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.     

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.     

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.     

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.     

Taxonomic serology of Amoeba proteus

The serological distances among six and eight strains of Amoeba proteus were determined. Quantitative immunological precipitation methods have the sensitivity and accuracy to detect the small differences in taxonomic relationships within this species. The data suggest that the _ShP strain might not belong to the proteus species and support the hypothesis that Amoeba indica is a strain of A. proteus. The invasion of a strain of A. proteus by infectious rod-shaped bacteria caused a diversion in taxonomic relationships with the original strain. Present serological findings are compared to previous data.     

Interactions between endoplasmic reticulum and nuclear membranes of different types of cells during repair of damaged Amoeba nuclei

Repair of amoeba nuclear envelopes that have been damaged microsurgically involves the association of pieces of endoplasmic reticulum with the damaged nuclear membranes. The capacity of endoplasmic reticulum of one type of cell to interact with the nuclear membranes of a different type was tested by placing the damaged nucleus of one kind of amoeba into the cytoplasm of another. Damaged nuclei from Amoeba proteus underwent repair in the cytoplasm of A. discoides or A. indica, as was the case in the reciprocal combinations of these nuclei and cytoplasms. In samples prepared 30 min after operation, heterologous endoplasmic reticulum was associated with holes in the nuclear membranes and appeared to fuse with the nuclear membranes at the margins of the holes. By 5 h after operation, almost all of the cells survived, and the nuclear membranes were largely intact, indicating that repair had occurred. In contrast, when an Amoeba dubia nucleus was damaged and placed in A. proteus cytoplasm there was no evidence of repair and many cells died within a few hours. The results indicate that endoplasmic reticulum and nuclear membranes from different types of cells can interact during repair of damaged nuclear membranes. There appears to be a specificity to this interaction, however, since in a combination of relatively dissimilar cells no association of endoplasmic reticulum with damaged nuclear envelopes was observed and repair did not occur.     

Karyotyping of <Emphasis Type="Italic">Amoeba proteus</Emphasis>

In this paper, we have developed a method to prepare spreads of mitotic chromosomes of Amoeba proteus and described the process of Amoeba proteus karyotyping. This protocol allows obtaining spread chromosomes with a characteristic pattern of chromomeres on individual chromosomes. It is shown that, in the metaphase of mitosis, amoebas of strain B (one of the type strains of A. proteus in the Amoebae Cultures Collection of the Institute of Cytology, Russian Academy of Sciences) contain 27 pairs of chromosomes. It is established that the pattern of chromomeres is a chromosome-specific feature. A typical karyogram and image bank of DAPI- and YoYo1-banded individual chromosomes of A. proteus, strain B composed of five different spreads of mitotic cells are presented.     

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.     

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).     

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.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : I. On the Particulate Nature of the DNA-Containing Elements

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 µ particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H₃-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.     

Optical tomography analysis of <Emphasis Type="Italic">Amoeba proteus</Emphasis> chromatin organization at various cell cycle stages

An optical tomography investigation of the nuclear cycle in large freshwater amoebae Amoeba proteus has been performed for the first time. Nuclei of cells from a synchronized culture were stained with DAPI and examined using a confocal laser scanning microscope. Detailed analysis of three-dimensional images of the intranuclear chromatin at different stages of the nuclear cycle has been performed. The materials obtained, in combination with the published data, allow for a completely new representation of the dynamics of the structural organization of the A. proteus nucleus during the cell cycle. Two-stage interphase and mitosis of a special type not matching any of the known types in the existing systems of classification of mitosis were found to occur in amoebae. Amplification of chromosomes and/or fragments thereof supposedly occurs during the cell cycle, which is consistent with the available data on nuclear DNA hyperreplication during the cell cycle of A. proteus. The number of chromosomes can vary at different stages of the cycle because of amplification, this being a putative reason for the discordant reports on the number of chromosomes in this species. The elimination of “excess” DNA mainly occurs during the transition from prophase to prometaphase. Finally, specific features of chromosome behavior during mitosis allow conclusion to be drawn that many, if not all, chromosomes are of a holocentric type.     

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.     

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.     

Regulation of nuclear DNA synthesis in amoeba interspecific hybrids

Interspecific hybrids between A. proteus and A. indica which have different durations of nuclear S periods have been produced by reciprocal nuclear transfer after enucleating the host cells. The duration of nuclear DNA synthesis was studied in the clones of these interspecific hybrids and parental stocks by ³H-thymidine autoradiography. These studies showed that nuclear S period of the hybrids changed to that characteristic to the nuclear component of the parental cell from which the hybrid's original cytoplasm was derived. The results of these studies were interpreted as evidence for cytoplasmic regulation of the rate of chromosome replication.     

The Karyotype of <Emphasis Type="Italic">Amoeba borokensis</Emphasis> from a “<Emphasis Type="Italic">proteus</Emphasis>-like” Amoeba Group (Amoebozoa: Euamoebida)

The karyotype of Amoeba borokensis was studied for the first time. At the metaphase of mitosis, this species has a haploid set of chromosomes (n = 27). This is exactly half of the diploid karyotype in Amoeba proteus (strain B). At first glance, it confirms the idea that has recently appeared that one species arises from another via multiple changes in chromosome number. However, comparative cytogenetic analysis revealed that four orthologous chromosomes from haploid sets of these two species were not distinguished by the pattern of DAPI-stained bands. It shows that the genetic distance between A. proteus and A. borokensis is higher than was believed earlier and these two species have diverged from each other. Analysis of data on the life cycles of A. proteus and A. borokensis shows that a kind of so-called “cyclic polyploidy” takes place in “proteus-like” amoebae. “Cyclic polyploidy” has recently been considered as an alternative to the sexual process for genetic recombination in agamic protists from different macrotaxons.     

Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.     

The role of nucleus and cytoplasm in the inheritance of multiplication rates in Amoeba proteus cultured at different temperatures.

In Amoeba proteus the inheritance of the multiplication rate is controlled exclusively by the nucleus in successive cell generations. This result has been obtained with four different types of nucleo-cytoplasmic ‘hybrids’ at three different temperatures, 10 °, 17 °, and 25 °C. No contribution of the cytoplasm towards the inheritance of this character has been found.     

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.