Inducement of wound motility in intact giant algal cells

Experimental Cell Research 145 (1983) 63-69

Copyright ~ 1983by AcademicPress, Inc. All rightsof reproductionin any form reserved 0014-4827/83/050063-07502.00/0

Inducement of Wound Motility in Intact Giant Algal Cells JOHN W. La CLAIRE II Department of Botany, University of Texas, Austin, TX 78712, USA

SUMMARY The cytoplasm (with its organelles) of intact cells of Ernodesmis verticillata (Chlorophyta) can be induced to contract in the presence of calcium ionophores and phenothiazine antipsychotics. The cell contents mimic wound-healing contraction if a combination of 10 ~tM A23187 and 10 pJVl chlorpromazine (or trifluoperazine) is present in a Ca2§ medium. The average incubation time is approx. 50 min for contraction. It is imperative that only fresh solutions of ionophores and phenothiazines are used, because stock solutions only 3 h old virtually double the response time of these cells. Separately, neither the ionophore nor the phenothiazines will induce contraction. The addition of 1.0 mM La 3+ completely prevents induction of motility. With trifluoperazine, equimolar X-537A will substitute for A23187, but it takes three times as long to induce contraction. Motility cannot be induced in CaZ+-free media (containing 5.0 mM EGTA). It therefore appears that Ca 2+ fluxes are responsible for triggering v~ound contraction in these giant algal cells. An influx of calcium ions from the external medium is suggested as being at least partially involved.

The cytoplasmic motility phenomenon of cellular wound healing has been investigated in a small, but varied group of organisms. Necessarily this work has focused on giant cells, including those of Amoeba proteus [12, 29, 30], amphibian eggs [1, 9, 11, 18], and a few coenocytic green algae [14, for review]. Severing the end from a cell of the green alga Ernodesmis induces a rapid, longitudinal contraction of the cytoplasm from the cut, and a centripetal contraction of the cytoplasm that closes the wound [14]. Both contractile events involved have been considered cell motility phenomena [15], and collectively are termed "wound motility" herein. A few investigations have shown that cellular wound healing requires free Ca 2+ in the external medium, and it has been postulated that wounding may induce an increase in Ca 2§ permeability of the plasma membrane [9, 15]. This influx of Ca 2§ would therefore trigger contractile events involved in sealing the wound. If this hypothesis is correct, then promoting Ca 2+ leakage into non-wounded cells should elicit wound-type motility of the cytoplasm. Numerous studies have shown that the insertable divalent cationophore A23187 transports Ca 2+ across a variety of cytomembranes [5, 21, 24 for reviews]. Another antibiotic, |asalocid (X-537A) has also been shown to transport Ca 2+ but it is much less specific for calcium ions than is A23187 [21, 22, 24] and Ca 2§ transport is much slower [13]. These ionophores therefore provide powerful agents for removing Ca 2+ concentration gradients across membranes. Another approach for equilibrating Ca 2+ across membranes is to inhibit Ca 2+ pumps that establish and maintain these gradients. Despite affecting many cellular functions, the phenothiazine tranquilizers inhibit (Ca2++Mg2§ by antagonizing calmodulin activation of the pumps [31, 33] or by inhibiting the 5-831818

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J . W . La Claire H

Exp Cell Res 145 (1983)

p u m p s directly [e.g., 8, 10, 25]. T h e p r e s e n t investigation e m p l o y e d i o n o p h o r e s and p h e n o t h i a z i n e s to d e t e r m i n e w h e t h e r w o u n d motility could be i n d u c e d in intact cells o f the c o e n o c y t i c green alga Ernodesmis verticillata.

MATERIALS

AND METHODS

Unialgal cultures of Ernodesmis verticiUata were maintained as previously described [14]. The standard experimental medium consisted of 0.375 M NaCI+0.008 M KCI+0.011 M CaClz+0.019 M MgC12+0.01 M piperazine-N,N'-bis-(2-ethanesulfonic acid) (PIPES buffer, disodium salt) with the pH adjusted to 7.2 with 1.0 N HCI. Salt concentrations and tonicity (810 mOsm/kg) were essentially equal to those of the sea water growth medium [28]. The Ca2§ medium differed only in having 0.005 M ethyleneglycol-bis-(f~-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) replacing CaCI2, and in having 0.391 M (total) NaC1 to bring the tonicity to 810 mOsm/kg. The pH was adjusted to 7.2 with 10 N NaOH. Control experiments with 0.391 M NaC1 in the standard medium showed no differences from those with 0.375 M NaC1. Stock solutions (1.0-5.0 mM) of A23187 (Calbiochem and Eli Lilly & Co.) and X-537A (HoffmanLaRoche Inc.) were prepared separately in dimethylsulfoxide (DMSO), and 0.1-1.0 mM chlorpromazine or trifluoperazine (Smith, Kline & French) stock solutions were made in the standard or Ca2§ free medium (the latter without any EGTA to prevent precipitation). Test solutions were prepared by adding 0.1 ml of stock solution(s) to the standard medium or to the CaZ+-free medium at a final volume of 10 ml. Final concentrations of ionophores and phenothiazines tested were 10-50 IxM (+ 1% DMSO) and 1.0-10 IxM respectively. Higher concentrations of ionophores precipitated from solution. LaCI3 (1.0-2.0 mM) was dissolved directly in the 10 ml of test solution. The pH of test solutions was 7.20+0.03 and the tonicity was 810+5 mOsm/kg (excluding DMSO). All reagents were obtained from Sigma Chemical Co., unless otherwise noted. Individual cells were rinsed in standard medium or CaZ§ medium for 10 min and then pipetted into Petri dishes (60 mm diameter, 15 mm high) containing the appropriate test solution, two cells per dish. All cells were periodically monitored for contraction through a Zeiss DRC Stereomicroscope under very low illumination, since phenothiazines are photolabile [26].

RESULTS In the p r e s e n c e o f c a 2+ motility w a s not i n d u c e d with incubation in either o f the i o n o p h o r e s or either o f the p h e n o t h i a z i n e s alone, at the c o n c e n t r a t i o n s tested. D M S O (1%)' b y itself or in c o n j u n c t i o n with a n y o f these s u b s t a n c e s also p r o m o t e d no visible c h a n g e s . H o w e v e r , if 10 ~tM A23187 ( + 1 % D M S O ) w a s c o m b i n e d with 10 ~tM c h l o r p r o m a z i n e (CPZ) or trifluoperazine (TFP), the c y t o p l a s m s u d d e n t l y split into t w o or three pieces. L o n g i t u d i n a l c o n t r a c t i o n o f the c y t o p l a s m w a s first e v i d e n t near the a p e x and/or the base o f a cell, w h e r e a faint, d a r k - g r e e n b a n d o f a g g r e g a t e d c h l o r o p l a s t s d e v e l o p e d prior to breakage. T h e individual s e g m e n t s o f c y t o p l a s m t h e n c o n t r a c t e d up to a few m m f r o m e a c h o t h e r and c o n c u r r e n t l y b e g a n to close centripetally w h e r e t h e y had separated f r o m e a c h o t h e r (fig. 1). E x e m p l a r y d a t a a p p e a r in table 1. With A23187, the a v e r a g e time that c o n t r a c t i o n o c c u r r e d was at a p p r o x . 50 min (after the start o f incubation) with either C P Z or T F P , but times varied greatly e v e n within single e x p e r i m e n t s . L o w e r c o n c e n t r a t i o n s o f i o n o p h o r e s did not i n d u c e c o n t r a c t i o n , but p h e n o t h i a z i n e s w o u l d infrequently and i r r e p r o d u c i b l y be effective d o w n to 1.0 ~tM if g r e a t e r a m o u n t s o f A23187 w e r e used (e.g., 50 ~tM). L a 3+ (1.0-2.0 mM) a d d e d to this milieu c o m p l e t e l y p r e v e n t e d c o n t r a c t i o n i n d u c e m e n t in all cells tested. W h e n T F P w a s p r e s e n t , X - 5 3 7 A w o u l d substitute for A23187, but the time required for c o n t r a c t i o n w a s m o r e than three times that with A23187, on the

Exp Cell Res 145 (1983)

Motility induction in a green alga

65

Fig. 1. An Ernodesmis cell (a) 1 min after being placed in standard medium +10 IxM CPZ+10 ~tM A23187+ 1% DMSO. A faint band of aggregated chloroplasts (arrows) developed at the apex and base at (b) 45, (c) 60 min respectively. The cytoplasm split into three segments at 62 min and contraction was quite evident by (d) 75 rain. In addition, the basal and apical portions had begun to close centripetally within (e) 90 min after immersion in the test solution, x9. Fig. 2. Small Ernodesmis cell (a) 1 min after being placed in Ca2§ medium +10 ~tM CPZ+I0 IxM A23187+1% DMSO. Holes developed in the cytoplasm and it began to collapse from the cell wall after (b) 120 min. No longitudinal contraction occurred. x22.

average, and a l o w e r p e r c e n t a g e of cells r e s p o n d e d (table 1). X-537A c o m b i n e d with C P Z would not induce contraction at 10 ~tM each. Identical e x p e r i m e n t s with these agents in Ca2+-free m e d i u m did not induce contraction, but with both 10 ~tM A23187 and CPZ (or TFP) present, large holes d e v e l o p e d in the c y t o p l a s m , and it collapsed shortly thereafter (fig. 2). N e v e r t h e less, this collapse was not a c c o m p a n i e d by any longitudinal contraction. The timing of these events was s o m e w h a t similar to that of contraction in the p r e s e n c e of Ca 2§ (table 1). Sample data c o m p a r i n g the effectiveness vs the age of test solutions a p p e a r in table 2. I f stock solutions w e r e left at r o o m t e m p e r a t u r e (in the dark) for 3 h before use, the a v e r a g e time required to induce motility a p p r o x i m a t e l y doubled. Utilizing 6-h-old stock solutions elicited an e v e n longer delay in contraction. DISCUSSION The present study was p r o m p t e d b y the discovery that wound-induced cytoplasmic motility in the marine alga Ernodesmis requires ATP and exogenous free-

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J.W.

Zxp Cell Res 145 (1983)

L a Claire H

Ca 2§ , and that La 3+ reversibly blocks this movement [15]. Since L a 3+ is known to inhibit transmembrane Ca 2§ fluxes [7, i9, 32], it seemed likely that motility could possibly be induced in intact cells by promoting Ca 2+ fluxes. The obvious agents for this task are the divalent cationophores. It was initially surprising that either A23187 or X-537A alone would not induce motility, although 10-50 ~tM A23187 does inhibit saltation of chloroplasts as revealed with time-lapse microcinematography (unpublished observations). It was hypothesized that CaZ+-extrusion mechanisms (i.e., pumps) might be keeping pace with ionophore-promoted leakage. Since greater concentrations of ionophores were not soluble in the high ionic strength media necessary for this organism, phenothiazines were added because they are known to reduce calcium pump activity directly and indirectly [8, 10, 25, 31, 33]. The results with most combinations of either ionophore with either phenothiazine provide circumstantial evidence that CaZ+-extruding/sequestering pumps may be responsible for the inability of ionophores alone to induce contraction. The observations that neither CPZ nor TFP alone would induce motility, could be explained by the fact that phenothiazines can apparently decrease Ca 2+ permeability in membranes [16]. Since Ernodesmis is a marine alga, bathed in approx. 10 mM Ca 2+ [28], it would not be surprising to find that Ca z+ pumps exist and are very active in this organism.

Table 1. Wound-contraction inducement of intact Ernodesmis cells a Time of contraction (min)

Treatment

No. of cells contracted/ cells tested

Standard medium

0/10

-

-

+DMSO

0/10

-

-

+A23187+DMSO

0/10

-

-

+CPZ

0/10

-

-

+CPZ+DMSO

0/10

-

-

+ CPZ + A23187 + DMSO

22/25

+CPZ+A23187+DMSO+

1.0 mM

L a 3+

21-77

51

0/10

-

-

+TFP

0/10

-

-

+TFP+DMSO

0/10

-

+ TFP + A23187 + DMSO

22/24

15-90

0/10

+TFP+X-537A+DMSO

7/10

+CPZ+X-537A+DMSO

0/25

-

-

medium

-

49

+X-537A+DMSO

CaZ+-free

89-270

177

0/10

-

-

+DMSO

0/10

-

-

+A23187+DMSO

0/10

-

-

+CPZ

0/10

-

-

+CPZ+DMSO

0/10

-

+ CPZ + A23187 + DMSO

0/20b

+TFP

0/10

-

+TFP+DMSO

0/10

-

+ TFP + A23187 + DM SO

0/10b

a 10 IxM c h l o r p r o m a z i n e c

Range

(CPZ),

trifluoperazine

28-120 c

26--75r

(TFP), A23187 & X-537A.

Holes developed in cytoplasm followed by collapse. Times that holes and collapse first evident. No contraction occurred.

78 c 48 c

Final [DMSO],

1%.

Exp CellRes 145 (1983)

Motility induction in a green alga

Regardless, it is clear that I0 ~tM A23187/X-537A plus 10 ~tM CPZ/TFP will induce cytoplasmic motility in intact cells, and that this motility closely resembles that in wounded cells, even in manifesting one or two faint bands of aggregated chloroplasts in the cytoplasm prior to breakage [cf 14]. The failure of CPZ plus X-537A to initiate contraction might be due to a combination of the lower specificity of X-537A [24] and the lesser potency of CPZ [8] compared to the other agents. The need for fresh ionophore and phenothiazine solutions is quite apparent from table 2. The latter are known to be photolabile [26], but storing either at room temperature even for short periods also appears to reduce potency dramatically. Best results were consistently obtained with freshly-made solutions in the present study. Lanthanum ions bind to A23187 [20] and prevent A23187 effects [17]. However, La 3§ also displaces Ca 2+ from membrane-binding sites, inhibits Ca 2§ transport and blocks Ca 2+ channels [see refs above]. That La 3+ prevents induction by A23187 plus CPZ, could be due to any or all of these known effects, especially since 1.0 mM La 3§ alone prevents motility in wounded cells [15]. The data herein strongly suggest that wound contraction in Ernodesmis is triggered directly or indirectly by Ca 2+ fluxes. Since X-537A transports Ca 2§ much more slowly than does A23187 [13], the great increase in incubation time needed with X-537A implies that Ca 2+ fluxes are indeed responsible for inducing motility. Furthermore, it would appear that an influx of Ca 2§ from the medium (+an efflux from internal stores or sites) is responsible, since intact cells cannot be induced in a Ca2+-free medium. Additional information will be necessary to determine the absolute directionality of Ca 2+ movements during wounding or

Table 2. Contraction time (rain) vs age o f stock solutions a Age of solutionsb Dish

Cell

Fresh

3-h-old

6-h-old

1

a

b

20 30

86 122

85 103

2

a b

25 63

75 158

87 135

3

a b

20 28

49 141

85 97

4

a b

69 -r

53 54

66 83

5

a b

30 90

62 113

112 132

42

91

99

a 1.0 mM A23187 and trifluoperazine in DMSO and standard medium respectively. Final concentrations, 10 ~M A23187/TFP and 1% DMSO in standard medium. b Stock solutions maintained in the dark at room temperature (23~ r Cell did not contract during 3 h observation period. Not included in computing ~.

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J . W . La Claire H

Exp Cell Res 145 (1983)

inducement, due to the inherent difficulties in discerning the significance of extracellular Ca 2§ in whole cells [2], and due to the complexity in general of Ca 2+ m o v e m e n t s in cells [3, 4, 23]. But Ca2+-free media did not prevent A23187-induced activation o f various animal eggs [27] or ionophore-mediated cessation of c y t o p l a s m i c streaming in plant ceils [6], and those results were given b y the authors as support for internal pools of Ca 2+ being involved in regulating those p r o c e s s e s . T h e r e f o r e , triggering of w o u n d motility in Ernodesmis m a y well utilize e x o g e n o u s calcium in addition to, or instead of sequestered stores, as Gingell [9] suggested for healing of Xenopus eggs. The fact that fenestrations and collapse occurred only w h e n both types of drugs were present, after similar incubations in Ca2+-free medium, suggests that internal pools m a y be leaking out and causing collapse at this point. The similarity in incubation times for these events to o c c u r is w e a k evidence that sequestered Ca 2§ might also be involved in triggering motility w h e n exogenous freeC a 2+ is p r e s e n t . The long delay in triggering m o v e m e n t m a y indicate that a massive flow of Ca 2+ is n e c e s s a r y or that the threshold concentration of Ca 2+ is high. H o w e v e r , preliminary data indicate that the threshold [free C a 2§ required is approx. 10 -7 M in Ernodesmis [15], so a sudden and m a j o r calcium flux appears to be m o r e significant for initiating contraction. This hypothesis would c o r r e s p o n d to the overall i m p o r t a n c e of w o u n d motility in preventing cuts or punctures f r o m killing giant-celled organisms like Ernodesmis. W h e r e a s minor fluxes of Ca 2§ initiate developmental events in cells [27], m a j o r Ca 2+ fluxes resulting f r o m severe injury m a y be n e c e s s a r y to initiate w o u n d healing. Permeable cell-motility models of Ernodesmis are being d e v e l o p e d in the a u t h o r ' s laboratory which should provide excellent tools for investigating the role and the exact threshold value of Ca 2§ in wound-induced motility, and for determining which subcellular structures are potentially involved in this p h e n o m e n o n . Such studies should lead to a better understanding o f w o u n d healing at the cellular level of organization. Sincere gratitude is expressed to Professor J. A. West (University of California, Berkeley, Calif.) for the original Ernodesmis isolates, and to Drs R. L. Hamill (Eli Lilly & Co. Laboratories, Indianapolis, Ind.), H. Green (Smith, Kline & French Laboratories, Philadelphia, Pa), and W. E. Scott (HoffmanLaRoche, Inc., Nutley, N. J.) for generous gifts of A23187, trifluoperazine and X-537A respectively. Thought-provoking discussions with Professors S. J. Roux, Jr and G. A. Thompson, Jr are also extremely appreciated. This work was supported by US NSF Grant PCM 81-17815.

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Motility induction in a green alga

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Received September 2, 1982 Revised version received December 4, 1982

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