The Unique Value of Protozoa in Membrane Investigation* Introductory Remarks by the Convener

SYNOPSIS. The constantly changing shape and surface area/volume ratio of Chaos and Amoeba provide sufficient evidence for continuous membrane activity during locomotion, endocytosis, and cell division. Factors influencing membrane intake and renewal are discussed on the basis of data from tracer experiments and from studies on fine structure. The concept of permanent pinocytosis as an essential part of locomotion is discussed, together with the differences between induced and permanent pinocytosis and the factors which might be responsible for the “induction” of permanent pinocytosis. The large saprobiotic ameba, Pelomyxa palustris differs from Chaos and Amoeba in membrane distribution. P. palustris is generally monopseudopodial, and changes in its shape are more limited. The mucous coat, less pronounced in this species, is most prominent at the very active uroid, the site of endocytosis. The membrane renewal cycle in Pelomyxa has not been studied to date.     

Modelling of fluid-phase endocytosis kinetics in the amoebae of the cellular slime mouldDictyostelium discoideum. A multicompartmental approach

Fluid-phase endocytosis (pinocytosis) kinetics were studied inDictyostelium discoideum amoebae from the axenic strain Ax-2 that exhibits high rates of fluid-phase endocytosis when cultured in liquid nutrient media. Fluorescein-labelled dextran (FITC-dextran) was used as a marker in continuous uptake- and in pulse-chase exocytosis experiments. In the latter case, efflux of the marker was monitored on cells loaded for short periods of time and resuspended in marker-free medium. A multicompartmental model was developed which describes satisfactorily fluid-phase endocytosis kinetics. In particular, it accounts correctly for the extended latency period before exocytosis in pulse-chase experiments and it suggests the existence of some sorts of maturation stages in the pathway.     

Caffeine, an inhibitor of endocytosis in Dictyostelium discoideum amoebae

The effect of the trimethylxanthine, caffeine, was examined on the growth and endocytosis pathways of the vegetative amoebae of the cellular slime mold Dictyostelium discoideum. Caffeine at concentrations of 1.5-3 mM was found to inhibit axenic growth, fluid-phase pinocytosis, and secretion of lysosomal enzymes. Cell viability was unaffected by incubation for 16 hours with 5 mM caffeine but decreased markedly thereafter. Phagocytosis of the bacterium Escherichia coli by Dictyostelium amoebae was also inhibited by caffeine, although at concentrations twofold to threefold higher. Caffeine rapidly entered into amoebae to reach an equilibrium between extracellular and intracellular concentrations, and it was not appreciably metabolized by Dictyostelium. Inhibition of growth and endocytosis was reversible upon removal of the drug and was partially counteracted by 10 mM adenosine. As caffeine discharged intracellular calcium stores in Dictyostelium (Abe et al., 1988), its inhibitory effect on endocytosis could result from the perturbation of calcium homeostasis. In agreement with this hypothesis, the cation La3+ (10 microM), a Ca2(+)-transport inhibitor, also strongly reduced fluid-phase pinocytosis.     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

Embryonic gut differentiation in nematodes: endocytosis of macromolecules and its experimental inhibition

During embryogenesis of Caenorhabditis elegans cytoplasmic components are transferred from nongut cells into the developing gut primordium and an exo/endocytosis mechanism has been hypothesized (Bossinger and Schierenberg 1992). To test endocytotic activity of the gut primordium, we compared the uptake of different fluorochrome-conjugated marker molecules in two nematode species, C. elegans and Cephalobus spec., which differ in the pattern of early cleavage and cell-cell communication. We found no uptake of dextran (as a marker for pinocytosis) but rapid internalization of 30-fold larger transferrin molecules (as a marker for receptor-coupled endocytosis) into the differentiating gut primordium in both nematodes. The two studied species differ with respect to when this process starts. While the uptake of macromolecules in the fast developing C. elegans is first observed at a stage when essentially all cells of the hatching juvenile have been generated, in the slow developing Cephalobus endocytosis begins during the early proliferation phase when only two gut precursor cells are present. We found that the polysulfated hydrocarbon dye trypan blue and the cationic amphiphilic drug chlorpromazine both inhibit endocytosis into the gut primodium.     

RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION IN AMOEBA PROTEUS

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 either in the substratum or in solution.     

Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.     

Comparison of pinocytosis and phagocytosis in Acanthamoeba castellanii

Acanthamoeba, with high rates of phagocytosis and pinocytosis of the non-concentrative type, offers favorable experimental material for investigation of similarities and possible differences in these two modes of uptake. Phagocytosis was measured by the rate of uptake of latex beads and pinocytosis by the rate of uptake of radioactive inulin and albumin. The effects of the metabolic inhibitors NaN₃, NaCN, NaF, iodoacetate, 2,4-dinitrophenol and cold were found to be identical on both forms of endocytosis. Both endocytic processes were suppressed by inhibitors of aerobic metabolism and low temperature and were not appreciably affected by inhibitors of glycolysis. The cells recovered capacity to endocytose after exposure to all these compounds except 2,4-dinitrophenol, which was irreversibly toxic. Endocytosis and O₂ consumption were measured as a function of temperature. Below 5 °C both phagocytosis and pinocytosis ceased; between 9 and 15 °C uptake was less than 10% that at 29 °C. From 16 to 29 °C uptake was a linear function of temperature for both pinocytosis and phagocytosis. Curves for O₂ consumption and endocytosis both showed breaks at about 16 °C. Concanavalin A (ConA) inhibited both types of endocytosis more than 50% at concentrations as low as 5 μg/2 × 10⁵ cells/ml. Pinocytosis and phagocytosis were also measured simultaneously in the same cells. Increasing the rate of phagocytosis suppressed pinocytosis, but the combined volume of the two forms of uptake was essentially constant. In contrast, the estimated combined surface intake varied over a two-fold range. These data show no differences between phagocytosis and pinocytosis of the non-concentrative type, and suggest that control of the rate of endocytosis is determined by the volume of an internal compartment. The volume of this compartment, estimated by measuring the volume of latex beads that “saturate” the phagocytic mechanism, amounted to about 500 μm³/cell or roughly 15% of the cell volume.     

Indirect immunofluorescent staining of the microtubules in interphase and mitotic amoebae of the myxomycetePhysarum polycephalum

Summary The microtubules ofPhysarum amoebae have been decorated with rat antibodies against yeast tubulin. The indirect fluorescent staining observed in interphase amoebae and in flagellated amoebae is consistent with the three-dimensional reconstructions previously deduced from electron microscopic studies. Mitotic amoebae exhibit a pattern of fluorescence which is similar to that exhibited by mammalian cells and is consistent with the previous electron microscopic studies, except that we also observe pole-pole microtubule fibers during metaphase and anaphase and the presence of a typical midbody during cytokinesis. The various types of tripolar mitosis which are observed suggest that there is a regulatory mechanism allowing the formation of pseudo-bipolar mitotic apparatuses in amoebae possessing more than two mitotic centers during mitosis. The mitotic center, located in the middle of the centrosphere, is not fluorescent after staining of the monoasters induced with taxol suggesting the absence of tubulin in the mitotic center.     

Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment.

We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).     

Effects of induced pinocytotic activity and extreme temperatures on the morphology of golgi bodies in Amoeba proteus

Summary The morphology of the Golgi apparatus of Amoeba proteus can be influenced by substances inducing pinocytotic activity as well as by extreme temperatures. During the ingestion of a solution of 0.5% egg white the number of Golgi bodies decreases from 100% measured in control cells to 82% measured in cells showing induced pinocytosis. Simultaneously the ratio of the surface area of the cisternae at the proximal face to that of the vesicles at the distal face of single dictyosomes remains constant (1.74–1.72).The decrease and increase of the temperature of the culture medium to 4° C and 30° C respectively, causes the disappearance of most of the dictyosomes. After keeping the cells for 3–10 h at these temperatures the number of Golgi bodies was only 5–10% of the controls. A continued treatment with cold or warm culture medium leads to a partial reorganization of dictyosomes. After 15 h the number of Golgi bodies counted per cell returned to 57% at 4° C and 38% at 30° C. The ratio of the surface area of the Golgi cisternae to the surface area of the Golgi vesicles also alters under the influence of extreme temperatures. The values measured after treating the cells for 3 h, 4 h 10 h and 15 h at 4° C and 30° C amounted to 0.75, 0.85, 1.14 1.53 and 0.93, 0.38, 0.88, 1.60, respectively, compared to 1.72 of control amoebae.The different values of the ratio of the surface area of cisternae to that of vesicles indicate that there are strong morphological changes of single dictyosomes.     

Possible regulation of cation-induced pinocytosis in Amoeba proteus by phospholipase A

We have studied the effects of exogenous phospholipids and compounds which are known to alter the activity of phospholipase A (PLA) on Ca2+-dependent, Na+-induced pinocytosis in Amoeba proteus. The PLA-inhibitors mepacrine, p-bromophenacyl bromide (pBPB) and Rosenthal's inhibitor depressed pinocytosis. Normal pinocytotic intensity was restored by the addition of Ca2+ or picomolar concentrations of lysolecithin. Very low concentrations of lysophospholipids and different molecular species of lecithins increased the capacity for pinocytosis in starved amoebae. The effect of the lecithins but not of the corresponding lysolecithins was abolished by PLA-inhibitors. Also, the restoration of the pinocytotic capacity of starved amoebae by melittin and mastoparan, which are known to stimulate PLA, was inhibited by mepacrine and pBPB. Isolated amoeba plasma membranes contain phospholipase A1 and A2 activity and the amoebae secrete a lipid (PRF, pinocytosis regulating factor) which has lysolecithin-like effects on pinocytosis. The enzyme activities and the release of PRF were markedly decreased by the PLA-inhibitors. Our observations support the hypothesis that PRF is a lysophospholipid that may constitute a signal for the formation of pinocytotic channels in the initial stages of pinocytosis. The phospholipase A activity of the amoeba must therefore be assigned an important role in the regulation of the Ca2+-dependent, cation-induced pinocytosis.     

Pinocytosis and locomotion in amoebae

Summary Different methods were used to demonstrate the existence of Ca⁺⁺-binding sites (Ca⁺⁺-bs) at the plasma membrane ofAmoeba proteus. In pinocytoting animals the number (indicated by the average distanced in nm) and size (average longitudinal axiss in nm) of Ca⁺⁺-bs at the cytoplasmic surface of the cell membrane were significantly increased (d=162±15;n=41 ands=93±5;n=47) in comparison to controls (d=208 ±21;n=37 ands=59±8;n=45). The ratio of P: Ca obtained by X-ray microanalysis was in the range of 1.5. The differences observed in the two experimental groups of amoebae are explained by conformational changes in the molecular structure and an increased Ca⁺⁺-permeability of the plasma membrane during induced pinocytosis.Microplasmodia of the acellular slime moldPhysarum polycephalum investigated for comparison were found to have no Ca⁺⁺-bs at the interior cell surface.     

Observations on induced amitosis in Acanthamoeba

Methods are described for the induction of amitotic cell division in Acanthamoeba rhysodes. Induced amitotic cell division in this organism is similar to normal cytokinesis in many respects, however, the nucleus is partitioned during its interphase state so that the daughter products of amitosis are not viable. It is proposed that the induction of amitotic cell division causes the amoeba to produce the normal cytoplasmic components responsible for cell division in the absence of nuclear mitosis. This is not a normal stage in the amoeba's life cycle and it appears to be a genetic defect unique to this strain of Acanthamoeba.Evidence is presented that induction of amitotic cell division requires protein synthesis but not ribonucleic acid synthesis. Further, induced amoebae require a period of adhesion to a foreign substrate before they are capable of amitosis. The pattern of amitotic cell division could be interpreted as a segregation of discrete cytoplasmic units, generated during induction.     

Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells [published erratum appears in J Cell Biol 1990 Mar;110(3):859]

Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF- stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.     

Depolarization of the cell membrane causes inhibition of cell locomotion and pinocytosis inAcanthamoeba castellanii andAmoeba proteus

Summary 10 M CCCP protonophore in an acidic medium causes depolarization of the cell membrane and immediate cessation of locomotion inAcanthamoeba castellanii andAmoeba proteus. In the basic media there is no depolarization or inhibition of cell locomotion. Other depolarizing agents (alkali cations, crown molecules) also stop locomotion and induce pinocytosis in amoeba. Pinocytotic uptake of horseradish peroxidase byAcanthamoeba castellanii is increased by 69% in the presence of CCCP in the medium at pH 5.7 but is not influenced at higher pH values. This might indicate that both amoeboid locomotion and pinocytosis are controlled by membrane potential.     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium