J. Cell Set. 71, 37-50 (1984) 37Printed in Great Britain © The Company of Biologists Limited 1984

REGULATION OF CELL SHAPE IN EUGLENA

GRACILIS

II. THE EFFECTS OF ALTERED EXTRA- AND INTRACELLULARCa2 + CONCENTRATIONS AND THE EFFECT OF CALMODULINANTAGONISTS

THOMAS A. LONERGANDepartment of Biological Sciences, University of New Orleans - Lakefront, NewOrleans, Louisiana 70148, U.SA.

SUMMARY

When cultures of Euglena gradlis Z., normally grown in medium containing 180jUM-Ca2+, areresuspended in Ca2+-free medium cells assume round shapes within lOmin, from which theyrecover slowly when Caz+ is returned to the cultures. Cultures grown in 10/JM-Ca2+ do not displaythe typical circadian rhythm in cell shape even though the photosynthesis and cell division circadianrhythms are unaffected. Elevating intracellular Ca levels by the addition of the Ca2+ ionophoreA23187 prevents cells from undergoing the two daily shape changes characteristic of growth-synchronized cultures, but does not alter the ability to maintain the cell shapes found at the timeof ionophore addition. When the calmodulin inhibitors trifluoperazine or chlorpromazine are addedto cultures, the cells always respond by rounding. Cells are not able to maintain any cell shape otherthan spherical in the presence of these inhibitors and therefore cannot change shape throughout thedaily cycle as is found in the control populations.

INTRODUCTION

Euglena gradlis cells can display at least three types of shape changes. The shapechanges most often attributed to Euglena are the various swimming and rapid cell-rounding movements collectively referred to as metaboly (Arnott & Walne, 1966;Bovee, 1982). Cells may also respond to various types of physical or chemical treat-ments by a rapid, usually reversible rounding, commonly called the shock response(Bovee, 1982; Murray, 1981). The most recent shape-change pattern reported forEuglena is a slow (hours) shape transition in growth-synchronized populations(Lonergan, 1983). Cells in culture, synchronized by light/dark cycles, display twoshape changes per 24 h. Cells during the dark portion of the cycle are round and havean average cell length of 22 /im. Five hours into the light period the cells have elon-gated to lengths averaging 30 ^m. Throughout the last half of the light cycle, and theentire dark portion of the cycle, the cells progressively become rounder and shorteruntil the beginning of the next cycle. These shape changes are controlled by thebiological clock and involve oxidative phosphorylation, photosynthesis and presum-ably cytoskeletal proteins.

The molecular basis of the contractile apparatus has not been reported for any ofthe three shape changes. While the existence of calmodulin has been reported inEuglena (Klee, Crouch & Richman, 1980), actin microfilaments and myosin have not

38 T. A. Lonergan

been reported. Whether any of the shape changes in Euglena involve an actomyosinor spasmin-based contractile system remains to be elucidated. Because bothactomyosin and spasmin contractile systems are Ca2+-sensitive, the ability to altercalcium levels as a probe to perturb contractility could be an important tool. Thepredictability of the shape changes in the clock-controlled system previously reportedrepresents a time-scale amenable to study, whereas a system for the study of the rapidmetaboly shape changes has never been described.

This research was intended to begin an investigation of how extra- and intracellularCa2+ levels affect the biological clock-controlled shape changes. The roles of Ca2+ inregulating cellular functioning are diverse. Calcium is known to affect plasma mem-brane integrity and function (Carafoli & Crompton, 1978), mitochondrial ion trans-port and ATP production (Carafoli & Crompton, 1978), photosynthesis (Barr,Troxel & Crane, 1983) and cytoskeletal integrity (Dedman, Brinkley & Means, 1979;Tash et al. 1980), all of which are functions thought to be involved in regulatingEuglena cell shape (Lonergan, 1983).

One of the predominant approaches to the study of the clock-controlled shapechange is the exposure of cells to various chemical and physical treatments that alterthe physiology or morphology (e.g. the cytoskeleton) of the cells. It was suspectedafter preliminary results that some of the treatments, for example, altering externalCa2+ levels, altered cell shape by inducing a shock response, thus precluding ananalysis of the slower clock-controlled shape change. The finding that changes inCa levels can induce shape changes is significant for two reasons. First, in vivo celltreatments are possible; and second, numerous cytoskeletal and contractile proteinfunctions are influenced and regulated by Ca2+ levels. This paper represents a con-tinuation of the attempt to relate the three types of shape changes and to determinewhich treatments used to study clock-control of cell shape cause shock responses. Inaddition, a preliminary study of the effects of calmodulin antagonists on the shapechanges is reported.

MATERIALS AND METHODS

Cell culturesEuglena gracilis Z. was cultured as previously reported (Lonergan, 1983). Samples from liquid

stocks were added to 250 ml flasks containing 200 ml liquid medium. Cultures were exposed to a 10 hlight, 14 h dark cycle with a light intensity of 300 fiEinsteins m~2 s"1 while magnetically stirred andaerated with filter-sterilized room air at a rate of 0-5 1 min"1. Samples of the culture were removedby syringe and centrifuged in a clinical centrifuge for 30 s. The cell pellet was resuspended in a dropof 10% formalin (3%, v/v, final concentration) to fix the cells for photography. Cells werephotographed and measurements of cell length recorded as previously reported (Lonergan, 1983).

Statistical analysisTo determine whether there were significant differences in the cell length profile for two

different times of day or after the use of an inhibitor, the percentages of the population residing inthree size classes, 15-24, 25-34 and 35-44 /im in length, were determined for each population. Atest for statistical differences in these percentages was performed as previously reported (Lonergan,1983).

Cell shape in Euglena 39

Resuspension experimentsFor some experiments, cells were resuspended in a 10 JZM-Ca2+ medium prepared by quantitative

addition of a stock CaC^ solution. Normal Ca2+ concentration is 180 im. The concentrations ofCa2+ was not monitored during the experiment. Cells were pelleted by centrifugation atapproximately 500 #, washed twice in the new medium for 1 min and resuspended in 20 ml of themedium. The medium was aerated at a rate of 0-1 lrnin"', which also kept cells suspended. Cellswere removed from the medium with a syringe, fixed and processed for photography.

Use of inhibitorsThe Ca2+ ionophore A23187 was purchased from Sigma and dissolved in dimethylsulphoxide

(DMSO) before addition to the medium (0-1 %, v/v, final concentration DMSO). Concentrationsof DMSO as high as 1 % did not affect the cell-shape changes reported in this study (Lonergan,1983). Trifluoperazine and chlorpromazine were kindly supplied by Smith Kline and FrenchLaboratories and were dissolved in growth medium. Cell cultures were analysed for shape changesthe day before the addition of any inhibitor and used as controls. Inhibitors were added to 20 ml ofcells removed from the main culture at two different times of day. One group of cells was exposedto the appropriate inhibitor at ST 0900 (standard time, 9 am) representing dawn or 'lights on'. Cellswere removed from the culture, fixed and photographed at the time of inhibitor addition, ST 0900,2 h later at ST 1100 (standard time, 11 am), which represents a 2 h exposure to the inhibitor, andat ST 1400 (standard time, 2 pm), which represents a 5 h exposure. Untreated control cells were alsoexposed to inhibitors starting at ST 1400 (5 h into the light period), and analysed at ST 1700 (8 hinto the light period, 3h exposure to the inhibitor) and ST 2000 ( l h into the dark period, 6hexposure). Cell-shape profiles were constructed at each of these times to ascertain the response ofthe cell shape changes to the presence of the inhibitor.

Photosynthesis and cell density measurementsIn some instances the rate of photosynthetic oxygen evolution and cell number/ml culture were

determined, as previously reported (Lonergan, 1983), throughout the light/dark cycle.

RESULTS

Effect of lowering extracellular Ca2+ concentration on cell shape

The role of Ca2+ in the Euglena cell-shape rhythm was initially investigated byattempting to culture cells in Ca2+-free medium. The growth medium typically usedin these studies contained 180/4M-Ca2+. Cellswere centrifuged, washed twice inCa2+-free medium and then resuspended in Ca2+-free medium. Fig. 1A shows cellsgrown in 180fiM-Ca2+, while Fig. 1B shows a characteristic cell-shape profile. Atstandard time 1400 (ST 1400), which represents 5h into the light portion of thelight/dark cycle, 24% of the cells are 15-24/im in length, 52% are 25-34pm and24% are 35-44 ^m. Resuspension of cells into Ca2+-free medium for 10 min resultsin a rounding of cells (Fig. lc). Fig. ID shows that only 5 % of the cells are 35-44[imin length, while 47 % of the cells are 25-34/im and 15-24/zm. Further investigationrevealed that the centrifugation, washing and resuspension steps induce a statisticallysignificant rounding. Fig. 1E indicates that the gross morphology of the cells haschanged, while Fig. IF indicates that the cells have rounded in comparison to thecontrol population in Fig. 1B. The cells recover from the shape changes induced bywashing and centrifugation within 30 min (data not shown). These results indicate,

40 T. A. Lonergan

*

' * -

'--ft s

a- C>

u

^ K «

30H

2 20

<5 1 0 -

15 30 45I S 1 124% 52% 24%

30-

20-

10- 10

47% 47% 5% 36% 50% 14%

Cell length

Fig. I. The effect of resuspending cells in Ca2+-free medium, A. Photomicrograph of cellsremoved from a culture at ST 1400 (5 h into the light cycle). X275. B. Cell-shape profilefor a culture at ST 1400. The percentages represent the proportions of the populationfound in the size classes 15-24, 25-34, and 35-44 pm length, c. Photomicrograph of cellsresuspended in Ca2+-free growth medium for 10 min. D. Cell-shape profile for cells resus-pended in Ca2+-free medium. Cells were washed and centrifuged twice in Ca2+-freemedium before the final resuspension. E. Photomicrograph of cells washed and centrifugedtwice in normal growth medium and then resuspended in normal growth medium, F. Cell-shape profile of cells washed and resuspended in normal growth medium.

however, that the cell rounding observed with resuspension in Ca2+-free medium ispartially the result of the physical manipulations used in the resuspension.

The same rounding of cells occurs when morning cells, which are predominantlyround and approximately 15—20/im long, are resuspended briefly in Ca2+-free

Fig. 2. Cell-shape changes, photosynthetic capacity and cell number for cells grown in alow-Ca2+ medium. Cells were grown in Cramer & Myers (1952) medium (180/iM-Caz+)for 6 days. Cells were removed from this control culture five times during the light/darkcycle (ST 0900 or dawn, 1100, 1400, 1700 and 2000 or 30 min into the dark period) todetermine the cell-shape profile (top panel). The percentages represent the proportions ofthe population found the size classes 15-24, 25-34, 35—44/*m length. Cells were removedfrom this culture and resuspended in growth medium containing 10 /un-Ca2+ and culturedfor 9 additional days. The cell-shape profile for the culture during the eighth day of low-Ca2+ culturing is shown in the middle panel. Photosynthetic capacity, as measured by therate of oxygen evolution, and cell density were determined for days 7, 8 and 9 of low-Ca2+ culturing (bottom panel).

Cell shape in Euglena

l equnu lle3

Fig. 2

42 T. A. Lonergan

medium and then returned to normal medium (data not shown). The normal size-class distribution for a control at this time of day is 66% of the cells 15-24/im long,24% 25-34/zm long and 6% 35-44/im long. A lOmin Ca2+-free wash results in apopulation that has an average of 93 % of its cells 15—24 jtim long, 7 % 25—34 /im longand no cells in the longest size class. The population returns to its normal profilewithin 2h of its resuspension in normal growth medium.

Another treatment used to lower extracellular Ca2+ was the addition of 1 miw or10mM-EGTA (in 0 1 M - P I P E S , pH6-5) to the cultures. Whereas cells respond toresuspension in medium without Ca2+ by rounding within 5—10 min, cells exposed toEGTA round gradually over a 30 min period. After 30 min, however, the cell-shapeprofile for EGTA-treated cells is the same as that shown in Fig. ID for resuspensionin medium without Ca2+ (data not shown). Recovery from EGTA-induced roundingwas not studied.

The rounding induced by lowering Ca2+ levels was specific for Ca2+ removal.Similar experiments in which cells were resuspended in medium without Mg2*revealed only a resuspension shock similar to that shown in Fig. 1E,F, from which thecells recovered within 30 min (data not shown). No cell rounding corresponding tothat observed in the absence of Ca2+ was seen in the Mg2"1"-deficient medium.

The third approach used to study the effect of extracellular Ca2+ levels was toresuspend cells in a growth medium containing 10 ̂ M-Ca2+ as opposed to the normal,180/iM-Ca2+. Fig. 2, day 8 (second horizontal panel) shows the cell profiles for theeighth day of growth in 10 /ZM-Ca 2+. The rhythm in cell shape (Fig. 2, top horizontalpanel) does not persist during any of the 8 days of low-Ca2+ culturing and the profileis similar to the profile of normally cultured cells at dawn (Fig. 2, control 0900). Themajority of cells (62-77 % of the population) are 15-24 /zm in length throughout theentire day. The daily rhythm in cell division and photosynthetic capacity, however,are unaffected by the low-Ca2+ culture conditions (Fig. 2, bottom panel). The rateof oxygen evolution shows its typical peak in the mid-light period, while cell divisionoccurs during the first half of the dark period, resulting in a step-wise doubling of cellnumber every 24h, as has been previously reported (Lonergan, 1983). The persis-tence of the daily cell division and photosynthesis rhythms indicates; first, that thecells are physiologically competent in these culture conditions; and second, that therhythm in cell shape has an optimum Ca2+ concentration different from that of therhythms of cell division and photosynthesis. This culture condition could thereforebe used to disrupt cell shape without disrupting all physiological processes dependent

Fig. 3. The effect of the calcium ionophore A23187 on cell length. The top panelrepresents the cell-shape profiles for the culture the day before the experiment. Thefollowing day, two 20-ml samples were removed from the culture and placed in two culturetubes. Middle row: at ST 0900 (dawn), 40 J1M-A23187 (final concentration), dissolved inDMSO (0 - l% (v/v), final concentration) was added to one sample. Cell length wasfollowed in that population at ST 1100 (2 h exposure) and ST 1400 (5 h exposure). Bottomrow: at ST 1400, 40/OH-A23187 (final concentration) was added to the second sample andcell length was followed in that population at ST 1700 (3 h exposure) and ST 2000 (6hexposure). The percentages represent the proportions of the population found in the sizeclasses 15-24, 25-34 and 35-44^m length.

Cel

l le

ng

th (

pm

)

44 T. A. Lonergan

upon Ca2+. While the free intracellular Caz+ concentration lorEuglena has never beenreported, it would be expected to be 100-to 1000-fold lower than the 10/ZM-Ca2+ usedin the modified growth medium (Caswell, 1979).

Effect of raising intracellular Ca2+ levels on cell shape

The only method feasible for increasing intracellular Ca2+ levels in these culturesis the addition of Ca2+ ionophores to the medium. The Ca2+ ionophore A23187 wasadded to cultures at two different times in the cell-shape rhythm. The addition of theionophore would be expected to equilibrate the Caz+ levels in cellular compartmentswith that in the growth medium (180^M-Ca2+). When 40^M-A23187 is added toround cells in the morning (Fig. 3, middle row), the cells stay round and do notelongate throughout the morning and afternoon as expected (Fig. 3, control, top row).When added to cells in mid-afternoon (Fig. 3, bottom row) the cells stay long and donot round as expected throughout the last half of the light cycle (Fig. 3, top row).These results imply that high intracellular Ca2+ levels either disrupt some processinvolved in the cell-shape changes or uncouple the cell-shape changes from thebiological clock. The possibility that the ionophore influences the shape changes byinhibiting ATP synthesis, which is required for both shape changes, has been con-sidered (see Discussion).

Involvement of calmodulin in determining cell shape

The observation that changes in Ca2+ level disrupt cell shape and the findings ofother investigators that the calcium-binding protein calmodulin may be involved indetermining the integrity of the cytoskeletal components, suggested that calmodulinmight mediate the effects of Ca2+ reported here. Calmodulin has been reported inEuglena, and it is generally considered a ubiquitous regulatory protein in eucaryoticcells. The involvement of calmodulin can be ascertained by using calmodulin-inhibiting drugs such as trifluoperazine and chlorpromazine. The validity of con-clusions from such studies does rely on the specificity of these drugs in inhibitingcalmodulin. When either trifluoperazine or chlorpromazine is added to round cells inthe morning (Fig. 4, middle row), the cells round and assume cell lengths shorter thanusually seen at that time of day. The cell profile for a culture shortly before dawn isshown in Fig. 4 at ST 0900. Cells 15-24 jzm long constitute 60-70% of the cellpopulation for that experiment while 30 % of the cells are 24—35 jum long. Addition

Fig. 4. The effect of the calmodulin inhibitor trifluoperazine on cell length. The top panelrepresents the cell-shape profiles for the culture the day before the experiment. Thefollowing day, two 20-ml samples were removed from the culture and placed in two culturetubes. Middle row: at ST 0900 (dawn), lOOjUM-trifluoperazine dissolved in growthmedium was added to one sample. Cell length was followed in that population at ST 1100(2h exposure) and ST 1400 (5 h exposure). Bottom row: at ST 1400, 100^vi-trifluoperazine was added to the second sample and cell length was followed in thatpopulation at ST 1700 (3h exposure) and ST 2000 (6h exposure). The percentagesrepresent the proportions of the population found in the size classes 15-24, 25-34, and35-44nm length.

Cell shape in Euglena 45

o o

S 8 2

jeqixinu

Fig. 4

46 T. A. Lonergan

of the calmodulin inhibitors results in 99% of the population being in the 15-24/imlong size class and only 1 % of the population in the 24-35 fxm long size class after a2h exposure to the inhibitor (ST 1100). When the inhibitors are added to long cellsin the afternoon (Fig. 4, bottom row), the cells again become round and stay roundthroughout the afternoon. Thus, in the presence of these calmodulin inhibitors, cellsdo not maintain their shape and respond by changing to round cells about 15-25 jumin length.

DISCUSSION

Calcium is involved in the operation and/or regulation of many cellular processes.It is difficult to alter extra- or intracellular Ca2+ levels and specifically describe all ofthe physiological and biochemical processes affected. Calcium is undoubtedly invol-ved in determining the integrity and functioning of the various cytoskeletal proteinsthought to dictate Euglena cell shape and mediate cell-shape changes. The intentionof this research is to alter extra- and intracellular Ca2+ levels and determine how suchalterations could be used to study the role(s) of Ca2+ in affecting the cell-shape rhythmreported in Euglena (Lonergan, 1983).

Physical manipulations such as centrifugation and resuspension of Euglena cellsinduce a rapid cell rounding (Fig. 1E-F), from which the cells recover in approximate-ly 30 min. Thus, the manner in which cells are handled should be considered as apossible source of error resulting from experimental design. Several shock responseshave been reported for Euglena, including the effects of reducing cation levels inculture (Bovee, 1982), and a response to bright illumination during microscopicexamination (Murray, 1981). A shock response to ethanol has been reported byLonergan (1983) and a rounding response to glutaraldehyde has been observed butnot studied (Lachney & Lonergan, unpublished observations). Such shock responsespreclude the use of certain treatments to study the slower clock-controlled shapechanges because no mechanism to account for the magnitude of the shock responsein relation to the slower shape-change response has been demonstrated. It is alsosuspected that formaldehyde slightly alters the cell-shape profile during the fixation,although the effect is not as great as observed with glutaraldehyde. When the cell-shape profile for formaldehyde-fixed cells, taken from the middle of the light period,is compared to that of unfixed cells at the same time (Lachney & Lonergan,unpublished observations) the profiles are nearly identical. However, whenformaldehyde-fixed cells from the end of the dark period are compared to unfixed cellsthe profiles are different, with more round cells being observed among theformaldehyde-fixed cells. This difference in the cell's response to formaldehyde atdifferent times of day implies that there is a rhythmic component in the effectivenessor efficiency of fixation. The nature of this response has not been investigated, butrepresents an area of research that should be investigated in the future.

The maintenance of a particular cell shape is sensitive to the extracellular Ca2+

concentration. A sudden reduction in Ca2+ concentration causes cells to becomeround and decrease in length (Fig. 1C,D), regardless of the cell shape at the start of

Cell shape in Euglena 47

the treatment. It is not known whether the lowered extracellular Ca2+ level altersplasma membrane properties by changing levels of bound Ca2+, or if it causesintracellular Ca2+ to leak out because of an induced diffusion gradient. The low-Ca2+-induced rounding occurs at any time during the light/dark cycle and is reversible overa recovery period of several hours. A shock response resulting from a reduction inextracellular cations was reported by Bovee (1982). Murray (1981) also reported theeffects of altered Ca2+ concentrations on Euglena cell shape, but detergent-lysed cellswere used, so direct comparisons cannot be made. Batten & Anderson (1981) reportedthat cultured ovarian granulosa cells become round in response to deprivation of bothCa2+ and Mg2"1". Removal of Mg2"1" from Euglena cultures does not induce rounding.

Cell-shape changes induced by removal of Ca2+, and the recovery from such treat-ments, could be used as a way of studying the role of Ca2+ in determining cytoskeletalintegrity. Alterations in the free cytoplasmic concentration of Ca2+ may exert an affecton cell shape at the level of microtubule and/or microfilament polymerization. Forexample, when cells round during a Ca2+-induced shape change, if an alteration inmicrofilament orientation occurs concomitantly then the relationship betweenmicrofilaments and the various shape changes can be studied. When cells that wereinduced to round by removal of Ca recover from the rounding by the addition ofCa2+, normal cell shapes are recovered. Such a recovery is currently being used as atool to study the cytoskeletal components in Euglena without the use of chemicalinhibitors such as cytochalasin, which disrupts microfilament polymerization but alsohas secondary effects on cell metabolism. Virtually all cytoskeleton-related proteinsare influenced in some manner by Ca2+, making studies of its role imperative in astudy of the mechanism of shape changes.

The shape-change rhythm can be dissociated from biological clock control byculturing cells in 10^M-Ca2+ instead of the usual 180jiM-Ca2+ While 10/iM-Ca2"1"would be estimated to be 100- to 1000-fold higher than the free intracellular Ca2+

concentration (Caswell, 1979), the cells remain round as long as they are exposed to10/iM-Ca2+. The low-Ca2+ culturing does not disrupt photosynthesis, cell division,or biological clock control over these two processes (Fig. 2). The low-Ca2+ culturingmust either arrest the cell-shape changes or specifically uncouple the shape changesfrom control by the biological clock. It has been reported by Kempner & Miller (1972)that cell division rates are unaffected by culturing Euglena cells in a Ca2+ concentra-tion as low as 0 - 7^M. Cell culturing in low-Ca2+ medium could be used as a milddisrupting agent that stops cell shape changes but allows other aspects of physiologyto occur unaffected, a situation rarely, if ever, to be achieved by chemical treatmentof cells (Lonergan, 1983).

When chemicals blocking oxidative phosphorylation are added to synchronizedEuglena cultures, the cells remain in the same population cell-shape profile as thatfound at the time of addition of the chemical (Lonergan, 1983). Thus, when oxidativephosphorylation is blocked, cells maintain their shape indefinitely and cannot changetheir shape. The addition of the Ca2+ ionophore A23187 gives the same pattern ofresults (Fig. 3). When A23187 is added to cultures presumably the extra- andintracellular Ca2+ concentrations are equilibrated, thus significantly raising the

48 T. A. Lonergan

intracellular Ca2+ concentrations. The results presented in Fig. 3 could be interpretedin two ways. First, it is possible that high intracellular Caz+ levels allow cells tomaintain their shape but disrupt some step(s) involved in changing shape. However,it has been reported that A23187 disrupts oxidative phosphorylation (Reed & Lardy,1972). If such a disruption of ATP production occurs in Euglena in the same con-centration range of this ionophore, then the blockage of cell-shape changes by A23187cannot be attributed unambiguously to the effects of a higher intracellular Ca2+

concentration on cytoskeleton-related proteins. The clock-controlled shape changesare dependent on ATP from oxidative phosphorylation (Lonergan, 1983). Whethercell-shape changes induced by the presence of A23187 result from Caz+-related per-turbations of cytoskeletal proteins will only be revealed by techniques like indirectimmunofluorescence, by which these protein complexes can be visualized. For exam-ple, it has been found that microtubule integrity is unaffected in vivo by the presenceof A23187 and is therefore not sensitive to elevated Caz+ levels (Lachney & Lonergan,unpublished results). Investigators using other systems have examined the effects ofaltering Ca2+ levels in the presence of A23187. Volvox cells exposed to A23187 in thepresence of DMSO undergo contraction (Viamontes, Fochtman & Kirk, 1979). Bat-ten & Anderson (1981) reported that cultured ovarian granulosa cells also round in thepresence of A23187 (dissolved in DMSO), with the retraction of filopodia and theeventual detachment of the cells from the culture dish. Murray (1981) exposed Eug-lena cells to A23187 and observed cell rounding, but the cells had also been exposedto detergent and therefore did not represent a comparable in vivo system. The lackof cell rounding in the presence of A23187 in the rhythmic Euglena cell-shape systemrepresents a deviation from other reported systems. However, no other system has yetbeen reported to have cell shape controlled by the biological clock and this mayrepresent a major difference in how Ca2+ control of cell shape is co-ordinated. Itwould be of interest to lower the intracellular Ca2+ level and observe the effect(s) oncell shape. To perform this type of experiment in vivo it would be necessary to reducethe Ca2+ concentration in the growth medium to a value below that of free intracellularCa2+ and then lower the intracellular Ca2+ concentration by introducing an ionophorethat equilibrates extra- and intracellular Ca2+. This would be complicated becauseexposure of cells to 10/iM-Ca2+, which is probably a concentration at least 100-foldgreater than free intracellular Ca2+, itself induces rounding (Fig. 2).

Whether calmodulin is involved in determining cell shape is also a complex ques-tion. Calmodulin could be involved in many systems affecting cell shape in additionto its suspected role in regulating the polymerization/depolymerization ofmicrotubules and microfilaments (Dedmanet al. 1979; Tashe* al. 1980). It is gener-ally believed that a change in free Ca2+ levels will activate or inactivate calmodulin,which then exerts its regulatory role, e.g. a Ca2+-dependent stimulation ofmicrotubule depolymerization. The involvement of calmodulin was implicated by theuse of calmodulin inhibitors. The validity of such a study relies completely on thespecificity of the inhibitor for calmodulin. It has been shown in spinach chloroplaststhat the calmodulin inhibitor trifluoperazine inhibits photosystem II activity (Barr,Troxel & Crane, 1982). This does not necessarily mean that calmodulin is involved

Cell shape in Euglena 49

in regulating photosynthesis, as has been suggested by some investigators (Burris &Black, 1983). It could be interpreted that trifluoperazine and related inhibitors bindto chloroplastic components other than calmodulin, i.e. they may have secondaryeffects. Such considerations are important in this system because the round-to-longshape change requires the operation of the photosynthetic light reactions (Lonergan,1983), while the long-to-round transition does not. The response of the cells totrifluoperazine or chlorpromazine is very similar to that seen with lowered extra-cellular Ca2+ — a rounding of cells regardless of the cell shape at the time of additionof inhibitor. Cell rounding when calmodulin inhibitors are added is slower than theCa2+-induced rounding, taking about an hour to complete. For this reason the res-ponse to these inhibitors is not considered to be a shock response, which is charac-terized as a rounding that occurs within minutes of the chemical or physical perturba-tion. A major difference, however, is the extent of the cell rounding. While Ca2+

deprivation causes cell rounding (Fig. 1C,D), the cell rounding in response totrifluoperazine is much greater, with virtually 100% of the cells having cell lengthsunder 25 jUM (Fig. 4).

The molecular basis of contractility in Euglena has not been resolved for any of thethree types of shape changes. The existence of contractile proteins that interact withthe pellicle/microtubule complex has been proposed by several investigators (Lefort-Tran, Bre, Ranck & Pouphile, 1980; Hofmann & Bouck, 1976) as an explanation forthe fibrils associated with the pellicle complex and viewed by electron microscopy.The chemical nature of the fibrils has not been determined, but the sodium dodecyl-sulphate/polyacrylamide gel analysis of pellicle proteins by Hofmann & Bouck (1976)indicates the presence of proteins corresponding in molecular weight to actin andmyosin. Current investigations in this laboratory using indirect immunofluorescencedirected against cytoskeletal components have examined the role of microtubules aswell as the role of microfilaments. The observation that a change in Ca2+ concentra-tion can induce a shape change from which the cells recover is being used as a majorprobe of the sites of Ca2+ regulation in the contractile process.

REFERENCES

ARNOTT, H. J. & WALNE, P. L. (1966). Metaboly in Euglena granulata. J. Phycol. 2 (suppl.): 4a(abstract).

BARR, R., TROXEL, K. S. & CRANE, F. L. (1982). Calmodulin antagonists inhibit electron trans-port in photosystem II of spinach chloroplasts. Biochem. biophys.Res. Commun. 104, 1182-1188.

BARR, R., TROXEL, K. S. & CRANE, F. L. (1983). A calcium-selective site in photosystem II ofspinach chloroplasts. PL Physiol. 73, 309-315.

BATTEN, B. E. & ANDERSON, E. (1981). Effects of Ca+2 and Mg+2 deprivation on cell shape incultured ovarian granulosa cells. Am. jf. Anat. 161, 101-114.

BOVEE, E. C. (1982). Movement and locomotion mEuglena. In The Biology of Euglena, vol. 3 (ed.D. E. Buetow), pp. 143-168. New York: Academic Press.

BURRIS, J. E. & BLACK, C. C. JR (1983). Inhibition of coral and algal photosynthesis by Ca2+-antagonist phenothiazine drugs. PI. Physiol. 71, 712-715.

CARAFOLI, E. & CROMPTON, M. (1978). The regulation of intracellular calcium. In Current Topicsin Membranes and Transport, vol. 10 (ed. F. Bronner&A. Kleinzeller), pp. 151-216. New York:Academic Press.

50 T. A. Lonergan

CASWELL, A. H. (1979). Methods of measuring intracellular calcium. Int. Rev. Cytol. 56, 145-181.CRAMER, M. & MYERS, J. (1952). Growth and photosynthetic characteristics of Euglena gracilis.

Arch. Mikrobiol. 17, 384-402.DEDMAN, J. R., BRINKLEY, B. R. & MEANS, A. R. (1979). Regulation of microfilaments and

microtubules by calcium and cyclic AMP. Adv. cyclic Nucleotide Res. 11, 131-173.HOFMANN, C. & BOUCK, G. B. (1976). Immunological and structural evidence for patterned

intussusceptive surface growth in a unicellular organism. A postulated role for submembranousproteins and microtubules. J. Cell Biol. 69, 693-715.

KEMPNER, E. S. & MILLER, J. H. (1972). The molecular biology of Euglena gracilis. VII. Inorgan-ic requirements for a minimal culture medium. J. Protozool. 19, 343-346.

KLEE, C. B., CROUCH, T. H. & RICHMAN, P. G. (1980). Calmodulin. A. Rev. Biochem. 49,489-515.

LEFORT-TRAN, M., BRE, M. H., RANCK, J. L. & POUPHILE, M. (1980). Euglena plasma mem-brane during normal and vitamin B12 starvation gTowth._7. Cell Sri. 41, 245-261.

LONERGAN, T. A. (1983). Regulation of cell shape in Euglena gracilis. I. Involvement of thebiological clock, respiration, photosynthesis, and cytoskeleton. PI. Physiol. 71, 719-730.

MURRAY, J. M. (1981). Control of cell shape by calcium in the Euglenophyceae. J. Cell Sri. 49,99-117.

REED, P. W. & LARDY, M. A. (1972). A23187: A divalent cation ionophore. J. biol. Chem. 247,6970-6977.

TASH, J. S., MEANS, A. R., BRINKLEY, B. R., DEDMAN, J. R. & Cox, S. M. (1980). Cyclic

nucleotide and calcium regulation of microtubule initiation and elongation. In Microtubules andMicrotubule Inhibitors (ed. M. DeBrabander& J. DeMay), pp. 269-279. Amsterdam: Elsevier/North-Holland Biomedical Press.

VIAMONTES, G. I., FOCHTMAN, L. J. & KIRK, D. L. (1979). Morphogensis in Volvox: Analysisof critical variables. Cell 17, 537-550.

(Received 20 February 1984-Accepted 18 April 1984)