Structure of filose amoeba <Emphasis Type="Italic">Rhogostoma minus</Emphasis> Belar 1921 (Cryomonadida,Cercozoa) cell

The structure of the unicellular cell of filose amoeba, Rhogostoma minus Belar, 1921, is studied. Zoospores, cysts, and multinuclear plasmodia have not been found. The cell is covered by a thin shell made out of organic matter. Narrow and branched pseudopodia arise from the pseudostome. The vesicle-shaped nucleus, endoplasmic reticulum, microbodies, and Golgi apparatus are of a usual structure. The oval-shaped mitochondria carry tubular cristae. No flagellar apparatus, fibrillar structures, or extrusive organelles have been found. The amoeba feeds on bacteria. The phylogeny of R. minus in regard to other filose amoebas and flagellates is discussed.     

Multiple Alleles and Other Factors Affecting Plasmodium Formation in the True Slime Mold Physarum polycephalum Schw*

SYNOPSIS. The life cycle of the true slime mold Physarum polycephalum includes 2 vegetative stages: the multinucleate coenocytic plasmodium and the uninucleate amoeba. A clone of amoebae established from a single spore does not normally yield plasmodia. Plasmodia are formed when amoebae from particular clones are mixed; thus plasmodium formation is said to be controlled by a ‘mating-type’ system. Previous work by the author with a sample of P. polycephalum derived from a single source revealed that 2 mating types were present and were determined by a pair of alleles at 1 locus. The present paper reveals the presence of 2 more mating types in a sample of P. polycephalum derived from a different source and provides evidence that these are determined by 2 alleles at the same locus as the other 2. Evidence for the presence of other inherited factors affecting plasmodium formation, the mode of action of these factors and possible explanations for the occurrence of plasmodia in single-spore cultures are also discussed.     

A new model of reticulopodial motility and shape: evidence for a microtubule-based motor and an actin skeleton

Cytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticollaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility-arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high-voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin-induced bodies. All aspects of cytochalasin-altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine-phalloidin; this staining is not observed in cytochalasin-treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin-based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule-dependent processes.     

Multiple Fission in Allogromia sp., Strain NF (Foraminiferida): Release,Dispersal, and Ultrastructure of Offspring1

The release, dispersal, and ultrastructure of juveniles arising through multiple fission in the benthic foraminiferan Allogromia sp., strain NF (Lee & Pierce, 1963) has been examined by light and electron microscopy. An extensive reticulopodial network participates in the dispersal of fully differentiated young as they emerge from the fragmented parental test. During the earliest stages of release, offspring are of two classes—aroused and unaroused. Unaroused juveniles, which have not extended pseudopods, attach externally to the network and are transported bidirectionally along its surface. Aroused juveniles, which have extended pseudopods and are in protoplasmic continuity with the network, move quickly to the periphery of the network. Within 24 h, juveniles establish a communal “feeding reticulum” in which dispersed individuals are in protoplasmic continuity with neighbors via a common reticulopodial network. At the ultrastructural level, the cell body cytoplasm of unaroused juveniles contains numerous patches of a paracrystalline material, which disappears as their pseudopodia are extended to join the communal feeding reticulum. This paracrystalline material therefore appears to be a temporary reservoir of precursors required for pseudopod construction.     

Reversal of motory polarity ofAmoeba proteus by suction

Summary When a glass capillary is introduced into the posterior body region ofA. proteus and its orifice is maintained inside the flowing mass of endoplasm, an applied suction force invariably initiates the reversal of streaming direction. This initial effect depends as well on the negative pressure value as on the terminal diameter of the pipette. Further transformations of configuration of pseudopodia are due to mixed effects of the direct application of sucking force and of the active response of amoeba to the new situation. When the sucking pipettes are applied to the outer cell surface, probably only a fraction of the negative pressure may be transmitted to the cell interior. The portion of cell periphery exposed to negative pressure acting from outside is still capable to contract. As a result, when amoeba as a whole is progressively sucked into the capillary, it manifests a clear active escape behaviour.Study supported by the Research Project II. 1 of the Polish Academy of Science.     

Digestion of prey in foraminifera is not anomalous: A correlation of light microscopic, cytochemical, and hvem technics to study phagotrophy in two allogromiids

Correlative light, high-voltage electron and conventional electron microscopic methods were used to investigate digestion in two allogromiid foraminiferans, Allogromia sp., strain NF, and A. laticollaris Arnold. Microscopic observations showed that bacterial prey are phagocytosed by reticulopodia and are transported to the allogromiid cell body within blister-like phagosomes. Larger prey (algae, diatoms) are transported along the reticulopodial surface and are either stored extrathalamously or phagocytosed at the oral opening (peduncle). Studies of allogromiids optimally fixed and labeled with an extracellular-space label (colloidal thorium) showed that phagocytosed prey are completely enclosed by a plasma membrane envelope; this finding was corroborated by a serial-section three-dimensional reconstruction of the oral zone of one allogromiid. Cytochemical staining for acid phosphatase showed that lysosomes are absent from reticulopods but abundant in the cell body, particularly in the oral zone cytoplasm. We conclude that digestion in allogromiid foraminiferans is accomplished by a vacuole-based digestive apparatus and not by extracellular digestion within a lacunary system, as has been suggested in earlier studies.     

Building a plasmodium: Development in the acellular slime mould Physarum polycephalum

The two vegetative cell types of the acellular slime mould Physarum polycephalum - amoebae and plasmodia - differ greatly in cellular organisation and behaviour as a result of differences in gene expression. The development of uninucleate amoebae into multinucleate, syncytial plasmodia is under the control of the mating-type locus matA, which is a complex, multi-functional locus. A key period during plasmodium development is the extended cell cycle, which occurs in the developing uninucleate cell. During this long cell cycle, many of the changes in cellular organisation that accompany development into the multinucleate stage are initiated including, for example, alterations in microtubule organisation. Genes have been identified that show cell-type specific expression in either amoebae or plasmodia and many of these genes alter their pattern of expression during the extended cell cycle. With the introduction of a DNA transformation system for P. polycephalum, it is now possible to investigate the functions of genes in the vegetative cell types and their roles in the cellular reorganisations accompanying development.     

A light microscopical investigation of amoebal and plasmodial mitosis in the MyxomyceteEchinostelium minutum

Summary Light microscopical observations on mitosis in living material of the amoebal and plasmodial phases of the MyxomyceteEchinostelium minutum de Bary (orderEchinosteliales) are reported for the first time. The uninucleate amoebal cell undergoes centric, open spindle mitosis whereas the multinucleate plasmodium exhibits acentric, closed spindle mitosis.     

Identification of three genes expressed primarily during development in Physarum polycephalum

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation. Received: 21 June 1999 / Accepted: 17 August 1999     

The autowave electromechanical activity of the <Emphasis Type="Italic">Physarum polycephalum</Emphasis> plasmodium

The aim of this work is to clarify the role of the electrical activity of the Physarum polycephalum plasmodium in the control of the contractile activity and self-organization of the directed locomotion. This single-celled organism with a non-excitable membrane is a classic object that is used in studies of amoeboid motility. Its patterns of motor behavior and signal systems are common for many tissue cells. The presence of 50 mM KCl in an agar substrate under half of a separate plasmodial strand strongly inhibits the formation of the frontal zone and leads to sharp morphological polarization of the strand, which suggests the involvement of electrical processes in the autowave self-organization of the plasmodial structure. The gigantic sizes of the plasmodium make it possible to record its electrical activity simultaneously at different parts of the cell. It has been established that potentials and currents at parts of the plasmodium that are distant from each other oscillate synchronously and differ only in the shape of the signals, probably due to differences in the phases or the number of excited harmonics. We recorded currents (~50 pA) of single ion channels of the plasmodial membrane using the classical local voltage-clamp method. It has been found that the oscillation spectrum of the current that is generated by the plasmodium has high-frequency fluctuations, which are probably connected with periodic detachments of the membrane from the cytoskeleton during the formation and growth of the pseudopodia. It has been also shown that neomycin, a substrate inhibitor of phospholipase C, prevents oscillations of both the mechanical and electrical activity of the plasmodium. This is consistent with its well-established ability to inhibit mechanosensitive Ca²⁺ channels, which are apparently present in the plasmodial membrane. These data indicate the presence of a general signal system that is linked with the dynamics of the membrane- cytoskeleton association, which could be involved in the galvano- and chemotaxis of amoeboid cells.     

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     

OBSERVATIONS ON “VAMPYRELLA PENULA-STYLODINIUM SPHAERA” AND THE ULTRASTRUCTURE OF THE REPRODUCTIVE CYST

“Vampyrella-Stylodinium,” an artificial name for a predaceous organism of uncertain taxonomic position, has at least three distinct phases in its life history: the amoeboid phase, both free-floating and attached; the feeding cyst or immobile phase; and flagellated gymnodinoid swarmers. The orange free-floating amoeba has unbranched, filose pseudopodia and several contractile vacuoles. When feeding on the filamentous green alga Oedogonium, the pseudopodia shorten and rearrange. After dissolution of part of the Oedogonium cell wall, the amoeba ingests the host protoplast. Then a stalked reproductive cyst may form. This cyst changes color from green to light orange as it matures. At the time of excystment, the cyst has a smooth outer wall, a spinose inner wall, and a well-delineated phagocytic vacuole. As this vacuole moves from its central position to the cyst's periphery, the walls rupture and 2-4 amoebulae emerge. With TEM observations, the reproductive cyst is shown to be multinucleate. Each nucleus is eukaryotic in organization and possesses one nucleolus. Mitochondria have tubular cristae and no structures unique to the division Pyrrhophyta are observed. Although this stage of the life history does not have a dinokaryotic nucleus, the gymnodinoid swarmers that can emerge from the reproductive cyst, do. Like other parasites which have been assigned to the division Pyrrhophyta, “Vampyrella-Stylodinium” does not conform well to the generalized concept of a dinoflagellate.     

Microtubule-dependent reticulopodial motility: is there a role for actin?

We summarize our recent immunocytochemical characterization of the reticulopodial cytoskeleton of two allogromiid foraminifers and our pharmacologic dissection of its motility. The reticulopodial microtubule cytoskeleton stained with an antiserum to brain microtubule-associated protein 2. Polymeric actin was localized in the reticulopodia by rhodamine-phalloidin staining. Microtubule inhibitors reversibly inhibited all aspects of motility; cytochalasins induced altered morphology and disorganization of motility but did not inhibit pseudopodial movements or intracellular transport. Simultaneous application of KCN and salicylhydroxamic acid (an alternative oxidase inhibitor) rapidly blocked all movement, indicating that motility is dependent on metabolic energy and that an alternative oxidative pathway functions in allogromiids. Micromanipulation and laser microsurgical experiments revealed tension throughout the reticulopodium. Our results suggest that microtubules are active components of the reticulopodial motile machinery. Actin may mediate substrate adhesion, whole-cell locomotion, pseudopodial tension, and coordination of the microtubule-based motility.     

Surface transport properties of reticulopodia: do intracellular and extracellular motility share a common mechanism?

The reticulopodial networks of the foraminiferan protozoans Allogromia sp., strain NF, and A. laticollaris display rapid (up to 11 microns/second) and bidirectional saltatory transport of membrane surface markers (polystyrene microspheres). Electron microscopy shows that microspheres adhere directly to the reticulopodial surface glycocalyx. A videomicroscopic analysis of this phenomenon reveals that microsphere movement is typically independent of pseudopod extension/withdrawal and that particles of different sizes and surface properties display similar motile characteristics. The motile properties of surface-associated microspheres appear identical to those of saltating intracellular organelles. Indeed, in some instances the surface-attached microspheres appear transiently linked in motion to these underlying organelles. Our observations suggest that, in reticulopodia, surface transport of microspheres and intracellular transport of organelles are driven by a common mechanism.     

Structure,function, and formation of extrusive organelles — microtoxicysts in the rhizopodPenardia cometa

Summary A study of the structure, function, and development of extrusive organelles (microtoxicysts) in the unusual bacteriovorous rhizopodPenardia cometa is performed. Microtoxicysts are located in the cortical cytoplasm of the cell body and in special thickenings on reticulopodia. Similar types of extrusomes have been observed in some cercomonads. The microtoxicysts are organized as membrane-bound vesicles of a complex form. An oviform-conical axial element lies inside each vesicle and is directed with its narrower end towards the plasma membrane. An internal cylindrical tube occupies the central part of the axial element; it is turned out as the organelle is shot out. The extrusomes ofP. cometa originate from and develop in derivates of the endoplasmic reticulum, the initial diameter of proextrusome vesicles is twice the diameter of the mature organelles. At late stages of maturation the microtoxicysts adopt their characteristic form and orientation. The mode of construction, ejection and development is compared with that in some other carnivorous and bacterivorous protists.     

Fine Structure and Development of Pilosporella chapmani (Microspora: Thelohaniidae) in the Mosquito,Aedes triseriatus (Say)1

ABSTRACT. New information on the life cycle and fine structure of Pilosporella chapmani, a microsporidium of the mosquito Aedes triseriatus, is presented. Pilosporella chapmani is shown to have two sporulation sequences, one of them being involved in transovarial transmission. One sequence, involving meiosis and production of a moniliform sporogonial plasmodium, occurs in the larval fat body, resulting in eight uninucleate, spherical, and fully developed spores. The other occurs in oenocytes of adult mosquitoes and results in isolated, binucleate, elongate, and thin-walled spores. Also, for the first time, metabolic products are shown to be expelled into the surrounding host tissues through the wall of the sporocyst.     

Ultrastructure of the membrane attachment sites of the extrusomes of Ciliophrys marina and Heterophrys marina (Actinopoda)

Summary The actinopods Ciliophrys marina and Heterophrys marina both have membrane bounded extrusomes attached to their cellular and axopodial membranes. The extrusomes of C. marina, the muciferous bodies, are fairly simple in structure and contain a homogeneous osmiophilic substance. Their attachment site is characterized by a rectangular array of freeze fracture particles in the cell membrane. The extrusomes of H. marina, the conicysts, are more complex and contain a two-part osmiophilic body. The attachment site of conicysts is characterized by a rosette of 8 freeze fracture particles very similar to the 9-particle rosette found at the mucocyst attachment sites in Tetrahymena. Furthermore, intracytoplasmic bridges connect the conicyst and cell membrane faces, and a specialized fibrillar structure is found on the cell membrane in the region of conicyst attachment. The various possible roles for such particle arrays are discussed and their presence in virtually all extrusomes is predicted.Supported by USPHS GM01021 and HL13849     

Cellular and molecular analysis of plasmodium development in Physarum.

The development of an amoeba into a plasmodium involves extensive changes in cellular organisation and gene expression. The genetic basis of a number of recessive mutations that block plasmodium development has been elucidated. The stage at which development becomes abnormal has been determined for all the mutants, as has the terminal phenotype. In order to investigate the changes in gene expression that accompany plasmodium development, a cDNA library has been made using RNA isolated from cell populations in which development was occurring.     

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.     

Ultrastructure and Description of a Fungus-Feeding Amoeba,Trichamoeba mycophaga n. sp. (Amoebidae,Amoebea), from Australia1

ABSTRACT. An amoeba isolated from a wheatfield and a forest soil in Australia has been identified as Trichamoeba mycophaga n. sp. Trophozoites of this amoeba are palmate to elongate and measure 45–136 μm in length and 25–94 μm in width. Amoebae in continuous locomotion may be limax with a villous-bulb uroid. Both the lobose pseudopodia and the advancing margin of a limax trophozoite bear an ectoplasmic crescent. The plasma membrane is coated with an electron-dense amorphous layer ca. 100 nm thick. Endoplasm is granular with elongate to bipyramidal crystals and contains bacterial endosymbionts. Trophozoites have a single, spherical to oval nucleus, 4–10 μm in diameter, which contains a centrally located, spherical to oval nucleolus, 2.8–5.0 μm in diameter. The nucleoplasm contains aggregations of filaments distributed radially within the nuclear membrane. Cysts are 21–60 μm in diameter, with ecto- and endocyst walls separated by an amorphous layer.