Lysosomal membrane proteins of Amoeba proteus, as studied with monoclonal antibodies.

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomes from fusing with them.     

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies.

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.     

Reversible changes in size of cell nuclei isolated from Amoeba proteus: role of the cytoskeleton.

Micrurgically isolated interphasal nuclei of Amoeba proteus, which preserve F-actin cytoskeletal shells on their surface, shrink after perfusion with imidazole buffer without ATP, and expand to about 200% of their cross-sectional area upon addition of pyrophosphate. These changes in size may be reproduced several times with the same nucleus. The shrunken nuclei are insensitive to the osmotic effects of sugars and distilled water, whereas the expanded ones react only to the distilled water, showing further swelling. The shrinking-expansion cycles are partially inhibited by cytochalasins. They are attributed to the state of actomyosin complex in the perinuclear cytoskeleton, which is supposed to be in the rigor state in the imidazole buffer without ATP, and to dissociate in the presence of pyrophosphate. Inflow of external medium to the nuclei during dissociation of the myosin from the perinuclear F-actin may be due to colloidal osmosis depending on other macromolecular components of the karyoplasm.     

Studies on changes in the nuclear helices of Amoeba proteus during the cell cycle.

The fine structure of the nuclei of synchronously growing cell population of Amoeba proteus was studied at I-h intervals during the interphase. This study showed that the nuclear helices undergo increases in their number at certain stages during interphase. These changes were found to correlate with ultrastructural changes occurring in the nucleoli.     

Migration of newly-synthesized RNA during mitosis. IV. Content of labelled RNA in nuclei before and after mitosis in Amoeba proteus

Amoeba proteus were incubated with ³H-uracil for 3 h. Thereupon RNA synthesis was blocked by actinomycin D and the population separated into dividing and non-dividing cells. Nuclei were isolated from cells of both groups and their RNA radioactivity was measured by means of autoradiography. The amount of label in the nuclei of non-dividing (interphase) cells was found to be equal to the sum of labels in both nuclei of daughter cells shortly after division. It is concluded that labelled RNA leaves the nucleus at the onset of mitosis and returns to the nuclei of daughter cells immediately after its termination.     

Protein migration from transplanted nuclei in Amoeba proteus. II. An electrophoretic study

Recently, we showed that protein migration from a nucleus transplanted into a host amoeba involved two classes of nuclear proteins. The chemical character of these proteins has now been investigated using isoelectric focusing and SDS gel electrophoresis. The proteins which migrated into the host cytoplasm from the transplanted nucleus have a range of isoelectric points (pI) between 7.0 and 7.6, and a molecular weight (MW) range of 9 000–110 000 D. This class is likely to be involved with the nucleocytoplasmic transfer of RNA. The second class of migratory proteins had a lower MW and pI range; the majority were between 11 000 and 45 000 D, with pIs between 5.9 and 7.0. This class of migratory proteins exhibited a shuttling character, possibly functioning as cytoplasmic regulators of nuclear activities.     

DNA hyperreplication in the nuclei of Amoeba borokensis in the cell cycle]

The relative nuclear DNA contents were determined cytofluorometrically for several groups of synchronized Amoeba borokensis at the early and late interphase. In some groups of these amoebae the nuclear DNA content by the end of the interphase exceeded more than twice that measured 1 h after division, when DNA in amoebic nuclei already being synthesized. This means that the extra DNA was synthesized in the nuclei of amoebae of these particular groups. In other amoebic groups the nuclear DNA content checked at the end of the interphase did not exceed the doubled 1 h level. Thus, in these amoebae the quantity of the synthesized extra DNA was less than that in the former groups, or the extra DNA was not synthesized at all.     

Relationship of the DNA content in the nuclei of Amoeba proteus to the food regimen]

The relative DNA content of isolated Amoeba proteus nuclei has been measured by cytofluorometry. With the amoeba strain studied, the generation time is roughly equal to 48 hours at 25 degrees C, and with the presence of food in the medium. After the synchronous divisions, amoebae were maintained in the medium either with or without food organisms (Tetrahymena pyriformis). DNA contents in the nuclei of both the amoebae groups were measured within 4 and 48 hours after division. Before 16 hours, the nuclear DNA contents did not differ in either group. Starting from 20 hours, the DNA amount in fed amoebae exceeded that in starved animals. On the whole, the differences in DNA quantity increased by a 48th hour after division, when the nuclei of the former contained 145% DNA of the latter. The results obtained suggest that the DNA synthesis in amoeba nuclei may proceed during the whole interphase, and that during the second half of interphase the content of DNA may depend on the feeding intensity in amoebae. After refeeding the starved animals, DNA contents in their nuclei increased to reach the same level as in the constantly fed amoebae seen in the end of interphase.     

The Protein Amino Acid Compositions of Amoeba proteus,A. discoides,A. dubia and Pelomyxa carolinensis*

SYNOPSIS. A comparison has been made of the protein amino acid compositions of Amoeba proteus, A. discoides, A. dubia and Pelomyxa carolinensis. The protein amino acid compositions of each of these species differed in 1–3 of the following amino acids: arginine, aspartic acid-asparagine, threonine and glycine. The possibility of using these characteristics as acceptable genetic markers is discussed.