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RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION INAMOEBA PROTEUS
Cell Biology International, 1996, Vol. 20, No. 9, 635–641
RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION IN AMOEBA PROTEUS W. KŁOPOCKA, J. KOŁODZIEJCZYK, A. LOPATOWSKA and A. GRE q BECKI* Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str., Warszawa, Poland Accepted 28 May 1996
Induction of pinocytosis in Amoeba proteus is independent of adhesion. It is manifested by non-adhering floating specimens, as well as by amoebae moderately adhering and locomoting on the glass, or tightly attached to the polylysine-coated substratum. The formation of pinocytotic rosettes results in de-adhesion, at the beginning of pinocytosis on the glass, but at its end on the polylysine. It suggests an opposition between adhesion and cell shape transformation. Pinocytosis and adhesion are both inhibited, by an unknown mechanism, in the presence of gelatin ? 1996 Academic Press Limited either in the substratum or in solution. K: pinocytosis; adhesion; Amoeba proteus
INTRODUCTION Endocytosis in the free-living amoebae, as a cytoskeleton dependent function, seems to be correlated with the cell-to-substratum adhesion. In Amoeba proteus an increase of adhesion is required for phagocytosis, whereas during cation-induced pinocytosis these cells detach from the substratum (Mast and Hahnert 1935; Chapman-Andresen, 1962; Opas, 1981). A locomoting A. proteus adheres to the substratum by the tips of contact pseudopods protruding from the ventral surface of the cell (Bell and Jeon, 1963; Gre¸becki, 1976). These adhesion sites were also demonstrated by reflection interference microscopy in the same species (Haberey, 1971; Opas, 1978), and in Naegleria gruberi (Preston and King, 1978). This type of cell–substratum contact is found in the bipolar amoebae, polytactic and orthotactic, whereas the radial heterotactic forms of A. proteus are not attached (Gre¸becki and Gre¸becka, 1978). Normally-locomoting specimens of A. proteus spontaneously manifest a weak permanent pinocytosis at the uroidal region (Wohlfarth-Bottermann and Stockem, 1966). In the case of cation-induced pinocytosis, pinocytotic pseudopods with invaginated channels arise in other sites, with a parallel loss of locomotion and of the bipolar morphology (Klein and Stockem, 1979; Gre¸becka and *To whom correspondence should be addressed. 1065–6995/96/090635+07 $25.00/0
Kłopocka, 1985). At the culmination of pinocytosis amoebae have the form of regular rosettes (Chapman-Andresen, 1962, and others). It was our aim to clarify in the present study which components of the pinocytotic behaviour are correlated with the cell-to-substratum attachment and how are they mutually related: is de-adhesion a prerequisite or merely a result of cation-induced pinocytosis. We tried to approach this question by comparing pinocytosis of amoebae moderately adhering to the untreated glass with pinocytosis on more and less adhesive substrata. Amoebae, as the cultured metazoan cells, strongly adhere to surfaces coated with polycations: polylysine (King et al., 1982; Kołodziejczyk et al., 1995) or concanavalin A (Preston and O’Dell, 1980). In contrast, they fail to adhere to a polyanionic substratum: the gelatin gel (Kołodziejczyk et al., 1995).
MATERIALS AND METHODS Amoebae were cultured in Pringsheim medium 200 ìg/ml, MgSO4 · 7H2O [Ca(NO3)2 · 4H2O 20 ìg/ml, Na2HPO4 · 2H2O 20 ìg/ml, KCl 26 ìg/ ml, FeSO4 · 7H2O 2 ìg/ml] and fed on Tetrahymena pyriformis. On the third day after feeding 20–50 cells in 20 ìl of the medium were transferred to experimental substrata. All experiments were repeated 3–10 times, so that total cell numbers exceeded 100 in each situation. ? 1996 Academic Press Limited
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Cell Biology International, Vol. 20, No. 9, 1996
Cell Biology International, Vol. 20, No. 9, 1996
Experiments with amoebae normally locomoting on untreated glass were run on carefully cleaned coverslips. The enhanced adhesion was obtained on the coverslips incubated for 15 min in 1 mg/ml solution of poly--lysine (Sigma) in deionized water or 1 mg/ml concanavalin A (Sigma) in 1 acetic acid, followed by rinsing in Pringsheim medium. Non-adhesive substrata were produced by spreading on the coverslips 10% or 20% gelatin (LobeChemie) dissolved in Pringsheim medium, and allowing it to set at room temperature. Pinocytosis was induced by adding 10 ìl of NaCl or polylysine solutions in Pringsheim medium, to 20 ìl of the culture of amoebae. Non-pinocytotic rosettes were induced by choline (Serva) or acetylcholine chlorides (Sigma). The presence or absence of adhesion was tested in the whole samples by the gravitation method, and in the individual cells by provoking a local flow of the medium. The variable intensity of pinocytosis could not be precisely expressed in numbers of pinocytizing cells and numbers of the pinocytotic channels per cell, because the situation profoundly changed during the time of counting. It was therefore qualitatively estimated and the results are presented in the descriptive way. The whole samples were continuously scanned with a Teloval inverted microscope (Zeiss, Jena), and individual amoebae were surveyed in differential interference contrast of Pluta system with a Biolar microscope (PZO, Warszawa). Parts of the experiments were recorded on the tape with Hamamatsu C2400 camera system. RESULTS AND DISCUSSION Amoebae on the untreated glass Amoebae transferred to the untreated coverslips were allowed to re-adhere, recover the polytactic shape and start locomotion (Fig. 1A). Subse-
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quently, 125 m NaCl was added to the medium and simultaneously the coverslip was turned upside-down. It enabled testing the cell–substratum attachment during pinocytosis (Fig. 2). Within 5 min of application of the inducer all cells stopped locomotion, began to contract and formed pinocytotic channels. Concurrently, they were losing the contact with the substratum and falling down (Fig. 2A–D). This confirms the observations made by Opas (1981) by means of the reflection interference microscopy. De-adhesion occurred at an early stage of shape transformation, so that all pinocytotically active cells, rosette-shaped and producing channels, were detached from the glass (Figs 2E and F and 1B). The heterotactic forms, produced by agitating amoebae before the experiment, were exposed to the same pinocytotic inducer until they reattached and recuperated the locomotory shape. In spite of the absence of adhesion, all cells started pinocytosis. The onset of pinocytosis was however delayed and the channels were produced only about 10 min after induction, because during the first phase amoebae retracted their strongly extended heterotactic pseudopods (Fig. 3A). The unattached heterotactic specimens eventually produced rosettes with pinocytotic pseudopods, which were normal in shape and in number (Fig. 3B). The polytactic amoebae adhering to the glass and locomoting, were exposed in next experiments to 125 m of choline or acetylcholine chlorides, which after addition to the Pringsheim medium, provoke formation of the same rosette forms as Na + , but without pinocytotic channels (Kłopocka and Pomorski, 1996). In the present experiments, such non-pinocytotic rosettes developed within 5–10 min of induction and they also detached from the glass at the early stage of transformation. Other samples of cells normally adhering and migrating on untreated glass, were exposed to the solution of 250 ìg/ml of polylysine. This polycation
Fig. 1. A polytactic specimen of A. proteus (A), adhering to the substratum and migrating, 15 min after the transfer to the untreated glass; note the tail-front polarity (T–F). A pinocytotic rosette (B) produced by a similar polytactic specimen after 24 min in NaCl solution; channels seen in focus are indicated by arrows. Bar=100 ìm. Fig. 2. Detachment of polytactic amoebae in 125 m NaCl, from the untreated glass coverslip turned upside-down and fixed 2 mm above the bottom of the observation chamber. In the top focus (A–D), the adhering cells and former positions of those who fell down are marked by circles. The detached pinocytizing amoebae on the bottom are shown in E and F. Time lapse indicated in min. Bar=100 ìm. Fig. 3. A heterotactic form (A), non-adhering, 2 min after sedimentation on the glass; note its radial symmetry. The pinocytotic rosette (B) formed by a heterotactic amoeba, 28 min after addition of NaCl. Bar=100 ìm. Fig. 4. The pinocytotic rosette produced by a polytactic amoeba 210 min after addition of polylysine to the medium. Bar=100 ìm.
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Cell Biology International, Vol. 20, No. 9, 1996
Cell Biology International, Vol. 20, No. 9, 1996
added to the medium acted as inducer of pinocytosis in 100% of cells. However, the separate pinocytotic pseudopodia were absent, but instead the channels were formed on flat protuberances of cell periphery (Fig. 4). A similar morphology of pinocytosis was seen in solutions of concanavalin A and Alcian blue. It seems therefore to be a product of polycations, different from the effects of monovalent ions. Important differences in cell adhesiveness were observed in the presence of these two types of inducers: in contrast to the Na + treatment, in the polylysine solution many amoebae still adhered to the glass during pinocytosis and they detached from the substratum at its advanced stage. In this case, consequently, the de-adhered population was mostly composed of late pinocytotic specimens. The experiments on the untreated glass have demonstrated that: (1) pinocytosis may be induced as well in the floating cells as in the substrateadhering amoebae, (2) it is produced regardless of the original morphological difference between the bipolar polytactic specimens and the radial heterotactic ones, (3) amoebae really de-adhere at the beginning of Na-induced pinocytosis, but remain longer attached after induction of pinocytosis by polycations which promote cell-to-substratum adherence and (4) formation of the non-pinocytotic rosettes also leads to de-adhesion.
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stage, 5–10 min after the addition of the inducer (Fig. 5B). When they were examined in the upsidedown position, the majority detached from the polylysine substratum much later, than from the untreated glass (compare Fig. 6A–C with Fig. 2A–D). As a result, the population of cells detached from the upper surface was mostly composed of postpinocytotic rosettes (Fig. 6D). A few rosettes, however, remained adhering to the polylysine substratum long after the pinocytosis has been accomplished (Fig. 6E and F). Similar effects were produced by addition of 250 ìg/ml polylysine, instead of NaCl, to the medium of amoebae adhering to the polylysinecoated glass. Coating the glass with concanavalin A also increases adhesiveness of Amoeba proteus, as well as it was earlier demonstrated for Naegleria gruberi by Preston and O’Dell (1980). On this substratum, as on the polylysine, the Na-induced pinocytosis is well manifested, whereas the shape changes are delayed and de-adhesion occurs at the end of pinocytosis. This indicates that de-adhesion is not a precondition of pinocytosis, although in NaCl on the untreated glass it occurs at a very early stage. The late formation and irregular shape of the rosettes on surfaces coated with polylysine or concanavalin A demonstrate that a partial de-adhesion, required for shape regulation, becomes difficult on polycationic substrata.
Amoebae on the polylysine-coated glass Amoebae on the gelatin gel As it was earlier described (Kołodziejczyk et al., 1995), amoebae strongly adhere to the positively charged polylysine substratum, are flattened and move slower than normally. Also under these conditions almost all cells produced abundant pinocytotic channels in 125 m NaCl solution. The shape transformation was slower than on the untreated glass. First, channels appeared in the still flattened individuals with distinct traces of bipolar symmetry: preferentially at the former uroid (Fig. 5A) or at both poles (the polarity of channels formation on the glass was earlier studied by Gre¸becka and Kłopocka, 1985). The cells on the polylysine only gradually arrived at the rosette
Amoebae transferred to gels formed by 10% or 20% gelatin dissolved in Pringsheim medium, neither adhere nor locomote (Kołodziejczyk et al., 1995). They are heterotactic at the beginning (Fig. 7A), but later they withdraw pseudopods and look like rosettes (Fig. 7B). In our experiments with this substratum, the pinocytosis was also strongly inhibited, regardless of the kind of inducer: 125 m NaCl or 250 ìg/ml polylysine solution. After induction amoebae took the regular rosette shape (Fig. 7C). Nevertheless, only in a few of them 1–3 pinocytotic channels could be found, in contrast to >20 channels per cell on the untreated glass.
Fig. 5. Na-induced pinocytosis in amoebae adhering to the polylysine-coated glass: early channels appearing within 5 min at one body pole (A), and a fully developed rosette after 210 min. (B) Arrows point to channels. Bar=100 ìm. Fig. 6. Detachment of amoebae from polylysine in 125 m NaCl, tested in the same manner as in Fig. 2. Gradual de-adhesion and falling down from the top surface (A–C), the group of pinocytotic and postpinocytotic rosettes on the bottom (D), and a very late detachment of two post-pinocytotic rosettes from the upper surface (E and F). Bar=100 ìm. Fig. 7. Non-adhering heterotactic (A) and rosette-like amoebae (B) on gelatin gel, and a NaCl-induced rosette on the same substratum, regular in shape but without pinocytosis. Bar=100 ìm.
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Two carbohydrate gels, 0.5% agar and 0.25% pectin, were tested as controls. Adhesion and locomotion were normal on the pectin, and defective on the agar, but in both cases amoebae were capable of pinocytosis. Inhibition of pinocytosis on gelatin gels can be explained neither by the absence of adhesion nor by the altered cell shape, since the heterotactic amoebae not adhering to glass manifested normal pinocytosis. Pinocytosis was also unaffected on carbohydrate gels. Thus, the inhibition seems to be related to some properties of the gelatin. It became, therefore, necessary to clarify whether it is specifically provoked by the contact with gelled gelatin in the substratum. Effects of gelatin in solution Gelatin was added, in final concentration of 3.3%, to the medium of amoebae normally adhering and migrating on the untreated glass. It provoked de-adhesion, gradually affecting all cells within 5–10 min. The detaching amoebae took rosette-like forms (without pinocytosis). The gelatin solutions, ranging from 1% to 3.3% completely blocked the pinocytotic reaction of amoebae to NaCl and polylysine, if they were applied simultaneously with these inducers, or 5 min earlier. If they were applied 5 min after NaCl or polylysine, i.e. at the culmination of pinocytosis, they provoked regression of the pinocytotic channels. In all situations amoebae kept the form of non-pinocytotic rosettes, always detached from the glass in NaCl, but sometimes re-adhering in the presence of polylysine. The threshold concentration of gelatin, inhibiting pinocytosis of 250% of cells, was established at 0.33%. These results suggest that the inhibition of pinocytosis by gelatin gels may not depend on the cell contact with this substratum, but on the presence of gelatin in the surrounding solution and/or on a modification of ionic composition of medium by this polyanion.
GENERAL CONCLUSIONS Initiation of cation-induced pinocytosis in A. proteus is independent of the adhesion to the substratum. Pinocytosis may be induced in the cells floating over the glass without adhesion, as well as in cells normally migrating on glass with a moderate adhesion, and also in amoebae strongly adhering to the glass coated with polycations. It means
Cell Biology International, Vol. 20, No. 9, 1996
that the capacity of amoebae for starting pinocytosis is also independent of the original cell shape: radial in heterotactic amoebae, bipolar in the normal polytactic forms, and flatly spread on the strongly adhesive substrata. De-adhesion, therefore, is not a prerequisite but a result of pinocytotic activity. It seems to be related to the cell shape changing into the rosette form, but independent of membrane internalization, since the non-pinocytotic rosettes also lose link with the substratum. Our results suggest an opposition between the cytoskeletal forces involved in the alteration of shape and the resistance of cell–substratum adhesion sites. Therefore, with a moderate adhesion to the untreated glass the Na-induced shape changes are quick and amoebae detach at the beginning of pinocytosis, whereas in the presence of polycations which increase cell adherence to the substratum, the formation of rosettes is delayed and they de-adhere at the later stages, or after the end of pinocytosis. The suppressive effect of gelatin on pinocytosis should not be, therefore, regarded as a result of the failure of adhesion in the presence of this polyanion. The reasons why adhesion and pinocytosis are both hampered by gelatin are under study. ACKNOWLEDGEMENT This study was partly supported by the grant 0453/p2/93/04 from the Committee of Scientific Research (KBN). REFERENCES B LGE, J KW, 1963. Locomotion of Amoeba proteus. Nature 198: 675–676. C-A C, 1962. Studies on pino cytosis in amoebae. C R Trav Lab Carlsberg 33: 73–264. G¸ L, KŁ W, 1985. Relationship between the surface distribution of membrane reserves and the polarity of pinocytosis in Amoeba proteus. Protistologica 21: 207– 213. G¸ A, 1976. Coaxial motion of the semi-rigid cell frame in Amoeba proteus. Acta Protozool 15: 221–248. G¸ A, G¸ L, 1978. Morphodynamic types of Amoeba proteus: a terminological proposal. Protistologica 14: 349–358. H M, 1971. Bewegungsverhalten und Untergrundkontakt von Amoeba proteus. Mikroskopie 27: 226–234. K CA, P TM, M RH, G C, 1982. The cell surface in locomotion—studies on the role of cell-substrate adhesion. Cell Biol Int Rep 6: 893–900. K HP, S W, 1979. Pinocytosis and locomotion of amoebae. XII. Dynamics and motive force generation during induced pinocytosis in Amoeba proteus. Cell Tissue Res 197: 263–279.
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KŁ W, P P, 1996. Cytoplasmic calcium transients in Amoeba proteus during induction of pinocytotic and non-pinocytotic rosettes. Acta Protozool 34: 169–175. KŁ J, KŁ W, L A, G¸ L, G¸ A, 1995. Resumption of locomotion by Amoeba proteus readhering to different substrata. Protoplasma 189: 180–186. M SO, H WF, 1935. Feeding, digestion and starvation in Amoeba proteus (Leidy). Physiol Zool 8: 255–272. O M, 1978. Interference reflection microscopy of adhesion of Amoeba proteus. J Microsc 112: 215–221.
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O M, 1981. Effects of induction of endocytosis on adhesiveness of Amoeba proteus. Protoplasma 107: 161–169. P TM, K CA, 1978. An experimental study of the interaction between the soil amoeba Naegleria gruberi and a glass substrate during amoeboid locomotion. J Cell Sci 34: 145–158. P TM, O’D DS, 1980. The cell surface in amoeboid locomotion: behaviour of Naegleria gruberi on an adhesive lectin substrate. J Gen Microbiol 116: 515–520. W-B KE, S W, 1966. Pinocytose und Bewegung von Amöben. II. Permanente und induzierte Pinocytose bei Amoeba proteus. Z Zellforsch 73: 444–474.
RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION IN AMOEBA PROTEUS W. KŁOPOCKA, J. KOŁODZIEJCZYK, A. LOPATOWSKA and A. GRE q BECKI* Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str., Warszawa, Poland Accepted 28 May 1996
Induction of pinocytosis in Amoeba proteus is independent of adhesion. It is manifested by non-adhering floating specimens, as well as by amoebae moderately adhering and locomoting on the glass, or tightly attached to the polylysine-coated substratum. The formation of pinocytotic rosettes results in de-adhesion, at the beginning of pinocytosis on the glass, but at its end on the polylysine. It suggests an opposition between adhesion and cell shape transformation. Pinocytosis and adhesion are both inhibited, by an unknown mechanism, in the presence of gelatin ? 1996 Academic Press Limited either in the substratum or in solution. K: pinocytosis; adhesion; Amoeba proteus
INTRODUCTION Endocytosis in the free-living amoebae, as a cytoskeleton dependent function, seems to be correlated with the cell-to-substratum adhesion. In Amoeba proteus an increase of adhesion is required for phagocytosis, whereas during cation-induced pinocytosis these cells detach from the substratum (Mast and Hahnert 1935; Chapman-Andresen, 1962; Opas, 1981). A locomoting A. proteus adheres to the substratum by the tips of contact pseudopods protruding from the ventral surface of the cell (Bell and Jeon, 1963; Gre¸becki, 1976). These adhesion sites were also demonstrated by reflection interference microscopy in the same species (Haberey, 1971; Opas, 1978), and in Naegleria gruberi (Preston and King, 1978). This type of cell–substratum contact is found in the bipolar amoebae, polytactic and orthotactic, whereas the radial heterotactic forms of A. proteus are not attached (Gre¸becki and Gre¸becka, 1978). Normally-locomoting specimens of A. proteus spontaneously manifest a weak permanent pinocytosis at the uroidal region (Wohlfarth-Bottermann and Stockem, 1966). In the case of cation-induced pinocytosis, pinocytotic pseudopods with invaginated channels arise in other sites, with a parallel loss of locomotion and of the bipolar morphology (Klein and Stockem, 1979; Gre¸becka and *To whom correspondence should be addressed. 1065–6995/96/090635+07 $25.00/0
Kłopocka, 1985). At the culmination of pinocytosis amoebae have the form of regular rosettes (Chapman-Andresen, 1962, and others). It was our aim to clarify in the present study which components of the pinocytotic behaviour are correlated with the cell-to-substratum attachment and how are they mutually related: is de-adhesion a prerequisite or merely a result of cation-induced pinocytosis. We tried to approach this question by comparing pinocytosis of amoebae moderately adhering to the untreated glass with pinocytosis on more and less adhesive substrata. Amoebae, as the cultured metazoan cells, strongly adhere to surfaces coated with polycations: polylysine (King et al., 1982; Kołodziejczyk et al., 1995) or concanavalin A (Preston and O’Dell, 1980). In contrast, they fail to adhere to a polyanionic substratum: the gelatin gel (Kołodziejczyk et al., 1995).
MATERIALS AND METHODS Amoebae were cultured in Pringsheim medium 200 ìg/ml, MgSO4 · 7H2O [Ca(NO3)2 · 4H2O 20 ìg/ml, Na2HPO4 · 2H2O 20 ìg/ml, KCl 26 ìg/ ml, FeSO4 · 7H2O 2 ìg/ml] and fed on Tetrahymena pyriformis. On the third day after feeding 20–50 cells in 20 ìl of the medium were transferred to experimental substrata. All experiments were repeated 3–10 times, so that total cell numbers exceeded 100 in each situation. ? 1996 Academic Press Limited
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Cell Biology International, Vol. 20, No. 9, 1996
Cell Biology International, Vol. 20, No. 9, 1996
Experiments with amoebae normally locomoting on untreated glass were run on carefully cleaned coverslips. The enhanced adhesion was obtained on the coverslips incubated for 15 min in 1 mg/ml solution of poly--lysine (Sigma) in deionized water or 1 mg/ml concanavalin A (Sigma) in 1 acetic acid, followed by rinsing in Pringsheim medium. Non-adhesive substrata were produced by spreading on the coverslips 10% or 20% gelatin (LobeChemie) dissolved in Pringsheim medium, and allowing it to set at room temperature. Pinocytosis was induced by adding 10 ìl of NaCl or polylysine solutions in Pringsheim medium, to 20 ìl of the culture of amoebae. Non-pinocytotic rosettes were induced by choline (Serva) or acetylcholine chlorides (Sigma). The presence or absence of adhesion was tested in the whole samples by the gravitation method, and in the individual cells by provoking a local flow of the medium. The variable intensity of pinocytosis could not be precisely expressed in numbers of pinocytizing cells and numbers of the pinocytotic channels per cell, because the situation profoundly changed during the time of counting. It was therefore qualitatively estimated and the results are presented in the descriptive way. The whole samples were continuously scanned with a Teloval inverted microscope (Zeiss, Jena), and individual amoebae were surveyed in differential interference contrast of Pluta system with a Biolar microscope (PZO, Warszawa). Parts of the experiments were recorded on the tape with Hamamatsu C2400 camera system. RESULTS AND DISCUSSION Amoebae on the untreated glass Amoebae transferred to the untreated coverslips were allowed to re-adhere, recover the polytactic shape and start locomotion (Fig. 1A). Subse-
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quently, 125 m NaCl was added to the medium and simultaneously the coverslip was turned upside-down. It enabled testing the cell–substratum attachment during pinocytosis (Fig. 2). Within 5 min of application of the inducer all cells stopped locomotion, began to contract and formed pinocytotic channels. Concurrently, they were losing the contact with the substratum and falling down (Fig. 2A–D). This confirms the observations made by Opas (1981) by means of the reflection interference microscopy. De-adhesion occurred at an early stage of shape transformation, so that all pinocytotically active cells, rosette-shaped and producing channels, were detached from the glass (Figs 2E and F and 1B). The heterotactic forms, produced by agitating amoebae before the experiment, were exposed to the same pinocytotic inducer until they reattached and recuperated the locomotory shape. In spite of the absence of adhesion, all cells started pinocytosis. The onset of pinocytosis was however delayed and the channels were produced only about 10 min after induction, because during the first phase amoebae retracted their strongly extended heterotactic pseudopods (Fig. 3A). The unattached heterotactic specimens eventually produced rosettes with pinocytotic pseudopods, which were normal in shape and in number (Fig. 3B). The polytactic amoebae adhering to the glass and locomoting, were exposed in next experiments to 125 m of choline or acetylcholine chlorides, which after addition to the Pringsheim medium, provoke formation of the same rosette forms as Na + , but without pinocytotic channels (Kłopocka and Pomorski, 1996). In the present experiments, such non-pinocytotic rosettes developed within 5–10 min of induction and they also detached from the glass at the early stage of transformation. Other samples of cells normally adhering and migrating on untreated glass, were exposed to the solution of 250 ìg/ml of polylysine. This polycation
Fig. 1. A polytactic specimen of A. proteus (A), adhering to the substratum and migrating, 15 min after the transfer to the untreated glass; note the tail-front polarity (T–F). A pinocytotic rosette (B) produced by a similar polytactic specimen after 24 min in NaCl solution; channels seen in focus are indicated by arrows. Bar=100 ìm. Fig. 2. Detachment of polytactic amoebae in 125 m NaCl, from the untreated glass coverslip turned upside-down and fixed 2 mm above the bottom of the observation chamber. In the top focus (A–D), the adhering cells and former positions of those who fell down are marked by circles. The detached pinocytizing amoebae on the bottom are shown in E and F. Time lapse indicated in min. Bar=100 ìm. Fig. 3. A heterotactic form (A), non-adhering, 2 min after sedimentation on the glass; note its radial symmetry. The pinocytotic rosette (B) formed by a heterotactic amoeba, 28 min after addition of NaCl. Bar=100 ìm. Fig. 4. The pinocytotic rosette produced by a polytactic amoeba 210 min after addition of polylysine to the medium. Bar=100 ìm.
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Cell Biology International, Vol. 20, No. 9, 1996
Cell Biology International, Vol. 20, No. 9, 1996
added to the medium acted as inducer of pinocytosis in 100% of cells. However, the separate pinocytotic pseudopodia were absent, but instead the channels were formed on flat protuberances of cell periphery (Fig. 4). A similar morphology of pinocytosis was seen in solutions of concanavalin A and Alcian blue. It seems therefore to be a product of polycations, different from the effects of monovalent ions. Important differences in cell adhesiveness were observed in the presence of these two types of inducers: in contrast to the Na + treatment, in the polylysine solution many amoebae still adhered to the glass during pinocytosis and they detached from the substratum at its advanced stage. In this case, consequently, the de-adhered population was mostly composed of late pinocytotic specimens. The experiments on the untreated glass have demonstrated that: (1) pinocytosis may be induced as well in the floating cells as in the substrateadhering amoebae, (2) it is produced regardless of the original morphological difference between the bipolar polytactic specimens and the radial heterotactic ones, (3) amoebae really de-adhere at the beginning of Na-induced pinocytosis, but remain longer attached after induction of pinocytosis by polycations which promote cell-to-substratum adherence and (4) formation of the non-pinocytotic rosettes also leads to de-adhesion.
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stage, 5–10 min after the addition of the inducer (Fig. 5B). When they were examined in the upsidedown position, the majority detached from the polylysine substratum much later, than from the untreated glass (compare Fig. 6A–C with Fig. 2A–D). As a result, the population of cells detached from the upper surface was mostly composed of postpinocytotic rosettes (Fig. 6D). A few rosettes, however, remained adhering to the polylysine substratum long after the pinocytosis has been accomplished (Fig. 6E and F). Similar effects were produced by addition of 250 ìg/ml polylysine, instead of NaCl, to the medium of amoebae adhering to the polylysinecoated glass. Coating the glass with concanavalin A also increases adhesiveness of Amoeba proteus, as well as it was earlier demonstrated for Naegleria gruberi by Preston and O’Dell (1980). On this substratum, as on the polylysine, the Na-induced pinocytosis is well manifested, whereas the shape changes are delayed and de-adhesion occurs at the end of pinocytosis. This indicates that de-adhesion is not a precondition of pinocytosis, although in NaCl on the untreated glass it occurs at a very early stage. The late formation and irregular shape of the rosettes on surfaces coated with polylysine or concanavalin A demonstrate that a partial de-adhesion, required for shape regulation, becomes difficult on polycationic substrata.
Amoebae on the polylysine-coated glass Amoebae on the gelatin gel As it was earlier described (Kołodziejczyk et al., 1995), amoebae strongly adhere to the positively charged polylysine substratum, are flattened and move slower than normally. Also under these conditions almost all cells produced abundant pinocytotic channels in 125 m NaCl solution. The shape transformation was slower than on the untreated glass. First, channels appeared in the still flattened individuals with distinct traces of bipolar symmetry: preferentially at the former uroid (Fig. 5A) or at both poles (the polarity of channels formation on the glass was earlier studied by Gre¸becka and Kłopocka, 1985). The cells on the polylysine only gradually arrived at the rosette
Amoebae transferred to gels formed by 10% or 20% gelatin dissolved in Pringsheim medium, neither adhere nor locomote (Kołodziejczyk et al., 1995). They are heterotactic at the beginning (Fig. 7A), but later they withdraw pseudopods and look like rosettes (Fig. 7B). In our experiments with this substratum, the pinocytosis was also strongly inhibited, regardless of the kind of inducer: 125 m NaCl or 250 ìg/ml polylysine solution. After induction amoebae took the regular rosette shape (Fig. 7C). Nevertheless, only in a few of them 1–3 pinocytotic channels could be found, in contrast to >20 channels per cell on the untreated glass.
Fig. 5. Na-induced pinocytosis in amoebae adhering to the polylysine-coated glass: early channels appearing within 5 min at one body pole (A), and a fully developed rosette after 210 min. (B) Arrows point to channels. Bar=100 ìm. Fig. 6. Detachment of amoebae from polylysine in 125 m NaCl, tested in the same manner as in Fig. 2. Gradual de-adhesion and falling down from the top surface (A–C), the group of pinocytotic and postpinocytotic rosettes on the bottom (D), and a very late detachment of two post-pinocytotic rosettes from the upper surface (E and F). Bar=100 ìm. Fig. 7. Non-adhering heterotactic (A) and rosette-like amoebae (B) on gelatin gel, and a NaCl-induced rosette on the same substratum, regular in shape but without pinocytosis. Bar=100 ìm.
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Two carbohydrate gels, 0.5% agar and 0.25% pectin, were tested as controls. Adhesion and locomotion were normal on the pectin, and defective on the agar, but in both cases amoebae were capable of pinocytosis. Inhibition of pinocytosis on gelatin gels can be explained neither by the absence of adhesion nor by the altered cell shape, since the heterotactic amoebae not adhering to glass manifested normal pinocytosis. Pinocytosis was also unaffected on carbohydrate gels. Thus, the inhibition seems to be related to some properties of the gelatin. It became, therefore, necessary to clarify whether it is specifically provoked by the contact with gelled gelatin in the substratum. Effects of gelatin in solution Gelatin was added, in final concentration of 3.3%, to the medium of amoebae normally adhering and migrating on the untreated glass. It provoked de-adhesion, gradually affecting all cells within 5–10 min. The detaching amoebae took rosette-like forms (without pinocytosis). The gelatin solutions, ranging from 1% to 3.3% completely blocked the pinocytotic reaction of amoebae to NaCl and polylysine, if they were applied simultaneously with these inducers, or 5 min earlier. If they were applied 5 min after NaCl or polylysine, i.e. at the culmination of pinocytosis, they provoked regression of the pinocytotic channels. In all situations amoebae kept the form of non-pinocytotic rosettes, always detached from the glass in NaCl, but sometimes re-adhering in the presence of polylysine. The threshold concentration of gelatin, inhibiting pinocytosis of 250% of cells, was established at 0.33%. These results suggest that the inhibition of pinocytosis by gelatin gels may not depend on the cell contact with this substratum, but on the presence of gelatin in the surrounding solution and/or on a modification of ionic composition of medium by this polyanion.
GENERAL CONCLUSIONS Initiation of cation-induced pinocytosis in A. proteus is independent of the adhesion to the substratum. Pinocytosis may be induced in the cells floating over the glass without adhesion, as well as in cells normally migrating on glass with a moderate adhesion, and also in amoebae strongly adhering to the glass coated with polycations. It means
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that the capacity of amoebae for starting pinocytosis is also independent of the original cell shape: radial in heterotactic amoebae, bipolar in the normal polytactic forms, and flatly spread on the strongly adhesive substrata. De-adhesion, therefore, is not a prerequisite but a result of pinocytotic activity. It seems to be related to the cell shape changing into the rosette form, but independent of membrane internalization, since the non-pinocytotic rosettes also lose link with the substratum. Our results suggest an opposition between the cytoskeletal forces involved in the alteration of shape and the resistance of cell–substratum adhesion sites. Therefore, with a moderate adhesion to the untreated glass the Na-induced shape changes are quick and amoebae detach at the beginning of pinocytosis, whereas in the presence of polycations which increase cell adherence to the substratum, the formation of rosettes is delayed and they de-adhere at the later stages, or after the end of pinocytosis. The suppressive effect of gelatin on pinocytosis should not be, therefore, regarded as a result of the failure of adhesion in the presence of this polyanion. The reasons why adhesion and pinocytosis are both hampered by gelatin are under study. ACKNOWLEDGEMENT This study was partly supported by the grant 0453/p2/93/04 from the Committee of Scientific Research (KBN). REFERENCES B LGE, J KW, 1963. Locomotion of Amoeba proteus. Nature 198: 675–676. C-A C, 1962. Studies on pino cytosis in amoebae. C R Trav Lab Carlsberg 33: 73–264. G¸ L, KŁ W, 1985. Relationship between the surface distribution of membrane reserves and the polarity of pinocytosis in Amoeba proteus. Protistologica 21: 207– 213. G¸ A, 1976. Coaxial motion of the semi-rigid cell frame in Amoeba proteus. Acta Protozool 15: 221–248. G¸ A, G¸ L, 1978. Morphodynamic types of Amoeba proteus: a terminological proposal. Protistologica 14: 349–358. H M, 1971. Bewegungsverhalten und Untergrundkontakt von Amoeba proteus. Mikroskopie 27: 226–234. K CA, P TM, M RH, G C, 1982. The cell surface in locomotion—studies on the role of cell-substrate adhesion. Cell Biol Int Rep 6: 893–900. K HP, S W, 1979. Pinocytosis and locomotion of amoebae. XII. Dynamics and motive force generation during induced pinocytosis in Amoeba proteus. Cell Tissue Res 197: 263–279.
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O M, 1981. Effects of induction of endocytosis on adhesiveness of Amoeba proteus. Protoplasma 107: 161–169. P TM, K CA, 1978. An experimental study of the interaction between the soil amoeba Naegleria gruberi and a glass substrate during amoeboid locomotion. J Cell Sci 34: 145–158. P TM, O’D DS, 1980. The cell surface in amoeboid locomotion: behaviour of Naegleria gruberi on an adhesive lectin substrate. J Gen Microbiol 116: 515–520. W-B KE, S W, 1966. Pinocytose und Bewegung von Amöben. II. Permanente und induzierte Pinocytose bei Amoeba proteus. Z Zellforsch 73: 444–474.