ELECTRON MICROSCOPY OF MITOSIS IN A RADIOSENSITIVE GIANT AMOEBA

Various aspects of the ultrastructure of the dividing nuclei in the large radiosensitive amoeba Pelomyxa illinoisensis are demonstrated. Evidence of nuclear envelope breakdown is presented, and membrane fragments are traced throughout metaphase to envelope reconstruction in anaphase and telophase. Annuli in the nuclear envelope and its fragments are shown throughout mitosis. During metaphase and anaphase some 15 to 20 mitochondria are aligned at each end of the spindle, and are called polar mitochondria. The radioresistant amoebae Pelomyxa carolinensis and Amoeba proteus do not have polar mitochondria, and Pelomyxa illinoisensis is unique in this regard. The shape of the P. illinoisensis interphase nucleoli differs from that in the two radioresistant species, and certain aspects of nucleolar dissolution in the prophase vary. Helical coils in the interphase nucleoplasm are similar to those in the radioresistant amoebae. A "blister" phase in the flatly shaped telophase nuclei of P. illinoisensis is described which is interpreted to be the result of a rapid nuclear expansion leading to the formation of the normal spherical interphase nuclei.     

Physical properties of blue-green algae: Ultracentrifugation and electron microscope studies

Summary The Feulgen-positive and envelope-free nucleoplasm ofAnabaena andGloeocapsa contains DNA filaments. When ultracentrifuged it is displaced between the maze of photosynthetic lamellae to form a continuous, flattened layer in the centripetal half of the cell. The absence of a mesosome and lack of attachment of the nucleoplasm to the plasmalemma make the method of duplication and separation of the genome to the progeny an enigma. In the centripetal end of ultracentrifugedAnabaena, the photosynthetic lamellae are somewhat stretched and sometimes broken. This condition suggests that the cortical positioned photosynthetic lamellae are tightly connected in several places along their surfaces to an undisplaced cortical cytoplasmic layer. It is uncertain whether or not they are also directly attached to the plasmalemma. A vesicle is often observed in the centripetal end of the cell; it probably represents for the most part, the contents of distorted intralamellar spaces. In ultracentrifugedGloeocapsa, the photosynthetic lamellae show little displacement. They appear attached, probably at their ends to the plasmalemma and, likeAnabaena andNostoc, they possess phycobilisomes which are not greatly affected by ultracentrifugation. Cells usually survive a centrifugal force greater than required to separate most nonliving protein systems. When subcultured, a redistribution of the displaced components usually occurs and growth is resumed.     

Characterisation of the Rac/PAK pathway in Amoeba proteus

Summary. Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5–6) than the control cells (9–10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells. Correspondence and reprints: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur ulica, 02-093 Warsaw, Poland.     

The Free Amino Acid Levels of Pelomyxa carolinensis,Amoeba dubia and Proteus*

SYNOPSIS. The free amino acid extracts of 3 species of the free-living amoebae, Pelomyxa carolinensis, Amoeba dubia and A. proteus, were studied. Both the free amino acid patterns and levels in each species were different. Altho it appears that the free amino acid patterns were not altered by culture routines or starvation, the free amino acid levels were altered by these factors. The possibility of using free amino acid levels as a criterion for recognizing genetic differences is discussed.     

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.     

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.     

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.     

Karyotypic instability of endoprophase and mitotic cells of Amoeba sp. strain Cont from the “proteus-type” group (Amoebozoa,Euamoebida, Amoebidae)

We performed karyotyping of Amoeba sp. strain Cont. Based on the results of a cytological analysis, we concluded that the chromosome number of Amoeba sp. strain Cont in mitosis was unstable. In all cases they appeared to be hypergaploid (the basic chromosome number is 30), with monosomy of all chromosomes except four shortest ones. The presence of “extrachromosomes” in the nucleus could prolong until the beginning of the anaphase. It was only then that they were ejected from the nucleus and the euploidy (haploidy) was restored. The stage of endoprophase nucleus was revealed in the cell cycle of Amoeba sp. strain Cont. This stage has not yet been found in other amoebae from the “proteus-type” group that had been previously studied (A. proteus strain B and A. borokensis). The maximum number of endoreplication rounds in the strain Cont amoebae nuclear cycle was 4 or 5. The regular extrusion of chromosomes from the nucleus into the cytoplasm occurred in each of the endoreplication rounds. Comparative cytological analysis of A. proteus strain B, A. borokensis and Amoeba sp. strain Cont karyotypes indicated that strain Cont, though rather close to the former two amoebae, is actually a distinct species.     

Actin dynamics in Amoeba proteus motility

Summary. We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid. Correspondence and reprints: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, ulica Pasteura 3, 02-093 Warszawa, Poland.     

Strain-Specific Proteins of Symbiont-Containing Amoeba proteus Detected by Two-Dimensional Gel Electrophoresis1

Two-dimensional gel electrophoresis was used to compare the protein composition of normal (D strain) and symbiont-containing (xD strain) A. proteus. Over 500 different peptide spots could be identified on a typical gel of cytosol proteins. In addition to differences in a few minor peptides, one prominent polypeptide with an isoelectric point of about 5.5 and a molecular weight of about 29,000 was present in the cytoplasm of symbiont-containing amoebae and in the endosymbionts but not in D-strain amoebae. When endosymbionts were removed from xD-strain amoebae, the xD-specific polypeptide also disappeared, suggesting that it is produced by the endosymbionts.     

A study on histones in amoeba nuclei by indirect immunofluorescence method

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

The Feeding of Amoeba proteus on Paramecium aurelia*

SYNOPSIS. Density of prey (Paramecium aurelia) and predator (Amoeba proteus) were varied while volume of inorganic medium was kept constant. Variations in density of prey had little effect on the rates of feeding and reproduction of the amoebae; but with increasing predator density the amoebae captured the paramecia less rapidly and ingested fewer before dividing, altho division size did not change appreciably. Therefore, amoebae of a low density population with a constant food supply carry more nutritional reserves from generation to generation than do those in a denser population.     

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.     

Vertical Distribution of Potentially Pathogenic Free-Living Amoebae in Freshwater Lakes1

The vertical distribution of thermotolerant (37°C and 45°C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic paniculate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by participates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P < 0.005) numbers of FLA were isolated from two paniculate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe²⁺ state was predominant. In this midsummer period, 13 Naegieria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.     

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.     

Orientation of interphase chromosomes as detected by Giemsa C-bands

The orientation of Giemsa C-bands has been studied in mitotic and interphase cells of Allium cepa, A. sativum and of Aloe vera. The C-bands in these three species are located at the telomeres, secondary constriction region of the nucleolar chromosomes and the centromeric regions, respectively. Observations in A. cepa and Aloe indicate clearly that the interphase chromosomes are non-random in their orientation and possibly maintain their telophase configuration through the attachment of telomeres and perhaps of kinetochores with the nuclear membrane. Electron micrographs of onion cells also reveal that certain heterochromatic segments are associated with the nuclear membrane. — The nucleolar interstitial C-bands in A. sativum remain free in the nucleoplasm and may come close to each other due to heterochromatic attraction. Such a heterochromatic attraction is also evident between telomeric regions and between centromeres. However, a two by two attachment could not be noticed. A diagrammatic representation of the orientation of interphase chromosomes has been presented.The major part of this work was presented at the First International Congress on Cell Biology, Boston, Sept. 5–10, 1976 (Platform Session 36, J. Cell Biol. 70, 418a (1976)     

ULTRASTRUCTURAL STUDY ON SPORE WALL MORPHOGENESIS IN LYCOPODIUM CLAVATUM (LYCOPODIACEAE)

Spore wall morphogenesis of Lycopodium clavatum was observed by transmission electron microscopy. The spore plasma membrane indicates the reticulate spore sculpture shortly after meiosis. The mature spore wall of this species consists of two layers, inner endospore and outer exospore. There is no perispore in the sporoderm of this species. The exospore formation begins during the tetrad stage; and this layer is divided into two distinct sublayers, an outer lamellar layer and an inner granular layer. The lamellar layer is formed on the sculptured spore plasma membrane. Additional lamellae attach to this layer in a centripetal direction. For that reason, this layer may be derived from spore cytoplasm. The granular layer is formed only in the proximal region following lamellar layer formation, and it also may be derived from spore cytoplasm. The endospore is formed lastly and seems to be derived from spore cytoplasm as well. Accordingly, the spore sculpture of this species may be under the genetic control of the spore nucleus.