Metabolic requirements for interactions between nuclear and cytoplasmic membranes in the repair of damaged Amoeba nuclei.

Amoeba nuclear envelopes were damaged using microsurgery, and metabolic requirements for the steps in their repair were studied, and my placing the cells in a solution containing one of several metabolic inhibitors. The first step in repair, the association of pieces of endoplasmic reticulum with holes in the nuclear membranes, appears to be a passive process since it was not affected by inhibitors of energy production, RNA synthesis, or protein synthesis. In contrast, fusion of pieces of endoplasmic reticulum with the nuclear membranes at the margins of the holes was blocked by KCN and dinitrophenol, indicating that membrane fusion requires energy derived from respiration, but RNA and protein synthesis inhibitors did not prevent fusion of pieces of endoplasmic reticulum with the nuclear membranes. The subsequent completion of repair and restoration of intact nuclear membranes was almost completely blocked by inhibitors of respiration, and it was reduced in the presence of actinomycin and emetine, suggesting that in addition to a requirement for energy, some later steps in the repair of the nuclear membranes require RNA and protein synthesis.     

Pattern of DNA synthesis in nuclei of feeding and starving Amoeba proteus : A cytofluorometric study

The DNA content in isolated nuclei of Amoeba proteus was determined for each of the three groups of synchronized amoebae over different intervals after division. Several nuclei of each amoeba group were fixed 1 h after division, before the amoebae were fed. About h after division, some amoebae in each group were given food (Tetrahymena pyriformis), while the rest were left starving. Samples of the nuclei of fed and starved amoebae were fixed 24 h and (in different groups) 42–55 h after division. In each group from 22 to 48% of the fed amoebae had divided prior to the last nuclei fixation. Starved amoebae did not undergo division. In all three amoeba groups the nuclear DNA content of fed cells by the end of interphase had increased to 280–300% the value for 1 h amoebae. The nuclear DNA content of starved amoebae of all three groups was also increased, and in two groups it exceeded the initial level more than two-fold. However, in all three groups, it was lower than that of fed amoebae. In all the groups the nuclear DNA content in fed amoebae grew after 24 h, i.e. during the second half of interphase, the increase accounting for from 11 to 48% of the total increase. The hypothesis is put forward that the increase in the nuclear DNA content during the cell cycle of Amoeba proteus is the result of two processes: (1) one-time replication of the DNA of the whole genome; and (2) repeated replication of some part of the DNA. In amoebae the relation of the pattern of nuclear DNA synthesis to the diet is considered.     

Nuclear Lethal Effect and Nucleocytoplasmic Incompatibility Induced by Endosymbionts in Amoeba proteus1

ABSTRACT. The role of bacterial endosymbionts in the acquisition of new phenotypic characters was studied by transplanting nuclei from an uninfected strain of Amoeba proteus into the enucleated cytoplasm of a symbiont-carrying strain. After 1–10 cell cycles, the nuclei were tested for two characters: compatibility with uninfected and infected cytoplasm, and their lethal effect against amoebae of the uninfected parent strain. A significant number of transplanted nuclei displayed both of the new phenotypic traits after a few divisions in the infected cytoplasm. Thus the influence of these endosymbionts on the nucleus of A. proteus was virtually instantaneous.     

Maintenance and regeneration of cytoplasmic organelles in hybrid amebae formed by nuclear transplantation

The fine structure of cytoplasmic organelles was studied in hybrid amebae that were formed by transplanting the nucleus from one kind of ameba into the cytoplasm of a different type of ameba. Four different species or strains of amebae were utilized; Amoeba proteus, A. discoides, A. dubia, and A. amazonas. The fine structure of A. discoides was indistinguishable from that of A. proteus, but A. dubia and A. amazonas had distinctive features. To determine if the fine structure of cytoplasmic organelles in hybrid cells is like that of the nuclear parent or the cytoplasmic parent, nuclei were transferred between A. proteus and A. amazonas, which differ in their cytoplasmic ultrastructure, especially in the morphology of mitochondria. The cytoplasm of these hybrids resembled most closely that of the cytoplasmic parent. The degree to which different heterologous nuclei are capable of maintaining normal cytoplasmic organization was then studied by replacing an A. proteus nucleus with a nucleus from one of the other kinds of amebae. An A. discoides nucleus maintained A. proteus cytoplasm for several weeks. A nucleus from A. dubia or A. amazonas was able to maintain A. proteus cytoplasm for a few days, but then the cytoplasm began to show ultrastructural changes characteristic of enucleated amebae. The ability of a heterologous nucleus to promote the regeneration of Golgi bodies and endoplasmic reticulum after their decline in the absence of the nucleus was tested by transplanting a heterologous nucleus into A. proteus cytoplasm that had been enucleate for 5 days. A. discoides was the most effective heterologous nucleus donor but was less successful than normal A. proteus. Insertion of a nucleus from either A. amazonas or A. dubia resulted in “reactivation” of the cytoplasm with extension of pseudopods and resumption of motility but little or no regeneration of cell organelles. Thus, the ability of a heterologous nucleus both to maintain and to regenerate normal features of cytoplasmic organelles varied with the type of heterologous nucleus and was related to the degree of morphological resemblance between the donor and recipient cells. The role of the nucleus in motility and its role in regeneration of cytoplasmic organelles were dissociated from one another in the case of some hybrid renucleates.     

THE CYTONUCLEOPROTEINS OF AMEBAE : I. Some Chemical Properties and Intracellular Distribution

Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.     

Binding characteristics of receptors for phagocytosis on the surface of Amoeba proteus

Summary Binding of the tripeptide n-formylmethionyl-leucylphenylalanine (NFMLP) to phagocytic receptors on the surface of Amoeba proteus was examined. Peptide-binding is reversible and demonstrates saturation kinetics. The receptors for phagocytosis are internalized by a temperature-sensitive process with indications that the receptors are recycled. The amoeba is capable of down-regulating its receptors for phagocytosis in response to constant external peptide levels, and also increasing the number of surface receptors in response to food deprivation. On the basis of competition studies, there is evidence that Amoeba proteus has separate surface receptors for both pinocytosis and phagocytosis.     

Selective Effects of Enucleation and Transfer of Heterologous Nuclei on Cytoplasmic Organelles in Amoeba proteus*

SYNOPSIS. The ultrastructural changes in the cytoplasm of lethal hybrids obtained by nuclear transplantation between different strains of Amoeba proteus were compared with those of enucleated amebae. It was found that, whereas the Golgi complex and glycocalyx degenerated first in enucleated cells, mitochondria and endosymbiotes became abnormal first in the hybrids. The selective effects are attributed to the presence of nucleic acids in the mitochondria and endosymbiotes and hence to the different interactions they would have with the nuclear genome.     

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.     

The Fine Structure of Four “Species” of Amoeba*

The fine structure of Amoeba discoides, Amoeba dubia, and Amoeba amazonas was studied and compared with that of Amoeba proteus. The different kinds of amebas showed general similarities but differed in the ultrastructural details of their organelles. With respect to fine structure, A. discoides was indistinguishable from A. proteus, while both A. dubia and A. amazonas had distinctive features. The nuclei of all had a prominent honeycomb-like fibrous lamina, but A. dubia differed from the others in the distribution of nucleoli within the nucleus. The mitochondria of A. amazonas were unusual in having a variable pattern of cristae, some being plate-like and others tubular. Golgi bodies in A. amazonas had a greater proportion of vesicles and a smaller number of cisternae than those of the others, while Golgi bodies in A. dubia had highly flattened cisternae without a lining of filamentous material such as is found in the other types. The plasma membrane of A. dubia also lacked the prominent filamentous cell coat common to A. proteus and other amebas. The relation between the Golgi apparatus and the cell coat and the significance of the degree of development of the cell coat for pinocytosis and other phenomena is considered. The experimental use of these cells, including the formation of hybrids by nuclear transplantation is discussed.     

Diacylglycerol as a component of the signal-coupling pathway during the initiation of endocytosis in Amoeba proteus

In an attempt to define the transmembrane-signal pathway used to couple external phagocytotic signals with effectors in the cell interior, the effects of diacylglycerol (DG) and related substances were examined in Amoeba proteus. DGs are highly chemotactic, readily attracting amoebae when presented in a glass micropipette. Addition of DG (10^-6 M) to the medium elicits rapid shape changes in the amoeba and the formation of large phagosomes. Monacylglycerol and 1,3-diacylglycerol were much less effective in eliciting phagosome formation. On the assumption that DG was stimulating phosphokinase C (PKC) activity in the amoeba, the effect of phorbol myristate acetate (PMA), a known activator of PKC activity i other cell systems, was assessed in the amoeba. PMA (10^-7 M) alone was capable of bringing about shape changes in amoebae as well as stimulating the formation of phagosomes. These observations suggest that PKC is involved in the signal-coupling associated with the onset of phagocytosis. On the other hand staurosporine and H-7, inhibitors of PKC activity in some cell systems, did not inhibit the phagocytic uptake of Tetrahymena by A. proteus. It may be then that DGs in the amoeba interact directly with elements of the cytoskeleton causing phagosome formation, although a role for PKC in the initiation of phagocytosis in the amoeba cannot be ruled out at this point.     

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.     

Ultrastructure of tapetal cell ontogeny inBeta

Summary Tapetal cell development and degeneration in anthers ofBeta vulgaris L. were studied with the electron microscope. Tapetal cells become differentiated from sporogenous cells early in anther ontogeny. The tapetal nuclei divide mitotically; binucleate tapetal cells contain relatively little endoplasmic reticulum and otherwise resemble meristematic cells of higher plants. There follows an increase in endoplasmic reticulum and by the time the sporogenous tissue has entered meiotic prophase, the tapetal cells have differentiated the usual characteristics of secretory cells. Degenerative changes begin to appear in tapetal cells after meiosis of the sporogenous tissue. Such changes include loss of inner tangential and anticlinal walls, degeneration of tapetal nuclear envelopes, disruption of the plasmalemma, and changes in the cytoplasmic organelles. Coated tubules are associated with tapetal nucleoli during degenerative stages and the tubules persist after tapetal nuclei have degenerated. Tapetal cell cytoplasm disappears completely by the stage of microspore mitosis.     

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.     

A Monoclonal Antibody Against a Symbiont-Synthesized Protein in the Cytosol of Symbiont-Dependent Amoebae1

A monoclonal antibody was obtained against a 29-kD polypeptide in the cytosol of a symbiont-bearing strain (xD) of Amoeba proteus and was used to determine the distribution of the antigen in amoebae. The 29-kD polypeptides (xD protein) are produced by bacterial endosymbionts that are necessary for the survival of host xD amoebae. Results of indirect immunofluorescent and electron-microscopic immunogold-labeling studies showed that the xD protein was present diffusely in the amoeba cytoplasm as well as in the symbiotic bacteria. The native protein containing 29-kD polypeptides was purified using an immunoaffinity column prepared with the monoclonal antibody and its molecular weight was determined to be 87,000.     

Symbiont-Induced Strain-Specific Lethal Effect in Amoebae

The cytosol fraction of a symbiont-bearing strain of Amoeba proteus exerted a lethal effect when injected into symbiont-free amoebae of the original strain. The lethal factor appeared to be a protein with a molecular weight of over 200,000. While the effect of the lethal factor on the nucleus was reversible, the host cytoplasm was permanently damaged so that it could not form a viable cell when combined with a normal nucleus.     

The interaction of nuclear and cytoplasmic damage after treatments with toxic chemicals

An attempt is made to show how the interaction of different degrees of nuclear and cytoplasmic damage may contribute to the ultimate whole cell damage by a chemical. It suggests that cytoplasmic, as well as nuclear damage, may be important in the action of chemical carcinogens. Using Amoeba proteus as a single cell model where nuclear and cytoplasmic damage can be separated by micrurgy, the mortality curves for the nucleus, the cytoplasm and the whole cell are examined after four different treatments: exposure to N-methyl-N-nitroso urethane, a potent carcinogen in mammalian systems; exposure to ββ₁ (dichlorodiethyl) methyl amine, an alkylating agent used in chemotherapy; exposure to methylmercury chloride, a very toxic organo-metal; and irradiation with X-rays. These illustrate how different relative nuclear/cytoplasmic sensitivities contribute to the death of the cell. The evidence for nuclear and cytoplasmic damage after treatment with the N-methyl-N-nitroso urethane is detailed, and possibilities of nuclear repair after the four different types of treatment examined. Work on Amoeba proteus makes no attempt to assess separately changes in structure or activity of any one of the cells many enzyme systems, but looks at the balance between nuclear and cytoplasmic damage as a whole.     

Nuclear transfer studies of amoeba proteus after the inhibition of cell division by injection of non-homologous cytoplasm

Following the injection of the post-microsomal supernatant fraction of $\text{Amoeba discoides}$ cytoplasm into $\text{A.proteus}$, cell division is inhibited in at least 90% of the recipient cells. Nuclear transfers were performed to determine the site of inhibition in these injected cells. When nuclei from injected, inhibited cells one day after injection were transferred into new $\text{A.proteus}$ cytoplasms, 62% of the transfers divided. This ability to promote division declined with the length of time between transfer and the original_ injection. However, when nuclei from $\text{A.proteus}$ were transferred into injected, inhibited cytoplasms, only a low number of cells divided, comparable to the number obtained after the injection operation only, namely less than 10%. Thus although many nuclei could recover from inhibition, it was not possible to restore the cytoplasms of inhibited cells by new nuclei.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : III. Further Studies on the Nature of the DNA-Containing Elements

The application of electron microscope autoradiography to Amoeba proteus cells labeled with tritiated thymidine has permitted the identification of morphologically distinct particles in the cytoplasm as the sites of incorporated DNA precursor. The particles correspond to those previously described from light microscope studies, with respect to both H³Tdr incorporation and distribution in centrifugally stratified amoebae. Ingested bacteria differ from the particles, in morphology as well as in the absence of associated label. Attempts to introduce a normal particle labeling pattern by incubating amoebae with labeled sediment derived from used amoeba medium failed. The resultant conclusion, that the particles are maintained in the amoeba by self-duplication, is supported by the presence of particles in configurations suggestive of division.     

Mass isolated ameba nuclei. I. Isolation procedure and determination of macromolecular composition and RNA polymerase activity

A rapid method for mass isolation of intact nuclei from Amoeba proteus has been developed. By this procedure, which includes a novel way of recovering nuclei during the filtration step, about 40% of the nuclei in the starting suspension of 4 to 5 × 10⁶ cells can be recovered within 70 min. A typical suspension of purified nuclei consists of 93% intact nuclei, 5.6% food-waste pellets, 0.4% membranes, and 1.0% extracellular debris.     

Development of a nuclear polyhedrosis virus in midgut cells and penetration of the virus into the hemocoel of the armyworm,Pseudaletia unipuncta

The development of a nuclear polyhedrosis virus (NPV) in larval midgut cells of the armyworm, Pseudaletia unipuncta, is similar to that of other NPV. In the nucleus, the envelopes around the nucleocapsids seem to be derived de novo or from the inner layer of the nuclear envelope wich forms cisternae, blebs, or infoldings. The nucleocapsids are also enveloped by synhymenosis during passage through the nuclear membrane, the cell membrane, or the endoplasmic reticulum membrane. Both enveloped and unenveloped nucleocapsids may enter the cytoplasm through the nuclear pore or budding through the nuclear membrane. From the cytoplasm the virions may enter the hemocoel through the basal cell and basement membranes or through the endoplasmic reticulum, intercellular space, and the basement membrane.