Wound-healing motility in the green alga - Springer Link

Planta

Planta (1982)156:466-474

9 Springer-Verlag 1982

Wound-healing motility in the green alga Ernodesmis: calcium ions and metabolic energy are required John W. La Claire II Department of Botany, University of Texas, Austin, TX 78712, USA

Abstract. Wounding a giant cell of the marine alga Ernodesmis verticillata (K/itzing) Borgesen (Chlorophyta) induces two concomitant motility phenomena: longitudinal contraction of the protoplasm away from the wound site, and centripetal contraction of the cut end around the central vacuole. Healing is complete within 30 min of wounding. Mechanical extrusion of the protoplasm into the medium with a teasing needle is followed by contraction of the protoplasm into numerous spherical protoplasts within 60 rain. Utilizing a simple defined medium, it is shown that motility is almost completely inhibited by the absence of exogenous free Ca 2+, with 5.0 mM ethylene glycol bis-(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid present. This inhibition is reversible by rinsing the cells with CaZ+-containing medium. Similarly, extruded cytoplasm fails to exhibit motility in C a 2 +-free medium. The threshold concentration of exogenous free Ca 2+ is approx. 10 - 7 M for wound-induced contraction. The ions Ba 2+, Cd 2+ and Sr z+ will substitute for C a 2+, but the rate of contraction is one-half that with C a 2 + present. Although darkness has no inhibitory effect, lower temperature (15 ~ C), cyanide, or micromolar amounts of phosphorylation uncouplers reversibly slow protoplasmic motility in wounded cells and extruded cytoplasm. Carbonylcyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenylhydrazone are especially potent inhibitors. These results indicate that cellular wound healing utilizes metabolic energy and requires exogenous free Ca 2 +, implying that motility in Ernodesmis is a true contractile process. Since 1.0 mM Abbreviations." CCCP = carbonylcyanide m-chlorophenylhydra-

zone; DMSO=dimethylsulfoxide; DNP=2,4-dinitrophenol; EGTA=ethylene glycol bis-(fl-aminoethyl ether)-N,N,N',N'tetraacetic acid; FCCP = carbonylcyanide p-trifluoromethoxyphenylhydrazone

0032-0935/82/0156/0466/$01.80

La 3 + completely and reversibly prevents contraction, it is postulated that Ca 2 + fluxes may actually trigger motility. Key words: Calcium and motility/wound healing - Cell repair - Chlorophyta - Ernodesmis- Motility - Wound healing.

Introduction

Plant motility encompasses a wide variety of movements, including cytoplasmic streaming, chloroplast movements, amoeboid locomotion, cytokinesis, gliding motility, etc. (for recent reviews: Wagner 1979; Allen 1981; Dillon 1981; Park 1981). Many of these are believed to be true contractile events, i.e., to involve contractile proteins and to require free Ca 2+ and ATP. Among the most widely investigated plants exhibiting motility are the giant-celled charophytes (Allen and Allen 1978; Seitz 1979) and the syncytial slime molds (Kamiya 1981), primarily because of their large size and the magnitude of their cytoplasmic streaming. In a recent study, several green algae in the order Siphonocladales were found to display fairly rapid protoplasmic movements involved in wound healing, and these movements are taken as manifestations of cell motility (La Claire 1982). The large size of the cells and the rate of motility make them highly suitable for investigating these motility phenomena and also for studying wound repair at the cellular level of organization. The present study of Ernodesmis was undertaken to determine w h e t h e r C a 2 + and ATP, which are both necessary in most contractile processes, are also required in wound-induced motility in this alga.

J_W. La Claire II : Cell motility in Ernodesmis requires C a 2 + and energy

Materials and methods Culture techniques and experimental conditions. Culture conditions, growth media and manipulations were identical to those previously described (La Claire 1982). The standard experimental medium was an aqueous solution containing 0.375 M NaCI, 0.008 M KC1, 0.011 M CaC12, 0.019 M MgC12 and 0.01 M 1,4piperazine-diethanesulfonic acid (PIPES buffer, disodium salt), with the pH adjusted to 7.2 with 1 N HC1. The molar concentrations of salts, the final tonicity (810 mOsm k g - 1 ) and the ionic strength approximate those of 28 ~ o sea water (Sverdrup et al. 1942), in which the organism was grown. The calcium-free medium was similar to the standard one, except that 5.0 mM ethylene glycol bis-~-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) was substituted for CaCI2, and the tonicity was increased to 810 mOsm kg- 1 by adjusting the NaCI to 0.391 M. The pH was also adjusted to 7.2 with 10 N NaOH. The cells were incubated in this medium for 10 rain prior to wounding to remove any Ca 2+ adsorbed to the celI walls. They were then transferred to fresh calcium-free medium and wounded. To test the ability of various divalent cations to replace Ca 2+, 0.011 M BaCI2, CdCI2, SrCI2, MgC12 or ZnC12 was substituted for CaCI2 in the standard medium. Cells were placed in the standard medium for 5 min, in calciumfree medium for 15 rain to removed any Ca 2+ from the walls, and then into the test solution for 5 min to equilibrate the cells before wounding. For calcium threshold determination, a series of Ca-EGTA (" caIcillm buffer ") solutions were prepared similar to the Ca 2 +free medium, but with appropriate CaC12 amounts added to maintain 10 -3, 10 .4 ..., 10 .9 M "free Ca 2+" in solution, at pH 7.4 (Reed and Bygrave 1975). The cells were preincubated for 10 rain to acclimate them to test concentrations of free Ca 2+, and then were transferred to dishes of fresh solutions and wounded. Individual cells were placed in 10 ml of medium in glass Petri dishes (60 mm diameter, 15 mm high) at 23 ~ C (equivalent to the growth temperature). The experiments at 15~ were performed similarly, but the small Petri dishes were placed inside large Petri dishes (150 mm diameter, 15 mm high) containing chilled water. The lower temperature was monitored and maintained by adding ice. Dark experiments (and incubation in the dark) were performed in a photographic darkroom and the cells were wounded and periodically monitored as below, with a VG-9 green filter (Carl Zeiss, New York, N.Y., USA) over the light source. The bases of individual cells were carefully and cleanly excised with Vannas ultra-microdissecting scissors (Roboz Surgical Instrument Co., Washington, D.C., USA) under a Zeiss DRC Stereomicroscope. In some cases, the protoplasm was then extruded with a curved teasing needle into the medium on a microscope slide with a depression, and examined through a Zeiss WL compound microscope. Transferral of wounded cells from one medium to another was performed by carefully pipettiag the cells through two or more changes of the standard medium. Inhibitors and other reagents. Stock solutions of 2,4-dinitrophenol (DNP, 1.0 raM) were prepared in the standard medium, and 10 mM stock solutions of carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) were made separately in dimethylsulfoxide (DMSO). Dilution series of these uncouplers were prepared, in steps of 10, down to 0.1 gM in the standard medium or DMSO. Combinations of DNP and CCCP were made by adding 0.1 mt of both the 1.0 mM stocks to 9.8 ml of the standard medium. The final DMSO concentration was 1% in all

467

test solutions containing CCCP or FCCP. Sodium salts of azide, cyanide (MCB Manufacturing Chemists, Cincinnati, Oh, USA) and arsenate (Fisher Scienfif]c Co., Dallas, Tex. USA) were added directIy to the standard medium, at concentrations up to 5.0 mM, and incubation in these three inhibitors was performed in the dark. Ruthenium red (Alfa Products, Danvers, Mass., and Sigma Chemical Co., St. Louis, Mo., USA) was purified according to Luft (1971). Stock solutions (1.0 mM) of freshly purified ruthenium red were made in the standard medium. The LaC13 (1.0-2.0 mM) was also added directly to the 10 ml of the standard medium. All reagents were obtained from Sigma Chemical Co. unless otherwise noted.

Results Effects o f ions. W o u n d h e a l i n g in t h e s t a n d a r d m e d i u m w a s i d e n t i c a l t o t h a t o b s e r v e d in sea w a t e r ( L a C l a i r e 1982). T h e p r o t o p l a s m o f cells w o u n d e d a f t e r a 10 m i n i n c u b a t i o n in t h e s t a n d a r d m e d i u m contracted longitudinally several millimeters from t h e c u t e n d d u r i n g t h e first 10 m i n a f t e r e x c i s i o n ( F i g . 1), a n d t h e h o l e b e t w e e n t h e v a c u o l e a n d t h e medium closed centripetally. Control experiments using the standard medium with extra NaC1 (0.391 M t o t a l ) a l s o s h o w e d n o r m a l c o n t r a c t i o n . H o w e v e r , a f t e r t h e s a m e i n c u b a t i o n in calcium-free m e d i u m , w o u n d e d cells s h o w e d p r a c t i c a l l y n o c o n traction from the cut or closure, although the prot o p l a s m b e g a n t o c o l l a p s e l a t e r a l l y a f t e r 30 m i n ( F i g . 2), p r o b a b l y b e c a u s e o f t u r g o r loss. C o n t r a c t i o n c o u l d b e r e s t o r e d if, 10 r a i n a f t e r w o u n d i n g in t h e c a l c i u m - f r e e m e d i u m , t h e cells w e r e t r a n s ferred through two changes of standard medium. Within seconds, the protoplasm began to contract longitudinally from the wound and close around t h e v a c u o l a r c a v i t y , o r it s p l i t i n t o t w o o r m o r e f r a g m e n t s w h i c h c o n t r a c t e d ( F i g . 3). Cells s i m i l a r l y w o u n d e d a n d t r a n s f e r r e d to f r e s h c a l c i u m - f r e e m e dium (instead of the standard one) did not exhibit c o n t r a c t i o n . O b v e r s e l y , t h e p r o t o p l a s m o f cells c u t in s t a n d a r d m e d i u m a n d t r a n s f e r r e d t o c a l c i u m free m e d i u m 10 m i n a f t e r w o u n d i n g , c e a s e d c o n traction within 10-15 min after transfer. Contract i o n w a s a g a i n r e s t o r e d i f t h e s e cells w e r e t h e n t r a n s f e r r e d b a c k to s t a n d a r d m e d i u m ( n o t s h o w n ) , s h o w i n g t h a t C a 2§ w a s r e q u i r e d f o r c t 3 n t r a c t i o n . I n t a c t cells i n c u b a t e d in t h e c a l c i u m - f r e e m e d i u m for similar periods of time displayed no morphological changes or any passive collapse. T h e t h r e s h o l d c o n c e n t r a t i o n o f free C a z+ a p p e a r s t o b e a p p r o x . 10 . 7 M , s i n c e o n l y t h o s e cells w o u n d e d in s o l u t i o n s c o n t a i n i n g g r e a t e r t h a n 10 . 7 M e x o g e n o u s free C a 2+ e x h i b i t e d a n y l o n g i tudinal contraction and centripetal closure of the p r o t o p l a s m . C e l l s w o u n d e d in 10 . 8 o r 10 - 9 M free Ca 2 + behaved identically to those in the Ca 2 +-free medium.

Figs. 1-3. Wounded cells of Ernodesmis. Time (in rain) after wounding/extrusion or transfer shown in upper left of each panel. Cut end of cells is on lefthand side. x 11. Fig. 1. A cell wounded (after 10 min preincubation) in the standard medium. The protoplasm contracted 4 mm from the cut within 10 min and the open end (arrows) closed around the central vacuole, becoming smooth 60 min after wounding. Fig. 2. Wounded cell in Ca2+-free medium ( + 5 . 0 r a m EGTA), cut after 10 min preincubation in the medium. The cell contents contracted very little from the cut, and began to collapse passively after 30 rain. Fig. 3. Intact cell (T=0) cut in Ca2+-free medium after 10 rain preincubation. The wounded cell was transferred through two changes of standard medium ( + C a 2+) after 10 min, and contraction and closure were evident 5 min after the transfer. The protoplasm in this cell split into two pieces and the portion near the cut end contracted and closed at both ends

Figs. 4-7. Extruded protoplasm (Figs. 4, 5, 7) and wounded cell (Fig. 6) of Ernodesmis. Time (in rain) after wounding/extrusion or transfer shown in upper left of each panel. Fig. 4, Protoplasm of a severed cell mechanically extruded into the standard medium after 10 min preincubation in the medium. Within 60 min most fragments contracted into spherical protoplasts of varying sizes, x 103. Fig. g. Protoplasm of a severed cell mechanically extruded into Ca2+-free medium after 10 rain preincubation in the medium. Virtually no spheration or contraction was evident 60 rain after extrusion, x 105. Fig, 6. Cell cut in the standard medium with 1.0 m M La 3 + added. Very little contraction from the severed end (on left) was evident by 30 min. The cell was then transferred through two changes of standard medium ( - L a 3+) and both contraction and closure began within 15 min. In this cell both ends of the protoplasm contracted after the La a+ was removed, x 11. Fig. 7. Protoplasm extruded into the standard medium with 1.0 m M La 3 + added. Very little contraction/spheration occurred within 60 rain. x 105

J.W. La Claire II : Cell motility in Ernodesmis requires Ca 2 + and energy

Protoplasm of wounded cells that was mechanically extruded into the standard medium formed numerous spherical protoplasts of variable sizes within 60 min (Fig. 4). If the cell contents were extruded into calcium-free medium (after 10.15 min incubation before extrusion), virtually no spheres resulted (Fig. 5). Substituting equimolar concentrations of B a 2 +, Cd 2 + or Sr ~+ for C a 2 + in the standard medium permitted contraction in wounded cells, but the rate of contraction was approximately one-half that with calcium present (not shown). The healing rate with Cd 2 + appeared to be somewhat faster than with Ba 2+ or Sr 2+. Mg 2+ and Zn 2 + did permit some contraction but the protoplasm usually did not completely heal the wound. Cells excised in the standard medium with 1.0.2.0 m M La 3+ added displayed essentially no protoplasmic contraction (Fig. 6). No preincubation in La 3 +-containing medium was necessary to elicit this inhibition, and if the La 3 + was added after cutting the cells, contraction ceased very shortly thereafter. If after 30 min these cells were then transferred to standard medium without added La 3+, contraction slowly resumed 10-15 min afterward, and the open end closed normally (Fig. 6). Frequently, contraction was evident at both ends of the protoplasm. Extruded cell contents showed little or no spheration in the presence of La 3 + (Fig. 7). Although the La 3 + inhibition indicates that calcium fluxes are involved in contraction, calcium pumps and antiporter mechanisms are probably not involved because ruthenium red (1.0-100 gM) purified from either source had no inhibitory effect on contraction in wounded cells or in extruded cytoplasm (not shown).

Effects of darkness, uncouplers and low temperature. Photosynthesis is not essential for contraction since darkness alone did not inhibit contraction in cut cells, even after dark incubations (before cutting in the dark) of up to 16 h. Although azide (1.0-5.0 mM) only slightly decreased contraction

471

in the dark, oxidative phosphorylation may provide ATP for contraction since 5.0 m M cyanide inhibited contraction 50-90% if cells were incubated for more than 6-8 h in the dark. Substratelevel phosphorylation might also be a partial source of ATP because arsenate (5.0 mM) slightly inhibited contraction, but only after incubations (in the dark) of 8 h or more. Utilizing phosphorylation uncouplers (even in the light) provided further evidence that ATP production is necessary for wound healing. Contraction was inhibited to a certain extent by D N P (10-100 gM), and D N P greatly decreased spheration of extruded cytoplasm. Both CCCP and FCCP (in standard m e d i u m + l % DMSO) were especially potent inhibitors of protoplasmic contraction, at concentrations of 0.1-100 ~tM. The degree of inhibition decreased at 0.1-1.0 gM, and effects were not detectable at or below 0.01 gM. Control cells wounded/extruded in standard medium + 1% DMSO alone showed normal contraction. All uncouplers appeared to be more effective if cells were incubated in uncoupler-containing solutions for 10-30 min prior to wounding. Some mottling of the cytoplasm was evident after these incubations also (Fig. 9). Combinations of D N P and CCCP (both 10 gM) distinctly inhibited contraction, especially after a 30 min incubation period (Figs. 9, 10). Although some contraction was obvious, the rate was greatly decreased (compare Figs. 1 and 9) and the cells usually did not heal completely in the presence of any uncouplers. These effects were also reversible by transferring the cells to standard medium. Extruded cell contents from similarly incubated cells also displayed some degree of contraction, but usually only the tiniest fragments formed smooth spheres while the larger masses remained very irregularly shaped (Fig. 10). The addition of 1.0 m M ATP (sodium salt) to the uncoupler-containing medium did not appear to reverse the inhibitory effects. Decreasing the overall rate of metabolism definitely decreased the rate of wound healing. Cells

Figs. 8-10. Wounded cells (Figs. 8, 9) and extruded cytoplasm (Fig. 10) of Ernodesrnis. Time (in min) after wounding/extrusion or transfer shown in upper left of each panel. Severed end of cut cells is on lefthand side of each panel, Fig. 8. Severed cell in standard medium at 15~ after 2 min preincubation at this temperature. The rate of contraction and closure slowed greatly (compare Fig. 1), and all motility ceased 90 rain after wounding prior to complete closure of the wound (arrow). The cell was then transferred to the same medium at 23~ (seventh panel) and within 10 rain the contraction resumed and the open end closed. Within 12 h ( T = 720) it expanded to fill the diameter of the old cell wall. (Note: the orientation of this cell was changed to show the open end of the protoplasm.) x 11. Fig. 9. Cell wounded in standard medium + 10 ~tM DNP + 10 gM CCCP + 1% DMSO after 30 min preincubation. Mottled appearance of cytoplasm was evident following incubation ( T = 1). The rate of contraction was slowed (compare Fig. i) and complete wound closure had not occurred at 60 min after wounding (arrow), when contraction ceased. • 11. Fig. 10. Cell contents extruded (following 30 min preincubation) into standard medium + 10 gM DNP + 10 ~tM CCCP + 1% DMSO. Some contraction occurred by 60 min after extrusion, but only the smallest fragments were spherical (compare Fig. 4). • 126

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J.W. La Claire II : Cellmotilityin Ernodesmisrequires C a

equilibrated for 2-5 rain in standard medium at 15 ~ C and cut, displayed contraction initially, but the rate of contraction was much slower and it virtually ceased after approx. 90min, before healing was completed (Fig. 8). If the cells were then warmed to room temperature (23 ~ C) contraction resumed within 10 min, and the cells healed shortly thereafter (Fig. 8), demonstrating the reversibility of low-temperature inhibition. Discussion

The wound-healing process in Ernodesmis appears to involve at least two contractile events. Longitudinal contraction of the protoplasm (within the cell wall) results in a rapid, unidirectional movement of the cytoplasm and organelles away from the severed end of the cell. Concurrently, the open end of the protoplast (i.e., that closest to the original cut end of the cell) contracts centripetally, leading to the eventual closure of protoplasm around the vacuolar cavity. Both types of contraction are much more noticeable with time-lapse microcinematography, and they are manifested by a sudden aggregation of chloroplasts and other organelles throughout the cell, and especially at the site of wound closure. These contractile events (as well as the rounding up of extruded cytoplasm) appear to be induceable cell motility phenomena which occur in response to wounding. Wound-induced motility in Ernodesmis is quite distinct from cytoplasmic streaming or saltatory movements of organelles. Saltation of chloroplasts in this organism can only be detected with timelapse microcinematography; and, similar to rotational streaming in charophytes (Williamson 1975; Higashi-Fujime 1980; Tominaga and Tazawa 1981 ; Williamson and Ashley 1982), it ceases when the concentration of free Ca 2 + is greater than some apparent threshold value (unpublished observations). However, wound motility in Ernodesmis can only be induced when the concentration of free Ca 2 + is greater than the apparent threshold value of 10 -7 M free Ca 2+. In this respect, its motility resembles muscle and the majority of nonmuscle motility phenomena currently known. Wound healing has been investigated in very few organisms at the cellular level of organization, including eggs of Xenopus (Gingell 1970; Bluemink 1972) and axolotl (Luckenbill 1971), and in Amoeba proteus (Szubinska 1971, 1978; Jeon and Jeon 1975), where centripetal closure via contractile mechanisms was reported. Furthermore, in Xenopus and Amoeba, closure was also noted to be sensitive to Ca 2+. Another coenocytic green

2+

and energy

alga, Bryopsis hypnoides (Caulerpales) deposits a large proteinaceous plug at the wound site and the cytoplasm undergoes some contraction and centripetal closure near the wound (Burr and West 1971; Burr and Evert 1972). Also, extruded cytoplasm of B. plumosa forms numerous viable spheres (Tatewaki and Nagata 1970), similar to Ernodesmis. Finally, Ishizawa et al. (1979) reported that wound-induced protoplasmic spheration in Boergesenia forbesii (Siphonocladales) also requires Ca 2 + in the medium. The fact that several of these studies also implicated microfilaments (actin?) in wound healing perhaps indicates that a similar contractile (actomyosin-mediated?) mechanism is universally involved in the process of cellular wound repair. Despite the apparent ubiquity of actin in plant cells, myosin has only been isolated from two flowering plants (Ohsuka and Inoue 1979; Vahey and Scordilis 1980) and from the charophyte Nitella flexilis (Kato and Tonomura 1977). There is also evidence that myosin may exist in Chara corallina (Williamson 1979). Currently, biochemical investigations of the Siphonocladales are underway to determine what contractile proteins may be present and potentially involved in wound-induced motility. The inhibitory effects of a Ca 2 +-free medium on motility must be interpreted cautiously. The difficulty in determining the roles of extracellular calcium in cellular events is primarily due to the alteration of intracellular pools of calcium by Ca 2 +-free media (Borle 1978; Carafoli and Crompton 1978). However, the inhibition with La 3+ indicates that transmembrane fluxes of Ca 2 + are necessary for motility in Ernodesmis, since lanthanum ions are known to displace Ca 2+ from membrane binding sites and to inhibit Ca 2 + fluxes and transport (for recent reviews: Weiss 1974; Martin and Richardson 1979; dos Remedios 1981). It seems likely that extracellular Ca 2 + may be flowing into the cytoplasm upon wounding rather than being released from intracellular pools because La 3 + is not believed to be transported across most membranes (dos Remedios 1981). Ruthenium red is known to inhibit active Ca 2 + transport due to Ca2+-ATPases (Carafoli and Crompton 1978) and to decrease passive influxes o f Ca 2+ involving antiporter systems (Hinnen et al. 1979) in some organisms. The lack of ruthenium-red inhibition of Ernodesmis motility indicates that if Ca 2 + is indeed entering from the external medium, a calcium channel might be the route of entry after wounding. This is supported by the ability of La 3+ to block such calcium channels (Borle 1981; dos Remedios 1981). Gingell (1970)

J.W. La Claire II: Cell motility in Ernodesmis requires Ca 2 + and energy

proposed that a wound-induced increase in Ca 2 + permeability of the plasma membrane was also responsible for triggering cytoplasmic closure of wounds in Xenopus eggs. It would be premature to speculate further in this regard, but future work with more specific calcium entry blockers may help determine whether such Ca 2 § channels exist in Ernodesmis. Although the apparent threshold level for wound contraction (10 .7 M) is somewhat similar to those reported for other motility phenomena (Stebbings and Hyams 1979), this value should be considered a preliminary one. It cannot be assumed that the cytoplasmic concentration of free Ca 2§ is equal to that of the medium, since dyeexclusion experiments indicate that the plasma membrane is intact in wounded cells (unpublished observations). Also the condition of internal calcium-regulating membrane systems and their role in contraction are unknown. Permeable cell models of Ernodesmis will be essential for the precise determination of the calcium threshold level for wound healing. The need to incubate cells in Ca 2 § medium prior to wounding, for complete inhibition of motility is probably due to adsorption of calcium to the cell wall. The calcium-binding ability of plant cell walls has been documented in flowering plants (e.g. Stassart et al. 1981), in a charophyte (Gillet and Lefebvre 1980) and in the siphonocladalean alga Valonia utricularis (Kessler 1980). At nearneutral pH values, EGTA preferentially chelates Ca 2+ over other divalent cations (Schmid and Reilley 1957), so the incubation in 5.0 mM EGTA may be necessary to chelate this pool of calcium. Adsorbed Ca 2+ may also explain the 10-15 min delay in cessation of motility in wounded cells transferred from the standard medium to the calcium-free one. The ability of Ba 2 § and Sr 2 + to replace Ca 2 + was also reported for eggs of Xenopus (Gingell 1970). The slower rates of healing in the presence of these cations or Cd 2 § imply that calcium cannot fully be replaced by other cations and hence Ca z § is probably required for normal motility in Ernodesmis. It appears that ATP is also necessary for motility, since lowered temperature, cyanide, and phosphorylation uncouplers markedly inhibit contraction. Similar low-temperature inhibition has been reported for motility in Boergesenia (Ishizawa et al. 1979). Photosynthesis does not seem to be essential for contraction, in view of the complete lack of dark inhibition. However, if respiration is also inhibited with cyanide (in the dark), insufficient ATP

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is apparently produced and contraction is greatly decreased. It is not clear why azide did not elicit the same inhibition that cyanide did, even with similar incubations. The fact that arsenate with darkness reduces motility indicates that substratelevel phosphorylation might also provide ATP for contraction. However, arsenate is also known to interfere with electron-transport-coupled phosphorylation (e.g. Lien and San Pietro 1981), so its absolute site of action in Ernodesmis is not yet known. It is well known that DNP, FCCP and CCCP are potent uncoupling agents of both oxidative and photophosphorylation (Good and Izawa 1966; Jagendorf 1975; Lehninger 1975), and FCCP/CCCP are quite effective in reducing motility in Ernodesmis, even at submicromolar concentrations. The absence of complete inhibition by the uncouplers (or combinations thereof) indicates that enough ATP may be present in the cytoplasm to permit at least the initial movements. The fact that cells wounded at lower temperatures continue to exhibit some motility for 90 min (presumably when ATP utilization surpasses production) supports this hypothesis, as does the need for long incubations in cyanide to achieve inhibition. Perhaps the increased uncoupler inhibition following incubation results in partial depletion of internal ATP stores, and this may relate to the cells' mottled appearances. Clearly, the effects of uncoupling agents on whole cells should be interpreted carefully, but the collective data with cyanide, low temperature, and uncouplers do indicate that ATP is utilized during normal wound healing in Ernodesmis, as has been reported in other motility phenomena in algae (e.g. Tendel and Haupt 1981; H/ider 1982). However, the inability of exogenous ATP to decrease this inhibition is probably a consequence of its lack of permeability. Attempts are now underway to develop permeable, cell-motility models of Ernodesmis to probe more accurately the roles and required concentrations of Ca 2 § and ATP. These and concurrent ultrastructural studies should greatly aid in elucidating the mechanisms by which cell motility occurs in this organism, and should contribute to the general understanding of cellular wound healing. Sincere gratitude is expressed to Professor J.A. West (University of California, Berkeley, USA) for the original isolates of Ernodesrnis. This work was supported by National Science Foundation grant PCM 81-J7815.

References Allen, N.S., Allen, R,D. (1978) Cytoplasmic streaming in green plants. Annu. Rev. Biophys. Bioeng. 7, 497-526

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J.W. La Claire II: Cell motility in Ernodesmis requires Ca z + and energy

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