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 organelle 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. Pollen tubes used for the microscopic examined as reported (Kohno & Shimmen, 1988). Briefly, observation were returned to the original suspension, and the pollen tubes were homogenized in the homogenization buffer whole suspension was put in a mortar containing 2 ml of the and the homogenate was introduced into internodal cells of ATP extraction medium (5 mM-CaCl2, 7-5% (v/v) perchloric Nitellopsis obtusa or Nitella axillifonnis whose native endo- acid, 30mM-Hepes) and was homogenized on ice with a pestle. plasm had been inactivated and removed. The motility of the The pH was then adjusted to 7-4 with 2M-K0H. All the pollen tube organelles was quantified by measuring the ratio of procedures from the microscopic observation to the neutraliz- moving organelles to the total organelles at the inner surface of ation of the homogenate were carried out within 15 min. The the chloroplasts of characean cells (Kohno & Shimmen, 1988). extract was stored at — 20 °C. ATP was analysed by the firefly- The effect of the pollen tube homogenate on the movement of

502 T. Kohno and T. Shimmen 100 5-0 « 100 o 4-0 I

3-0 'g> "o I 50 a 2-0 I c 1-0 c 6 8 CO « I

7 6 5 4 3 30 60 90 pCa Time (min)

100 B Fig. 2. The time course of Ca2+ inhibition of cytoplasmic streaming. Pollen tubes were incubated in A23187 medium of 2+ 39 1-OX1O~°M-Ca (pCaS-0) (D). After 30min, samples were transferred to A23187 medium of 1-5X1O"6M-Ca2+ (pCaS-8) I (A) or Mxl0~8M-Ca2+ (pCa8-0) (•). Streaming pollen 2 tubes were counted within S min at each treatment time. Bars £ 50 represent ± s.E.M. 1 (Fig. 1A). Methanol at 1 %, which was used for dissolv- CQ ing A23187, did not have any effect on cytoplasmic streaming. The decrease in the percentage of streaming L 2+ 0 pollen tubes at [Ca ] lower than 1-5X10~6M (pCa5-8) might result from the side effects of extremely low 8 7 6 5 4 3 extracellular [Ca2+], as is evident in the increase in the pCa ratio of burst pollen tubes (Fig. IB). In support of this 2+ hypothesis was the observation that lowering the extra- Fig. 1. The effect of [Ca ] on cytoplasmic streaming, ATP 2+ content and cell bursting. Abscissa, pCa= —log[Ca2+]. A. cellular [Ca ] in the absence of A23187 also induced Streaming pollen tubes/unburst pollen tubes Xl00% (O) bursting of pollen tubes and the cessation of cytoplasmic and the ATP content (•). B. Burst pollen tubes/total pollen streaming (data not shown). tubes Xl00%. Bars represent the S.E.M. Pollen tubes were The cessation of cytoplasmic streaming progressed treated in A23187 medium at various [Ca2+] for 30min. from the tip to the base in A23187 medium of l-0xl(T5M-Ca2+ (pCa5-0). The percentage of stream- skeletal muscle myosin on characean actin bundles was studied. ing pollen tubes decreased to 10% after a 30 min incu- Latex beads coated with rabbit skeletal muscle myosin were bation and to 1% after a 45 min incubation (Fig. 2). prepared following the method of Shimmen & Yano (1984) with Cytoplasmic streaming did not recover when pollen tubes slight modifications. The mixture of myosin and poly-L-lysine- that had been incubated at 1-OX1O~5M-Ca2+ (pCa5-0) coated beads in high ionic strength medium (0-5 M-KC1, 20 mM- for 30 min were transferred to A23187 medium of Pipes, pH7-0, adjusted with KOH) was diluted 10-fold with 6 2+ 8 2+ l-5xlO~ M-Ca (PCa5-8) or Mxl0~ M-Ca cold deionized and distilled water. Subsequently, myosin- ( Ca8-0)(Fig.2). coated beads were collected as a pellet following centrifugation P at 11 000 g for 5 min and mixed with the pollen tube homogen- Effect ofCa2+ on ATP content ate. The ATP content remained constant when the [Ca2+] was maintained at 1-5X10~6M (pCa5'8) or higher Results (Fig. 1A). These values were almost the same as those obtained for untreated pollen tubes 2+ Effect of Ca on cytoplasmic streaming (3-37 ± 0-39nmolmg"1 pollen). It is evident that the Ca had a great influence on cytoplasmic streaming in inhibition of cytoplasmic streaming at higher [Ca2+] was pollen tubes, when pollen tubes were treated in A23187 not caused by a change in the ATP content. The decrease medium for 30min (Fig. 1A). At 1-SX 1(T6M-Caz+ in the ATP content at [Ca2+] lower than 4-2xlO~7M (pCa5-8), 62% of the unburst pollen tubes showed (pCa6-4) (Fig. 1A) is presumably attributed to the cytoplasmic streaming. At l-0x l(T5M-Ca2+ (pCa5-0), increase in the number of burst pollen tubes (Fig. IB). however, only 1 % of the unburst pollen tubes showed cytoplasmic streaming. At higher [Ca2+], less than 1 % of Effect of Ca2+ on actin filament organization the unburst pollen tubes showed cytoplasmic streaming. Before studying the effect of Ca2+ on actin filament At lower [Ca2+], the percentage of streaming pollen organization, we examined the effect of fluoro-staining tubes decreased but seemed to reach a minimum at 25 % procedures. Untreated pollen tubes were stained with rh-

Cytoplasmic streaming in pollen tubes 503 ph using both a fixing and a non-fixing method. The medium of [Ca2+] lower than 1-5X1O~6M (pCa5-8) was appearance of actin filaments stained with rh-ph similar to that of untreated pollen tubes (Fig. 4A,G,H). depended on the staining procedure (Fig. 3). A large In pollen tubes treated with A23187 medium of number of thin straight actin filaments were observed in l-0xl0"5M-Caz+ (pCa5-0), however, thin actin fila- the non-fixed pollen tubes, whereas in fixed pollen tubes ments disappeared concomitantly with the appearance of actin filaments were always wavy, thick and fewer in fragmented filaments or aggregates of filaments number. Since both methods may modify the structures (Fig. 4C-F,I). In these experiments, actin filaments of cytoskeleton differently, we employed both methods to were stained after 30min treatment in A23187 medium. study the effects of Ca2+ on actin filament organization. In the next experiment, the same pollen tube in A23187 Using both methods, the organization of actin fila- medium of 1-Ox 10~5 M-Ca2+ (pCa5-0) was continuously ments in most of the pollen tubes treated with A23187 observed and the actin filaments were stained by the non-

Fig. 3. The effect of staining methods on the structure of actin filaments. A-C. Non-fixing method. A. Pollen grain and base of the tube. Many actin filaments emerged from the grain to the tube. B. Pollen tube near the base where the vacuole is highly developed. Thin straight actin filaments are seen. C. Pollen tube near the tip. Many thin straight actin filaments are distributed throughout the whole cytoplasm. D,E. Fixing method. D. Pollen grain and the base of the tube. Actin bundles emerge from the grain. The grain has high background fluorescence. E. Pollen tube. Wavy actin bundles are seen. Bars, lOjUm.

504 T. Kohno and T. Shimmen Fig. 4. The effect of Ca2+ on actin filament organization in Lilium pollen tube and grain. A-F. Non-fixing method. A. Pollen tube incubated in A23187 medium of 1-SXlO"6 M-Ca2+ (pCa5-8) for 30min. Many thin straight actin filaments were seen. B. Pollen tubes were incubated in 1-0x10" M-Ca2+ (pCaS'O) and actin filaments were stained just after confirming the cessation of cytoplasmic streaming. Many thin straight actin filaments remain. C-F. Pollen tube and grain incubated in A23187 medium of l-0xl0~5 M-Ca2+ (pCaS-0) for 30min. Actin filaments in pollen tube (D-F) and grain (C) are fragmented. G-I. Fixing method. Pollen tube incubated in A23187 medium of 1-1 X 10"S M-Ca2+ (pCa8-0) (G) and of 1-SX 10~°M-Ca2+ (pCa5-8) (H) for 30min. The structure of actin filaments is similar to those of untreated pollen tubes (see Fig. 3E). 1. Pollen tube incubated in A23187 medium of l-0xl0~5M-Ca2+ (pCa5-0) for 30min. Actin filaments are fragmented in a manner similar to those stained by the non-fixing method. Bars, fixing method just after the cessation of cytoplasmic Thus, actin filament fragmentation occurs only at higher streaming (Fig. 4B). In this case actin filaments were [Caz+] where cytoplasmic streaming was inhibited. affected to some extent but the fragmentation was not significant. Fragmentation in A23187 medium of Effect of Ca2+ on putative pollen tube myosin l-0xl0~5M-Ca2+ (pCa5-0) progressed with time. In We previously reported that pollen tube organelle move- addition, actin filament fragmentation progressed from ment along characean actin bundles was inhibited by the tip to the base. The ratio of pollen tubes with Ca2+ at 10~ M and that the inhibition was partly fragmented actin filaments at the base was determined at reversible (Kohno & Shimmen, 1988). To study the various times after the initiation of the incubation in reversibility of the Ca2+ inactivation of putative pollen s 2+ A23187 medium of l-0x l(T M-Ca (PCa5-0) (Fig. 5). tube myosin in situ, pollen tubes were successively After a 60min incubation, actin filament fragmentation treated at l-9xl(T4M-Ca2+ (pCa3-7) for 30min and occurred in 87% of the unburst pollen tubes. Actin then at 1-5X10~6M (pCa5-8) for 30min and were filament fragmentation was significantly blocked by de- homogenized in Caz+-free homogenization buffer. When 2+ creasing the [Ca ] from l-OxKT^M (PCa5-0) to the homogenate was introduced into characean cells, 6 8 1-5X1(T M (PCa5-8) or Mxl(T M (PCa8-0) (Fig. 5). pollen tube organelles moved along characean actin

Cytoplasmic streaming in pollen tubes 505 100 Velocity (/ans ) 1 2 3

-Ca 5 3 .5 50 +Ca2

Fig. 7. The effect of pollen tube homogenate on the movement of latex beads coated with rabbit skeletal muscle myosin along characean actin bundles in the absence ( —Ca2+) or presence (+Ca2+) of Ca2+ (3-2Xl(T5M, pCa4-5). Bars represent the S.D. 30 60 90 Time (min) myosin were mixed with the pollen tube homogenate and Fig. 5. The time course of actin filament fragmentation. introduced into characean cells in the absence or presence Pollen tubes were incubated in A23187 medium of of Ca2+ (3-2xl(T5M, pCa4-5). The velocity of latex 5 2+ l-0xl(T M-Ca (pCa5-0) (•). At 30min samples were beads coated with skeletal muscle myosin was not affec- transferred into A23187 medium of l-5xlO~6M-Ca2+ 2+ 8 2+ ted by Ca (Fig. 7), indicating that the pollen tube (pCa5-8) (A) or MX1CT M-Ca (PCa8-0) (•). The ratio homogenate does not affect characean actin bundles in of pollen tubes in which actin filament fragmentation progressed to the base is shown. Bars represent the S.E.M. this reconstituted system.

Discussion Moving organelles (%) 0 50 The present study unambiguously shows that Ca2+ 5 pCa5-8, 60min inhibits cytoplasmic streaming in pollen tubes at 10~ M levels (Fig. 1A). The ATP content was scarcely affected 2+ 6 pCa3-7, 30 min at [Ca ] higher than 1-5X10~ M (PCa5-8) (Fig. 1A). T Since the inhibition of cytoplasmic streaming occurred at pCa5-8, 30min these [Ca2+] (Fig. 1A), it is concluded that the inhibition of cytoplasmic streaming was not due to the decrease in 2+ Fig. 6. The motility of pollen tube organelles along ATP. It is strongly suggested that Ca is a physiological characean actin bundles after Ca2+ treatment in situ. Bars regulator of cytoplasmic streaming in pollen tubes. Since represent the s.D. Pollen tubes were treated in A23187 cytoplasmic streaming in pollen tubes may be supported medium of l-5xlO~6M-Ca2+ (pCa5-8) for 60 min, or in by the sliding of putative pollen tube myosin (Kohno & 4 z+ A23187 medium of l-9xlO" M-Ca (PCa3-7) for 30min Shimmen, 1988) along actin filaments (Franke et al. 6 2+ and then in A23187 medium of l-SxlO" M-Ca (pCa5-8) 1972; Mascarenhas & Lafountain, 1972; Condeelis, 1974; for 30 min. They were homogenized in homogenization buffer 2+ Perdue & Parthasarathy, 1985; Kohno & Shimmen, without Ca and introduced into tonoplast-free Nitellopsis 1988), it is possible that actin-linked regulation and/or cells by intracellular perfusion. myosin-linked regulation (Lehman & Szent-Gyorgyi, 1975) may be the mechanism responsible for the Ca bundles. The motility was almost equal to that of inhibition of cytoplasmic streaming in pollen tubes. organelles isolated from pollen tubes treated at 1-5X1O~6M-Ca2+ (pCa5-8) for 60min (Fig. 6), indi- Involvement of actin-linked regulation cating that the motility of 'myosin' attached to pollen tube Actin filaments in pollen tubes have been stained with organelles (Kohno & Shimmen, 1988) was not irrevers- fluorescent dyes conjugated to phalloidin by many ibly inactivated in situ by treating pollen tubes in A23187 workers (Parthasarathy et al. 1985; Perdue & Parthasar- 4 2+ medium of 1-9X 1(T M-Ca (PCa3-7). athy, 1985; Heslop-Harrison et al. 1986; Pierson et al. 1986a,b). In all reports, however, fixing methods were Effect of pollen tube homogenate of characean actin employed. In these procedures the disappearance of actin bundles filaments due to fixation may occur (Parthasarathy et al. Since actin filaments were fragmented in pollen tubes by 1985; Kakimoto & Shibaoka, 1987; Lloyd, 1987; Traasef increasing cytoplasmic [Ca ] (Fig. 4), pollen tubes al. 1987). Recently, Traas et al. (1987) and Kakimoto & might contain actin filament severing factor(s). There- Shibaoka (1987) reported that the appearance of actin fore, pollen tube homogenate may affect characean actin filaments is different in cells prepared by the fixing bundles in a Ca +-dependent manner. We examined the method from cells prepared by the non-fixing method. effect of the pollen tube homogenate on the motility of The non-fixing method reveals additional features not skeletal muscle myosin along characean actin bundles, seen using the fixing method: for example, the cortical both of which are insensitive to Caz+ (Shimmen & Yano, network of actin filaments in cultured plant cells, which 1985, 1986). Latex beads coated with skeletal muscle disappears during cell division in the fixing method, is

506 T. Kohno and T. Shimmen present throughout all stages of the cell cycle (Traas et al. The characean actin bundles might be protected from 1987; Kakimoto & Shibaoka, 1987). Thus, before study- fragmentation by the stabilizing effect of phalloidin. ing the effects of Ca2+ on actin filament organization in However, the movement of latex beads coated with pollen tubes, we tested the effects of staining procedures skeletal muscle myosin along characean actin bundles was on actin filaments. We also observed the disappearance of not affected by pollen tube homogenate in the presence of thin actin filaments after fixation (Fig. 3). We used the Ca2+, even when the characean actin bundles were not homogenization buffer that was used to isolate the active treated with phalloidin (data not shown). The characean translocator (myosin) associated with organelles from actin bundles in the present reconstituted system are pollen tubes (Kohno & Shimmen, 1988) in conjunction insensitive to the actin-filament severing factor of pollen with the non-fixing method, which is expected to be tubes. This may be due to the different organization of suitable for the preservation of not only myosin but also actin filaments, the different sensitivity to the severing actin filaments. The organelle movements in pollen tubes factor of actin filaments or the dilution of the severing are mostly straight except at the tip. This is in good factor by homogenizing pollen tubes. agreement with the straight arrangement of actin fila- The critical [Ca2+] for myosin inactivation (10~SM) ments observed by the non-fixing method. However, we (Kohno & Shimmen, 1988) is close to that for the cannot exclude the possibility of artifacts occurring in the inhibition of cytoplasmic streaming in situ (Fig. 1). non-fixing method. Therefore, we employed both Therefore, myosin inactivation also seems to be a cause of 2+ methods to observe the effect of Ca on the actin the inhibition of cytoplasmic streaming by Ca2+ in situ. filament organization in pollen tubes. The inactivation of myosin by Ca2+ is reversible. This We found that Ca2+ induces an irreversible fragmen- was the case in treatments both in vitro (Kohno & tation of actin filaments in pollen tubes. This phenom- Shimmen, 1988) and in situ (Fig. 6). The ratio of motile enon was observed in both the fixing and the non-fixing organelles along characean actin bundles was 12 % after methods. Since the [Ca2+] effective in inhibiting cyto- treatment with high [Ca2+] in vitro (Kohno & Shimmen, plasmic streaming (Fig. 1A) was consistent with that 1988). On the other hand, it was 53 % after high [Ca2+] needed for the fragmentation of actin filaments (Fig. 5), treatment with in situ (Fig. 6). This difference may be actin filament fragmentation seems to be a cause of the due to dilution of some component(s) necessary for Ca2+ inhibition of cytoplasmic streaming. Observations reactivation of myosin after homogenization of pollen that both inhibition of cytoplasmic streaming and actin tubes. The treatment of pollen tube organelles with Ca2+ filament fragmentation progressed from the tip to the after homogenization may also result in the digestion of base also support this possibility. myosin by Ca2+-activated proteases, which are released Actin filaments could be stained with rh-ph even after as a result of disruption of the intracellular compartmen- long treatments with Ca2+. Since phalloidin binds to F- tation (Moriyasu & Tazawa, 1987). actin but not G-actin (Barak et al. 1980), it is suggested 2+ that actin filaments are not completely depolymerized to Mechanism ofCa inhibition G-actin or degraded. The [Ca2+] dependency of the actin It is concluded that the Ca2+ inhibition of cytoplasmic filament fragmentation observed in pollen tubes is similar streaming in pollen tubes is due to both actin fragmen- to that caused by severing proteins in animal cells and tation and myosin inactivation. The irreversible Ca2+ Physarum (Stossel et al. 1985; Pollard & Cooper, 1986). inhibition of cytoplasmic streaming is probably due to the Therefore, some protein(s) that are capable of severing irreversible fragmentation of actin filaments. actin filaments may be present in pollen tubes. In animal cells, dual Ca2+ regulation of the actomyosin system has been reported in several instances (Lehman & Szent-Gyorgyi, 1975). In this case, actin-linked regu- Involvement of myosin-linked regulation lation is mediated by troponin-tropomyosin. In Phy- We previously reported (Kohno & Shimmen, 1988) that sarum the possibility of a dual regulation of the actomyo- pollen tube organelle movement along characean actin sin system has also been suggested (Kohama & Kendrick- bundles was inhibited at [Caz+] higher than 4-0xl0~6M Jones, 1986). The inhibition may result from the direct (pCa5-4) and argued that myosin associated with pollen binding of Ca2+ to myosin (Kohama & Kendrick-Jones, tube organelles has Ca2+ sensitivity. Since it was found in 1986) and also from the fragmentation of actin filaments the present study that pollen tubes seem to contain an by the Ca2+-sensitive protein, fragmin (Hasegawa et al. actin-filament severing factor, the possibility arose that 1980; Sugino & Matsumura, 1983). Our results suggest the Ca2+ inhibition of the reconstituted movement of that cytoplasmic streaming in pollen tubes is also under pollen tube organelles results from the effect of the dual regulation by Ca2+. severing factors on characean actin filaments. However, In this paper and a previous one (Kohno & Shimmen, this possibility was excluded, since the movement of 1988), we have separately examined the effects of Ca2+ on beads coated with skeletal muscle myosin along characean ATP, actin filaments and myosin, all of which are actin bundles was not affected by pollen tube homogenate responsible for the occurrence of cytoplasmic streaming. in the presence of Ca2+ (Fig. 7). It is concluded that For the further elucidation of the mechanisms of cyto- Ca2+ regulation is linked to the myosin of pollen tube plasmic streaming in pollen tubes, biochemical studies organelles. In our previous work (Kohno & Shimmen, are needed. Generally, the purification of contractile 1988), and in Fig. 6 of the present study, we pretreated proteins from plant cells is difficult. Preparation of characean actin bundles with phalloidin to stabilize them. demembranated cell models of pollen tubes has been

Cytoplasmic streaming in pollen tubes 507 unsuccessful to date (not shown). Our present approach HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1987). An analysis of will be plausible for studying the mechanism and regu- gamete and organelle movement in the pollen tube of Secale cereale L. PL Sci. 51, 203-213. lation of cytoplasmic streaming in plant cells. HESLOP-HARRISON, ]., HESLOP-HARRISON, Y., CRESTI, M., TIEZZI, A. & CIAMPOUNI, F. (1986). Actin during pollen germination. J Physiological significance Cell Sci. 86, 1-8. In intact pollen tubes a gradient of [Ca2+] exists, from IWANAMI, Y. (1959). Physiological studies of pollen. J. Yokohama the tip to the base (Reiss & Nobiling, 1986- Nobiling & Municipal Univ. 116 (c-34, Biol.-13), 1-137. 2+ 6 JOHNSON, J. D. & NOGUEIRA ARANJO, G. DE C. (1981). A simple Reiss, 1987).The[Ca ]atthetipis9xl0" M(pCa7-0) method of reducing the fading of lmmunofluorescence during (Nobiling & Reiss, 1987). Since the spatial resolution of microscopy. J. Immun. Meth. 43, 349-350. 2+ this measurement was 30 [im in diameter, the [Ca ] KAKIMOTO, T. & SHIBAOKA, H. (1987). Actin filaments and measured should represent an average value for this area. microtubules in the preprophase band and phragmoplast of tobacco Furthermore, the contribution of the very tip should be cells. Protoplasma 140, 151-156. small due to the smaller cell diameter. [Ca2+] at the very KAMIYA, N. (1981). Physical and chemical basis of cytoplasmic streaming. A. Rev. PI. Physio!. 32, 205-236. tip may be much higher than 9xlO~8M (pCa7-0). If this 2+ KAMIYA, N. (1986). Cytoplasmic streaming in giant algal cells: a were the case, the Ca regulation of cytoplasmic stream- historical survey of experimental approached. Bot. Mag. Tokvo. 99, ing would be possible at the very tip. Actually, at the tip 441-467. area, the motility of organelles is very low and random KILMARTIN, J. V. & ADAMS, A. E. M. (1984). Structural (Iwanami, 1959; Heslop-Harrison & Heslop-Harrison, rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J. Cell Biol. 98, 922-933. 1987). In the present method, we could not observe any KOHAMA, K. & KENDRICK-JONES, J. (1986). The inhibitory Ca2+- clear structure of actin filaments at the tip area. Recently, regulation of actin-activated Mg-ATPase activity of myosin from it was reported that only short actin filaments are Physarum polycephalum plasmodia. J. Biochem. 99, 1433-1446. observed at the tip area of pollen tubes olNicotiana alata KOHNO, T. & SHIMMEN, T. (1987). Ca2+-induced F-actin in electron micrographs, using the rapid-freeze fixation fragmentation in pollen tubes. Protoplasma 141, 177-179. KOHNO, T. & SHIMMEN, T. (1988). Accelerated sliding of pollen and freeze substitution methods (Lancelle et al. 1987). 2+ 2+ tube organelles along Characeae actin bundles inhibited by Ca . Ca inhibition of cytoplasmic streaming may play a role J. Cell Biol. 106, 1539-1543. in inducing the polarized accumulation of organelles at LANCELLE, S. A., CRESTI, M. & HEPLER, P. K. (1987). the tip region. This localized accumulation would then Infrastructure of the cytoskeleton in freeze-substituted pollen tubes result in tip growth. of Nicotiana alata Protoplasma 140, 141-150. LEHMAN, W. & SZENT-GYORGYI, A. G. (1975). Regulation of muscular contraction. Distribution of actin control and myosin We thank Professor M. Tazawa and Dr R. Wayne for their control in the animal kingdom.^, gen. Phvsiol. 66, 1-30. valuable discussion and critical reading of this manuscript. We LLOYD, C. V. (1987). The plant cytoskeleton: the impact of also thank Dr S. Chaen for providing rabbit skeletal muscle fluorescence microscopy. A. Rev. PI. Phvsiol. 38, 119-139. myosin. MASCARENHAS, J. P. & LAFOUNTAIN, J. (1972). Protoplasmic This work was partly supported by a Grant-in-Aid for streaming, cytochalasin B, and growth of the pollen tube. Tissue £f Scientific Research from the Ministry of Education, Science Cell 4, 11-14. and Culture, Japan. MCKERRACHER, L. J. & HEATH, 1. B. (1986). Polarized cytoplasmic movement and inhibition of saltation induced by calcium-mediated effects of microbeams in fungal hyphae. Cell Motil. 6, 136-145. References MIMURA, T., SHIMMEN, T. & TAZAWA, M. (1983). Dependence of the membrane potential on intracellular ATP concentration in BARAK, L. S., YOCUM, R. R., NOTHNAGEL, E. A. & WEBB, W. W. tonoplast-free cells of Nitellopsis obtusa. Planta 157, 97-104. (1980). Fluorescence staining of the actin cytoskeleton in living MORIYASU, Y. & TAZAWA, M. (1987). Calcium-activated protease in cells with 7-nitrobenz-2-oxa-l,3-diazole-phallacidin. Proc. natn. the giant alga Chara australis. Protoplasma 140, 72-74. Acad. Sci. U.SA. 77, 980-984. NOBILING, R. & REISS, H.-D. (1987). Quantitative analysis of CONDEELIS, J. C. (1974). The identification of F actin in the pollen calcium gradients and activity in growing pollen tubes of Lilium tube and protoplast of Ajriaryllis belladonna. Expl Cell Res. 88, longiflorum. Protoplasma 139, 20-24. 435-439. PARTHASARATHY, M. V., PERDUE, T. D., WITZTUM, A. & ALVERNAZ, DICKINSON, D. B. (1968). Rapid starch synthesis associated with J. (1985). Actin network as a normal component of the increased respiration in germinating lily pollen. PI. Phvsiol. 43, cytoskeleton in many vascular plant cells. Am. J. Bot. TZ, 1-8. 1318-1323. DOREE, M. & PICARD, A. (1980). Release of Ca2+ from intracellular PERDUE, T. D. & PAJCTHASARATHY, M. V. (1985). In situ localization pools stops cytoplasmic streaming in Tradescantia staminal hairs. of F-actin in pollen tubes. Eur. j. Cell Biol. 39, 13-20. Experientia 36, 1291-1292. PIERSON, E. S., DERKSEN, J. & TRAAS, J. A. (1986o). Organization FRANKE, W. W., HERTH, W., VAN DER WOUDE, J. & MORRE, D. J. of microfilaments and microtubules in pollen tubes grown in vitro (1972). Tubular and filamentous structures in pollen tubes: or in vivo in various angiosperms. Eur.J. Cell Biol. 41, 14-18. possible involvement as guide elements in protoplasmic streaming PlERSON, E. S., WlLLEKENS, P. G. M., MAESSEN, M. M. & and vectorial migration of secretory vesicles. Planta 105, 317-341. HELSPER, J. P. F. G. (19866). The effect of lectins on germinating HASEGAWA, T., TAKAHASHI, S., HAYASHI, H. & HATANO, S. (1980). pollen of Lilium longiflorum. I. Effect on pollen tube growth and Fragmin: a calcium ion sensitive regulatory factor on the formation organization of microfilaments. Acta bot. Neerl. 35, 249-256. of actin filaments. Biochemistry 19, 2677-2683. POLLARD, T. D. & COOPER, J. A. (1986). Actin and actin-binding HEPLER, P. K. & CALLAHAM, D. A. (1987). Free calcium increases proteins. A critical evalulation of mechanisms and function. A. during anaphase in stamen hair cells of Tradescantia. J. 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508 T. Kohno and T. Shim?nen 131, 244-246. levels of Ca2+.J. Cell Biol. 96, 199-203. 2+ RODRIGUEZ, J. & DEINHARTD, F. (1960). Preparation of a TAKAGI, S. & NAGAI, R. (1986). Intracellular Ca concentration semipermanent mounting medium for fluorescent antibody studies. and cytoplasmic streaming in \'allisneria mesophyll cells. PI. Cell Virology 12, 316-317. Physiol. 27, 953-959. SHIMMEN, T. & YANO, T. (1984). Active sliding movement of latex TAZAWA, M. & SHIMMEN, T. (1987). Cell motility and ionic relations beads coated with skeletal muscle myosin on Chara actin bundles. in characean cells as revealed by internal perfusion and cell models. Protoplasma 121, 132-137. Int. Rev. Cytol. 109, 259-312. SHIMMEN, T. & YANO, T. (1985). Ca2+ regulation of myosin sliding TAZAWA, M., SHIMMEN, T. & MIMURA, T. (1987). Membrane control in the Characeae. A. Rev. PI. Phvsiol. 38, 95-117. along Chara actin bundles mediated by native tropomyosin. Proc. TOMINAGA, Y., WAYNE, R., TUNG, H. Y. L. & TAZAWA, M. (1987). Japan Acad. 61(B), 86-89. Phosphorylation-dephosphorylation is involved in Ca2+-controlled SHIMMEN, T. & YANO, T. (1986). Regulation of myosin sliding along cytoplasmic streaming in characean cells. Protoplasma 136, Chara actin bundles by native skeletal muscle tropomyosin. 161-169. Protoplasma 132, 129-136. TRAAS, J. A., DOONAN, J. H., RAWUNS, D. J., SHAW, P. J., STAIGER, C. J. & SCHLIWA, M. (1987). Actin localization and WATTS, J. & LLOYD, C. W. (1987). An actin network is present in function in higher plants. Protoplasma 141, 1-12. the cytoplasm throughout the cell cycle of carrot cells and STOSSEL, T. P., CHAPONNIER, C, EZZELL, R. M., HARTWIG, J. H., associates with the dividing nucleus. J. Cell Biol. 105, 387-395. JANMEY, P. A., KWIATKOWSK], D. J., LIND, S. E., SMITH, D. B., WOODS, C. M., POLITO, V. S. & REID, M. S. (1984). Response to SOUTHWICK, F. S., YIN, H. L. h ZANER, K. S. (1985). chilling stress in plant cells II. Redistribution of intracellular Nonmuscle actin-binding proteins. A. Rev. Cell Biol. 1, 353-402. calcium. Protoplasma 121, 17-24. SUGINO, H. & MATSUMURA, F. (1983). Fragmin induces tension reduction of actomyosin threads in the presence of micromolar {Received 1 July 1988 - Accepted 4 August 1988)

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