Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

[Enzymes of the phosphogluconate pathway in amebas]. / Fermenty fosfogliukonatnogo puti u ameb.

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases (G6PD and 6PGD) are revealed in Amoeba proteus by electrophoresis in polyacrylamide gels, thus proving the availability of the phosphogluconic pathway in amoebae. 6PGD is marked as a single band, and G6PD shows multiple banding. When an amoebic homogenate is obtained using Triton-100, a supplementary form of G6PD extracted from membranes of some cell organelles (presumably mitochondria) becomes apparent. Hexose-6-phosphate dehydrogenase seems to be absent and therefore all the G6PD forms found may be specific G6PDs proper.

Indirect immunofluorescence localization of ponticulin in motile cells.

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.

Preparation of cultured and isolated cells for X-ray microanalysis.

Various electron microscopical preparation techniques are compared with regard to the preservation of the intracellular element distribution as determined by X-ray microanalysis in scanning and scanning transmission electron microscopy. By use of chemical agents for fixation and dehydration ions are redistributed and washed out. This is also true for freeze-substitution. Whole cells are prepared by cryofixation followed by freeze-drying. Interference of intracellular measurements by extracellular elements can be avoided by appropriate washing the cells before cryofixation. The washing medium has to be carefully selected in order to avoid distortions of the original intracellular element content. These problems are circumvented by the preparation of freeze-dried cryosections from cryofixed cells. This is demonstrated by data of the intracellular elemental composition in cultured cells (fibroblasts, Staphylococcus aureus bacteria) and in cells isolated from rat tissue (kidney papillary collecting duct and liver). Cryofixation of a single cell in a defined functional state is illustrated by results obtained from streaming Amoeba proteus cells, cryofixed under light microscopical control. The main conclusion is that X-ray microanalysis of cells in functional states requires cryofixation and cryopreparation techniques which have to be adapted to the particular cell biological problem to be investigated.

Assembly, disassembly, and movements of the microfilament-rich ridge during the amoeboflagellate transformation in Physarum polycephalum.

The amoeboflagellate transformation in Physarum polycephalum involves a series of dramatic changes in cell shape and motile behavior. This report describes the morphological and behavioral changes through which a synchronously transforming population of cells passes, stressing that, although there are a series of distinguishable stages, cells at all stages display striking plasticity. Our previous studies showed that amoeboflagellates transiently display a flattened motile extension--the ridge--that projects from a specific location on the cell surface and contains a laminar core densely packed with a series of crisscrossing arrays of actin microfilaments. Details are presented here concerning the movements of the ridge as well as the dynamics of ridge formation and disassembly in relation to other morphogenetic events of the transformation. The ridge forms at about the same time as transforming cells begin to elongate, propagates undulations parallel to the long axis of the cell as the transformation proceeds, and disassembles late in the transformation. Staining of fixed cells with the fluorescent probe rhodaminephalloidin shows that the actin of amoeboid cells is strikingly redistributed as the transformation proceeds. Amoeboflagellates contain most of the stainable actin in the ridge and in a ventral-posterior spot that may be a site of cell-substratum adhesion. These results provide additional insights into the possible functions of the ridge and the roles of actin during the amoeboflagellate transformation.

Purification and characterization of an ATP-sensitive actin gelation protein from Acanthamoeba castellanii.

A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.

Dependence of the mechanical properties of actin/alpha-actinin gels on deformation rate.

The cortical cytoplasm, including the cleavage furrow, is largely composed of a network of actin filaments that is rigid even as it is extensively deformed during cytokinesis. Here we address the question of how actin-filament networks such as those in the cortex can be simultaneously rigid (solid-like) and fluid-like. Conventional explanations are that actin filaments rearrange by some combination of depolymerization and repolymerization; fragmentation and annealing of filaments; and inactivation and reestablishment of crosslinks between filaments. We describe the mechanical properties of a model system consisting of actin filaments and Acanthamoeba alpha-actinin, one of several actin crosslinking proteins found in amoeba and other cells. The results suggest another molecular mechanism that may account for the paradoxical mechanical properties of the cortex. When deformed rapidly, these mixtures are 40 times more rigid than actin filaments without alpha-actinin, but when deformed slowly these mixtures were indistinguishable from filaments alone. These time-dependent mechanical properties can be explained by multiple, rapidly rearranging alpha-actinin crosslinks between the actin filaments, a mechanism proposed by Frey-Wyssling to account for the behaviour of cytoplasm long before the discovery of cytoplasmic actin or alpha-actinin. If other actin-filament crosslinking proteins behave like Acanthamoeba alpha-actinin, this mechanism may explain how the cortex recoils elastically from small rapid insults but deforms extensively when minute forces are applied over long periods of time.

Temperature-compensated ultradian variation in cellular protein content of Acanthamoeba castellanii revisited.

Synchronous cultures of the soil amoeba Acanthamoeba castellanii, established in the laboratory by selection procedures, show oscillations of total cell protein, as well as respiration. Previously, time series analysis by Aspect, a University of Warwick Computer Unit Program, showed an average period of 76 min. A reanalysis by least-squares rhythmometry revealed that, in general, the analyses accompanying the original paper and those in this replication agree very well. The periods extracted by the two approaches differ by only 1 to 5 min. In one case, when the original analysis did not detect a statistically significant period, however, the analysis used here does so. While the incubation temperatures used varied from 20 to 30 degrees C and cell division time increased from 7.8 hr at 30 degrees C to 16 hr at 20 degrees C, the metabolic bioperiodicity appeared to be temperature compensated. Decimation studies indicate that the sampling interval of 5 to 15 min must not be further reduced. It is promising to incorporate the study of ultradian metabolic rhythms into a broad spectral approach, i.e., for a concomitant assessment of ultradian, circadian, and infradian rhythms.

Isolation and identification of 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N-trimethyl)homoserine from the soil amoeba, Acanthamoeba castellanii.

A polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan Acanthamoeba castellanii. On the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (DGTS). Fast atom bombardment (FAB) mass spectra of DGTS are reported for the first time and are compared to the FAB mass spectra of phosphatidylcholines and the electron ionization (EI) and field desorption (FD) mass spectra of DGTS. Gas-liquid chromatographic-mass spectrometric (GLC-MS) analysis of the acyl chain composition of this lipid has shown that 87.5% consists of cis-9-octadecenoic acid. Plasma membrane isolated from this organism has shown that labeled DGTS appears in the plasma membrane but is not enriched in this fraction. DGTS has been isolated previously only from a limited number of green plants and one species of fungus. Identification of this lipid in Acanthamoeba indicates that this lipid is distributed among a diverse group of lower eucaryotes.

Crystallization of Acanthamoeba profilin-I.

Profilin-I, a protein that inhibits actin polymerization in Acanthamoeba castellanii, has been crystallized in a form suitable for high resolution x-ray analysis. The crystals have the symmetry of the space group C2 with lattice constants a = 110.4 +/- 0.2, b = 31.7 +/- 0.1, c = 33.5 +/- 0.1 A, beta = 112.2 degrees. They diffract to at least 2.0-A resolution. The asymmetric unit contains one 12,800-dalton monomer of profilin-I.

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin I by cross-linking actin filaments.

The actin-activated Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I was previously shown to be cooperatively dependent on the myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This observation was rationalized by assuming that myosin I contains a high-affinity and a low-affinity F-actin-binding site and that binding at the low-affinity site is responsible for the actin-activated ATPase activity. Therefore, enzymatic activity would correlate with the cross-linking of actin filaments by myosin I, and the cooperative increase in specific activity at high myosin:actin ratios would result from the fact that cross-linking by one myosin molecule would increase the effective F-actin concentration for neighboring myosin molecules. This model predicts that high specific activity should occur at myosin:actin ratios below that required for cooperative interactions if the actin filaments are cross-linked by catalytically inert cross-linking proteins. This prediction has been confirmed by cross-linking actin filaments with either of three gelation factors isolated from Acanthamoeba, one of which has not been previously described, or by enzymatically inactive unphosphorylated Acanthamoeba myosin I.

Isolation and partial characterization of a 110-kD dimer actin-binding protein.

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.

Preliminary characterization of an organism isolated from a case of Viluy encephalomyelitis indicates a protozoal, rather than viral, aetiology.

A microbial agent was isolated previously from a case of Viluy encephalomyelitis and named the 'KPN agent' after the initials of the patient. Here a detailed characterization of nucleic acids extracted from the purified KPN agent is presented. The agent contains both DNA and RNA, and has its own tRNAs and some other low-Mr RNAs, including 5S RNA. These findings, and the isolation of eukaryotic-type ribosomes, suggest that the KPN agent is not a virus, as believed before, but a more complex micro-organism, with protein-synthesizing capacity. The nucleotide sequence of the 5S RNA in the ribosomes of the KPN agent is identical with the sequence of 5S RNA of Acanthamoeba castellanii. The novel protozoan nature of the KPN agent is discussed in relation to other unusual properties of this micro-organism. Some implications of these results for the aetiology of Viluy encephalomyelitis are also discussed.

Amino acid sequence of the active site of Acanthamoeba myosin II.

We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.

Vacuolar pH is one factor that regulates hydrolase secretion.

Lysosomal hydrolases are continually secreted by Acanthamoeba as a consequence of membrane cycling between the vacuolar compartment and the cell surface. In pinocytosing amoebae acid hydrolases can be separated into two groups on the basis of their secretion kinetics. We have previously shown that in Acanthamoeba acid hydrolases are almost exclusively restricted to a single compartment, digestive vacuoles, and that pH-dependent differential binding of hydrolases to vacuolar membrane can account for the different rates of hydrolase secretion from this compartment. In this report we show that the hydrolase secretion pattern changes and that all of the hydrolases are released with the same kinetics after phagocytosis of yeast or in growth media supplemented with ammonium acetate or chloroquine, but not after phagocytosis of polystyrene beads. The changes in the pattern of hydrolase secretion correlate with changes in vacuolar pH. The vacuolar pH of pinocytosing amoebae and amoebae saturated with beads is about 4.8. This value is increased to 6.8 by accumulation of weak bases and to about 6.1 when digestive vacuoles are saturated with yeast. These results indicate that vacuolar pH modulates hydrolase transport and secretion.

Characterization of alpha-actinin from Acanthamoeba.

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii.

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.