J. Cell Sci. 22, I33-M7 (1976) 133 Printed in Great Britain STUDIES ON THE MECHANISM OF CIRCUS MOVEMENT IN DISSOCIATED EMBRYONIC CELLS OF A TELEOST, ORYZIAS LATIPES: FINE-STRUCTURAL OBSERVATIONS NOBORU FUJINAMI Laboratory of Developmental Biology, Zoological Institute, Faculty of Science, University of Kyoto, Sakyo-Ku, Kyoto bod, Japan SUMMARY The fine structure of lobopodia in dissociated embryonic cells of the freshwater fish, Orysias latipes, was observed with the electron microscope in order to understand the mechanism of the circus movements which they display. Dense material (granular or fibrillar) is present in the zone between the lobopodium and the endoplasm, as well as in the cortical layer around the cell circumference. The direction of lobopodial movement is related to the distribution of this dense material. The band between the lobopodium and the endoplasm is conspicuous and is connected to the cortical dense layer around the cell periphery at the advancing front of the lobopodium, while the dense material is usually almost absent beneath the cell membrane in the anterior region of the lobopodium. The band between lobopodium and endoplasm is blurred or disrupted near the hind end of the lobopodium, where the peripheral dense layer is well developed. In situ localization of actin/heavy meromyosin complexes in the cell showed that the dense material has actin-like properties. Cytochalasin B (05 /tg/ml) induced constriction of the neck of the bleb, shrinkage of the endoplasm, and herniation of the endoplasmic contents to the enlarged hemispherical bleb, and thus arrested the circus movement. On the basis of these results, an hypothesis concerning the mechanism of circus movement is proposed and discussed. INTRODUCTION Although the mechanism of cell motility has hitherto been investigated mainly in protozoa, knowledge of the motile mechanism of metazoan cells, such as leucocytes or cultured cells, is advancing in the light of recent biochemical and ultrastructural findings (Abercrombie, 1973). In the case of embryonic cells, their motile activity was described in detail in dissociated amphibian cells some thirty years ago (Holtfreter, 1946, 1947, 1948), but we still cannot elucidate its mechanism. Circus movement, which involves the pushing out of a hyaline pseudopodium and the propagation of this lobopodial bleb around the cell circumference, is one of the remarkable movements shown by isolated embryonic cells. This movement was also reported in vivo in deep cells of teleostean blastoderm (Trinkaus, 1973; Kageyama, 1976) and may be related, as Sirakami (1972) stated, to wave-like changes propagating in embryonic cell surfaces, such as surface contraction waves (Hara, 1971). Using a quantitative method for estimating the velocity of the movement (Sirakami, 134 N. Fujinami 1963; Satoh, Kageyama & Sirakami, 1976), circus movement in dissociated embryonic cells of a teleost, Oryzias latipes was reported in earlier papers on the general features of this type of movement (Fujinami & Kageyama, 1975) and its responses to various classes of biologically active agents (Fujinami, 1976). These studies suggest that similar mechanisms may be involved in circus movement and other motile activities such as hyaline cap formation of amoebae (Allen, 1973), peristaltic constrictions in barnacle eggs (Lewis, Chia & Schroeder, 1973), blebbing of the polar lobe in marine mud snail eggs (Conrad, 1973), and zeiosis of cultured cells (Ross, 1966; Price, 1967), supporting the view that circus movement may be regarded as a type of cell behaviour based on fundamental properties of the cell surface and the motile activities of the cell. The present study was undertaken to demonstrate the fine-structural features of cells showing circus movement, in order to understand the mechanism of this move- ment in embryonic teleostean cells. It was also interesting to see the effect of cyto- chalasin B, which causes rapid morphological changes in cells showing circus move- ment (Fujinami, 1976). The present study demonstrated remarkable changes in shape and ultrastructure of cells treated with cytochalasin B. MATERIALS AND METHODS Developing eggs of the orange-red variety of the cyprinodont fish, Oryzias latipes (Japanese Medaka), were used. The fertilized eggs, attached in masses to the abdomen of females, were collected each morning. The detailed procedures employed in obtaining a suspension of isolated cells from the early gastrula have been previously described (Fujinami & Kageyama, 1975). Briefly, eggs were dissected and blastoderms were mechanically dissociated into their constituent cells with watchmaker's forceps in Ca2+-free Yamamoto solution. Cell suspensions were washed and transferred to 35-mm plastic Petri dishes (Falcon Plastics). After the cells had settled and were loosely attached to the substratum, and were showing circus movement, the supernatant fluid was decanted and then fixative, described below, was gently poured into the dishes. For cytochalasin B treatment, cells were immersed in Ca2+-free Yamamoto solution containing 0-5 /<g/ml cytochalasin B (in 0025 % dimethyl sulphoxide) for 5 or 10 min at room temperature before fixation. After several attempts to obtain improved fixation of the ultrastructure of embryonic cells, simultaneous fixation with glutaraldehyde and osmium tetroxide was employed according to Hirsh & Fedorko (1968), with some modifications. Cells placed in the dishes were fixed in situ at 4 °C or room temperature in a freshly-made cold mixture of 2#5 % glutaraldehyde and 1 % 3 osmium tetroxide in 005 M phosphate buffer, pH 7-4, containing io~ M CaCl2 for 30 min. The fixed cells were rinsed with distilled water and prestained in 05 % uranyl acetate solution (dissolved in 50 % ethanol) for 30 min. Dehydration was carried out through a graded series of ethanol solutions and followed by Epon embedding. After Epon polymerization in the Petri dishes was completed, selected bits of flat-embedded dishes containing the fixed cells, trimmed and remounted on Epon blocks, were sectioned parallel (or perpendicular) to the plastic surfaces. Thin sections were stained with ethanolic uranyl acetate and lead citrate, and examined with a Hitachi HU-11D-1 electron microscope. For heavy meromyosin (HMM) treatment, cells were partially lysed by immersing in Ca2+-free Yamamoto solution containing sodium dodecyl sulphate (SDS) because glycerina- tion, the usual method of enabling HMM to penetrate into cells (Ishikawa, Bischoff & Holtzer, 1969), was unsuccessful. As soon as the cells in the dishes were immersed in io~3 M SDS solu- tion, the immersing solution was decanted and the cells were rinsed with Ca2+-free Yamamoto solution. The cells were then reacted with HMM (05—1 mg/ml) for 3 h at room temperature, rinsed with o-i M KC1 and prepared for electron microscopy as described above, except that Circus movement of embryonic cells 135 005 M cacodylate buffer, pH 7-2, was employed instead of phosphate buffer. The contents of partially lysed cells were also dispersed on carbon-coated Formvar films on electron-microscope grids, treated with HMM and negatively stained according to the method described by Perry (1975). HMM prepared from rabbit skeletal muscle was kindly supplied by Dr K. Maruyama, Department of Biophysics, University of Kyoto. It was stocked in 02 M sodium glutamate solution at —20 °C and diluted with 01 M KC1 immediately before use. RESULTS Cells showing circus movement A cell showing circus movement generally has a large lobopodium (Figs. 1, 2). The lobopodial wave usually propagates around the cell circumference in a plane more or less parallel to the substratum. Most of the lobopodium usually contains few mem- branous structures and is composed of ground plasm resembling that in the ectoplasm of amoebae (Komnick & Wohlfarth-Bottermann, 1965; Bowers & Korn, 1968). Polyribosomes are frequently observed in the lobopodium. Numerous small vesicles (approx. 0-2 /<m in diameter) stream into a specific region of the lobopodium and thus serve as an indicator of the direction of lobopodial propagation, because stream- ing of the endoplasmic particles into the lobopodial bulge occurs at the hind end, as described by Holtfreter (1946). Other cytoplasmic components, such as mitochondria and microtubules, are sometimes observed at the hind end of the lobopodium. A thin band (0-05-0-2 /<m wide) of finely granular or fibrillar dense material separates the lobopodium from the endoplasm as described in the electron-microscopic study of intact teleostean embryos (Lentz & Trinkaus, 1967). In some cells, the filamentous material in this zone is more prominent (Fig. 3). The filaments are approximately 6 nm wide and appear to be oriented parallel to each other. The band of granular or fibrillar material separating the lobopodium from the endoplasm is clearly observed near the advancing front of the lobopodium (Fig. 4). This band is continuous with a cortical dense layer of endoplasm around the circumference, which has been described in embryonic amphibian cells (Perry, 1975). In other words, the dense material in the separating band, together with the cortical dense layer at the advancing front of the lobopodium, envelops the endoplasm. On the other hand, the peripheral dense layer of the lobopodium is usually poorly developed or almost absent near the advancing front. Sometimes several small vesicles are observed to form a line above the separating band at the advancing front as shown in Fig. 4. The separating band is curved (Fig. 2) or