An Anterior-Posterior Gradient of Refractive Index in the Ameba and Its Significance in Ameboid Movement

Sustained locomotion in Amoeba proteus and Amoeba dubia results in the establishment of a measureable gradient of refractive index along the anterior-posterior axis of the cell, provided thickness of the specimens is kept constant by even compression under a selected coverglass supported by quartz beams of uniform diameter. The tail region of the ameba develops a higher refractive index, indicative of from 6 to 40 per cent more organic matter (expressed as protein) there than present in the front. This gradient fades on cessation of movement. The average protein concentrations in the crystal-free tails and fronts of 15 A. proteus were 3.9 and 3.4 per cent, respectively. In individual experiments, the tail-front difference ranged from one to eight times the accuracy of the method. Since the gradient of refractive index was shown not to result from extraction of water from the tail by the contractile vacuole, it was interpreted as displacement of water toward the anterior part of the cell during movement. It is suggested that contraction of the ectoplasm drives forward a "tide" of syncretic fluid, the anterior border of which is visible as the hyaline cap, which contains less than 1 per cent protein. The movement of the granular endoplasm into the hyaline cap would then complete the cycle by imbibition of the fluid tide. The theoretical positions of Pantin and of Dellinger have been combined in the proposal that ameba cytoplasm consists of a network of a contractile phase which is able to expel (by syneresis) a highly mobile fluid phase. Some other possible interpretations are discussed.     

The contractile basis of amoeboid movement : IV. The viscoelasticity and contractility of amoeba cytoplasm in vivo

The viscoelasticity and contractility of amoeba cytoplasm has been studied in vivo and in vitro. A gradient of increasing viscoelasticity and contractility was identified in the endoplasm of intact cells from the uroid (tail) to the fountain zone (tip of advancing pseudopod). Anterior endoplasm, as well as all of the ectoplasm, contracted in response to the microinjection of a threshold calcium ion concentration (ca 7.0 × 10⁻⁷ M). In contrast, there were only delayed weak contractions in the uroid endoplasm upon the microinjection of a threshold calcium ion concentration. Contractions induced in the ectoplasm by microinjecting the contraction solution readily caused the endoplasm to stream. However, the endoplasm at the tips of the extending pseudopods were also contractile and transmitted applied tensions. Furthermore, the microinjection of subthreshold calcium ion concentrations caused the loss of distinct endoplasmic structure and the cessation of streaming in both the uroid and the anterior third of the cell. In addition, the relationship between contractility and cytoplasmic streaming was characterized in “relaxed” cytoplasm placed in a gradient of calcium ion concentration inside quartz capillaries. The results of these experiments demonstrated that the mechanochemical conversion of endoplasm to ectoplasm caused the cytoplasm to become more structured and contractile. Therefore, physiological contractions are possible during and after the conversion of endoplasm to ectoplasm.     

Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

ELECTRON MICROSCOPY OF THE NUCLEAR MEMBRANE OF AMOEBA PROTEUS

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.     

鲢疯狂病病原体鲢碘泡虫营养体的超微结构观察

本文报道鲢碘泡虫营养体的超微结构。叙述了两层结构的原生质膜,外质中的胞饮管道,各种长短的分枝管道、芽体和泡状体,双层核膜具有核孔,在胞饮管道的内层,有很多线粒体、高尔基器、内质网、溶酶体、核糖体、脂粒及各种大小和作用未明的颗粒、微丝等等,但未见中心粒。     

A new myxosporean,Zschokkella leptatherinae n. sp. (Myxozoa: Myxidiidae), from the hepatic ducts and gall-bladder of Australian marine fishes

A new myxosporean,Zschokkella leptatherinae n. sp., was found in the hepatic ducts and gall bladder of five atherinid fish species,Leptatherina presbyteroides, Atherinosoma microstoma, Kestratherina brevirostris, K. esox andK. hepsetoides, collected at Dru Point, Margate in south-eastern Tasmania, Australia. This species has a round, ellipsoid or irregular plasmodium. The plasmodium is enclosed by a surface membrane and the cytoplasm is composed of an outer homogeneous ectoplasm and an inner coarse endoplasm with large clear areas, numerous vacuoles and spores which differentiate in the central area of the endoplasm. The characteristic metrical data of the myxosporean are as follows: spores 15.3 × 11.8 m; polar capsules 3.9 × 3.4 m. This is the thirdZschokkella species reported from Australia.     

REFORMATION OF NUCLEOLUS-LIKE BODIES IN THE ABSENCE OF POSTMITOTIC RNA SYNTHESIS

The dependence of nucleolar reformation on RNA synthesis that resumes in late anaphase or early telophase has been investigated in synchronously dividing Amoeba proteus. RNA synthesis was completely inhibited throughout all stages of mitosis and the early hours of interphase with high concentrations of actinomycin D. In such cells, nucleolus-like bodies that bind azure B and pyronin were apparent in the reformed nuclei. The bodies appear as dense, fibrous masses with loosely associated, finely fibrillar material. There are no characteristic granular regions in the reformed structures. It is suggested that the bodies probably represent mainly nucleolar protein and residual RNA which can bring about the reorganization of nucleoli in the absence of postmitotic RNA synthesis.     

Myxosoma pendula n. sp. (Protozoa,Myxosporida) from the Creek Chub Semotilus atromaculatus*

SYNOPSIS. A new species, Myxosoma pendula, was found surrounded by tissue fluid with granulocytes in pedunculated cysts formed from the mucous membrane of the gill arches of Semotilus atromaculatus collected in the Kewaunee River in Wisconsin. This species has a spherical vegetative stage with 2-layered ectoplasm composed of an outer homogeneous layer and an inner layer with canaliculi, and avoid spores that differentiate in the central area of the endoplasm.     

A Study of the Structure and Gliding Movement of Gregarina garnhami

SYNOPSIS The structure and gliding movement of Gregarina garnhami Canning, a eugregarine found in the midgut of the desert locust, Schistocerca gregaria , have been studied by light microscopy and transmission and scanning electron microscopy (EM). Ultrastructural studies revealed that the cytoplasm of G. garnhami is separated from the epicyte folds by a basal lamina. The pellicle consists of 3 membrane layers. At the tips of the epicyte folds there are 2 sets of longitudinally oriented filaments. An ectoplasmic network is present in the ectoplasm and the endoplasm contains numerous paraglycogen granules. The effect of cytochalasin B on G. garnhami was studied. Examination of scanning EM preparations of gliding and stationary gregarines yielded inconclusive results. In some instances the epicyte folds were thrown into waves; in others the folds were straight, regardless of treatment before fixation. Gregarina garnhami glides through its environment without any apparent deformation in shape. As it moves, a mucus trail is left behind it. Phase-contrast observations were made of centrifuged gregarines in which the endoplasm was displaced. Centrifuged gregarines continued to glide. Displacement of the endoplasm allows visualization of the epicyte folds in gliding animals. No lateral waves were seen in the epicyte folds of gliding centrifuged animals.     

Ultrastructure of a colonial radiolarian Collozoum inerme and a cytochemical determination of the role of its zooxanthellae

Collozoum inerme (Müller) is a colonial Radiolarian containing numerous cells bound in a common gelatinous matrix. The cells do not possess a skeleton as observed in many unicellular Radiolaria, but the cytoplasmic organization is similar. The cells are multinucleate and a complex system of cellular processes containing mitochondria, Golgi, and numerous vacuoles radiate out from the nuclear region. The endoplasm is connected to the ectoplasm across a double membrane boundary by thin cytoplasmic strands called fusules whose structure resemble those in unicellular Radiolaria. The ectoplasm contains a lacy network of vacuoles containing an osmiophilic substance. Rhizopodia emerge from the ectoplasmic sheath. Some are thin and densely granular. Larger diameter rhizopodia, containing less dense cytoplasm, sequester the zooxanthellae which present a typical dinoflagellate fine structure. Some of the zooxanthellae are apparently cultivated since they are sometimes observed dividing and persist in large numbers when colonies are cultivated under illumination for several weeks in the laboratory. However, colonies maintained in the dark have a decline in number of zooxanthellae and light microscopic examination shows they are being drawn into the ectoplasm of the radiolarian cells. Electron microscopic examination of zooxanthellae drawn into the ectoplasm sheath indicates they are digested. C. inerme is a remarkable example of a simple cellular aggregate that has exploited its colonial habit to culture algae and use them as food thus possibly enhancing the viability of the colony.     

Relationship between endoplasmic and ectoplasmic oscillations during chemotaxis ofPhysarum polycephalum

Summary The plasmodium ofPhysarum polycephalum usually migrates coordinately as one whole body even in a complicated environment. By measuring oscillation phenomena in endoplasm and ectoplasm separately during chemotactic process, we studied the mechanism of information processing to achieve such a coordination. (1) The interaction between endoplasmic oscillators was long-range, competitive according to the length of period, and fast (18 cm/min). Ectoplasmic one was short-range. (2) After a partial stimulation of attractant to the organism, the period at the stimulated portion decreased first, and a global phase gradient appeared in endoplasm. Then ectoplasm at the non-stimulated portion was entrained to the endoplasmic pattern, and the migration direction at each part changed in accordance with the phase gradient as a whole body. (3) When the endoplasmic interaction was interrupted, the above coordinated response was not observed. These facts suggest that two-layer coupled oscillator system composed of endoplasm and ectoplasm play important roles for such an information integration.     

Electron Microscopy of the Ectoplasm and the Proboscis in Didinium nasutum

SYNOPSIS. A morphological study on the ectoplasm and the proboscis in the ciliate Didinium nasutum , has been performed by means of an electron microscope. The ectoplasm and the endoplasm of Didinium are separated by a fibrous layer. In addition to the ciliary apparatus and the filament system, the ectoplasm is characterized by having ectoplasmic vacuoles enclosing cross-striated bodies and by having small rods surrounding the ciliary basal body.
The filament system is composed of 4 types of tubular filaments: primary filaments originating from the basal body, secondary ones coursing longitudinally along the cell periphery, tertiary ones going down in cylindrical arrays from the periphery of the proboscis into the endoplasm, and finally kinetosomal ones from the base of the basal body into the endoplasm through the newly found pore of the fibrous layer.
The fine morphology of the trichites in the proboscis is elucidated three-dimensionally and illustrated schematically. Moreover, the correlation among the small rod, ectoplasmic vacuole and trichite is discussed.     

A Highlights from MBoC Selection: Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions

For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell—the endoplasm—needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II–mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II–mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.     

THE CELL IN STARVATION: PHYSIOLOGICAL AND CHEMICAL OBSERVATIONS

Observations made on Amoeba proteus during total inanition revealed the following changes: Dry weight declined progressively, but at a decreasing rate to about 45 per cent of the initial levels when determined in surviving members of a dying population. Protein fell to about 70 per cent of the initial level. A hexane-alcohol extractable component fell during early starvation then rose to about its initial absolute level in the dying cells. While initially most of this component is probably lipide, it is not certain that other materials are not extracted during cell degeneration. Survival as a function of cell size was studied. No advantage in survival was apparent for any size class. Nucleate cell "halves" likewise showed no survival time differential, unlike a highly significant decrease in the survival of enucleate portions. The maintenance of the initial variance about the mean population weight (after hexane-alcohol extraction) during starvation, likewise supports the idea that survival depends largely on concentration parameters.     

AN ELECTRON MICROSCOPE STUDY OF CORTICAL STRUCTURES OF OPALINA OBTRIGONOIDEA

The pellicular framework of Opalina obtrigonoidea consists of numerous longitudinal ribs parallel to the kineties. These ribs lie erect on the cell surface, and each is composed of striated longitudinal fibers. A membrane covers the ribs and the ectoplasm between them. Flagella, of conventional structure, emerge from the ectoplasm between the ribs. The two central fibers of each flagellum end at the cell surface; the nine peripheral fibers continue for about 400 mµ into the cell to form an open tubular kinetosome. From the anterolateral curvature of each kinetosome arise two rows of fibrils, each fibril oriented perpendicular to the cell surface and about 150 A in diameter. The two rows converge anteriorly and probably meet the next adjacent kinetosome. Minute granules or tubules, arranged in oblique rows and at least sometimes accompanied by very fine fibers, lie at the surface of the ectoplasm but show no detectable connection with the kinetosomes. The whole flagellar apparatus of Opalina thus bears a general resemblance to the infraciliature of some holotrich ciliates, but the degree of evolutionary relationship between them remains uncertain.     

THE CONTENT AND RELATIVE BASE RATIOS OF RIBONUCLEIC ACID IN AMOEBA

The amount and relative base ratios of ribonucleic acid (RNA) in the nucleus and cytoplasm of Amoeba proteus and A. dubia, and of homospecies cells obtained by nuclear transfer with A. proteus, have been determined by microelectrophoresis. In A. proteus the average amounts of RNA in the nucleus and the cytoplasm were 134. micromicrograms and 2520. micromicrograms; in A. dubia the averages for the nucleus and cytoplasm were 67. micromicrograms and 1427. micromicrograms. The relative base ratio of RNA of the nucleus is similar to that of the RNA of the cytoplasm within a species, but the two species differed in this respect. Homospecies nuclear transfer did not affect the relative base ratio or amount of RNA.     

Rétablissement du cytoplasme et de la force motrice au cours de la restauration de la cyclose chezNitella après irradiation

Résumé En irradiant (500 kR) une fraction seulement du volume cellulaire de la cellule internodale deNitella, il est possible de distinguer au cours des phénomènes de restauration du mouvement cytoplasmique ceux qui dépendent de Pendoplasme (observation de la cyclose dans la zone protégée) et ceux qui dépendent de l'ectoplasme siège de la force motrice (observation de la cyclose dans la zone irradiée).Dans la zone protégée, la vitesse de la fraction endoplasmique irradiée se rétablit en fonction du temps suivant une équation du second ordre. L'augmentation du volume irradié diminue la rapidité du premier des deux processus de restauration et la quantité d'endoplasme qui reste altérée s'accroît.Dans la zone irradiée, le courant cytoplasmique est suspendu; il ne reprend que 4 minutes après le traitement alors que la couche chloroplastique se réorganise. Le glissement des inclusions qu'il charrie se fait un certain temps par saccades. La vitesse de la fraction endoplasmique protégée y augmente régulièrement témoignant ainsi du rétablissement de la force motrice. Le rétablissement a lieu suivant un schéma analogue à celui de Pendoplasme irradié sauf que ce phénomène y est plus rapide et que la force motrice du moins pendant les premières heures se rétablit intégralement. Ces résultats sont discutés (rôle des groupes SH, effet indirect d'empoisonnement, nature des lésions) et comparés à ceux obtenus pour des cellules irradiées in toto.

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Cytoplasm and motive force separated recovery during the re-establishment of the gyclosis in irradiatedNitella cells

Summary In these experiments onNitella internodal cells, only a fraction of the cellular volume is irradiated (by 500 kR). The restoring of the endoplasm (by measurement of the cyclosis in the shielded zone) and the restoring of the ectoplasm where the motive force is generated (by measurement of the cyclosis in the irradiated zone) can be examinated separately during the recovery of the cytoplasmic streaming.In the shielded zone, the plot on a semi-logarithmic scale of a typical rate of streaming-time curve shows that the speed of the irradiated endoplasm. increases again according to a second order equation. With increasing irradiated cellular volume, there are a gradual slowing down of the first processus of restauration and an increase of the definitively altered fraction of the endoplasm.In the irradiated zone, the cytoplasmic streaming stopped completely. About 4 minutes after the treatment, when the chloroplasts layer is reorganising, it exhibits a tendency to flow again. The carried inclusions slide then in jerks during several minutes. The rate of the protected fraction of the endoplasm increases regularly, proving the recovery of the motive force. An analysis of this processus versus time shows that it can be exprimed by a second order equation as that of the irradiated endoplasm. But it is faster and the motive force resumes until full recovery (at least during the first hours).These results are discussed (importance of SH-groups, indirect poisoning effect, kind of lesions) and compared with those observed in completely irradiated cells.

     

INFECTIVE ORGANISMS IN THE CYTOPLASM OF AMOEBA PROTEUS

Evidence from electron and phase microscopy is given which shows that infective organisms are present in the cytoplasm of Amoeba proteus. Vesicles containing living organisms have been observed after repeated washing and starvation of the amebae for a period of 2 weeks. Exposure to γ-radiation in conjunction with starvation, repeated washing, isolation of single amebae, refeeding with contaminant-free Tetrahymena, and clone selection has produced clones with reduced cytoplasmic infection. These findings are discussed in regard to the autoradiographic studies of other investigators on Amoeba proteus. The controversies over whether DNA and RNA are synthesized in the cytoplasm may be resolved by the finding of cytoplasmic infection.     

STRUCTURE AND DEVELOPMENT OF THE SIEVE-ELEMENT PROTOPLAST IN THE HYPOCOTYL OF PINUS RESINOSA

Hypocotyl tissue of Pinus resinosa Ait. was fixed in glutaraldehyde-paraformaldehyde and postfixed in osmium tetroxide for electron microscopy. Although young sieve cells contain all the components characteristic of young, nucleate cells, they can be identified early in their development. Increase in wall thickness occurs early and rapidly. Concurrently, the plastids, which already contain starch granules, form both crystalline and fibrillar inclusions. As the sieve cell approaches maturity, an extensive network of smooth, tubular endoplasmic reticulum (ER), which becomes mostly parietal in distribution, is formed. At maturity, massive aggregates of this ER occur on both sides of sieve areas. These ER aggregates are interconnected with one another longitudinally by the parietal ER. In addition, the mature, plasmalemma-lined sieve cell contains a degenerate nucleus, mitochondria, and intact plastids. Dictyosomes, ribosomes, and vacuolar membranes are lacking. P-protein is not present at any stage of development.