Mechanisms of the 14-3-3 protein function: regulation of protein function through conformational modulation.

Many aspects of protein function regulation require specific protein-protein interactions to carry out the exact biochemical and cellular functions. The highly conserved members of the 14-3-3 protein family mediate such interactions and through binding to hundreds of other proteins provide multitude of regulatory functions, thus playing key roles in many cellular processes. The 14-3-3 protein binding can affect the function of the target protein in many ways including the modulation of its enzyme activity, its subcellular localization, its structure and stability, or its molecular interactions. In this minireview, we focus on mechanisms of the 14-3-3 protein-dependent regulation of three important 14-3-3 binding partners: yeast neutral trehalase Nth1, regulator of G-protein signaling 3 (RGS3), and phosducin.

The cytoskeleton in the unique cell reproduction by conidiogenesis of the long-neck yeast Fellomyces (Sterigmatomyces) fuzhouensis.

The morphology of conidiogenesis and associated changes in microtubules, actin distribution and ultrastructure were studied in the basidiomycetous yeast Fellomyces fuzhouensis by phase-contrast, fluorescence, and electron microscopy. The interphase cell showed a central nucleus with randomly distributed bundles of microtubules and actin, and actin patches in the cortex. The conidiogenous mother cell developed a slender projection, or stalk, that contained cytoplasmic microtubules and actin cables stretched parallel to the longitudinal axis and actin patches accumulated in the tip. The conidium was produced on this stalk. It contained dispersed cytoplasmic microtubules, actin cables, and patches concentrated in the cortex. Before mitosis, the nucleus migrated through the stalk into the conidium and cytoplasmic microtubules were replaced by a spindle. Mitosis started in the conidium, and one daughter nucleus then returned to the mother via an eccentrically elongated spindle. The cytoplasmic microtubules reappeared after mitosis. A strong fluorescence indicating accumulated actin appeared at the base of the conidium, where the cytoplasm cleaved eccentrically. Actin patches then moved from the stalk together with the retracting cytoplasm to the mother and conidium. No septum was detected in the long neck by electron microscopy, only a small amount of fine "wall material" between the conidium and mother cell. Both cells developed a new wall layer, separating them from the empty neck. The mature conidium disconnected from the empty neck at the end-break, which remained on the mother as a tubular outgrowth. Asexual reproduction by conidiogenesis in the long-neck yeast F. fuzhouensis has unique features distinguishing it from known asexual forms of reproduction in the budding and fission yeasts. Fellomyces fuzhouensis develops a unique long and narrow neck during conidiogenesis, through which the nucleus must migrate into the conidium for eccentric mitosis. This is followed by eccentric cytokinesis. We found neither an actin cytokinetic ring nor a septum in the long neck, from which cytoplasm retracted back to mother cell after cytokinesis. Both the conidium and mother were separated from the empty neck by the development of a new lateral wall (initiated as a wall plug). The cytoskeleton is clearly involved in all these processes.

Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts.

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.

Disruption of the actin cytoskeleton in budding yeast results in formation of an aberrant cell wall.

A temperature-sensitive, conditionally lethal actin mutant of Saccharomyces cerevisiae, DBY 1693, was used to study, using light and electron microscopy, dysfunction of the actin cytoskeleton in the morphogenesis of the cell wall. Cells of this mutant strain survived at least 24 h at the restrictive temperature (37 degrees C). These cells showed isodiametric growth. Mutant cells accumulated vesicles, probably as a consequence of chaotic secretory transport caused by loss of polarity. A conspicuous morphological response to the dysfunction of actin was the formation of an aberrant wall over the whole surface of the isodiametrically-growing cell. This wall was of loose texture with protruding glucan microfibrils incompletely masked with amorphous matrix. It resembled the regenerating cell wall on the surfaces of yeast protoplasts. The localization of wall synthesis over the whole surface of temperature sensitive actin mutant cells was in accordance with an even distribution of submembranous actin in the form of patches (similarly to regenerating protoplasts). Delocalization of finger-like invaginations of the plasma membrane from the bud region to the whole surface of the growing cell was also found in mutant cells.

Ultrastructure of the cell wall of Schizosaccharomyces pombe following treatment with various glucanases.

The ultrastructure of isolated cell walls of Schizosaccharomyces pombe was studied by electron microscopy after treatment with the following purified enzymes: endo-beta-(1-->3)-glucanase, endo-beta-(-->6)-glucanase, and endo-alpha-(1-->3)-glucanase produced by Bacillus circulans; exo-beta-(1-->3)-glucanase and endo-beta-(1-->3)-glucanase produced by Schizosaccharomyces japonicus var. versatilis. The exo-beta-(1-->3)-glucanase had no detectable effect on the walls, but amorphous wall material was removed by action of the endo-beta-(1-->3)- and endo-beta-(1-->6)-glucanases of B. circulans to reveal a wall component consisting of densely interwoven microfibrils. The fibrils were hydrolyzed by treatment with the Schiz. japonicus endo-beta-(1-->3)-glucanase followed by B. circulans endo-alpha-(1-->3)-glucanase--suggesting that they were composed of -beta-(1-->3)-linked glucan and alpha-(1-->3)-linked glucan. The presence of a fibrillar component in untreated walls was evident after negative staining.

The influence of congo red on the cell wall and (1----3)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae.

Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1----3)-beta-D-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1----3)-beta-D-glucan elementary fibrils depicted by electron microscopy. The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.

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.

Lipids in yeast cell wall glucans.

Lipids isolated from glucans of Saccharomyces cerevisiae were analysed by thin-layer chromatography, infrared spectroscopy and gas-liquid chromatography. Localization of glucan lipid in the polysaccharide net was observed by light and electron microscopy. Despite using Manner's method for glucan preparation, the residue of acidic lipid occupied a central position in the treated cells. Analysis showed the presence of free fatty acids in which a relatively high content of vaccenic acid was detected.

[Secretory otitis media in children]. / Sekretorický stredousní zánet u detí.

In 1987-1988 323 children with conduction hypacusia were examined. Among the in 103 secretory otitis media was detected, i.e. 31.8%. The authors defined diagnostic and therapeutic approaches to the disease which may cause life-long anatomical and functional damage of the organ of hearing.

[Secretory otitis media and mastoiditis in children]. / Sekretorický stredousní zánet a mastoiditida u detí.

In 1984-1987 the authors followed up a selected group of 15 children with a long-standing history of secretory otitis media and latent mastoiditis. In there of them the condition was complicated by labyrinthitis. Mastoidectomy which was performed in 12 children contributed to the healing of chronic inflammatory changes in the middle ear.

Assembly of microfibrils in vivo and in vitro from (1----3)-beta-D-glucan synthesized by protoplasts of Saccharomyces cerevisiae.

Polymer chains of (1----3)-beta-D-glucan were dissolved with 1 M NaOH at 4 degrees C from native microfibrillar protoplast nets. The chains associated into microfibrils during NaOH neutralization or dialysis. In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide. X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens. They corresponded to those of paramylon. Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples. The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains. Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands. These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties. The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn. Since 6(1) helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane. The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic study of purified polysaccharide components glucans and mannan of the cell walls in the yeast Saccharomyces cerevisiae.

In this study electron-microscopic characteristics of platinum shadowed or negatively stained preparations of purified beta-(1----3)-D-glucan, beta-(1----6)-D-glucan, and mannan were examined. These polysaccharides were isolated from cell walls of the yeast Saccharomyces cerevisiae. While purified samples of beta-(1----6)-D-glucan and mannan proved to be amorphous in structure and homogenous in appearance, the purified beta-(1----3)-D-glucan, isolated and presented to us as alkali-insoluble yeast glucan A2, was not homogenous. It consisted of (i) fibrillar component, (ii) amorphous matrix, and (iii) chitin bud scars. The ultrastructure of beta-(1----3)-D-glucan present in the glucan A2 sample did not change after treatment with 0.5 M acetic acid at 75 degrees C for 2 hours. After treatment with 1 M NaOH for 3 days at 4 degrees C scar material was removed by centrifugation and after a subsequent acidification of supernatant with acetic acid both the microfibrillar and the amorphous components were still present. It was concluded that beta-(1----3)-D-glucan component consists of molecules probably differing in their physico-chemical properties such as D. P., the degree of branching, conformation, and that cannot be separated by the methods currently used for their isolation.

Papulacandin B: inhibitor of biogenesis of (1----3)-beta-D-glucan fibrillar component of the cell wall of Saccharomyces cerevisiae protoplasts.

The effect of papulacandin B on regenerating protoplasts of Saccharomyces cerevisiae was studied by light and electron microscopy. In liquid media it inhibited the biogenesis of (1----3)-beta-D-glucan fibrillar nets; as a result, the protoplasts did not grow polarly but only spherically. The effect was reversible. Instead of the nets the inhibited protoplasts synthesized only individual microfibrils soluble in hydroxide; these were not joined in the nets and were partially masked by amorphous material. The microfibrils disintegrated after lysis and did not maintain the shape of protoplasts. Protoplasts inhibited in solid media grew spherically up to 25 micron but they did not divide or revert, in spite of forming cell walls. These walls were amorphous and fragile and they disintegrated during preparation. Papulacandin B did not decrease the viability of protoplasts and did not interfere with their growth, biogenesis of alkali-soluble glucan microfibrils or amorphous wall matrix. It inhibited specifically the synthesis of alkali-insoluble branched (1----3)-beta-D-glucan, a necessary building unit required for the formation of the fibrillar component of the cell wall responsible for the cell wall shape, its rigidity and tensile strength.

Lysis of growing cells of Saccharomyces cerevisiae induced by papulacandin B.

Light and electron microscopy was used to study the effect of papulacandin B on Saccharomyces cerevisiae in the exponential growth phase. At 1-2 micrograms/mL cell division in the culture continued almost in parallel with the control, at 4 micrograms/mL cell proliferation was reduced and the culture contained some cells with 2-9 buds which were not separated from the mother cell by a septum, and at higher concentrations (8, 16 and 32 micrograms/mL) the proliferation stopped within 2 h. Cessation of proliferation was due to lysis of budding cells in the bud region including perforation of thinned cell wall (most often at the bud basis and sometimes at its apex), extrusion of cytoplasm and death of cell. Lysis was also observed in cells without visible buds. Dividing cells died without visible lysis.