Accelerated Sliding of Pollen Tube Organelles along

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca 2+ concentrations lower than 0.18 IxM (pCa 6.8) but was inhibited at concentration higher than 4.5 txM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca 2÷ through "myosin" inactivation. p OLLEN tubes show active cytoplasmic streaming. This cytoplasmic streaming may be responsible for the transport of substances and organelles indispensable for tip growth. When the cytoplasmic streaming is inhibited by cytochalasin B, tip growth stops (Mascarenhas and Lafountain, 1972; Franke et al., 1972), even though secretory vesicles are produced (Picton and Steer, 1981). Cytoplasmic streaming in pollen tubes has been suggested to be supported by the actomyosin system based on the following findings. The actin filaments emerge from the grain and traverse into the pollen tube to the tip. When pollen tubes are treated with cytochalasin B, actin filaments disappear and cytoplasmic streaming stops, whereas colchicine affects neither the actin filaments nor the cytoplasmic streaming (Mascarenhas and Lafountain, 1972; Franke et al., 1972; Condeelis, 1974; Perdue and Parthasarathy, 1985). Thus, the involvement ofactin filaments in the cytoplasmic streaming of pollen tubes has been well documented. However, the involvement of myosin in the cytoplasmic streaming of pollen tubes has not yet been established. The free Ca 2+ concentration was reported to be highest at the tip region and decreases towards the base (Reiss and Nobiling, 1986; Nobiling and Reiss, 1987). When the Ca 2+ gradient was disrupted, an accumulation of secretory vesicles at the tip region was disrupted and tip growth was inhibited (Herth, 1978; Reiss and Herth, 1979). Localized Ca 2+ was thought to limit the vesicle fusion at the tip area (Picton and Steer, 1981). On the other hand, we will suggest that the Ca 2÷ regulation of cytoplasmic streaming may be one of the key processes in controlling tip growth. Cell motility is generally studied by using isolated contractile proteins or demembranated cell models. Myosin isolations were done from characean cells (Kato and Tonomura, 1977) and from two species of higher plant cells (Ohsuka and Inoue, 1979; Vahey et al., 1982). Demembranated cell models were made only in some giant algal cells using the methods of tonoplast removal (Williamson, 1975; Tazawa et al., 1976; Fukui and Nagai, 1985) or plasma membrane permeabilization (Shimmen and Tazawa, 1983; La Claire, 1984). Recently, a novel system to study actomyosin-based motility has been developed. In characean internodal cells, bundles of actin filaments are fixed at the inner surface of the stationary chloroplast layer. It has become possible to induce sliding of foreign myosin along these actin bundles (Sheetz and Spudich, 1983; Shimmen and Yano, 1984). Foreign organelles also showed active movement (Shimmen and Tazawa, 1982; Adams and Pollard, 1986). Since it was demonstrated that actin filaments of characean cells lack Ca 2÷ sensitivity (Shimmen and Yano, 1986), myosin-linked Ca 2+ regulation can also be studied using this trans-situ method (Vale et al., 1984; Kohama and Shimmen, 1985). Since the Ca 2+ regulation of the cytoplasmic streaming may be a key process of tip growth, we examined the possibility of myosin-linked Ca 2÷ regulation of cell organelle movement in pollen tubes using CaZ+-insensitive characean actin bundles. Here we demonstrated that pollen tube organelles can move along characean actin bundles and the characteristics of this translocator were similar to those of skeletal muscle myosin. However, unlike skeletal muscle myosin, the movement of this translocator along actin bundles was much faster and was inhibited by Ca 2+ at physiological concentrations. © The Rockefeller University Press, 0021-9525/88/05/1539/5 $2.00 The Journal of Cell Biology, Volume 106, May 1988 1539-1543 1539 on July 1, 2017 jcb.rress.org D ow nladed fom Materials and Methods