adenosinetriphosphate, calcium and temperature...

J. Cell Sci. 3a, 67-86 (1978) 67Printed in Great Britain © Company of Biologists Limited 19J8

ADENOSINETRIPHOSPHATE, CALCIUM AND

TEMPERATURE REQUIREMENTS FOR THE

FINAL STEPS OF EXOCYTOSIS IN

PARAMECIUM CELLS

H. MATT, M. BILINSKI AND H. PLATTNER*Department of Pharmacology, University of Innsbruck,Peter-Mayr-Str. 1, ^-6020 Innsbruck, Austria

SUMMARYIn Paramecium cells a synchronized discharge of trichocysts (which involves only the final

exocytosis steps of membrane fusion, content discharge and membrane resealing) was achievedwith ATPase-blockers, Ca1+-ionophores, lipid solvents (including lysolecithin), polyethylene-glycol, anaesthetics (Dibucain) and cationic detergents (cetyltrimethylammonium bromide(CTMAB) and cetylpyridinium chloride (CPC)). Only Dibucain - and to some extentcationic detergents - can trigger exocytosis independently of extracellular Ca1+, possibly bymobilizing intracellular Caa+. The internal free [Ca*+] necessary for exocytosis can be estimatedto be > 10"* to io~* M. Membrane-free trichocyst contents were isolated by density gradientcentrifugation; they are converted from the contracted to the expanded state by Dibucain,CTMAB and CPC, and also by exogenous ATPase (Apyrase). Thus, it is possible to de-couplethe discharge (stretching) process from membrane-related phenomena. Since only the latterare inhibited by low temperature (o °C), membrane lipids probably have to be in a fluid statefor exocytosis to occur. At least 2 steps appear to be involved: when membrane fusion isinitiated, an independent matrix-bound system is activated for the synchronized stretchingprocess. The energy requirement for one discharge event is estimated to be about 14 x 10*ATP molecules.

INTRODUCTION

The following morphological details render Paramecium trichocysts very appro-priate material for the study of the final steps in exocytosis. (1) The secretory vesiclemembrane is permanently positioned as closely as ~ i 5 n m to the cell membrane(Plattner, Miller & Bachmann, 1973; Plattner, Wolfram, Bachmann & Wachter, 1975).(2) The multiplicity of these permanent attachment sites allows for an amplificationeffect during synchronous triggering. (3) The discharge involves stretching of thesecretory contents (the trichocyst) which can be conveniently followed in the lightmicroscope. When exocytosis is triggered this involves only the final exocytosis steps,i.e. membrane fusion, discharge of the contents and membrane resealing.

Trichocysts contain within their limiting membrane a highly ordered matrix(Bannister, 1972) composed mainly of one major protein (Steers, Beisson & Marchesi,1969) in a paracrystalline arrangement (Hausmann, Stockem & Wohlfarth-Botter-mann, 1972 a, b). Trichocyst discharge takes place explosively, probably within less

• Address for correspondence: Dr H. Plattner, at the above address.

68 H. Matt, M. Bilinski and H. Plattner

than a millisecond (Plattner, Bilinski & Vb"llenklee, unpublished results), accompaniedby about 7-fold stretching (Hausmann et al. 1972 a) of the contents. One can, there-fore, study parameters relevant for the stretching process, even with isolated,membrane-free trichocysts, and, thus, achieve a de-coupling of membrane fusion fromstretching (extrusion) phenomena.

As in other systems (Palade, 1975) the cell membrane and the membrane of the'extrusome' fuse during exocytosis (Plattner et al. 1973). Specialized membranestructures were recognized at the fusion sites in freeze-fracture studies (Bachmann,Schmitt & Plattner, 1972; Plattner et al. 1973). Experiments with ionophoretic Ca2+

injections (Plattner, 1974, 1976) not only revealed ultrastructural intramembraneouschanges during exocytosis; they also suggested that Ca2+-mediated 'stimulus-secretion-coupling', a phenomenon known from other secretory cell types (Douglas,1974; Rubin, 1974), occurs at the final exocytosis steps. These experiments wereaccompanied in Paramedum cells by formation of reaction products at some sites ofthe membranes involved and on some structural elements of the trichocyst contents(Plattner & Fuchs, 1975); as electron-microscopic X-ray microanalyses revealed thepresence of Ca and P in these reaction products, this pointed to the presence ofphosphate-splitting enzymes at well-defined sites. In more elaborate cytochemicalanalyses the occurrence of a Ca2+-dependent ATPase at the potential membranefusion sites was ascertained (Plattner, Reichel & Matt, 1977).

Information on energy- and temperature-requirements for exocytosis is scarce(cf. Meldolesi, Borgese, DeCamilli & Ceccarelli, 1978). These aspects, in conjunctionwith Ca2+-requirements, were analysed in the present system specifically for the finalsteps of exocytosis.

MATERIALS AND METHODS

Cell material

Paramecium tetrattrelia (formerly P. aurelia; Sonneborn, 1975), strain K+oi, was cultivatedat 26 °C strictly monoxenically with Enterobacter agglomerans added in a dried lettuce mediumand harvested in early stationary phase. Cultures were filtered through cheese cloth; with theuse of a sieve-plate filter (30-40 fim) bacteria were removed and cells concentrated to ~ io6

cells/ml immediately before use.

Chemicals

A23187 (Eli Lilly). Apyrase (Sigma). Adenosinetriphosphate (ATP, Tris salt; Sigma).Cetylpyridinium chloride (CPC; Merck). Cetyltrimethylammonium bromide (CTMAB;Merck), p-chloromercuribenzoate (Sigma). D600 (methoxyverapamil; Knoll). Dibucain(Nupercain; Ciba). EDTA (ethylenediamine tetraacetate; Merck). EGTA (ethyleneglycol-Wj[/?-aminoethyl ether]iV,iV'-tetraacetate; Sigma). TV-ethylmaleimide (NEM; Sigma). Fireflylantern extract (type FLE-50; Sigma). La8+ (chloride; Merck). Lettuce medium (Difco).Lysolecithin (L-a-lysophosphatidylcholine, type I; Sigma). Mersalylic acid (Sigma). Poly-ethyleneglycol (PEG, mol. wt. ~6ooo; Koch-Light). Salyrgan (Farbwerke H6chst). Valino-mycin (Calbiochem; Serva). X-537A (Hoffmann-LaRoche). All chemicals were of the highestpurity available.

ATP, Cai+ and temperature requirements for exocytosis 69

Measurement of cation concentrations

K, Na, Ca and Mg concentrations in the culture medium were determined with a Beckmanatomic absorption photometer, model 1248. For Ca or Mg, 'masking' with 1 % La,Os in1 % HNO, (final concentrations) was applied. K, Na were also determined by flame photo-metry and Ca, Mg by colorimetric methods. The concentrations of K, Na, Ca, Mg were1-70, 0-40, 0-16 and 0-14 rrtM, respectively, in the cell suspensions used.

ISOLATION OF TRICHOCYSTS AND PELLICLESCULTURES: Paramecium tetraurelia monoxenicin Difco medium, early stationary phase.Homogenization medium = HM: 2o mM Tris/malaatepH70, + 0-1 M KCI, 1 mM EDTA, 1 mM ATP.Centrifuges: Heraeus Christ Minifuge (20'C)for<6000, swing-out-rotor.

culture5 ml cells+ 5 ml HM

10 min x1200 rev/mi n

\

concentrate 250 mlculture (30—40 /imsieve plate) to2x105 cells/5 ml

bacteriacontractedtrichocystssmallpelliclefragmentspellicles

discard

V.^'A.- If-

discardsupernat.

Teflon pestlehomogenizer15x200 rev/min

I 15 x

y—*«

1

>K — 5 min x

3000 rev/min

\resuspendpellet in3 ml HM

\

3 h x1500rev/min I1

3 mlhomogenate+ 3 ml HM

discard supernat.

-repeat 1 x - ^—

resuspend pellet in 1 ml HM

discontinuous! 1 ml 1-2M, 2 ml 1 8 Msucrose grad.' sucrose and 3 ml 2-1 M cushion

Fig. 1. Cell fractionation schedule for the isolation of contracted, membrane-freetrichocysts and trichocyst-free pellicles. Cells are homogenized in a medium (HM)containing 20 mM Tris/maleate pH 7-0 with o-i M KCI, 1 mM EDTA and 1 mMATP added. See also Anderer & Hausmann (1977). The gradient is made up withdifferent sucrose concentrations (1 ml 1-2 M, 2 ml i-8 M and 3 ml 2-1 M) in homo-genization medium. Centrifugation is performed at 20 CC in a Heraeus Christ Minifugewith a swing-out rotor.

Cell fractionation

Trichocysts were isolated according to Fig. 1. This method was developed on the basis ofdata presented by Hoffmann-Berling (1961) and Anderer & Hausmann (1977). Homogenizationwas performed with a loosely (~o-25-mm clearance) fitting Teflon pestle. On a I - 2 / I - 8 Msucrose interface and in the presence of 1 mM EDTA and 1 mM ATP we obtained puretrichocysts which were in a contracted state and devoid of a membrane envelope.

7o H. Matt, M. Bilinski and H. Plattner

Light-microscopic studies on exocytosis triggering

Triggering was monitored with a Reichert light microscope, model Diavar. io-^tl dropletscontaining ~so cells were analysed and the time required to trigger exocytosis in all cells wasregistered. For long time periods samples were stored in a moist chamber. The microscope wasequipped with a Nomarski-type optical system and a temperature-controlled object holder,which can be heated or cooled; the actual specimen temperature was measured with a thermo-couple. Potential trigger compounds were tested and their concentration adjusted so thatcomplete triggering was achieved just within a ~3-min period (20 °C, pH ~7 , free [Ca1+]0~o-i HIM). For experiments at o °C cell suspensions were rapidly cooled by shooting themin a fine jet, using 0-5 atm. (50-5 kNm"1) pressure, through a ~iso-//m wide pipette on toan object holder at o °C, since the usual cooling procedures were too slow and provokedmassive trichocyst discharge. Spraying did not damage the cells. When re-warmed( ~ 20 °C/min) after 3 min at o °C they regained their full motility and the response to thetrigger compounds at 20 °C was almost fully re-established. This allowed us to analysetemperature effects on fully viable cells at 20 and o °C.

Isolated contracted trichocysts (contents) with 1 mM EDTA and 1 mM ATP added werealso exposed to trigger compounds at 20 and o °C. Apyrase (E.C. 3.6.1.5.), Sigma type I frompotatoes, was applied at 0-33 % pH 67 + 3-3 mM Ca1+, following the procedure of Molnar &Lorand (1961).

Fig. 2. Calibration curve for ATP determination by liquid scintillation counting.Superposed is a set of data pairs obtained from untriggered and Dibucain-triggeredcells.

ATP assaysIn principle the method of Strehler & Totter (1952) was followed.Preparation of samples. The effect of every substance added to viable cells was checked by

light microscopy. For each triggering procedure we prepared at least 4-10 pairs of triggeredand untriggered aliquots. The simultaneous, pairwise comparison of stimulated and non-

ATP, Caz+ and temperature requirements for exocytosis 71

stimulated cells under directly comparable conditions (e.g. same time schedule, same chemicalsin varying sequence, same ionic milieu and pH, etc.) was crucial to circumvent artifact hazards.Residual bacteria accounted for less than 1 % of the total ATP measured.

Firefly lantern extracts. 50 mg extract were dissolved in 5 ml o-2 M Tris/HCl pH 7-4. After12-18 h at 20 °C the extract was centrifuged and filtered (Millipore 0-2 fim). 0-2 ml, containing20 mM Mg8+, were added to each 2-ml sample. The final [Mg*+] in the assay was invariably5-8 mM, i.e. within a range we found for maximal activation.

Calibration curves. They were obtained from Tris-ATP solutions in 0-2 M Tris/HCl,pH 7-4, with a final [Mg1+] of 5-8 mM. For each type of experiment we ascertained that anycompound added did not affect the calibration. See Fig. 2.

Scintillation counting was performed with a Packard Tricarb liquid scintillation spectrometer,model 3375, using the 8H-canal, 50% coincidence and a detector temperature of 8 °C; 20 8after addition of firefly lantern extract, samples were counted for o-i min. The timing was preciseenough to minimize any effect of impulse rate decay. The final pH value of the samples, deter-mined immediately after counting, was 7-50 ± 0-02. This lies within the optimal pH range whichwas between 7-2-7-8 under our experimental conditions.

Determination of free phosphate (Pi)

As a control for ATP-hydrolysis measurements by scintillation counting we assayed alsothe corresponding P,-increase according to Taussky & Shorr (1953).

Protein measurementThe method of Lowry, Rosebrough, Farr & Randall (1951) was used in the Eppendorf

manual modification.

Respiration measurements

The oxygen pressure (pOJ was measured at 25 °C with a Gilson oxygraph, model K.I.C.,equipped with a 2-ml chamber and a Clark electrode.

Electron microscopy

Cells and isolated cell fractions were analysed in a conventional electron microscope. Afterfixation in 2% glutardialdehyde and washing (o-i M cacodylate buffer pH 7-0), isolated cellfractions were either spread on coated support grids (with or without 1 % aqueous uranylacetate) or processed by routine methods (1 % OsOa; acetone dehydration; Durcupan ACM-Fluka-embedding) to ultrathin sections, which were contrasted for 20 min with 7-5 % aqueousunbuffered magnesium uranyl acetate and for 3 min with lead citrate, pH 12-0.

Freeze-fracturing was carried out routinely as indicated previously (Plattner et al. 1973),i.e. without any pretreatment, and with the use of a Balzers BAF 300 unit at —100 °C. Somepellets were cooled at ~ 20 °C/min till frozen (to provoke massive trichocyst discharge) andthen plunged into liquid nitrogen.

From estimates of the cell volume and surface (by light microscopy), of the possible numberof trichocysts (from freeze-fracture data) and of the trichocyst volume (from electron micro-graphs of longitudinally cut organelles) we obtained a rough estimate of the relative volumefraction of trichocysts in an average cell (Table 2).

RESULTS

Experiments in vivo

Various chemical agents listed in Table 1 trigger the exocytosis of trichocysts veryefficiently: (1) ATPase-inhibitors (p-chloromercuribenzoate, mersalylic acid, La3"1",iV-ethylmaleimide, Salyrgan), (2) the Ca2+-antagonist D600, (3) Ca2+-transportingionophores (X-537A, .£123187), (4) lipolytic agents (lysolecithin, acetone), (5) the

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ATP, Caz+ and temperature requirements for exocytosis 73

fusogenic compound polyethyleneglycol, (6) the local anaesthetic Dibucain (Figs. 3, 4)and (7) the cationic detergents cetyltrimethylammonium bromide (CTMAB) andcetylpyridinium chloride (CPC). With most compounds the triggering effect dependson the availability of extracellular Ca2+ (Cajj+), at least in the concentrations presentin the medium (see below); exceptions are La3+ and Dibucain, while D600, CTMABand CPC gave variable results. With X-537A and A23187 [Ca2+]o has to be enhancedto accelerate exocytosis. With all compounds used trichocyst discharge is precededby a strong ciliary reversal reaction. Only with La3+ is ciliary reversal rare and allciliary movement is, in contrast to other agents, rapidly abolished.

None of these compounds is effective at o °C (Table 1), even though cells fullyrespond to these agents after re-warming to 20 °C, first by ciliary reversal and thenby trichocyst exocytosis.

Table 2. Biophysical and biochemical parameters of Parameciumtetraurelia cells

Cell vol. i zx io- 'mlNo. of trichocysts per cell 4000Relative vol. of trichocysts 8 %Protein/cell n-o x io~9 gProtein fraction of a cell 9-2 %ATP concentration 125 mM ( = 7 x io~*g/g protein

= 063 x 10-3 g/ml)ATP content/cell 15 x io~M molATP loss during triggering ~ 60 %

m , , , . , ,. , 15 x io"1* x 6022 x io° x 60ATP hydrolysed per one tnchocyst discharge =

100x4000

= 14 x 10* ATP molecules/event.

To check for a specific Ca2+ effect on exocytosis triggering we administered inanother approach different ionophore-cation combinations (Fig. 5). Ionophore experi-ments were designed to reverse the high [K+]r and low [Ca2+]rvalues which deter-mine the normal surface potential in Paramecium cells (Naitoh & Eckert, 1974).Values of [K+, Na+, Ca2+, Mg2+]0 which are constantly around 1-70, 0-40, 0-16 and0-14 mM in the culture medium, were correspondingly diluted 2 or 3 times in theseexperiments unless added in excess. K+ is unable to exert a trigger effect, even whenvalinomycin is incorporated and [K+]o then raised to levels exceeding those assumedfor [K+]i (~20 mM; Naithoh & Eckert, 1974), provided Cao2"1" is chelated by EDTAor EGTA. Both X-537A and A23187 are about 7-8 times more effective with exo-genous Ca2+ than with Mg2+.

Temperature effects on trichocyst discharge were studied in different ways. Veryrapid cooling to o °C, performed as indicated in Methods, abolishes the reaction toany trigger compound (Table 1) and, concomittantly, does not entail spontaneoustrichocyst discharge. However, slow cooling (~ 20 to 30 °C/min) always results inmassive exocytosis as a temperature of about 12-10 °C is reached (Fig. 6). Warming~8o °C/min) results in a similar reaction. Within a temperature range (26-12 °C

H. Matt, M. Bilinski and H. Plattner

Figs. 3, 4. Nomarski interference differential contrast micrographs from Parameciumcells. X75.

Fig. 3. Normal cells.Fig. 4. Cells massively triggered with Dibucain. Abundant expelled trichocysts

are present.

addition ofions, 10 mM

ionophore I time required forincubation (25*C) triggering at 20"C

1COin

00

C O

CDCO '

c

Ca++

Mg++

K+

Ca ++

Mg+ +

K+

Ca+ +

Mg++

K+

1 mM EDTA

1mM EDTA + 50 mMK+1little trigger

cells immobilized H 10 min «no trigger

§ • !

Fig. 5. Effect of different ionophores in the presence of 10 mM Ca*+, Mg1+ or K+ on thetriggering of trichocyst exocytosis. Hatched zones indicate the variation range of thetime required for a complete effect. The data indicate a specific effect of Ca1+ forexocytosis.

tested) where spontaneous trichocyst expulsion is rare, exocytosis mediated byionophoretic Ca2+ injection is temperature dependent (Fig. 6).

When exocytosis is triggered by slowly cooling the cells to o °C and the pelletsare then freeze-fractured, they contain abundant free, i.e. discharged, stretchedtrichocysts. Those membrane-intercalated particles which are normally randomlydistributed in the cell membrane ('h-type' particles; see Plattner et al. 1975), becomemostly clustered by lateral segregation as in Tetrahymena membranes (Speth &Wunderlich, 1973). The regular double 'rings' of cell membrane particles which

ATP, Ca2+ and temperature requirements for exocytosis 75

surround every trichocyst attachment site (Plattner et al. 1973), persist; they becomeonly slightly displaced so that small groups of particles no longer lie within an idealring (Fig. 7). 'Central granule patches' ('fusion rosettes') become rare or absent.Nevertheless, one never sees an open exocytosis canal within these exocytosis sites.These, evidently, undergo resealing, even when the temperature is progressivelyreduced.

When the ATP content of cells is measured after massive synchronous trichocystexocytosis, the ATP-content is significantly reduced in comparison with untriggeredcontrols (Figs. 8, 9). This holds true for all the different triggering procedures, exceptfor polyethyleneglycol, notwithstanding its strong triggering capacity. Values of

2 0 -

u 10-

0 J 10 min 2 min

B•time

Fig. 6. Effect of temperature on the X-537A + Ca1+-mediated exocytosis of trichocysts.As the temperature is decreased the time required to achieve complete exocytosis in-creases (hatched zones indicating experimental variations). At temperatures around12-10 °C and below, massive spontaneous exocytosis takes place, i.e. withoutchemicals added. A, ionophore X-537A incorporation; B, temperature adaptation;C, trigger phase.

controls prepared in different ways are not significantly different from each other,regardless of whether trichocyst discharge is inhibited by the addition of Mg2+ orwhether cells are inactivated with trichloroacetic acid. The ATP loss obtained withdifferent types of trigger agents was slightly different; this could be due to slightlydifferent amounts of trichocysts expelled; so far, there are unfortunately no means fora more precise quantitation of trichocyst exocytosis and we always aimed at a maximaltrigger effect under light-microscopical control.

As a control for the involvement of ATPase-activity during chemically (Dibucain)induced exocytosis we measured whether there occurs a concomitant increase offree phosphate (Pt). In all experiments Pj increased in parallel with the hydrolysis ofATP.

76 H. Matt, M. BiUnski and H. Plattver

At 22 °C a Paramecium cell consumes o-2i x io"12 mol Oa per min. Massivesynchronous trichocyst expulsion was not paralleled by enhanced respiration (Fig. 16)under any experimental conditions used. Nevertheless, even within a triggeringperiod of io s cells could compensate for some of the ATP consumed during exo-cytosis. Therefore, the ATP-consumption determined after triggering might be anunderestimate. pOt measurements also indicate that cells were fully viable during thetrigger phase.

Fig. 7. Freeze-cleaved Paramecium cell membrane (cytoplasmic face) containing atrichocyst attachment site, which is surrounded by a double ' ring '-structure of mem-brane-intercalated particles. This material was slowly frozen (~ 20 °C/min) whichwas accompanied by massive trichocyst discharge. Neither 'fusion rosette' particlesnor an exocytosis canal are present (indicating membrane resealing). Some of thering particles are laterally displaced. Shadowing from bottom to top. x 170000.

Experiments with isolated trichocysts

Trichocysts were separated from pellicles in a sucrose gradient, supplemented withEDTA and ATP (Fig. 1). Pure trichocysts (Fig. 10) are obtained which are devoidof the membrane and of the thin 'outer lamellar sheath' (Bannister, 1972); theycontain the 'inner lamellar sheath' and the whole matrix in a contracted state (Figs. 11,12). Isolation of trichocysts without addition of ATP resulted in a variable percentageof stretched trichocysts (Bilinski & Plattner, unpublished observations).

As shown in Table 1 and Figs. 13-15, isolated trichocysts can be stretched withDibucain, CTMAB and CPC. Stretching is also achieved with exogenous ATPase(Apyrase). Trichocysts respond to all these agents at o °C as well as at 20 °C.


-58-6

T1-58-2

T1-61 8 - 3 5-6

l+l

NEM D-600 X-537A-^Ca++ Acetone PEG Dibucain CTMAB

Fig. 8. Change of ATP-concentration after massive triggering of trichocyst exocytosisby various compounds at 20 °C. Values shown within columns are percentages.NEM, JV'-ethyl-maleimide; PEG, polyethyleneglycol; CTMAB, cetyltrimethyl-ammonium bromide. The ATP-values of triggered cells are normalized to 100 %(full bar — 1-25 mM = 15x10"" mol/cell; see Table 2). To judge experimentalvariations see data obtained in parallel experimental series with CTMAB and data setsfrom Fig. 2. The error probability (a) was determined by a parameter-free one-sidedWilcoxon test (N = no. of samples): NEM (a <| o-i %, N = 20); D600 (a < o-i %,N = 20); X-S37A + Cas+ (a < o-i %, A = 14); acetone (a ^ o-i %, N = 8); PEG(a = o-i%, N = 20); Dibucain (a < o i % , JV = 20); CTMAB (a < o-i%, N = 20).Bars indicate standard deviations.

DISCUSSION

Paramecium cells contain a free internal Ca2+ concentration ([Ca2+]i) of < io~7 M(Naitoh & Eckert, 1974), i.e. significantly lower than [Ca2+]o ( I ^ X I O ^ M ) . Iono-phoretic Ca2+ injections indicated that an increase of free [Ca2+]i stimulates exocytosisin this system (Plattner, 1974; Plattner & Fuchs, 1975). These observations were nowextended to different triggering procedures which - except for a few - depend uponthe availability of Cao2+ (Table 1). With all triggering procedures used a ciliary re-versal reaction, which is initiated by a [Ca2+]i of ~ icr"8 M (Naitoh & Kaneko, 1972)precedes exocytosis. Free [Ca2+]i necessary for the final exocytosis steps must, there-fore, be in the range of io"6 to IO~*M. Interestingly, fusion of isolated secretorygranules needs just about io~B M Ca2+ (Dahl & Gratzl, 1976).

The surface potential of paramecia is determined by a low [Ca2+]i and a high[K+]i of ~2omM (Naitoh & Eckert, 1974). When the K+-selective ionophorevalinomycin (Pressman, 1976) is incorporated and [K+]o raised to > [K+]t (afterchelation of Ca<,2+) cells become immobilized, but no exocytosis takes place (Fig. 5).Thus, reversal of the K+-gradient, which participates in the surface potential forma-tion, does not suffice to initiate exocytosis. This is in line with the experience ofelectrophysiologists (Ogura, personal communication). Among the other ionophoresused, only A23187 is rather selective for bivalent cations, while X-537A transports

6 CEL 3a


also K+ and other monovalent cations (Pressman, 1976). Both promote trichocystdischarge considerably more with increased [Ca2+]0 than with Mg2+ or K+ added.These experiments underscore the fact that a [Ca2+]i-increase is the crucial event forthe final exocytosis steps.

-60'C

20"C

o-c -835

•

-73

20'C-

Fig. 9. Change of ATP content (full bar ^ 100 % ^ 15 x 10-" mol/cell; see Table 2)when trichocyst exocytosis is produced by slow cooling or heating of the cell suspen-sions; for cation-concentrations in the medium see Methods. Values are percentagesand bars indicate standard deviations. The error probability is a <Z c i % in both cases(N = 8); see Fig. 8.

A variety of ATPase inhibitors trigger exocytosis (Table 1), but only when freeCao2+ is present. This means that the Ca2+ influx over the cell surface would no longerbe compensated by the Ca2+-pumping system(s) of the cell surface. As to be expected,cells are not triggered by other inhibitors which attack only selective types of ATPases(100/ig/ml oligomycin; 5 min ouabain), which inhibit nucleotide transport (5 mMatractyloside or carboxyatractyloside) or the hydrolysis of monophosphates beyondpH 7 (5 mM L( + )tartrate or L-tetramisole) (Plattner, unpublished results).

D600 acts as a potent inhibitor to potential-dependent Ca2+-influx (Fleckenstein,Nakayama, Fleckenstein-Grun & Byon, 1975) and thus stops the secretory activityin a variety of cells (cf. Carafoli, Clementi, Drabikowski & Margreth, 1975). Itstriggering effect on paramecia was, therefore, unexpected. However, at the relativelyhigh concentrations used (575 fiM), this compound acts rather like a local anaestheticand releases bound Ca2+ (Dorrscheidt-Kafer, 1977). In fact, the local anaestheticDibucain also strongly triggers trichocyst (Table 1) or mucocyst discharge (Tetra-


hymena: Satir, 1977), possibly by liberating membrane-bound Ca2+ (Feinstein, 1964;Nicolson, Smith & Poste, 1976). This could explain why triggering takes place alsoin the absence of free Cao2+. It is uncertain whether the cationic detergents usedwould exert a similar effect when incorporated into membranes. La3+ also triggersregardless of the [Ca2+]0. La3+ inhibits various Ca2+ pumps; as it seems to penetratecertain cells (Batra, 1973) it could also augment free [Ca2+]i by depressing intracellularCa2+-segregating systems.

10Fig. 10. Survey of a grid mesh covered by trichocysts isolated in the contracted state

by density gradient centrifugation according to Fig. 1. x 1300.

The fusogenic lipid lysolecithin (Lucy, 1974) also induces exocytosis, but commonlipid solvents, like acetone (Table 1), ethanol, diethylether, chloroform, etc., do aswell. As their effect always depends upon Cao2+ they might trigger not simply byperturbation of the membrane lipids but also by a simultaneous Ca2+-influx. Con-comitantly, according to Ahkong, Fisher, Tampion & Lucy (1975), cell fusion in-duced by various lipid-soluble fusogenic agents also depends upon Ca0

2+. It isextensively documented that Ca2+ influx from outside stimulates exocytosis in varioussystems (Douglas, 1974; Rubin, 1974). It remains open, to what extent intracellular

6-a

l l H. Matt, M. BiHnski and H. Plattner

11

Figs, i i , 12. Trichocysts isolated by density gradient centrifugation; ultrathinsection. The membrane is missing, x 55000.

Fig. 11. Note the absence of the 'outer lamellar sheath' and the presence of the'inner lamellar sheath' (arrows) (c.f. Bannister, 1972). The whole matrix is in acontracted state.

Fig. 12. Cross-section through the trichocyst body. Some fuzzy material (arrow)covers part of the trichocyst matrix.

ATP, Caz+ and temperature requirement-s for exocytosh


Ca2+ stores, like 'calcium-storing vacuoles' (Plattner & Fuchs, 1975) or mitochondria,would contribute to a [Ca2+]i-increase for trichocyst exocytosis under physiologicalconditions.

1 min

Fig. 16. Oxygraphic measurements of O, consumption before and during massive tri-chocyst exocytosis mediated by addition of Ca1+ (at arrow) to cells previously incu-bated with X-537A. There is no change of the />O2-slope during massive exocytosis.A, ionophore X-537A incorporation; B, trigger phase; C, cell death. In these experi-ments no attempts were made to wash out the trigger compounds for allowing the cellsto survive.

As Table 1 shows, none of the trigger compounds becomes effective when cells arevery rapidly chilled to o °C. This holds true also for those agents, like Dibucain,which transform isolated trichocyst contents from the contracted to the expandedstate even at o °C. We assume, therefore, that the temperature sensitivity of theexocytosis process in vivo is due to a membrane effect. At o °C not only Ca2+-pumpingsystems (Browning & Nelson, 1976) but also Ca2+ channels for Ca2+ entry appear tobe blocked in paramecia.

When slowly cooled to ~ 12 to 10 °C or below, paramecia display considerabletrichocyst exocytosis. Could an increase of free [Ca2+]i again be involved? One couldargue that solidification of the membrane lipids (their phase transition point could bearound 12-10 °C but was not determined precisely) could entail a deactivation of themembrane-bound Ca2+-ATPase system (Seelig & Hasselbach, 1971) which thenwould fail to maintain the normal, low [Ca2+]i. We found morphological evidence byfreeze-cleaving that in these experiments membranes are (or again become tempo-rarily) sufficiently fluid for undergoing subsequent resealing. Above the criticaltemperature region, Ca2+-ionophore-mediated exocytosis depends directly from thetemperature. We presume that this pattern of temperature-dependence is analogous


to the triphasic temperature response of Ca2+-dependent miniature endplatepotentials in neuromuscular junctions (Duncan & Statham, 1977).

Too little is known about the biochemistry of membranes involved in exocytosisin paramecia to make it worth while to try to project into our system all the detailedknowledge on membrane fusion which has been obtained by analysing well-definedsynthetic systems. All our evidence underscores the necessity for lipids to be in afluid state, as in cell fusion experiments (Ahkong et al. 1973; Papahadjopoulos,Poste & Schaeffer, 1973; VanDerBosch, Schudt & Pette, 1973).

At what steps is energy needed when a trichocyst is discharged from a Parameciumcell? In their well-known review article Poste & Allison (1973) indicate reasons whylocally bound ATP would have to be hydrolysed at the membrane contact sites. IfCa2+ for exocytosis would possibly be mobilized from internal pools such mechanismscould also require energy. One also has to envisage actin- or actomyosin-like materialsaround some secretory granules (Palade, 1975) and around trichocysts (Bannister,1972). Similar material is visible on the ~i5-nm-wide contact zone between thecell membrane and a trichocyst (Plattner et al. 1973, 1975, 1977), which could act asa ' mechanocomplex' postulated by Poste & Allison (1973). Similar structures wererecently recognized to 'tie' secretory granules to the cell membrane in differentsystems. The actin- or actomyosin-nature of such structures has not yet been provedin Paramecium cells. In other systems actin and myosin bind to secretory granulemembranes in vitro (Burridge & Phillips, 1975) and display Ca2+-ATPase activity(Clarke & Spudich, 1977). It is unknown whether such structures are candidates forenergy transduction during exocytosis of trichocysts, but they could indeed facilitatein different ways the attraction of membranes to be fused. The trichocyst tip isflanked by a tightly fitting microtubular collar (Bannister, 1972; Plattner et al. 1973).In pituitary cells 'dynein-like crossbridges' connect the secretory granules withmicrotubules (Sherline, Lee & Jacobs, 1977). Although it is unclear on the one handwhether they display ATPase activity (cf. Sherline et al. 1977) and on the other handwhether such connecting structures exist on Paramecium trichocysts, it seems note-worthy, that we found calcium phosphate deposits in these regions, which neatlyreflected the arrangement of microtubules (Plattner & Fuchs, 1975).

The stretching of isolated trichocyst contents upon addition of Apyrase (Table 1)indicates that stretching possibly involves the hydrolysis of matrix-bound ATP. Thiscould be expected from earlier work (Hoffmann-Berling, 1961) and also explains, whycontracted trichocyst contents can be isolated only with ATP added (Anderer &Hausmann, 1977). We had previously obtained some indications by X-ray micro-analysis for a sudden Ca2+ influx into trichocysts under trigger conditions and for theoccurrence of phosphate-splitting sites within trichocysts (Plattner & Fuchs, 1975).To confirm this assumption spectrophotometric ATPase assays will have to be done.A note on ATPase activity of trichocyst contents was given by Hoffmann-Berling(1961). The impressive ultrastructural changes of matrix proteins upon discharge(Hausmann et al. 1972 a, b; Bannister, 1972) indicate that conformational changes actas a driving force for their expulsion. Since trichocysts can be stretched in theabsence of a boundary membrane, this is a strong argument against the osmotic

84 H. Matt, M. Bilinski and H. Planner

gradient theory (which presumes membrane-integrated particle aggregates at exo-cytosis sites to act as gap-junction analogues) proposed by Satir (1974), and in favourof our previous evidence against this theory which was obtained from tracer analyses(Plattner et al. 1975).

Trichocysts stretch explosively during discharge, i.e. within ~o-jms accordingto high-speed microkinematographic analyses (Plattner, Bilinski & Vollenklee, un-published results). Given the large extent of random coil organization in adrenergicvesicles (Smith & Winkler, 1967; Sharp & Richards, 1977) and the presumableabsence of expandable proteins in cholinergic transmitter vesicles (cf. Meldolesi et al.in the press) there does not appear to exist a common feature for extrusion. How-ever, exocytosis in mast cells (Douglas, 1974) and in nerve terminals (Heuser, 1977) isperhaps as rapid and vigorous.

In Paramecium cells exocytosis stimulation by all kinds of triggers strongly reducesthe ATP content of cells. For the only exception observed, polyethyleneglycol, wehave no explanation so far. As indicated in the Results section, the ATP-loss deter-mined after triggering might be somewhat underestimated. Evidently the wholeexocytotic process needs energy. In most systems some energy is invested into re-synthesis of secretory materials during triggering and for transcellular transport(Palade, 1975; Meldolesi et al. in the press). Data recently obtained for lacrymalgland cells by Herzog, Sies & Miller (1976) on the increase of O2 consumption duringexocytosis stimulation allow for some rough calculations. The basal O2 consumptionper unit time and cell volume (37 CC) is about twice as high as in Paramecium cells(22 °C). In contrast, the energy needed per one discharge event in paramecia appearsto be only a very small percentage of that which one can estimate for the lacrymalgland cells. Therefore, we believe that the energy required for the final exocytosissteps would be much smaller than that consumed during the preceding steps ofsecretory activity.

We thank Professor H. Benger and Dr F. Tiefenbrunner for making available to us somelaboratory equipment and several firms for gifts of chemicals (Eli Lilly: A23187; Hoffrnann-LaRoche: X-537A; Knoll AG: D600). Part of this work is from a PhD thesis by M. B. Thiswork was supported by the Osterreichische Fonds zur Fdrderung der wissenschaftlichenForschung.

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(Received 14 October 1977)

adenosinetriphosphate, calcium and temperature...

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