Cell Models as Tools to Study the Structural and Molecular Basis of Cytoplasmic Streaming in Characean Cells

Using phase-contrast microscopy, Kamitsubo (1966) first visualized the immobile fibrils seated on the inner surface of chloroplasts, which are embedded in the cortical gel that served as the structural entity against which the endoplasm moved. Using electron microscopy, Nagai and Rebhun (1966) revealed that the fibrils were composed of mocrofilaments. Since heavy meromyosin (HMM) (Kersey et al. 1976) and subfragment one (S1) (Williamson 1974) isolated from rabbit skeletal muscle myosin bound to the filaments and formed typical arrowhead structures directed opposite to the original cytoplasmic flow, the microfilaments were identified to be actin filaments.

The presence of myosin in the endoplasm of Nitella was first suggested by Chen and Kamiya (1975) who showed that when the endoplasm pretreated with an SH-reagent, N-ethylmaleimide (NEM), it is incapable of moving when it is brought into contact with the intact cortical gel. When the vacuole is rapidly perfused with a medium containing no ATP, the organelles remain attached to the cortical fibers. The organelles can be gradually freed from the fibers when either ATP or inorganic pyrophosphate is introduced into the cell. Williamson (1975) postulated that a myosin-like protein might be the component that links the organelles to the actin filaments. Higashi-Fujime (1980) provided more definitive evidence for the presence of myosin in the endoplasm by showing that when the endoplasm is squeezed out from a Nitella cell and used to coat a glass slide, in the presence of MgATP, actin cables slide along a glass surface at a rate similar to that of the cytoplasmic streaming in vivo. In the same functional assay, the exceptionally fast motor protein isolated from characean cells moved muscle F-actin at a rate of 60 mm s-1 (Higashi-Fujime and Sumiyoshi 2001). This is ten times faster than myosin isolated from skeletal muscle that can move muscle F-actin.

Studies of another cell model derived from characean internodal cells provide evidence for the importance of the interaction between actin filaments and myosin for motility (Kuroda and Kamiya 1975). This movement is assumed to be caused by interaction of actin filaments attached to the surface of the chloroplast with myosin in the endoplasm. Evidence in support of this assumption comes from studies that show that chloroplast rotation is stopped by NEM. However, when the surface membrane of the drop bathed in a Ca2+-free medium is ruptured with the tip of a fine glass needle in order to allow HMM to enter the droplet to replace the NEM-inactivated myosin, chloroplast rotation resumes.

Kuroda (1983, 1990) developed a "cut-open cell." To make this type of cell model, an internodal cell of Chara was cut open parallel to its long axis. When the cut-open cell, whose cell wall is in contact with the glass slide, is bathed in Ca2+-free medium, the tonoplast is ruptured spontaneously, and cytoplasmic streaming still takes place as in tonoplast-free cells. By contrast, cut-open cells prepared from cells pretreated with heat at 50°C lose their motile activity due to inactivation of heat-sensitive myosin (Kamitsubo 1981). However, streaming can be reconstituted following the application of HMM.

Shimmen and Tazawa (1982b) reconstituted cytoplasmic streaming by introducing an organelle suspension prepared from Chara cells into a Nitella cell whose endoplasm had been removed by intracellular perfusion. The Chara organelles immediately move along the Nitella actin cables, indicating that there is an association of "myosin" with the organelles. Sheez and Spudich (1983) succeeded in inducing the movement of HMM-coated fluorescent polymer beads that they had introduced onto the cut-open cell model.

The actin cables of characean internodal cells provided us with a functional assay for identifying myosin during isolation procedures. An alternative assay method was developed for the determination of isolated myosins. Yangida et al. (1984) observed the movement of single actin filaments stabilized with fluorescent phalloidin that interacted with soluble myosin fragments. Kron and Spudich (1986) observed that single actin filaments labeled with phalloidin exhibit ATP-dependent movement on a glass surface coated with skeletal myosin II. Since the rates of movement proved to be relatively independent of the type of actin, the system can be used as a quantitative myosin-movement assay with purified protein.

The method was applied to the isolation of myosin from lily pollen tubes (Kohno et al. 1991). A crude extract of lilly pollen tubes was brought into contact with the surface of a nitrocellulose-coated coverslip. When the assay solution containing rhodamine-phalloidin treated F-actin was introduced onto the coverslip, fluorescent images of actin filaments moved under a microscope, indicating that the crude extract contained myosin-like translocator. With aid of this motility assay, Yokota and Shimmen (1994) for the first time purified plant myosin. The molecular mass of the heavy chain of this myosin was 170 kDa and its ATPase activity was stimulated to 60 times by chicken breast actin. The velocity of fluorescent actin in the in vitro motility assay was similar to the in vivo velocity of the cytoplasmic streaming.

Again using the same motility assay, Chara myosin was isolated from the cytoplasm of cells whose vacuolar sap had been removed by vacuolar perfusion with a solution containing EGTA (Yamamoto et al. 1994). The molecular mass of Chara myosin is about 230 kDa, which is considerably larger than that of lily myosin. An antibody raised against this myosin did not recognize either smooth muscle myosin or myosin from lilly pollen tubes. Higashi-Fujime et al. (1995) also isolated myosin from Chara . Both myosins were soluble at low ionic strength, and their ATPase activities were stimulated 100-150 time by F-actin.

For more information about the progress of research on cytoplasmic streaming since the proposal of the "sliding theory" by Kamiya and Kuroda in 1956 (Kamiya and Kuroda 1956b), refer to the review article by Shimmen (2007).

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