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Mechanism of Ca Inhibition of Cytoplasmic Streaming in Lily Pollen Tubes

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Mechanism of Ca Inhibition of Cytoplasmic Streaming in Lily Pollen Tubes Mechanism of Ca inhibition of cytoplasmic streaming in lily pollen tubes TADASHI KOHNO and TERUO SHIMMEN Department of Botany, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan Summary Using a Ca2+ ionophore, A23187, the free Ca2+ was reversible. In the present study, the reversi- concentration ([Ca ]) in the cytoplasm of pollen bility was also demonstrated using an in situ Ca2+ tubes of Liliutn longiflorum was controlled from treatment. Organelles were isolated from pollen the cell exterior. At [Ca2+] higher than 1-OX1O~SM tubes that had been treated with high [Ca ] and (pCa5-0), cytoplasmic streaming was inhibited, A23187. They moved along characean actin bundles and the inhibition was irreversible. The ATP con- in Ca2+-free medium. tent did not change, but actin filaments were frag- It is concluded that Ca2+ inhibition of cytoplas- mented and formed aggregates. A subsequent de- mic streaming can be attributed to both inactivation crease in [Ca2+] almost stopped the progress of the of myosin and fragmentation of actin. The irrever- actin filament fragmentation, but filamentous actin sibility of Ca2+ inhibition in situ is attributed to the did not re-form from the fragmented actin. In a irreversible fragmentation of actin filaments. previous paper, we reported that pollen tube organ- elle movement along characean actin bundles 'was Key words: actin, Ca2+, cytoplasmic streaming, myosin, inhibited by Ca2+ at 10~sM levels and the inhibition pollen tube. Introduction filaments also run in the longitudinal direction. These filaments can be disrupted by cytochalasin B, which Cytoplasmic streaming is widely observed in plant cells results in the cessation of cytoplasmic streaming (Franke and Ca2+ is thought to be a regulator of cytoplasmic et al. 1972; Mascarenhas & Lafountain, 1972; Condeelis, streaming (Hepler & Wayne, 1985). In contrast to the 1974; Perdue & Parthasarathy, 1985). Therefore, it is Ca2+ activation of muscle contraction (Lehman & Szent- suggested that the tracks for cytoplasmic streaming in Gyorgyi, 1975), Ca2+ inhibits cytoplasmic streaming in pollen tubes are actin filaments. plant cells (Hepler & Wayne, 1985). This view has been In characean cells, it is likely that myosin is the mainly established in characean internodal cells (Kamiya, translocator (Kamiya, 1981, 1986; Tazawa & Shimmen, 1981, 1986; Tazawa & Shimmen, 1987; Tazawa et al. 1987; Tazawa et al. 1987), and it seems that myosin may 1987). Characean actin bundles in the tonoplast-free cell also be the translocator protein in higher plant cells. This are insensitive to Ca2+ (Shimmen & Yano, 1985, 1986); hypothesis is supported by the observation that organ- therefore, Ca2+ is thought to act upon myosin either elles from Ulium pollen tubes move along characean actin directly or indirectly. Available evidence suggests that bundles and the reconstituted movement is inhibited by Ca2+ regulates myosin indirectly through phosphoryl- pretreating pollen tube organelles with Af-ethylmaleimide ation (Tominaga et al. 1987). In other plant cells, it has or heat. In addition, cytoplasmic streaming in pollen also been shown that Ca2+ has an inhibitory role, by the tubes is also A'-ethylmaleimide- and heat-sensitive application of a Ca2+-selective ionophore (Herth, 1978; (Kohno & Shimmen, 1988). These results strongly Dore£ & Picard, 1980; Woods et al. 1984; McKerracher indicate that myosin is the translocator protein respon- & Heath, 1986; Takagi & Nagai, 1986). Furthermore, sible for cytoplasmic streaming in pollen tubes. microinjecting Ca2+ also inhibits cytoplasmic streaming In the actomyosin system, two mechanisms of Ca2+ in the stamen hair cells of Tradescantia (Hepler & regulation are well documented; actin-linked regulation Callaham, 1987). However, the mechanism of Ca2+ and myosin-linked regulation (Lehman & Szent- inhibition remains unknown, because of the difficulties Gyorgyi, 1975). In order to understand the mechanism involved in the purification of contractile proteins and the involved in the Ca2+ regulation of cytoplasmic streaming, preparation of demembranated cell models (Staiger & it is necessary to examine both possibilities. When pollen Schliwa, 1987). tubes are incubated in a medium containing A23187 In pollen tubes, active bidirectional cytoplasmic without Ca2+, most of the intracellular Ca2+ is lost (Reiss streaming occurs parallel to the longitudinal axis. Actin et al. 1983). Thus, the extra- and intracellular Ca2+ is Journal of Cell Science 91, 501-509 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 501 exchangeable in the presence of A23187. Herth (1978) flash method with an ATP photometer (Chemglow photometer reported that extracellular Ca2+ at lXlO~3M inhibited J4-7441; Aminco, Silver Spring, MD, USA). The ATP assay cytoplasmic streaming in pollen tubes treated with medium contained 50niM-Hepes, 50mM-MgSO4 and SOmM- A23187. In the present study, we determined the effect of K2SO4. The pH was adjusted to 7-4 with KOH (Mimura et al. 1983). Ca2+ at physiological levels on cytoplasmic streaming in To study the time course of the ratio of streaming cells to situ. We found that [Ca2+] at 10~5M inhibited cytoplas- unburst cells and the ratio of burst cells to total cells during the mic streaming in pollen tubes irreversibly. We previously A23187 treatment, pollen tubes were immobilized on a glass reported that the trans situ movement of pollen tube slide coated with poly-L-lysine and covered with A23187 me- organelles along characean actin bundles was reversibly dium. The occurrence of cytoplasmic streaming was examined + 5 inhibited by Ca^ at 10~ M (Kohno & Shimmen, 1988). at various times after the initiation of the A23187 treatment. In the present study, we demonstrate that the Ca2+ effect Approximately 100 unburst cells could be examined within on 'myosin' in situ is also reversible. By contrast, Ca2+ 5 min. irreversibly induces the fragmentation of actin filaments. We conclude that Ca2+ inhibits cytoplasmic streaming in Staining of actin filaments pollen tubes and that this regulation is linked to both For staining actin filaments, two methods were employed. myosin and actin. The irreversible inhibition may result (1) Fixing method. Pollen tubes were fixed in A23187 me- from the fragmentation of actin filaments. dium supplemented with 1 mM-dithiothreitol (DTT), WO/Jgrnl leupeptin, 3-7% (w/v) formaldehyde and 0-1% Some of these results have been reported elsewhere (w/v) glutaraldehyde at room temperature for 15 min. They (Kohno & Shimmen, 1987). were demembranated in phosphate-buffered saline (PBS) (137mM-NaCl, 2-7mM-KCl, l-5mM-KH2PO4 and 8mM- Na2HPO4, pH7-3) supplemented with 0-5% (w/v) Triton X- Materials and methods 100 at room temperature for 20min. Demembranated pollen tubes were washed with PBS and transferred to PBS containing 8 Pollen tube culture 6-6xlO~ M-rhodamine-phalloidin (rh-ph) (Molecular Probes INC) and incubated at room temperature for 2 h or at 4°C Pollen grains of Lilium longiflorum collected 2 days after overnight. After washing in PBS, pollen tubes were mounted in flowering were stored at 4°C and used within 3 days. Pollen gelvator (Rodriguez & Deinhartd, 1960) containing 01% grains were sown in glass vials containing culture medium that (w/v) p-phenylenediamine (Johnson & Nogueira Aranjo, included 260 M- or 290mM-sucrose, l-27mM-Ca(NO ) , 3 2 1981). Samples were observed with a microscope (Zeiss, 162//M-boric acid, 0-99mM-KNO 3-0mM-KH PO , pH5-2 3l 2 4 Photomicroscope III) equipped with epifluorescence optics (Dickinson, 1968). The glass vials were then placed on a shaker (G436 for excitation filter, FT510 for beam splitter and LP520 for l-5hto2-0hat25°C. for cut filter). (2) Non-fixing method. Pollen tubes were incubated in hom- Conttvl of cytoplasmic [Ca2+] ogenization buffer (Kohno & Shimmen, 1988) (5mM-EGTA, Pollen tubes were incubated in A23187 medium containing 6 mM-MgCl2, 30 mM-piperazine-iV,iV'-bis(2-ethanesulphonic 290mM-sucrose, 162 j/M-boric acid, 5 mM-ethyleneglycol-bis(/3- acid) (Pipes), 71 mM-KOH, 300mM-sorbitol, 1 % (w/v) casein, aminoethylether)-A^V,iV',iV'-tetraacetic acid (EGTA), 30mM- 1 f 1 mM-DTT, 100 jig ml" leupeptin, pH7-0) supplemented with iV-2-hydroxyethylpiperazine-A '-2-ethanesulphonic acid 0-01 % (w/w) saponin and 6-6x 10~ M-rh-ph at room tempera- (Hepes), S0^M-A23187 and various concentrations of CaCl2- 2+ ture for 30 min and then they were washed with the homogeniz- The pH was adjusted to 70 with KOH. The free Ca ation buffer. The homogenization buffer was the same as the concentrations were calculated as reported (Kohno & Shim- one that was used to isolate active myosin associated with men, 1988). The solution A23187 (5mM) in methanol was organelles from pollen tubes (Kohno & Shimmen, 1988). added to the A23187 medium and mixed gently. Vigorous Samples were mounted in gelvator containing p-phenylenedi- shaking resulted in the formation of precipitations, which were amine or in homogenization buffer containing 0-1 % (w/v) N- dissolved by sonication. propyl gallate (Kilmartin & Adams, 1984) and observed as described above. Observation of cytoplasmic streaming and analysis of To study the time course of the changes in the actin filament ATP content organization during A23187 treatment, pollen tubes were at- For analysing both the ratio of streaming pollen tubes and the tached to a glass slide coated with poly-L-lysine and covered ATP content, 4-7 mg of pollen grains was germinated as with A23187 medium, and actin filaments were stained by the described above and pollen tubes were collected using a glass non-fixing method at various times during A23187 treatment. microfibre filter (Whatman GF/A). They were incubated in About 100 pollen tubes were observed. 1 ml of A23187 medium for 30 min at room temperature. Part of the pollen tube suspension was put on a glass slide, and the Assay of motility of pollen tube oiganelles and myosin- occurrence of cytoplasmic streaming and the bursting of pollen coated beads along characean actin bundles tubes were examined with an inverted microscope (Olympus The motility of pollen tube organelles along actin filaments was IM) with X40 objective.
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