciliated in a part of G 1 during which the cells can arrest in suboptimal environmental conditions. Arrests in low serum or at high cell density also occur before centriole replication. These results suggest that deciliation and duplication of the centriole may occur near the time that quiescent cells become irreversibly committed to DNA synthesis. Certain centriole events may therefore be necessary before DNA synthesis can be initiated in 3T3 cells.

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin, and myosin

Abstract In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.

CONTRACTILE PROTEINS IN PLATELET ACTIVATION AND CONTRACTION

Our role in this annal is to review recent progress in characterizing platelet contractile proteins and to suggest how contractile proteins may participate in platelet activation and clot retraction. There has been a rapid expansion of knowledge about the molecular basis of cell motility since 1970, and platelets have been one of the important model systems. Readers interested in the exciting advances made in other aspects of the motility question can refer to recent books and reviews 1-3 on the subject.

Organization and Function of Stress Fibers in Cells in Vitro and in Situ

The purpose of this review is to present the tremendous body of research on stress fibers, which has grown exceedingly rapidly in the last 7 or 8 years, due to immunofluorescent techniques, with both a technical and functional perspective. The first section is primarily a chronology of technical innovations which have enabled better observation and characterization of stress fibers. This section also reviews the numerous contractile-associated proteins, actin-binding proteins, regulator proteins, and other proteins shown to localize to stress fibers in a characteristic distribution. The second section discusses the wide variety of roles for stress fibers that have been set forth, including cell spreading, cell adhesion, cell locomotion, contraction, isometric contraction, cell surface compartmentalization, differentiation, cell transformation, tumorigenicity, and morphogenesis. In order to help interpret the significance of the many purported roles of stress fibers, it is important to ask whether stress fibers in vitro are pure artifacts and whether stress fibers exist in cells in situ. This review demonstrates that the stress fiber is fundamentally a light microscopic term, and in order to avoid confusion with other microfilament bundle-containing structures seen in the electron microscope, such as circumferential microfilament bundles, contractile rings, microvilli, microspikes, and rootlet structures, it is necessary to establish criteria for the identification of stress fibers in situ. Finally, using these criteria, this paper presents two recently introduced models which exhibit stress fibers in cells in tissues: the fibroblast, called a scleroblast in the fish scale, and the endothelial cells of avian and mammalian vasculature.

Characteristics of lung pericytes in culture including their growth inhibition by endothelial substrate.

Pericytes and endothelial cells from the same sample of adult rat lung have been separately established in culture by use of selective growth media. The endothelial cells are positive and the pericytes negative for angiotensin-converting enzyme activity and tissue plasminogen activator. Morphologically in culture, the pericytes are similar to pericytes from bovine retina and other sites and show positive immunofluorescence to both human platelet (non-muscle) myosin and smooth muscle myosin. In this respect they resemble smooth muscle cells grown from the rat main pulmonary artery, but lack the myofilaments and dense bodies characteristic of muscle cells. Lung endothelial cells and fibroblasts are positive only for platelet myosin. Pericytes in culture demonstrate an unusual growth response to endothelial substrate, obtained by removing confluent endothelial monolayers with nonionic detergent or alkali. When plated onto this material at low density, pericyte growth is inhibited. By contrast, the substrate stimulates the growth of endothelial cells and has no effect on smooth muscle cells. Initial attachment of endothelial cells and pericytes to the substrate is similar.

The use of tannic acid in microtubule research

Publisher Summary This chapter describes a fixation method using tannic acid with glutaraldehyde for the study of microtubule structure and provides mechanism of the effect tannic acid. Various applications of the use of tannic acid to other ultrastructural studies are also discussed. Tannic acid is a natural product and a mixture of many types of polyphenolic compounds. Several mechanisms have been proposed to explain various effects of tannic acid observed in electron micrographs. Tannic acid acts as a fixative because it causes polypeptides with more than nine amino acids to precipitate, if the pH is optimally controlled. These polypeptides that precipitate with tannic acid are probably not covalently crosslinked because the precipitates can be resolubilized by altering pH or by removing tannic acid by dialysis. Covalent crosslinking may not occur in the biological specimen treated with tannic acid; tannic acid can stabilize certain biological structures, such as many types of membranes, interstitial structures, and isolated tubulin paracrystals induced by vinblastine.

Techniques for localizing contractile proteins with fluorescent antibodies

Publisher Summary This chapter presents the technique of antibody staining. Several features of fluorescent antibody technique contribute to its success in cell and developmental biology—namely, (1) specific antibodies to virtually any macromolecule can be produced experimentally, (2) the technique is very sensitive, and (3) the fluorescent images allow to map out the molecular anatomy of the cell to a resolution of about 0.5 μm. To visualize and identify myosin molecules within tissue culture, cells antibodies are made against human platelet myosin, labeled with fluorescent dyes, and used to stain tissue culture cells grown on a microscope coverglass. Antigen preparation is the most crucial step in conventional antibody production. Ideally an antigen should consist of a homogeneous macromolecule. In cases in which a given antigen is difficult to purify by conventional methods, it is sometimes possible to achieve purity by preparative polyacrylamide gel electrophoresis. The success of fluorescent antibody localization of cytoplasmic antigens depends on adequate specimen preparation. Usually this includes fixation to immobilize antigens in situ, followed by some treatment to make the plasma membrane permeable to antibody molecules.