Miroslav Gabriel

The influence of Congo red on the cell wall and (1 → 3)-β-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)-β-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)-β-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.

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.

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.

Actin cortical cytoskeleton and cell wall synthesis in regenerating protoplasts of the Saccharomyces cerevisiae actin mutant DBY 1693

The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of beta-(1-->3)-D-glucan wall microfibrils in yeast cells.

Cytochalasin D interferes with contractile actin ring and septum formation in Schizosaccharomyces japonicus var. versatilis.

The cells of Schizosaccharomyces japonicus var. versatilis responded to the presence of cytochalasin D (CD), an inhibitor of actin polymerization, by the disappearance of contractile actin rings (ARs) that had already formed and by inhibition of new ring formation. Actin cables disappeared. Actin patches remained preserved and became co-localized with regions of actual cell wall formation (at cell poles and at the site of septum development). Removal of the AR arrested formation of the primary septum and led to the production of aberrant septum protrusions in that region. Nuclear division was accomplished in the presence of CD but new ARs were not produced. The wall (septum) material was deposited in the form of a wide band at the inner surface of the lateral cell wall in the cell centre. This layer showed a thin fibrillar structure. The removal of CD resulted in rapid formation of new ARs in the equatorial region of the cells. This implies that the signal for AR localization was not abolished either by CD effects or by removal of an AR already formed. Some of the newly developed ARs showed atypical localization and orientation. In addition, redundant, subcortically situated actin bundles were produced. The removal of CD was quickly followed by the development of primary septa co-localized with ARs. Wall protrusions occurred co-localized with the redundant actin bundles. If these were completed in a circle, redundant septa developed. The AR is a mechanism which, in time and space, triggers cytokinesis by building a septum sequentially dependent on the AR. Aberrant septa were not capable of separating daughter cells. However, non-separated daughter cells subsequently gave rise to normal cells.

Formation, growth, and regeneration of protoplasts of the green alga,Uronema gigas

SummarySeparate protoplasts were obtained by the action of snail gut juice enzymes on the cell walls of the green algaUronema gigas. The cultivation of the protoplasts in mineral media caused only their enormous growth; in the presence of glucose a fibrillar network was formed on the surfaces of the growing protoplasts. Only after the addition of pectin the regeneration of the cell wall and the renewal of their morphogenesis could be observed.

Staining the nuclei in cells and protoplasts of living yeasts, moulds and green algae with the antibiotic lomofungin

Lomofungin, an antibiotic active against yeasts, mycelial fungi and bacteria imparts a red colour to living yeast, fungal and algal nuclei when it is present in the culture medium at concentration of 20–100 μg/ml. Staining is equally distinct at all stages of the cell cycle, including mitosis and meiosis. The coloured nuclei are plainly visible in the light microscope and the presence in them of lomofungin has been confirmed by electron microscopy.

A Method of Isolating Anucleated Yeast Protoplasts Unable to Synthesize the Glucan Fibrillar Component of the Wall

A mixture of nucleated and anucleated protoplasts was produced from log-phase Saccharomyces cerevisiae by the use of snail enzymes. The mixture was separated by centrifugation, and anucleated protoplasts were studied by means of light and electron microscopy. Anucleated protoplasts did not synthesize glucan fibrils even though they seemed to contain all other basic structures in their cytoplasm, and the structure of the plasma membrane was unchanged. This was in sharp contrast to ordinary nucleated protoplasts which synthesized glucan fibrils even after inhibition of protein synthesis by cycloheximide. The reason for this behaviour of anucleated protoplasts is not clear. Such anucleated yeast protoplasts represent the first example of uniform anucleated fungi produced by a reproducible method.

Karyokinesis and septum formation during the regeneration of incomplete cell walls in protoplasts of Schizosaccharomyces japonicus var. versatilis: a time-lapse microcinematographic study

Regeneration in flattened Schizosaccharomyces japonicus var. versatilis protoplasts was studied with regard to nuclear behaviour, septum formation and lateral wall regeneration. In the course of protoplast regeneration all stages of the cell cycle were realized. However, the time sequence was conserved only between nuclear division and the beginning of septum formation. From this it is suggested that the signal initiating septum formation was issued by the dividing nucleus. Its primary target was not the original cell wall. However, the wall, though incomplete, was an essential prerequisite for the signal to be put into operation, i.e. for septation to begin. Incomplete cell wall regeneration allowed cytokinesis but not reversion of protoplasts into cells with their genetically determined cylindrical cell shape.

Microtubules and Actin Cytoskeleton of Potentially Pathogenic Basidiomycetous Yeast as Targets for Antifungals

Background: The cytoskeleton was investigated as a potential target for the inhibition of cell division in Fellomyces fuzhouensis CBS 8243 related to Cryptococcus neoformans. Methods: Vincristine, vinblastine, paclitaxel, methyl benzimidazole-2-yl carbamate (BCM), thiabendazole, cytochalasins A, B and D and latrunculin A were added to yeast extract peptone dextrose medium containing cells, investigated by phase contrast and fluorescence microscopy, counted in a Bürker chamber and absorbance was measured. Results: Vincristine, vinblastine, paclitaxel, cytochalasins A, B and D transiently blocked proliferation. BCM disrupted microtubules and inhibited mitosis, but F-actin patches and cables persisted and neck-less conidia appeared without stalks. Latrunculin disrupted F-actin, cells became spherical, and stalks and necks degenerated; microtubules persisted, but mitosis, cytokinesis and conidiogenesis were blocked. The combined application of latrunculin and BCM disrupted F-actin and microtubules, and inhibited cells became spherical and did not divide. Conclusions: Microtubules and F-actin are effective targets for permanent inhibition of nuclear and cell division and conidiogenesis by BCM and latrunculin A.