Freshwater Amoebae Research Articles

Direct membrane effects of morphine and endorphins on amoeba proteus

Morphine, leu-enkephalinamide, met-enkephalin, α-neoendorphin and its Arg 8 1–8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 μM. Significant effects of leu-enkephalin and naloxone are obtained at 180 μM. All compounds have reached their maximal activity at 720 μM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds.

Direct observation of microtubule dynamics in Reticulomyxa: Unusually rapid length changes and microtubule sliding

AbstractMicrotubule dynamics has been studied extensively in vitro, but comparatively little information is available on the in vivo behavior of microtubules. Here we report on the assembly, disassembly, and sliding of microtubules in the giant freshwater amoeba, Reticulomyxa. We have found that treating the cell with 0.25% trypsin induces the rapid formation of exceedingly flat areas within the reticulopodial network, allowing for the direct observation of microtubule behavior by DIC optics and computer‐enhanced video microscopy. In flattened areas, microtubule sliding occurs at rates of between 1 and 6.5 μm/sec. The average rate of microtubule assembly is 1.6 μm/sec, while microtubule disassembly takes place at about 4 μm/sec and can reach up to 19.5 μm/sec. We also observed many cases where a microtubule forms a hairpin loop and eventually breaks, resulting in bidirectional disassembly from the point of breakage. Our observations demonstrate sliding of cytoplasmic microtubules in vivo. The high rates of microtubule assembly/disassembly in this cell type are difficult to reconcile with conventional views of association and dissociation processes at microtubule ends and suggest unconventional mechanisms for the growth and shrinkage of microtubules.

The effects of vasopressin and related peptides on osmoregulation in Amoeba proteus

We describe the effects of arginine-vasopressin (AVP) and five related peptides on the contractile vacuole, the osmoregulatory organelle of the fresh water Amoeba proteus. Arginine-vasopressin, lysine-vasopressin, and SKF 101926, a synthetic antagonist of vasopressin, cause a significant increase in the rate of output of the contractile vacuole. Deamino-vasopressin (dAVP), oxytocin, and arginine-vasotocin have no such activity, although dAVP interferes with the action of AVP when present in equimolar concentration. Relatively high concentrations are required and the effect of active peptides is readily reversible. When the normal, hypotonic medium (a synthetic pond water) is replaced by isotonic sucrose, the action of AVP on the vacuole is abolished. Thus vasopressin is believed to act by increasing permeability of the Amoeba plasma membrane to water.

An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa.

The rapid, vectorial, microtubule-associated transport of organelles is believed to be mediated by specific mechanochemical transducers. Recent studies of various metazoan cells have allowed the identification of novel microtubule-dependent translocator molecules capable of promoting microtubule gliding across glass surfaces and translocation of inert beads along microtubules. These translocators could be involved in force generation for directional organelle movements in vivo. Here we report the identification of a microtubule-binding protein with characteristics expected for an organelle translocator in the giant freshwater amoeba Reticulomyxa. This factor has an apparent relative molecular mass (Mr) of 440,000 (440K) and sediments at 20-22S in sucrose-density gradients. It binds to microtubules under conditions of ATP depletion, possesses an ATPase activity and is sensitive to ultraviolet-induced, vanadate-dependent cleavage. Although its pharmacological properties differ from those of axonemal dynein, it can be considered to be a variant of cytoplasmic dynein. The Reticulomyxa high-molecular-weight protein (HMWP) promotes rapid, bidirectional movement of latex beads along Reticulomyxa microtubules in vitro at an average speed of 3.6 micron s-1. This protein, therefore, is a likely candidate for a microtubule-dependent motor.

Responses of freshwater amoebae to salinity changes

1. 1. Salinity effects on freshwater amoebae were quantitatively studied. 2. 2. The locomotive velocity of Vannella simplex descended slowly in company with the decrease in the concentration of bathing saline. A sudden decrease in velocity took place in response to solutions with more than three times the salinity of the culture medium. 3. 3. Trichamoeba osseosaccus were found to be able to adjust themselves to increased salinity within several hours, and this process of adaptation was examined. 4. 4. A step-wise effect in adaptation was confirmed.

Cytoskeletal architecture and motility in a giant freshwater amoeba, Reticulomyxa.

Reticulomyxa is a large, multinucleated freshwater protozoan with striking intracellular transport. Cytoplasmic streaming and saltatory movements of individual organelles (at rates of up to 25 micron/sec) are observed within the naked cell body and the extensive reticulate peripheral network of fine cytoplasmic strands. As demonstrated by video-enhanced light microscopy, individual organelles move only when associated with cytoskeletal linear elements. The linear elements are composed of mixed colinear bundles of microtubules and actin filaments, which form the backbone of the reticulopodial network. The constant branching, sprouting, and fusion of network strands suggest unique membrane properties and an unusually dynamic cytoskeleton. The electrophoretic mobility of Reticulomyxa tubulins and the lack of crossreactivity with several antibodies known to react with many plant and animal tubulins suggest that they may differ from other tubulins more widely than might be expected. Reticulomyxa's large size, the rapidity and pervasiveness of the two forms of transport, and the simple and ordered cytoskeleton make the organism well suited for future studies on the mechanisms of intracellular transport.

The Extent of Algal and Bacterial Endosymbioses in Protozoa1,2

Long neglected has been the extensive and more or less intimate association of protozoa with a wide variety of other cells, either prokaryotic or eukaryotic in nature. Yet study of such relationships can provide important information concerning certain basic aspects of cellular evolution in general. A survey is offered here of the whole range of such symbiotic associations (i.e. with species of protozoa serving as hosts) with the purposes of drawing attention to the exciting possibilities of such research and of reviewing significant findings made to date. Because of the vastness of the overall field, examples and discussion are primarily limited to consideration of the following major studies: methanogenic bacteria in certain ciliates, bacterial endosymbionts of the large freshwater amoeba Pelomyxa palustris (itself an amazing organism from an evolutionary/phylogenetic point of view), the rod‐shaped bacteria found in Amoeba proteus, the “Greek‐letter” prokaryotes of Paramecium species, the xenosomes (sensu stricto) of the marine scuticociliate Parauronema acutum, and the diverse algal endosymbionts of similarly diverse protozoan taxa–ciliates, flagellates, radiolarians, acantharians, and foraminifera.

An Isolated Cytoskeletal Framework From Reticulomyxa: A New Model System For Intracellular Transport

Pharmacological and correlative light and electron microscopic studies suggest that the two major cytoskeletal systems, the microtubules (MTs) and microfilaments (MFs), are involved in the directed transport of intracellular organelles. However, the details of organelle-cytoskeletal interactions and force generation are not known, nor is it clear whether transport is solely a MT or MF-based phenomenon, a coordinated effort, or if each system supports different classes of motility. Here I describe an isolated cytoskeletal “framework” derived from an unusual species of freshwater amoeba that provides a model system in which to examine these questions. We are intending to use this preparation to study the interactions with and movement of both isolated native and artificial organelles.

The Effects of Low Temperatures on the Growth of Four Fresh-Water Amoebae (Protozoa: Gymnamoebia)

The Effects of Low Temperatures on the Growth of Four Fresh-Water Amoebae (Protozoa: Gymnamoebia)

Further studies on respiratory rates of freshwater amoebae (Rhizopoda, Gymnamoebia)

The respiration rates of axenically culturedAcanthamoeba castellanii (Gymnamoebia) and of the test organismTetrahymena pyriformis (Ciliophora) were compared using both Warburg and cartesian diver respirometer techniques at 20°C. Furthermore, the latter procedure was employed to measure the oxygen consumption of the amoebae,Polychaos fasciculatum, Saccamoeba limax, andAmoeba sp. The linear relationship between respiration rate and mean cell volume in amoebae was reassessed in view of these additional estimates. The importance of this regression in ecological studies is discussed.

Respiratory studies on two small freshwater amoebae

The changes in respiration rate and mean cell volume induced by temperature within the range 10°C-25°C were investigated in two small species of freshwater amoebae,Saccamoeba limax Page andVannella sp. Mean cell volume varied in response to temperature, with maxima at 20°C inVannella sp. (10.15× 10(3) (±1.80)μm(3) and 15°C inS. limax (9.08×10(3) (±0.93)μm(3). Respiration rate increased over the temperature range investigated. The highest rates and the greatest rate of increase between temperatures occurred inVannella sp. Q10 ranged between 0.12 and 1.33 inS. limax and between 1.77 and 7.36 inVannella sp. A regression of log oxygen uptake versus log cell volume incorporating the data of the present investigation and the data of other workers on amoeba respiration is presented, and the ecological significance and application of such data discussed.

The Use of Physiological Characterizations in the Classification of Five Species of Acanthamoeba

Physiological tests were devised and used to compare 15 strains representing five species of soil and freshwater amoebae assigned to the genus Acanthamoeba. The tests gave an acceptable level of reliability and the pattern of responses was not affected by the differing growth rates of the organisms. There was some degree of overlap between the strains, as shown by the high level of inter-species similarities in relation to those between strains of the same species. Previous classifications of Acanthamoeba, which are based solely on morphological criteria, do not adequately reflect the diversity of these amoebae.

An Illustrated Key to Freshwater and Soil Amoebae with Notes on Cultivation and Ecology

An Illustrated Key to Freshwater and Soil Amoebae with Notes on Cultivation and Ecology

Studies on the heavy spherical (refractive) bodies of freshwater Amoebae. I. Morphology and regeneration of HSBS in Chaos carolinense

The heavy spherical bodies ofAmoeba andChaos are refractive organelles, varying in size from ca. 10 μm in diameter, down to the limit of resolution of the light microscope. These inclusions have many characteristics in common with the phosphate-rich, «volutin» granules of other unicellular organisms, but differ from the latter in being constantly present in healthy growing amoebae, while in other organisms they appear only under conditions of nutritional imbalance. The fine structure of the HSBs ofAmoeba andChaos show that the organelles are membrane-bound, with an electron translucent periphery, and an electron dense inner core which sublimes in the electron beam, but which is stabilized by lead staining. During centrifugation in vivo, the HSBs collect at the centrifugal pole of the amoebae; under favourable conditions, a HSB-sack is formed at the heavy pole, and can be excised, leaving amoebae, normal in other respects, but containing only ca. 5% of the normal complement of HSBs. Using this method for obtaining practically HSB-free amoebae, the regeneration of HSBs was studied inChaos carolinense, by determining the number and size distribution of HSBs in a standard volume in fixed, whole mounts by phase contrast microscopy. It was found that although the number of HSBs per standard volume in both fed and starved amoebae returned to that found in control, unoperated,Chaos by 7 days after operation, the size distribution did not return to normal until ca. 50 days after excision of the HSBs, indicating that HSBs originate from precursors in the cytoplasm. Operated amoebae showed normal feeding and locomotory behaviour, and their division rate was similar to that of control amoebae. However, HSB-free amoebae were more prone to rupture at an air-water interface, presumably owing to their lower specific gravity.

Electron microscopy of Trichamoeba villosa and amoeboid movement

The fine structure of a limax type amoeba Trichamoeba villosa is described. The ultrastructural organisation of this amoeba is compared with that of other fresh-water amoebae. In materials, fixed normally, the plasma filaments are observed very frequently. They are located in definite arrangement. The filaments are seen in abundance in cytoplasm subjacent to the fringed uroid and are of inconsiderable density at the front. The filaments are also observed under the plasmalemma. They regularly converge in the endoplasm at certain intervals of different pitch. An attempt has been made to correlate these findings with one of the theories of amoeboid movement—contraction-hydraulic mechanism.

Stratification within centrifuged amoeba nuclei.

Two species of large, fresh water amoebae were ultracentrifuged and studied with the electron microscope. Emphasis was placed on the stratification of the nucleoplasm, including nucleoli, within the confines of the nuclear envelope during interphase. Three major strata were found in the nuclei of both amoeba species, namely the centripetal nucleoplasm, the middle chromatin stratum, and the centrifugal nucleolar mass. In the highly radioresistant A. proteus, the nucleolar mass separated into a centripetal electron-opaque layer and a centrifugal electron-lucent layer. The latter layer appears to be missing from the radiosensitive P. illinoisensis. The nature of these nucleolar layers and their possible relationship to differences in radiosensitivity between the two species of amoebae is discussed. The contents of the heavier of the two nucleolar layers in A. proteus might be resistant to radiation damage and may possess radiorestorative capacity.

Amoeba proteus : Studying the Contractile Vacuole by Micropuncture

Direct measurements of the freezing point depression of the protoplasm and of the fluid from the contractile vacuole of fresh-water amoebae showed that the fluid in the vacuole is distinctly hypoosmotic to the protoplasm. Fourteen samples of protoplasm from amoebae, placed in a medium with a milliosmolality of 7, had an average osmolality of 101 milliosmoles with a range of 73 to 116. Eleven samples of vacuolar fluid had an average osmolality of 32 milliosmoles, with a range of 24 to 38. It is suggested that the fluid may be isoosmotic to the protoplasm when secreted and that salt is subsequently reabsorbed, leaving the vacuolar fluid hypoosmotic to the protoplasm.

Amoeba lescherae (nov. spec.)—Its morphology, cytology, and life-history

ABSTRACT 1. The discovery of a new species of large, free-living freshwater amoeba is recorded, and a description of the adult and of its reproduction is given. Although provisionally called Amoeba lescherae, it may have to be placed in the genus Chaos. 2. A method of cultivating it in the laboratory on a diet of small flagellates and ciliates subsisting on a pabulum of wheat grains is detailed. By starting them in Petri dishes pure line cultures are shown to be easily obtainable. 3. The resting nucleus, discoid in shape, has been shown to bear a general resemblance to that of Amoeba discoides as well as Amoeba proteus Y. Division is mitotic, and occurs once in twenty-four hours under optimum conditions. In contrast to Amoeba proteus Y the separation of the daughter amoeba is facilitated by the mechanical support of minute fragments of floating debris. 4. The cytoplasmic contents include square prismatic crystals, nutritive spheres, fat globules, neutral red bodies, and mitochondria. 5. It has been shown that while the adult has a general resemblance to Amoeba proteusY the respective developmental stages of the two amoebae are in marked contrast. 6. Agametogony is seen to begin with the emission of ‘chromidia’ from the nucleus. These chromidia form the rudiments of the agametes which differentiate into cysts. Excystationis described. 7. Agametogony occurs more frequently in Amoeba lescherae than in Amoeba proteus Y, the intervals between the ‘depression’ periods being shorter in the former than in the latter. 8. The development of the nucleus from the newly hatched amoeba to its adult condition has been traced, and the immature nucleus has been contrasted with that of species of M a y o r e l l a.

The Culturing of Fresh-Water Amoebae in the Laboratory

The Culturing of Fresh-Water Amoebae in the Laboratory

The Amoebae Living in Man: A Zoological Monograph

THIS is a very valuable piece of work, bringing order and critical intelligence to bear in a field of study, which, already touched by many observers, has immensely increased in activity and importance during the war. The common freshwater Amoeba, living freely in natural pools, has many relatives, some of which have been distinguished by definite characters of the nucleus, form of pseudopodia, cysts, and other characteristics as “good” species and even assigned to distinct genera. But there has been no careful cytological study of the various species, though here and there important observations have been made. When to these forms are added those living in the soil, in sea-water, and, lastly, those parasitic in other animals, we find that there is quite a large group of these “amœboid” organisms which have been recorded from this or that habitat by observers who were hurried by other work or insufficiently trained in cytological methods. As a consequence, without sound method or criticism, specific and even generic names have been given to “Amœbæ?,” parasitic or free-living, and misleading sketches of them have been published. A perplexing confusion of inaccurate statements obscures the whole subject.