Modulation of fibroblast motility by a cytosolic extract of Cyanobacteria.

Proliferation and migratory behavior of L929 murine fibroblasts were shown to be modified in the presence of a cytosolic extract of Phormidium sp. (Cyanobacteria). The addition of Phormidium extract to the growth medium (Dulbecco's modified Eagle's medium) supplemented with 0.5% newborn calf serum increased cell proliferation. The effect was shown to be cell line specific. A quantitative analysis performed according to De Laat, Tertoolen, and Bluemink (1981, Eur. J. Cell Biol., 23, 273-279), showed that Phormidium extract was a potential aggregative effector for fibroblasts. Heating (100 degrees C, 4 min) inactivated the clustering effect of the extract, but the effect on cell proliferation was retained. A video analysis of cells after divisions showed that the extract activated cell migration in the same way as 5% serum did during the first 24 h of treatment. Between 24 and 48 h of treatment, cell migration in the presence of the extract was inhibited when compared to migration in 0.5 or 5% serum. We have shown that Phormidium extract may contain two or three kinds of effectors which acted as exogenous growth factors (allowing attachment and proliferation) and as modulator(s) of the cell migratory behavior (activator of migration in early times of the growth and inhibitor later).

Cytoplasmic membrane changes during adaptation of the fresh water cyanobacterium Synechococcus 6311 to salinity.

In this investigation, changes were characterized in cell structure and cytoplasmic membrane organization that occur when the freshwater cyanobacterium Synechococcus 6311 is transferred from 'low salt' (0.03 molar NaCl) to 'high salt' (0.5 molar NaCl) media (i.e. sea water concentration). Cells were examined at several time points after the imposition of the salt stress and compared to control cells, in thin sections and freeze fracture electron microscopy, and by flow cytometry. One minute after exposure to high salt, i.e. 'salt shock', virtually all intracellular granules disappeared, the density of the cytoplasm decreased, and the appearance of DNA material was changed. Glycogen and other granules, however, reappeared by 4 hours after salt exposure. The organization of the cytoplasmic membrane undergoes major reorganization following salt shock. Freeze-fracture electron microscopy showed that small intramembrane particles (diameter 7.5 and 8.5 nanometers) are reduced in number by two- to fivefold, whereas large particles, (diameters 14.5 and 17.5 nanometers) increase two- to fourfold in frequency, compared to control cells grown in low salt medium. The changes in particle size distribution suggest synthesis of new membrane proteins, in agreement with the known increases in respiration, cytochrome oxidase, and sodium proton exchange activity of the cytoplasmic membrane.

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells.

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.

Comparative freeze-fracture study of perialgal and digestive vacuoles in Paramecium bursaria.

In the endosymbiotic unit of Paramecium bursaria (Ciliata) and Chlorella sp. (Chlorophyceae) algae are enclosed individually in perialgal vacuoles, which do not show acid phosphatase activity and thus differ from digestive vacuoles. Both types of vacuoles have been studied by freeze-fracture. Perialgal vacuoles are nearly spherical; their membrane always fits tightly to the algal surface. The vacuole size and shape do not vary much. During division of the algal cell into four autospores the vacuole diameter only doubles. After autospore formation the vacuole invaginates around the algal daughter cells and divides. Newly formed perialgal vacuoles remain in intimate contact and exhibit characteristic attachment zones before final separation. The two fracture faces of perialgal vacuole membranes are homogeneously covered with intramembranous particles (IMPs) but rarely show signs of vesicles pinching off or fusing with the membrane, except during vacuole division. The P-faces bear more IMPs (3164 +/- 625 IMP/micron 2) than the E-faces (654 +/- 208 IMP/micron 2). The range of IMP density on both faces is enormous, suggesting that the membrane is not static. Membrane changes are supposed to occur simultaneously with the enlargement of the vacuole and to be caused by fusion with cytoplasmic vesicles, as the fractured necks on vacuole membranes may indicate. Digestive vacuoles in P. bursaria show significant variations in size, shape, membrane topography and IMP density, as well as signs of endocytic activity. Different vacuole populations are present in P. bursaria according to different feeding conditions: ciliates fed for a long time have small vacuoles with few IMPs (322 +/- 198 IMP/micron 2 on the E-faces, 1438 +/- 458 IMP/micron 2 on the P-faces), which are probably condensed digestive vacuoles, whereas organisms fed for a short time have larger vacuoles with highly particulate faces (680 +/- 282 IMP/micron 2 on the E-faces, 2701 +/- 503 IMP/micron 2 on the P-faces) and thus are supposed to be older vacuoles. The digestive vacuole membrane changes continuously. Compared to digestive vacuoles perialgal vacuoles are characterized by small size combined with high IMP density on the two fracture faces. Their IMP densities resemble those of old digestive vacuole membranes. However, it would be premature to conclude that membranes of perialgal and old digestive vacuoles are identical. Membranes of old digestive vacuoles are mainly derived from lysosomal material, which presumably does not contribute to the formation of perialgal vacuole membranes as is indicated by the small vacuole diameter; fusion with lysosomes would considerably enlarge it.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochemical studies of nuclear basic proteins in control and vitamin B12 starved Euglena.

In avitaminosis B12, Euglena gracilis Z is blocked in the cell cycle in the S/G2 phase. In these blocked cells, transcription and traduction go on and the amount of DNA is less than doubled and remains constant during the blockage. Chromatin clumps observed in situ with classical electron microscopic methods are always condensed in control cells but are not visualized in B12 starved cells. Two cytochemical reactions, ethanolic phosphotungstic acid and ammoniacal silver reaction, specific for lysine- or arginine-rich residues, are performed to reveal basic nuclear proteins of chromatin. With these two methods, control chromatin in situ always shows a condensed aspect, whereas the starved chromatin appears dispersed. These cytochemical differences might be considered to result from a different supramolecular organization of the two kinds of chromatin.

Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia.

The trichocysts of Paramecium tetraurelia constitute a favorable system for studying secretory process because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis. Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place. We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts. Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane.

Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: cytochemical determination of Ca2+-ATPase activity over performed exocytosis sites.

In different Paramecium tetraurelia strains the occurrence of a Ca2+-ATPase (or p-nitro-phenylphosphatase) activity at the preformed attachment and exocytosis sites of the secretory vesicles (trichocysts) was analysed by electron-microscopic cytochemistry and X-ray microanalysis. In conjunction with freeze-fracture studies it was found that only those strains, which contain rosette particles, display this Ca2+-ATPase activity (7S, K 40I, nd 9 (18 degrees C), while other strains (nd 6, nd 9 (27 degrees C), tam 38) are devoid of both these characteristics. The presence (absence) of rosette particles and of Ca2+-ATPase activity at the preformed exocytosis sites is correlated with the capability (incapability) of performing exocytosis in these strains. We discuss several possible interpretations of this structure-function correlation.

Control of membrane fusion in exocytosis. Physiological studies on a Paramecium mutant blocked in the final step of the trichocyst extrusion process.

Previous studies on exocytosis in Paramecium using mutants affecting trichocyst extrusion permitted us to analyze the assembly and function of three intramembrane particle arrays ("ring" and "rosette" in the plasma membrane, "annulus" in the trichocyst membrane) involved in the interaction between these two membranes. Using a conditional mutation, nd9, which blocks rosette assembly and prevents exocytosis at the nonpermissive temperature, we have analyzed the effect of temperature on the secretory capacity of nd9 cells. By combining several techniques (physiological studies, microinjections, inhibition of fatty acid synthesis, and freeze-fracture analysis) we demonstrate (a) that the product of the mutated allele nd9 is not thermolabile but that its activity is dependent upon temperature-induced changes in the membrane lipid composition and (b) that the product of the nd9 locus is a diffusible cytoplasmic component whose interaction with both plasma membrane and trichocyst membrane is required for rosette assembly and exocytosis. The data provide physiological evidence for the existence of a molecular complex(es) linking the two membranes and involved in the control of membrane fusion; we discuss the possible nature and function of these links.

Euglena plasma membrane during normal and vitamin B12 starvation growth.

Freeze-fracture and optical diffraction techniques were used to study the organization of the Euglena pellicle during the normal and replicative stages of the cell cycle and during vitamin B12 starvation. It was shown that the diffuse layer underlying the tripartite structure has a fibrillar structure. Despite the absence of homology in the 2 fracture faces of the pellicle, the EF striated and the PF particulate ones appear complementary as shown by optical diffraction studies; it must therefore be considered as a true membrane. The grooves are free from such particles and striations. They appear as a specific pattern of the cortex, different from the ridges in their structural organization and their replicative capacity as observed during vitamin B12 starvation. This notion is confirmed by the mode of pellicular growth which is characterized by 2 steps. The first occurs during the early replicative stage (pre-mitotic phase of the cell cycle) when the formation of a new ridge is correlated with the appearance of the 'minor' orientation of a 2-dimensional lattice on the EF and the PF faces and the spread of the particles over the PF face of the space between the old ridges. The second takes place during the lengthening of the ridges from the initiating posterior side (non-replicative stage). During this second step, the 'major' orientation of the lattice is preferentially observed in control cells and exclusively in starved cells. The striking differences between the grooves and the ridges is discussed, as well as the 2 modes of growth and their significance in morphogenesis.

Genetic analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst cycle in wild-type and mutant strains.

Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freeze-fracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles-trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (76 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18 degrees C), while in cells grown at the nonpermissive temperature (27 degrees C) the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses", also found in wild-type and in nd9 (18 degrees C) cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.