J Plant Res (2007) 120:31–43 DOI 10.1007/s10265-006-0061-0 CURRENT TOPICS IN PLANT RESEARCH The sliding theory of cytoplasmic streaming: fifty years of progress Teruo Shimmen Received: 18 August 2006 / Accepted: 22 November 2006 / Published online: 25 January 2007 Ó The Botanical Society of Japan and Springer 2007 Abstract Fifty years ago, an important paper ap- the motive force of the flow is located within the peared in Botanical Magazine Tokyo. Kamiya and streaming protoplasm itself or whether the driving Kuroda proposed a sliding theory for the mechanism of energy is produced in the protoplasmic system not in- cytoplasmic streaming. This pioneering study laid the volved in the streaming so that the moving protoplasm basis for elucidation of the molecular mechanism of is driven passively.’’ cytoplasmic streaming—the motive force is generated In characean cells, the sol endoplasm rotates in the by the sliding of myosin XI associated with organelles same direction with a constant velocity (rotational along actin filaments, using the hydrolysis energy of streaming). This stable streaming is suitable for quan- ATP. The role of the actin–myosin system in various titative analysis. Ewart (1903) published a figure plant cell functions is becoming evident. The present showing the streaming velocity to be highest in the article reviews progress in studies on cytoplasmic inner layers of the endoplasm and lowest at the inter- streaming over the past 50 years. face between the endoplasm and the ectoplasm. The figure also showed streaming in the cell sap, with the Keywords Actin Chara Cytoplasmic streaming highest velocity at a site close to the endoplasm. This Á Á Á Myosin Nitella figure was based on the idea that the motive force is Á generated in the flowing endoplasm. Kamiya and Ku- roda (1956) used cinematographic analysis to obtain an Sliding theory exact figure for the velocity distribution in cells of Ni- tella flexilis. In analyses using rhizoid and leaflet cells, Since its discovery by Corti (1774), cytoplasmic they did not find any appreciable velocity gradient at streaming (protoplasmic streaming) had been recog- the boundary (Fig. 1a). They expressed the situation as nized by many botanists. However, the mechanism of follows: ‘‘plasmasol layer (endoplasm) is actually not motive force generation remained unknown. In 1956, a streaming, but just slipping as a whole on the inner pioneering paper opened the door to the elucidation of surface of the cortical layer’’. The vacuole showed the the molecular mechanism of cytoplasmic streaming velocity distribution of a sigmoid pattern (Fig. 1a). (Kamiya and Kuroda 1956). The opening sentence of Kamiya and Kuroda centrifuged an internodal cell the paper was: ‘‘One of the basic questions concerning and collected the flowing endoplasm at the cell end. A the mechanism of protoplasmic streaming is whether cell fragment lacking a large central vacuole was pre- pared by ligation. The distribution of the streaming velocity in the cell fragment was sigmoid (Fig. 1b). They concluded that ‘‘the interaction of organized gel T. Shimmen (&) surface and sol phase produces the shearing force Department of Life Science, which brings about the interfacial slippage’’ (sliding at Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan the sol–gel interface; sliding theory). Because the e-mail: [email protected] viscosity of the endoplasm is high, it moves as a mass in 123 32 J Plant Res (2007) 120:31–43 Fig. 1 Velocity distribution of cytoplasmic streaming in N. flexilis cells. a Cell containing a large central vacuole. b Cell lacking a large central vacuole. w cell wall, g gel ectoplasm, s sol endoplasm, v vacuole (from Kamiya and Kuroda 1956) a cell having a large central vacuole, resulting in the absence of a velocity gradient in the endoplasm (Fig. 1a). Bundle of microfilaments at the sol–gel interface Kamiya and Kuroda (1956) suggested: ‘‘What comes into focus concerning the rotational movement in Nitella is the mechanism causing the slippage between Fig. 2 Tonoplast-free cell of Characeae. a Intact cell. b Cell with the sol–gel interface’’. In leaflet and internodal cells of artificial cell cap. c Tonoplast-free cell. d Cell lacking endoge- nous myosin. e Cell with exogenous organelles or myosin-coated Characeae, chloroplasts are anchored to the gel beads. After both cell ends were amputated, the vacuole was ectoplasm (Fig. 2a). (In most plants, chloroplasts are perfused with an artificial medium (vacuolar perfusion). When mobile and move in response to light conditions). The the perfusion medium contained a Ca2+ chelator, the tonoplast sol–gel interface thus corresponds to the inner surface disintegrated (c tonoplast-free cell). By the subsequent perfu- sion, the chemical composition in the cell is thoroughly of the fixed chloroplasts. controlled. Some of the endoplasmic organelles remained on It was then necessary to identify the structural entity the actin bundles and moved (not shown). Endogenous myosin is at the gel surface responsible for motive force gener- inactivated by N-ethylmaleimide (NEM) treatment and effuses ation. Within a decade, two pupils of Noburoˆ Kamiya out by strong perfusion (d). When organelles or myosin-coated beads are introduced by perfusion, the movement is reconsti- found it. Kamitsubo (1966) discovered cables on the tuted (e). CW cell wall, PM plasma membrane, Tp tonoplast, Vac inner surface of chloroplast, by light microscopy. At vacuole, En endoplasm, AF actin filament, Chl chloroplast the same time, Nagai and Rebhun (1966), using (modified from Shimmen 1988b) 123 J Plant Res (2007) 120:31–43 33 electron microscopy, found these cables to be bundles Demembranated cell models of microfilaments. Although the cables are located at the sol–gel Demembranated cell models have extensively con- interface, are they actually involved in generation of the tributed to studies on the regulatory mechanism of motive force? When a small part of the gel layer was cytoplasmic streaming. Two types of model have been illuminated with strong light under a microscope, developed. chloroplasts became detached from the gel layer and were swept away by the cytoplasmic streaming with the Tonoplast-free cell cables (Kamitsubo 1972; window technique). At the window, the cytoplasmic streaming was disturbed. Tazawa (1964) developed a vacuole-perfusion method. When the cell was incubated for one to several days, the After cutting both ends of an internodal cell of Char- cables were regenerated, resulting in recovery of aceae, the natural cell sap (vacuolar sap) is replaced the streaming. This observation unequivocally showed with an artificial cell sap by intracellular perfusion the involvement of the cables in cytoplasmic streaming. (Fig. 2b). This makes it possible to control the chemi- cal composition in the vacuole. The natural cell sap contains Ca2+ at mmol L–1 levels, which stabilizes the What are microfilaments? tonoplast. Williamson (1975) and Tazawa et al. (1976) added a Ca2+ chelator to the perfusion medium, lead- Microfilaments were suggested to be actin filaments. At ing to disintegration of the tonoplast (Fig. 2c). This that time neither anti-actin antibody nor phalloidin was disintegration causes disorganization of the cytoplasm. available. The only strategy available was demonstra- However, the actin bundles remain attached to the tion of the arrowhead structure formed by application inner surface of the chloroplast layers, and organelles of fragments of skeletal muscle myosin, heavy mero- or their aggregates continue streaming. The intracel- myosin (HMM) or sub-fragment 1 (S1). Two groups, lular chemical composition is thoroughly controlled by Williamson (1974) and Palevitz et al. (1974), showed subsequent perfusion. Although most organelles effuse the presence of microfilaments in the characean cells, out, some remain on the actin bundles and move (not which is decorated by HMM to form the arrowhead shown). structure. Because the cells were completely disorga- nized, the intracellular localization of the actin fila- Permeabilized cell model ments could not be shown. Palevitz and Hepler (1975) and Kersey and Wessells (1976) demonstrated the When the extracellular osmolarity is increased to a presence of actin cable at the sol–gel interface, the in- value higher than the intracellular osmolarity, plas- ner surface of chloroplasts. Kersey et al. (1976) further molysis is induced. If the plasomolyticum is suitable, showed the polarity of the actin filaments in the cell. protoplasts can survive. Shimmen and Tazawa (1983) The orientation of the arrowheads indicated that cyto- induced plasmolysis in Characeae cells by using an ice- plasmic streaming occurs in the direction of the barbed cooled medium containing a Ca2+ chelator. By plas- end of the actin filaments. This direction was the same molysis under such severe conditions, the plasma as that of myosin sliding along the actin filaments in membrane is irreversibly permeabilized but the tono- skeletal muscle. Cytoplasmic streaming was stopped by plast remains intact. Because the cytoplasm is sand- treatment with cytochalasin B, which inhibits poly- wiched between the tonoplast and the perforated merization of actin filament (Williamson 1972; Bradley plasma membrane, it maintains its organization to 1973), indicating involvement of the actin filaments in some extent. In this model, the chemical composition generation of the motive force. of the cytoplasm is controlled by exchanging the Actin filaments are observed in a variety of plants extracellular medium. (Staiger and Schliwa 1987), reflecting the ubiquitous occurrence of cytoplasmic streaming. In Arabidopsis ATP as an energy source thaliana, eight expressed functional actin genes are known. They are divided into two subclasses by their Although ATP had long been believed to be a direct phylogeny and their expression pattern. ACT2, ACT7, energy source of cytoplasmic streaming, evidence for and ACT8 are strongly expressed in vegetative tissues, this was lacking until the development of demembra- whereas ACT1, ACT3, ACT4, ACT11, and ACT12 are nated cell models of Characeae.