The plasma membrane of yeast protoplasts exposed to hypotonicity becomes porous but does not disintegrate in the presence of protons or polyvalent cations.

Protoplasts of Saccharomyces cerevisiae swelled, lysed and disintegrated when exposed to hypotonic solutions at neutral pH. At pH 4.5 or lower the hypotonically treated protoplasts did not disintegrate and they retained their intracellular proteins, nucleic acids and nucleotides. However, they became leaky for K+ and Ca2+, indicating that pores had been created in the surface membrane, relaxing the osmotic stress. Upon readjustment of pH to neutral, the hypotonically treated protoplasts released the intracellular content and disintegrated. Also, at low pH, protoplasts did not swell in isotonic ammonium acetate and were refractory to the permeabilizing effect of nystatin and to lysis with low concentrations of detergents. Protoplasts were similarly protected against lysis and disintegration by hypotonic treatment or by detergents, even at neutral pH, if the incubation media contained polyvalent cations, especially Zn2+, La3+, spermine, and Ca2+ chelated with EDTA. The protoplasts exposed to hypotonic stress at low pH did not respire and could not regenerate into viable cells. Effects of H+ and polyvalent cations on intramembrane forces acting between molecules of membrane phospholipids are considered along with possible changes in interactions between membrane proteins.

Ultrastructure of two secretory mutants of Saccharomyces cerevisiae as revealed by freeze-fracture technique.

Permissive and restrictive phenotypes of two secretory mutants of Saccharomyces cerevisiae, sec 1 and sec 18, were studied by freeze-fracture technique. The sec 1 mutant, in addition to accumulating secretory vesicles, was characterized by a disappearance of the plasma membrane invaginations and by an aggregation of intra-membrane particles in vacuolar membranes. A prolonged incubation of the cells at 37 degrees C led to pathological fusion of some vesicles with the plasma membrane. After the cells were transferred back to the permissive temperature the invaginations reappeared rapidly while the accumulated vesicles disappeared only after budding had been resumed. The sec 18 mutant, apart from having distended endoplasmic reticulum membranes, also lost the plasma membrane invaginations at 37 degrees C and regained them at 24 degrees C. The described ultrastructural changes are typical for the restrictive phenotypes and represent further manifestations of the pleiotropic effect of the respective sec mutations.

Disassembly of microtubules due to low intensity ultrasound.

HeLa and MCF cell monolayers growing on glass cover-slips were sonicated using continuous wave ultrasound at low intensity levels approaching diagnostic conditions. The disassembly of cytoplasmic microtubules immediately after sonication was demonstrated using indirect immunofluorescence. The recovery from exposure to ultrasound, i.e. the reassembly of microtubules, was observed in the cultured cells 2 h after the sonication was discontinued.

Ultrastructure of spores of Rhizopus nigricans after repeated freezing and thawing shocks.

Disintegration of nuclear envelopes is the only ultrastructural change detectable by freeze-etching in dormant spores of the mould Rhizopus nigricans, both dry and swollen, subjected to repeated freezing and thawing. The increase of the number of freeze-inactivated spores corresponds well with the increase of the number of damaged nuclei. This fact led us to formulate a hypothesis that the structure of the nucleus is the primary target of the freezing or thawing damage. As other biomembranes are not damaged it may be assumed that the disintegration of the nuclear membrane is probably secondary. No changes in ultrastructure of metabolically activated spores could be detected, in spite of the fact that the spores lost their germinative ability. Thus, the mechanism of the freeze injury may be different in dormant and growing spores.

Viability of spores after repeated freezing and thawing shocks.

Survival of spores of the fungus Rhizopus nigricans after repeated freezing and thawing was investigated. The cooling rate was 10(4) degrees C/min. Dry spores were fully inactive after 32 repeated shocks. About one-half of spores were killed after 8 repetitions. The water content did not change the resistance, swollen spores reacted to shocks much like dry ones. The sensitivity of spores to freezing-thawing shocks increased considerably when the spores changed from the dormant to the active state. Already after a 30 min cultivation of spores in the nutrient medium two freezing and thawings were sufficient for inactivation of 60% spores. After a 90 min cultivation one freezing and one thawing were sufficient to inactivate practically all spores.

[Cytoskeletal principles of the functional organization of cells]. / Cytoskeletální princip funkcní organizace bunky.

The paper provides basic information on cytoskeletal structures [microtubules, microfilaments and intermediate filaments] which form a very dynamic system in the cytoplasm and in specialized cellular structures. The cytoskeleton comprises also the membrane and nuclear skeleton. The cytoskeleton performs all motor functions of the cell, incl. intracellular transport, some morphogenic processes and it is the carrier of positional information in the cell and probably also the memory structure of non-genetic cell memory. The author mentions some molecular mechanisms of transformation of the chemical energy into mechanical energy. The article indicates also some perspectives of the application of findings in the sphere of medicine, in particular in the biology of tumour cells, in the pathogenesis of some diseases, in cytostatic treatment, in toxicology and in diagnostic applications.

Ultrastructure of Saccharomyces cerevisiae cells accumulating Golgi organelles.

Restrictive phenotype of sec 7 mutant of Saccharomyces cerevisiae was examined by freezefracture electron microscopy. In accordance with previous findings (NOVICK et al. 1981) dictyosomes and middle-size (200-600 nm) vesicles (Berkeley bodies) were found to accumulate. Dictyosomes are formed by aggregated flattened or dilated cisternae without associated secretory vesicles. After transfer to permissive conditions the dictyosomes disappear and are not detectable, just like in the wild type or in the permissive phenotype. Associated with the restrictive phenotype is an extended plasma membrane and tonoplasts with particle-free impressions.

Regeneration of yeast protoplasts. A freeze-etching study.

The submicroscopical structure of yeast protoplasts regenerating the new cell wall or merely its fibrillar component was studied by freeze-etching. No relation was found between the number and distribution of plasma membrane particles at various stages of regeneration. Hexagonal arrangement of the particles was found only solitarily even in protoplasts synthesizing intensely glucan microfibrils in liquid media. The fibrillar network on protoplasts grown in liquid medium or fibrillar groundwork of the cell wall on protoplasts grown in gelatine medium were exposed only after etching on etched faces. The microfibrils did not penetrate the outer leaflet of the unit membrane, which consequently indicates that no structural relation could exist between the fibrils and the plasma membrane particles. During conversion of cells to protoplasts, plasma membrane invaginations were arranged end-to-end to form prolonged furrows which persisted until cell wall regeneration had been completed. Then the long furrows broke into short units. Thus plasma membrane invaginations appear to be loca, rigid differentiations of the plasma membrane which may migrate laterally. Neither the plasma membrane nor the adjacent cytoplasm showed signs of reverse pinocytosis. The endoplasmic reticulum, which was hypertrophic during regeneration, consisted of extensive membranes, often parallel in arrangement. The cytoplasm frequently contained groups of small globular particles without characteristic localization.

Plasma membrane particles in yeast protoplasts.

Investigations of plasma membrane particles in Saccharomyces cerevisiae protoplasts are shortly reviewed. Particle arrangement into a hexagonal patterns is considered to reflect some minute changes in the biomembrane organization rather than functional differentiation. Plasma membrane invaginations are proved to be rigid structures capable of migrating in the fluid domain of the membrane. Density of the particles per unit area is related to the degree of membrane stretching which suggest that upon membrane extension new protein molecules are shifted towards the hydrophobic layer of the membrane.

Cell surface structures in osmotically fragile mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae characterized by osmotic fragility showed a marked fibrillar structure on the inner wall surface when studied by two electron microscopic techniques, i.e. freeze-etching of whole native cells and metal shadowing of isolated cell walls. The walls of the mutant cells were more permeable to macromolecules than were those of the wild-type parental strain. The synthesis and assembly of (1----3)-beta-D-glucan wall microfibrils studied in protoplasts of mutant cells were not impaired. It is suggested that the osmotic fragility of the mutant cells is related to the deficiency of the wall structure as a consequence of the srb1 mutation affecting biogenesis of the amorphous (glucan) component.