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.     

Pinocytose und Bewegung von Amöben

Zusammenfassung Zur Bestimmung des cytotischen Membran-Turnovers wurden morphometrische Messungen an über 60 Zellen der Art Amoeba proteus durchgeführt. Danach nehmen diese Amöben 0,14% ihrer Zelloberfläche pro Minute durch permanente Endocytose in das Cytoplasma auf. A. proteus benötigt also insgesamt 12 Std, um die gesamte Zellmembran während der normalen Bewegung einmal zu erneuern. Infolge des geringen Membranturnovers kann der permanenten Endocytose keine aktive Bedeutung für die Erzeugung der Bewegungstriebkraft zugesprochen werden. In Übereinstimmung mit dieser Vermutung ließ sich eine Abhängigkeit zwischen Fortbewegungsgeschwindigkeit und Endocytoseintensität nicht nachweisen.Entsprechende Messungen mit drei verschiedenen Endocytoseinduktoren ergaben für die induzierte Endocytose in Abhängigkeit von der verwendeten Substanz eine wesentlich höhere Ingestionsrate von 0,43–2,25%/min. Derartige Spitzenwerte können allerdings nur innerhalb eng begrenzter Zeiträume von 15–30 min erzielt werden. Vergleicht man dagegen die Membranaufnahme während der permanenten und induzierten Endocytose über längere Zeitintervalle (4–5 Std), so bleibt die induzierte Endocytose mit 0,05–0,12%/min in der Intensität deutlich hinter der permanenten Endocytose (0,14%/min) zurück. Eine Erhöhung der Temperatur auf 30° und eine Erniedrigung auf 15°C bringen beide Endocytoseformen zum Erliegen.Die permanente Endocytose muß bei Amöben neben der Phagocytose als der wichtigste Mechanismus zur kontinuierlichen Aufnahme gelöster und suspendierter Stoffe (bis zur Größenordnung von Bakterien) angesehen werden.

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Pinocytosis and locomotion of amoebae

Summary Cytotic membrane turnover of Amoeba proteus was morphometrically studied in more than 60 cells. The results obtained indicate that 0.14% of the total cell membrane area per minute is ingested by permanent endocytosis. Consequently during normal locomotion the total cell membrane area is renewed once within 12 hours.This rate is too low to play any role in the generation of motive force. No correlations were found between the rates of locomotion and permanent endocytosis.Comparative measurements on cells treated with three different substances inducing endocytosis reveal that induced endocytosis leads to an increased rate of membrane ingestion of 0.43–2.25%/min depending on the substance used. These high rates, however, are only maintained during short periods of time (15–30 min). When the rates are calculated on the basis of long periods of time (4–5 hours), it is obvious that induced endocytosis (0.05–0.12%/min) is less effective in long term membrane turnover than permanent endocytosis (0.14%/min). Endocytotic activity is completely abolished by both the increase and decrease in temperature to 30°C and 15°C respectively.In addition to discontinuous phagocytosis permanent endocytosis is an important mechanism for continuous ingestion of fluid including particles up to the size of bacteria.

Der Kultusminister des Landes Nordrhein-Westfalen unterstützte die Untersuchung aus Überschußmitteln des Westdeutschen Rundfunks.     

Pinocytose und Bewegung von Amöben

Zusammenfassung Während der aktiven Fortbewegung von Amoeba proteus zeigt das Uroid einen charakteristischen Formwandel, der aus vier Stadien besteht. Im Verlaufe dieser sich rhythmisch wiederholenden Phasen treten bei der normalen Wanderung der Zellen Pinocytosevorgänge auf, die lieht- und elektronenmikroskopisch durch die Entstehung schmaler Kanäle charakterisiert sind. Die Lebensdauer der schlauchförmigen Plasmalemm-Einstülpungen beträgt in der Regel nur wenige Minuten. Die Ingestion von Zellmembran am caudalen Pol von Amoeba proteus ist an die amöboide Bewegung der Zellen gebunden. Es wird vorgeschlagen, diesen Vorgang als Permanente Pinocytose zu bezeichnen, um ihn von dem schon länger bekannten Prozeß der induzierten Pinocytose zu unterscheiden. Die Plasmalemminvaginationen der permanenten Pinocytose sind cytomorphologisch mit den Kanälen der induzierten Pinocytose identisch. Unterschiede bestehen lediglich in quantitativer und topographischer Hinsicht.Das Schicksal der durch die permanente und induzierte Pinocytose aufgenommenen Zellmembran ist mit Ausnahme zeitlicher Unterschiede weitgehend identisch.In Übereinstimmung mit der Lokalisation der permanenten Pinocytose in der Uroidregion konnte ein von der Mucoidschicht bewirkter Transport größerer, an der Zelloberfläche adsorbierter Markierungspartikel an das Hinterende der Tiere festgestellt werden.Die an der Mucoidschicht der Zellmembran anhaftenden, aber von den Amöben nicht ingestierten Markierungssubstanzen werden an den Boden des Versuchsgefäßes abgegeben.

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Summary During active locomotion of Amoeba proteus, the uroid shows characteristic changes in shape and behaviour consisting of four different stages. In the course of these rhythmically repeated transformations of the uroid, pinocytotic processes can be demonstrated, which are bound to the normal locomotion of the cells. This sort of pinocytosis is characterised by the formation of small channels as revealed by light- and electron-microscopy. The tube-like invaginations exist only for a few minutes.The ingestion of cell membrane at the caudal pole of A. proteus is bound to the amoeboid movement. In order to distinguish this process from the well known induced pinocytosis, the term permanent pinocytosis is proposed. Cytomorphologically, the plasmalemminvaginations of induced as well as of permanent pinocytosis are identical. The differences found are of quantitative and topographic nature. With the exception of temporal differences, the fate of the cell membrane ingested by permanent and induced pinocytosis is identical as well.In agreement with the localisation of permanent pinocytosis in the uroid region, a transport of particles (adhering to the mucous layer of the cell membrane) to the uroid could be demonstrated.Tracer particles, transported in this way to the uroid, accumulate at the caudal pole of the cell. In the case they are not incorporated by the process of permanent pinocytosis, they are deposited to the bottom of the glass vessel.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft im Rahmen des Schwerpunktprogrammes Molekulare Biologie.     

Effects of induction of endocytosis on adhesiveness ofAmoeba proteus

Summary The adhesive behaviour ofAmoeba proteus during induction of endocytosis has been studied with interference reflection microscopy. Although phagocytosis and pinocytosis, jointly termed endocytosis, are closely related phenomena, it was found that the way they affect adhesiveness of amoebae differs substantially. Phagocytosis is accompanied by an increase in area of cell surface contacting the substratum, whereas during pinocytosis a sharp decrease of contact area is observed. The resistance to detachment increases in prey-stimulated and phagocytosing amoebae but declines as the pinocytosis is being induced. These results suggest that the influence of the induction of phagocytic activity on the cell periphery differs from that of induction of pinocytosis.     

Characterization of f-Met-Leu-Phe-stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry

N-formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.     

A Simple Method for Making Tetrahedra to Illustrate Stereochemicai Principles

Actin and myosin, the major proteins of the contractile complex actomyosin, have now been demonstrated to be important constituents of many eukaryotic cells. As in muscle, their role is primarily that of a contractile system. This system is thought to underly all aspects of cellular motility: locomotion, shape change, mitosis and meiosis, cell division, cytoplasmic streaming, organelle motion, endo-cytosis (pinocytosis, phagocytosis), and exocytosis.

We describe here a simple experimental system to demonstrate quantitatively aspects of motility and its regulation in the slime mould Physarum polycephalum     

Selective effects of the PKC inhibitors Ro 31-8220 and CGP 41 251 on PMN locomotion,cell polarity,and pinocytosis

Using two newly synthesized inhibitors, Ro 31-8220 and CGP 41 251, of protein kinase C (PKC), we analyzed: (1) how distinct PMN functions (shape changes, locomotion, pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) locomoting cells into non-locomoting highly pinocytic cells using PMA, and (3) PMA-stimulated cells showing marked pinocytosis into locomoting or into resting cells using Ro 31-8220. It is thus possible to selectively manipulate PMN function (resting state, locomotion, marked pinocytosis), indicating that there are different regulatory pathways. It was not possible to induce locomotion and marked pinocytosis simultaneously, indicating crosstalk between pathways. Ro 31-8220 inhibited PMA-induced shape changes (nonpolar cells) and pinocytosis, but not fNLPNTL-induced shape changes (polarity) and pinocytosis. At higher concentrations, Ro 31-8220 alone elicited cell polarity and chemokinesis, indicating that a constitutively active protein kinase is involved in maintaining the spherical shape of resting PMNs. Functional effects of another PKC inhibitor, CGP 41 251, on neutrophil function were strikingly different. CGP 41 251 selectively inhibited fNLPNTL-induced polarity and locomotion (but not colchicine or Ro 31-8220-induced polarity), and it failed to inhibit PMA-induced, stimulated pinocytosis and shape changes. Although the effects of Ro 31-8220 vs. CGP 41 251 on PMN function were strikingly different, the inhibition of profiles for constitutive and for fNLPNTL- or PMA-induced protein phosphorylation in intact PMNs showed only small differences, which could not yet be conclusively related to cell function. © 1994 Wiley-Liss, Inc.     

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (<2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t_1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 μM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.     

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.     

Degeneration of species-rich Calthion palustris hay meadows; some considerations on the community concept

Abstract. An overview of the vegetation history of Calthion palustris meadows is presented with special emphasis on the natural habitats of the character species. The response of meadow species under the influence of drainage was calculated using species-environment response curve techniques. The changes were monitored in permanent plots situated on sites with various intensities of drainage. The similarity with an undisturbed local reference type of the hay meadow community was calculated for all plots and years. On this basis a half-life time of the Calthion palustris stands could be assessed. The half-life time was small in the plots which are most affected by drainage. The response to drainage of character species of the Calthion palustris was very different. This imposes problems for some definitions of the community concept.     

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.     

Role of Contractile Microfilaments in Macrophage Movement and Endocytosis

PHAGOCYTOSIS of bacteria and other large particles and pinocytosis of colloids—two processes collectively termed endocytosis—are among the characteristic properties of macrophages. When mouse peritoneal macrophages in culture are observed by phase contrast microscopy, most small endocytotic vesicles (pinosomes) are seen to be formed in the region of ruffled membrane activity, usually in a pseudopod¹. The phase-lucent pinosomes move rapidly towards the Golgi region where they unite with phase-dense granules to form secondary lysosomes. Although there is evidence that both phagocytosis and pinocytosis in macrophages have a high temperature coefficient and require metabolic energy¹, the mechanism of endocytosis is unknown. Clearly, movement of the plasma membrane and directional movement of pinosomes is involved. During the past few years attention has been drawn to the apparent association in many cells between movement and the presence of contractile microfilaments of about 50 Â diameter^2,3. Some of these are actin-like and can bind heavy meromyosin to give distinctive “arrowhead” structures in electron micrographs⁴. One of us (S. de P., in preparation) has found that the peripheral or cortical cytoplasm of macrophages contains a network of microfilaments, some of which may be inserted into the plasma membrane. These filaments bind heavy meromyosin (Figs. 1 and 2) and details of their structure and disposition will be published later.     

The influence of concanavalin A and cytochalasin B on pinocytotic activity inAmoeba proteus

Summary Addition of concanavalin A to a suspension ofAmoeba proteus brings about cellular agglutination and the formation of what appear to be pinocytotic channels in cell surface projections. Although concanavalin A apparently brings about pinocytotic channel formation, it does not elicit bulk medium uptake or surface membrane turnover. Cytochalasin B brings about an initial cessation of locomotion and the development of a number of randomly distributed pseudopods. After a 30 to 45 minute exposure to cytochalasin B, the cells resume their normal appearance and pattern of locomotion. Cytochalasin B itself has no influence on inducing pinocytotic channel formation or membrane turnover, but when pinocytosis is induced with 0.01% alcian blue, pinocytotic activity is greatly intensified by the presence of cytochalasin B.     

Effects of the myosin inhibitor 2,3-butanedione monoxime (BDM) on cell shape, locomotion and fluid pinocytosis in human polymorphonuclear leucocytes

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC(50)=15.5 m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC(50)=8.4 to 10 m m). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid pinocytosis produced non-motile spherical cells (IC(50)=25 to 30 m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs.     

Size variation in Trebralia palustris (Gastropoda: Potamididae) of Iriomote Island,southern Japan,and its effect on some population characteristics

The growth stages of Trebralia palustris can easily bedistinguished by the presence of a varix on the last whorl and the shape ofthe outer lip. In a T. palustris population in the mangal of IriomoteIsland, southern Japan, juveniles are the most abundant and sub-adults arethe least abundant. The mean size of sub-adults is not different from that ofadults in sites with similar environmental conditions, but the mean sizesdiffer in different life environments. Sub-adult and adult snails in awell-developed mangrove stand of Rhizophora stylosa and Bruguiera gymnorrhiza with rich food resources are larger than those in arather open stand of short Avicennia marina trees with comparativelypoor food resources. The size of snails positively affected some population traitssuch as fecundity and the grazing rate of mangrove litter.     

Fine Structure and Taxonomic Position of the Giant Amoeboid Flagellate Pelomyxa palustris1,2

ABSTRACT Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 μm in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.     

Transportation mechanism for vanillin uptake through fungal plasma membrane

Protoplasts of the basidiomycete, Fomitopsis palustris (formerly Tyromyces palustris), were utilized to study a function of the fungal plasma membrane. Fungal protoplasts exhibited metabolic activities as seen with intact mycelial cells. Furthermore, the uptake of certain compounds into the protoplast cells was quantitatively observed by using non-radioactive compounds. Vanillin was converted to vanillyl alcohol and vanillic acid as major products and to protocatechuic acid and 1,2,4-trihydroxybenzene as trace products by protoplasts prepared from F. palustris. Extracellular culture medium showed no activity responsible for the redox reactions of vanillin. Only vanillic acid was detected in the intracellular fraction of protoplasts. However, the addition of disulfiram, an aldehyde dehydrogenase inhibitor, caused an intracellular accumulation of vanillin, strongly suggesting that vanillin is taken up by the cell, followed by oxidation to vanillic acid. The addition of carbonylcyanide m-chlorophenylhydrazone, which dissipates the pH gradient across the plasma membrane, inhibited the uptake of either vanillin or vanillic acid into the cell. Thus, the fungus seems to possess transporter devices for both vanillin and vanillic acid for their uptake. Since vanillyl alcohol was only observed extracellularly, the reduction of vanillin was thought to be catalyzed by a membrane system.     

Pinocytose und Bewegung von Amöben

Zusammenfassung Hyalodiscus simplex zeigt während der normalen Fortbewegung eine auf den Uroidbereich beschränkte permanente Endocytoseaktivität, durch die kontinuierlich Zellmembran und extrazelluläres Milieu ins Innere der Zelle aufgenommen wird. Nach unter-schiedlich langer Inkubation in einer 2.5% igen Thorotrastlösung kann die Markierungssubstanz bei ihrer Passage durch die Zelle morphologisch verfolgt werden. Das endocytierte Thorotrast gelangt in ein intrazelluläres Membransystem, welches für die Verdauung verantwortlich ist und dessen Gesamtoberfläche in der Größenordnung der Zelloberfläche liegt. Verschiedene, funktionell miteinander in Verbindung stehende Vakuolen stellen die charakteristischen Komponenten des intrazellulären Verdauungssystems dar.Die morphometrische Auswertung der Markierungsversuche hat gezeigt, daß der Membran-Turnover etwa 2% pro Minute beträgt. Hyalodiscus simplex benötigt demnach bei normaler Lokomotion zur vollständigen Regeneration der Zellmembran eine Zeit von 7,5–8 Std. Da die Oberfläche trotz permanenter Endocytoseaktivität auch nach unterschiedlich langer Thorotrastinkubation stets konstant bleibt, darf vermutet werden, daß die endocytotisch aufgenommene Zellmembran durch einen noch unbekannten Mechanismus wieder regeneriert wird.

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Pinocytosis and locomotion of amoebaeVII. Quantitative studies on membrane-turnover in Hyalodiscus simplex

Summary During locomotion Hyalodiscus simplex is active in permanent endocytosis at the uroid region. Thereby plasma membrane and extracellular medium are translocated into the cell interior. After different periods of incubation in a 2.5% solution of thorotrast the tracer can morphologically be followed during the passage through the cell. The ingested thorotrast is collected within a vacuolar system. This system is involved in intracellular digestion and composed of different types of vacuoles, which are structurally separated but functionally interconnected. Its total surface area approximately corresponds to the surface area of the entire cell. As demonstrated by morphometric analysis, the membrane turnover is about 2% per minute. Therefore, in the migrating amoebae 7.5–8 hours are required for the complete regeneration of the plasma membrane. Since the surface area of the cell remains constant despite of permanent endocytotic activity the ingested plasma membrane must be continuously regenerated by an unknown mechanism.

Prof. Dr. W. Stockem, Institut für Cytologie und Mikromorphologie der Universität Bonn, 53 Bonn 1, Gartenstr.61a. Das Landesamt für Forschung des Landes NRW unterstützte die Untersuchungen durch eine Sachbeihilfe.     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

The Fine Structure of the Cytostome of the Apostomatous Ciliate Hyalophysa chattoni*

SYNOPSIS. The fine structure of the organelles concerned with the ingestion of exuvial fluid by the trophont of the apostome ciliate, Hyalophysa chattoni, has been examined. One of the taxonomic characteristics of the order Apostomatida is that cytostomes of ciliates within the taxon are reduced and evolving toward astomy. When examined by electron microscopy the cytostome of H. chattoni appears as a small region of active pinocytosis which is continuous with a very large cortical area, the extended cytostome. The fine structure of the extended cytostome resembles that of the cytostomes of ciliates from other orders in that it is covered by a single membrane underlain with microtubular ribs. Beneath the extended cytostome are accumulations of peculiar organelles that may represent stored membrane for recycling during food vacuole formation. Associated with the site of pinocytosis is a complex fiber that may be contractile.