Pinocytosis and locomotion of amoebae

European Journal of

Europ.J.Protistol. 23,317-326 (1988)

PROTISTOLOGY

Pinocytosis and Locomotion of Amoebae XVII. Influence of Different Cations on Induced Pinocytosis in Amoeba proteus Wilhelm Stockem and Hans Peter Klein Institute of Cytology, University of Bonn, Federal Republic of Germany

SUMMARY The influence of various monovalent (Na", K+, Li"), divalent (Ca2+, S~+, Mn2+), trivalent (La3+, In3+, Ta3+ ), and polyvalent cations (egg albumin) on the pinocytotic activity of starved Amoeba proteus was studied by a modified channel-counting method. Accordingly, a distinct stimulationof pinocytosis is induced by all substances in the following order of intensity: Mn2+ < Ca2+ < S~+ < Li" < K+ < Na" < Ta3+ < In3+ < La3+ < eggalbumin. Equimolarmixtures of different cations exhibit a simple additive influence on the induction capacity with the exception of Ca2+ and Mn2+ which both inhibit pinocytosis of other inorganicor organic ions at mM-concentrations (1-150 mM); on the other hand, experiments using!LM-concentrations (10-0.001 um) delivered no perceptible effect of low externalCa2+ levels on inducedpinocytosis. Independent of the nature or substantial composition of the induction solution pinocytotic activityis also suppressed in a linearway beyond total molarities of 150-200 mM and completely restrained at concentrations of 600 mM. Specific ionophors (A 23187, valinomycin) and inhibitors (D-600, isoptinhydrochloride, amilorid, tetraethylamonium chloride) of ionic transport increase and decrease the rate of Ca2+ - and, to a lower extend, Na+- or K+-induced pinocytosis, respectively. In this connection, the general significance of endogeneous and exogeneous calcium for the control of membrane flow and actomyosin contraction is discussed.

Introduction Intense adsorption of external cations at the mucous layer of the plasma membrane of large amebas causes the rapid loss of cellular polarity and motile activity in conjunction with the formation of numerous pinocytotic channels in the entire cell periphery [11,29]. According to recent results, induced pinocytosis is not really significant for food-ingestion but rather serves as a mechanism to regenerate the affected cell surface and to survive strong external contamination as a particular menace to free living organisms [27]. Nevertheless, cation-induced pinocytosis has often been used as a model system to study the physiological significance of different chemical and physical parameters for the general course of endocytosis [12, 21,30,33]. Reliable indications exist according to which free as well as bound external and internal calcium ions have a crucial function for signal transmission and motive force generation during induced pinocytosis by changing the permeability of the plasma membrane and activating © 1988 by Gustav Fischer Verlag, Stuttg art

the contractile microfilarnent system, respectively [21,25, 27,31,36]. On this account, the present investigation was carried out with the aim to analyze the influence of experimentally caused alterations in the environmental and cytoplasmic Ca2+-concentration and to test the mutual effects of calcium with other ions. Material and Methods

a) Material Amoeba proteus (strain Princeton) was cultured in Chalkleymedium [17] and starved prior to induction of pinocytosis for 2 days. The different substances and conditions used for external application are listed in Table 1. All experiments were carried out at 22°C by using a special chamberwhichallowedthe easy and rapid exchange of solutions during microscopical observation with a differential interference contrast (DIC) microscope [17] .

0932-47 3918810023-0317$3.5010

318 . W. Stockem and H. P. Klein

b) Methods

Table 1. List of substances Substance

solvent

salts: NaCI KCl LiCI MnCl z

aqua bidest aqua bidest aqua bidest aqua bidest aqua bidest aqua bidest aqua bidest aqua bidest aqua bidest

s-o,

CaClz LaCl] InCl]

rso,

proteins: egg albumin

concentration pH 1-600 mM 1-600 mM 1-600 mM 1-600 mM 1-600 mM 1-600 mM 1-600 mM 1-600 mM 1-600 mM

10 mM acetate 0.5% = buffer 132mM

buffers: imidazol buffer Tris buffer Ca2+-chelator: EGTA sugar: sucrose drugs: colchicin vinblastin ionophors: A 23187 valinomycin antagonists: isoptin hydrochloride D 600-hydrochloride amiloride tetraethylammonium chloride control

6.4-6.8 6.4-6.8 6.4-6.8 6.4-6.8 6.4-6.8 5.8-6.4 6.4-6.8 6.4-6.8 6.4-6.8

Comparable amebas of the same culture exhibit a variable capacity for endocytosis ranging from completely inactive to highly active cells. Hence, in thepresent investigation only specimens showing distinct induced pinocytosis were taken into consideration. Themean number of pinocytotic channels was evaluatedaccording to a modified method originally applied byChapman-Andresen [11] andJosefsson [21]. Since allsubstances differ from one another in the duration of induced pinocytosis, channels were merely counted during the phase of optimum activity (Table 2). The mean channel number was then determined in a group of twenty amebas during five inspections at regular distances, e.g. during Ca2+-induced pinocytosis at distances of 20 seconds and K+-induced pinocytosis of 1 minute, respectively (compare Table 2).

4.5

aqua bidest aqua bidest

10-300 mM 7.0 10-300 mM 7.0

aqua bidest

2.5- 10 mM 7.2

aqua bidest

1-400 mM 6.8

aqua bidest aqua bidest

0.1- 1 mM 6.4-6.8 0.1- 1 mM 6.4-6.8

DMSO-aqua bidest aqua bidest

0.1 mg/ml

0.1- 1 mM 5.8-6.8

aqua bidest

0.1- 10 mM 5.8-6.8

aqua bidest

0.1- 10 mM 5.8-6.8

aqua bidest aqua bidest

0.1- 10 mM 5.8-6.8 0.1- 10 mM 5.8-6.8

Results

1. Morphology

aqua bidest

5.8-6.8

6.8

Induced pinocytosis represents a highly dynamicprocess mainly due to the contractile activity of a membranebound microfilament layer (Fig. 1, a-r). During the initial phase a broad surface invagination developswhich is then constricted in the apical region by a ringlike contraction of the actomyosin system (Fig. 1, b-e). Thereafter, the invagination elongates by the gradual extension of a hyaline pseudopodium (f-n) until a large endosome arises by disruption (Fig. 1, o-r). The topographical distribution and number of pinocytotic channels as indications for the intensity of endocytosis distinctly differ according to the chemical nature of the inducing substances. Whereas rnono-, tri- and polyvalent cations cause channel formation at the entire cell surface, intensified pinocytotic activity induced by divalent cations is mainly restricted to the uroid region. Corresponding differences exist with respect to the total duration of pinocytosis (Table 2) which largely corresponds to the inducing capacity of the tested substances.

2. Rate of pinocytosis

Table 2. Totalduration and phase of optimum induced pinocytosis Substance (concentration 150 mM) NaCI KCl LiCI CaClz MnCl z SrClz TaCl z InCl]

=

Totalduration of pinocytosis in min

Phase ofoptimum pinocytosis in min afterinduction

25-30 20-25 20-25 5- 6 8-13 12-17 15-20 15-20

5-12 5-10 5-10 3- 4 4- 8

5- 9 5-10 5-10

LaC!]

20-25

5-12

egg albumin Tris-buffer

25-30 25-30

5-13 5-14

In a first series of experiments the intensity of induced pinocytosis was measured (Fig. 2) by applying a modified channel-counting method (see material and methods). Highest rates with a maximum of about 60 channels during the optimum phase of activity (Table 2) were evaluated for polyvalent cationic egg albumin (Fig. 2 f, curve 1) and for trivalent cations in the succession La3+ > In3+ > Ta3+ (Fig. 2 b; curve for Ta3+ is not shown). A slightly decreased inducing capacity was determined for monovalent cations in the order Na + > K+ > Li" (Fig. 2 a), whereas a distinctly lower number of channels with values between 20 and 35 is induced by the divalent cations Sr+ > Ca2+ > Mn2+ (Fig.2c). Equimolar mixtures of monovalent, divalent and trivalent cations result in curves representing the respective statistical average of pinocytotic activity (Fig. 2 d) thus, demonstrating a simple additive effect of the inducing cations and a rather low specifity of the anionic receptor sites on the cell surface. In this connection it seems of

Influence of Different Cations on Induced Pinocytosis . 319

Fig. 1 a-r. Sequence of pictures (a-r) taken from A. proteus at a DIC-microscope (time interval between single pictures = 15-20 s) after induction of pinocytosis with 0.5% egg albumin. Note the distinct decrease in the diameter of the apical channel region (arrowheads in b-e) and the disruption phase during endosome formation (arrow in pl. Bar = 10 urn,

320 . W. Stockem and H. P. Klein

2a 60

NaCI Ch.No.

l\, Ii,'

50 40 30

I

20

I

---

KCI

b

LaCl3 InCI 3 _

60

LiCI ------ 50 40 30

I

20

10

10 50

150

250

350

C

550 mM

450

CaCI

2

250

350

550

mM

---

ea2++Mn2++Sr2+ ------

- - 60

_ - ___ 50

40

450

K+ +Na+ +L1+

Ch.No.

MnCI2 ~O

150

d

SrC I2

60 Ch.No.

50

La3++ln 3+

40

30

30

20

20

10

10 50

e 60 Ch.No.

150

250

350

450

mM

50

f

50mMNaCI ·SUCROSE

60

50

50

40

40

30

30

20

20

10

10 350

I J

mM

150

250

550 mM

Ch.No. -"">::-

~f~

O.~ Ea+

'~

r'

---

KCI

------

CaCI2

'-~J

\-

'\ \~I

~

"~I

50

150

250

350

450

mM

Fig.2 a-d. Intensity of pinocytois in A. proteus after induction with monovalent (a),trivalent (b), divalent (c),and equimolar mixtures of different equivalent cations (d). - e. Intensity of pinocytosis after induction with 50 mM NaCI as a function of increasing sucrose concentration. The sucrose was added to the NaCl-solution in doses of 10, 50, 100, 150 etc. to 550 mM. - f. Intensity of pinocytosis after induction with 0,5% egg albumin as a function of increasinginorganiccation-concentration.The different cations were added to the egg albumin solution in doses of 1, 10, 150, 300, 400 and 500 mM. Curve 1 indicates channel number (Ch. No) of pure egg albumin solution as a control. interest that all curves exhibit an optimum with a more or less linear increase of intensity between 0 mM and 150, and a corresponding decrease between 200 mM and 600 mM (Figs. 2 a-d, 3 d, 4 a-c, 5 a-c). The application of a 50 mM NaCI solution containing different concentrations of the non-inducing substance sucrose (Fig. 2 e) clearly reveals that suppression of pinocytotic intensity is

not due to a toxic influence of the different cationic inducers but caused by a rather simple osmotic effect.

3. Influence of high external calcium concentrations In a second series of experiments it was demonstrated that mM-concentrations (1-150 mM) of external Ca2+ as

Influence of Different Cations on Induced Pinocytosis . 321

3a 60

b

{>omM MnCI,

h.No.

60

~\

40

Jr \r,

30

,

1"

100mM NaCI

50

---

100mM CaCI

- -. 40

'~

30

20

20

10

10

150

50

250

C 60

350

LaCI 3+ 100mM NaCI

50

i

40

):-1" I

30

\,

__ _ _ __

,

"L. "1- ":::

r

150

mM

d

NaCI Ch .No.

---- - -

2

I mM COI·NaCI 50

---

1mM VIN ·NaCI - -

-- - .

40

' 1<,

30

"I ,

10

'" -,

150

li ,1-- r;.

50

60

100mM CaCI 2 -

20

50

mM

450

r~~CI'

Ch.Na .

J

MnCI2+ 100mM NaCI

CaCI2· 100mM LaCI3 50

rmM LaCI

Ch.No.

250

350

20 ~

.. '1

450

10

mM

50

150

250

350

450

mM

Fig. 3 a--\:. Intensity of pinocytosis in A. proteus after induction with different cationic salt solutions as a function of increasing concentrations of externalCa2+ (a), Mn2+ (b) and La3+ (c). CaCI2, MnCI2 and LaCI 3 wereadded to the 100 mM salt solutionsin doses of 1, 10,50, 100, 150,200,300 and 400 mM. - d. Intensity of pinocytosis after induction with NaCI to demonstrate the influence of 1 mM colchicin (COL) and 1 mM vinblastin (YIN).

well as Mn H (at least under laboratory conditions) have a unique function in the control of pinocytotic activity as comp ared with other cations (Figs. 2£, 3,4). When CaH , K+ and Na + are added in doses of increasing molarity to a 0.5% egg album in solution, only clacium causes a distinct inhibition of album in-induced pinocytosis in the physiological range, i.e., up to values in the tot al molarity of 150-200 mM. Corresponding results were obtained by addin g different concentrations of Ca2 + to 100 mM solutions of Mn H , La3+ and Na + (Fig. 3 a): whereas Ca H exerts no influence on MnH -induced pinocytosis, the pinocytot ic capacity of La3 + and Na+ is clearly suppressed. A similar effect is elicited by Mn 2+, thus pointing to an identical chemical efficacy of the two cations (Fig. 3 b). In contras t, the addition of La3+ at increasing concentrations to 100 mM solutions of Mn H , Nat and Ca H has no effect on the course of pinocytotic activity as induced by these cations (Fig. 3 c).

4. Influence of low external calcium concentrations In a third series of experiments the question was studied whether ltM-concentrations (10-0.001 ltM) of external

calcium have an influence on the induction of pinocytosis and which other ions may be involved in signal transmission for the initiation of this process (Fig. 4). When pinocytosis is induced by Ca2+ in the presence of the ionophor A 2318 7 the channel number is increased by more than 30% (Fig. 4 a), whereas control experiments show no effect of the ionophor on Sr'"-pinocytosis (Fig. 4 c). On the other hand, the Ca2 + -antagonisrs D 600 and isortin hydrochloride cause a significant suppression of Ca + -induced pino cytosis by more than 50% (Fig. 4 b). The significance of ltM-concentrations of external CaH on pinocytosis after indu ction with 150 mM NaCl was tested by emplo ying thre e different EGTA-buffers containing NaOH (Fig. 4 d), imidazol (Fig. 4 e) and Tris (Fig. 4 f). In all experiments a significant suppression or stimulation of Na +-pinocytosis by f.l.M-CaH -concentrations was not observed (Fig. 4 d-e, curves 1 and 2). However , ·differences exist when the EGTA-buffer is applied simultaneously with (curve 1) or prior to the 150 mM NaCl solution (curve 2). A preincubation with the EGTA-buffer always reduces the pinocytotic intensity to a level characteristic for the respective concentrations of NaOH, imidazol and Tris lacking EGTA (column 3). Followingly, all experi-

322 . W. Stockem and H. P. Klein

4a 60

d CaCI2

h.No.

A23187+CaC1 2

50 40

---

r

'r

"r r"

/

~

t

i

i1

}-

I

1

1

r2

10 - 5

10-

10-

10- 8 10-9 Mc.eet+J

f

I

f~l

1

I

I

30 ~

20 10

10 150

250

mM

b 60

50 40

/I-,

30 20

60 Ch.No.

e

CaCI2

h.No.

60 Ch.No.

D600+CaC 12 _ _ _

50

50

ISQ+CaC 12 ______ 40

40

30

30

20

20

10

f"' -:~ =-=l= =-!--""-~~,

10

/,

~'

I-

.::..;

~.

150

250

SrCI2

10- 8 10-

3D M[Ca H ]

6D Ch.No.

A23187+SrC/ _ _ _ 2

50

-12

f

C 60

10- 5 10- 6 10-7

mM

350

3~

~;

50

40

40

30

30

20

20

10

10

3

50

150

250

350

mM

10·

Fig. 4 a--e. Intensity of pinocytosis in A. proteus after induction with CaCh(a, b) andSrCh(c) to demonstrate the influence of theCaionophor A 23187 (a, c) and the Ca-antagonists D 600 and isoptin hydrochloride (ISO; b). - d-f. Intensity of pinocytosis after induction with 150 mMNaCl in EGTA-NaOH (d), EGTA-imidazol (e) and EGTA-Tris buffer (f) to demonstrate the influence oflow external Ca++-concentrations. Curve 1: simultaneous application of NaCI and EGTA buffer; curve 2: preincubation in EGTA buffer (5 min) and subsequent induction with NaCl; curve 3: induction capacity of NaOH, imidazol and Tris buffer without EGTA.

ments shown in Fig. 4 demonstrate that low external Ca2+ levelshave no essential influence on induced pinocytosis of

A. proteus. Corresponding experiments in which the influence of external Na" and K+ on the intensity of pinocytosis was

investigated deliver evidence for a similar significance of these monovalent cations as compared to Ca2 + (Fig. 5). The ionophor valinomycin increases K+-induced pinocytosis up to 50% (Fig. 5 a) but the antagonist tetraethylammonium chloride exhibits no significant suppression of

Influence of Different Cations on Induced Pinocytosis . 323

5a

VAL·KCI 50

-r/

40

b

KCI

60 Ch.No.

NaCI

60 Ch.No.

---

/1.. . . . .

AMI·NaCI 50

---

40

/

30 I

30

t

20

20

10

10 50

250

350

C

450 NaCI

60

150

mM

250

d

TEA·KCI 50

50 40

40

30

30

20

20

10

10 50

150

250

3

450

mM

KCI

h.No.

60

350

---

II

f /

50

150

250

350

450 mM

Fig. 5 a-d. Intensity of pinocytosis in A. proteus after induction with KCl (a, d) and NaCI (b, c) to demonstrate the influence of the ionophor valinomycin (VAL; a, c) and the antagonists amiloride (AMI; b) and tetraethylammonium chloride (TEA; d).

channel formation (Fig. 5 d). These observations are in good agreement with results on the influence of the same or respective substances on Na +-induced pinocytosis: although valinomycin again increases the intensity of Na +pinocytosis up to 35% (Fig. 5 c), a clear inhibiting effect of the antagonist amiloride was likewise not evaluated (Fig.

5 b). 5. Inhibition of induced pinocytosis by drugs In a last series of experiments, different drugs were tested that are known to interfere specifically with microtubule s (colchicin, vinblastin) or microfilaments (cytochalasin B). The evaluation of these results revealed no influence of colchicin and vinblastin or cytochalasin B (not shown) on Na+-pinocytosis even when applied at concentrations of 1 mM (Fig. 3 d). Since microfilaments are essentially involved in motive force generation for induced pinocytosis, negative results, at least with cytochalasin B, may be explained by a rather high impermeability of the plasma membrane of Amoeba proteus for this drug.

Discussion Notwithstanding the large number of quantitative investigations on the intensity and physiology of induced pinocytosis in Amoeba proteus [11-13, 21, 33] there exist some controversial results with respect to the inducing capacity of certain cations and the regulatory function of calcium. In opposition to other authors [14, 21] the present study revealed a slightly divergent efficiency in particular for monovalent inorganic salts with a higher capacity of Na + in comparison to K+ as inducer. Likewise, the proposal that Ca2+ and Mn2+ do not possess a pinocytosis inducing effect [23] or that IJ.M-concentrations of external Ca2+ stimulate induced pinocytosis [19] could not be confirmed by the results of this paper. Several reasons may explain these disagreements: (a) amebas from the same culture show a large heterogeneity by differing in the amount of externally bound calcium [27], the value of actual membrane potential [7] and the ability to exhibit induced pinocytosis [11]; (b) the errors expected in quantitative measurements by comparing different channel-

324 . W. Stockem and H. P. Klein

6A.Normal conditions

B. Pinocytosis conditions I I

1*

I

:OC*lPO I

--------~~~~

11*

inducer

OOOOO~

II

.

- I

I I

BS

III

-I@M Mf

ER

-

~: Mi

. \

.:1 .

cs:

I I

I

I

• =;(0CJ0):

I

I

-------

111*

I

C. Restitution

BS: Ca: binding sites

00000: polycations E£Xt>: Ca. Mn (£):

Na, K

CV: contractile vacuole Ca"

MIttie

~:

Ml: mucous layer

ER: endopl. reticulum Mf: microfilaments Mi: mitochondrium

Fig. 6. Schematic drawing to summarize the different events coupled with the induction of pinocytosis and restitution of normal conditions in A. proteus (for explanation see discussion).

counting methods are likely to be in the range of 20% [11]; c) many buffers used in various studies for the application of inorganic cations differ in the material composition (e.g. EGTA or EDTA) so that variable mutual effects of single substances (e.g. calcium and magnesium) on the intensity of pinocytosis cannot be excluded [26]. However, in spite of the mentioned dissimilarities most values of the present investigation are in good agreement with the data of other laboratories when considered from a rather general point of view. Accordingly, the efficiency of equivalent cations to induce pinocytosis largely depends on the hydrated size of the ions in a contrary sense [12], whereas (with the exception of divalent cations) the induction capability of heterovalent ions directly follows the order of their net positive charges. This mainly corresponds to observations of Josefsson et al. [23] according to which the ability of different cations to reduce the negative membrane potential by binding to the mucous layer strictly complies with the capacity to induce pinocytosis. Experiments by preparing equimolar mixtures of equivalent cations reveal a simple additive effect of the tested inducers, thus pointing to a rather low specifity of the external receptors and binding sites. Nevertheless, induced

pinocytosis is not an "all-or-none" response to, above all, positively charged substances but controlled in a distinctly graded manner by different external and internal parameters [13]. Most effective are changes in the molarity of inducing solutes with a linear increasing number of channels up to 150-200 mM and a clear inhibition of pinocytosis beyond this concentrations. Chapman-Andresen [10] explained this inhibitory effect by a specific toxicity of the applied ionorganic salts. This is, however, in contrast to the present results on A. proteus, because all inducing substances show the same characteristic biphasic response of pinocytosis intensity with a minimum at 8 and 600 mM, respectively. Experiments using the non-inducer sucrose rather point to an unspecific osmotic effect so that declarations about the intensity of pinocytosis and the regulative function of physiological parameters such as external calcium are merely inconclusive at molarities exceeding 200mM. Besides osmolarity, there is clear evidence that high external calcium levels play a central role in controlling the initial step of induced pinocytosis [8, 18, 21, 28, 30, 31]. Concentrations in the mM range (1-150 mM) result in fast binding of Ca 2 + at the neoinositol-linked anionic

Influence of Different Cations on Induced Pinocytosis . 325 phosphate groups [1], thus maintaining a rather high stability of the plasma membrane as indicated by membrane potentials of -60 mV to -80 mV and a membrane resistance with maximum values of 100-150 megaohms [7, 9, 23]. Intensive pinocytotic activity starts, when externally bound Ca2+ is substituted by cationic substances [16] and distinct changes in the electrical and physiological properties of the plasma membrane occur (Fig. 6 A, B) as evident by an increase in both the membrane thickness and conductance. An external cation concentration of 8 mM decreases the membrane potential to -30 mV and is sufficient to induce pinocytosis [11,23]. The electrophysiological results on pinocytosing amebas are in good agreement with measurements of Batueva [4, 5] and Bingley [6] on normallocomoting cells which exhibit a membrane potential of -70 mV at the front and -30 mV at the uroid so that suitable conditions for a permament pinocytotic activity, as always present in moving A. proteus [35], exist only in the posterior cell body region. The question whether external Ca2+ also controls the second and third step of induced pinocytosis, i.e., participates in signal transmission and activation of the microfilament system to form membrane invaginations has been the subject of several studies [21,26,30,31]. The observation of Johansson and Josefsson [19] that the passage of external ci+ from the cell surface into the cell interior inhibits pinocytosis is in contrast to the present results because the ionophor A 23187 increases Ca-pinocytosis by more than 30%, whereas the Ca-antagonists D-600 and isoptin hydrochloride cause a decrease in Ca-induced pinocytotic activity of about 50%. A possible explanation of these contradictions is delivered by recent observations of Klo~ocka and Grebecka [26] according to which external Ca + inhibits pinocytosis only in the presence of i.e., different results largely depend on the application of EGTA or EDTA as Ca-buffers, On the other hand, the finding that pinocytosis in A. proteus is inducible by inorganic cations even at very low external Ca2+ concentrations, i.e., in the range of 10-0,001 f.lM, may also be explained by the possibility of releasing calcium from different intracellular stores. In this connection it seems important to study in future experiments the question whether the influx of additional external calcium is necessary to sustain induced pinocytosis over long periods of time. Corresponding conclusions can be drawn from the results about a significance of Na" and K+ for signal transmission. Moreover, since it has been possible to induce pinocytosis by the microinjection of polyamines, i.e., without the participation of any external stimulation [15], it still remains obscure how the signal for membrane flow is transmitted from the external into the internal compartment. According to comprehensive results recently obtained by Josefsson and his coworkers it cannot be excluded that other substances, such as membrane stabilizing drugs [24] lysolecithin [2], biogenic amines and opioids [22], antibiotics [20] or a pinocytosis-regulating factor [3] play an important modifying role for induced pinocytosis in A. proteus.

Mi+'

In contrast to the question which external parameters control the process of signal transmission during induced pinocytosis, all investigations agree, however, in the significance of intracellular Ca2+ for the activation of the contracile apparatus and, hence, motive force generation for channel formation. Several Ca-accumulating systems such as internal membrane-attached binding sites [34], smooth endoplasmic reticulum [32] and mitochondria [16] represent sources to attain physiological Ca2+-concentrations necessary to induce actomyosin contraction (Fig.6B). Finally, the mechanism by which cytoplasmic and plasma membrane conditions are restored to the physiological situation (Fig. 6 C) so that pinocytosis terminates are also largely unexplained. So far, there is no experimental evidence at all for the participation of a plasma rnembran-bound active extrusion mechanism in regulating the cytoplasmic Ca2+ level of A. proteus. Although the proposed role of the contractile vacuole in Ca2+ homeostasis (Fig. 6 C, Cv) is highly speculative and not based on any concrete results further experiments should take this possibility into consideration.

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Key words: Induced pinocytosis - Specifity of membrane receptors - Influence of cations - Significance of Ca2+ Amoeba proteus Wilhelm Stockem, Institut fur Cytologie der Universitat Bonn, Ulrich-Haberland-Stralse 61a, 5300 Bonn 1, FRG