Augustin Svoboda

MICROTUBULE-DRIVEN NUCLEAR-MOVEMENTS AND LINEAR ELEMENTS AS MEIOSIS-SPECIFIC CHARACTERISTICS OF THE FISSION YEASTS SCHIZOSACCHAROMYCES-VERSATILIS AND SCHIZOSACCHAROMYCES-POMBE

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.

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.

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.

The effect of cycloheximide (actidione) on cell wall synthesis in yeast protoplasts

In yeast protoplasts formation of the formation of cell wall matrix can be blocked with cycloheximide in contrast to the formation of fibrillar groundwork of cell wall. This indicates that biosynthesis of these two structural components follows different pathways and may be controlled by different mechanisms.

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.

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

Summary.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.

Structural rearrangement of the actin cytoskeleton in regenerating protoplasts of budding yeasts

In Saccharomyces cerevisiae cells the actin cytoskeleton is present as actin dots in the bud and around the septum, i.e. in areas of intensive cell wall synthesis, and as actin cables, which are loose bundles along the longitudinal cell axis. However, the apparently asymmetrical pattern of actin no longer persisted after protoplasting, when the cables disappeared and dots were evenly distributed under the whole protoplast surface. This pattern was maintained during regeneration of a new cell wall all over the protoplast surface, thus providing evidence of a relationship between the new wall formation and the presence of a regular arrangement of actin dots. The completed cell wall allowed the protoplast to bud and produce a normal daughter cell. However, before the walled protoplast began to bud, actin dots accumulated at the site of bud emergence and actin cables appeared, extending to the cytoplasm. Later, actin dots accumulated in the growing bud, forming a ring in the neck, and actin cables passed to the bud. Completion of the protoplast-to-cell reversion was preceded by restoration of the normal actin cytoskeleton.

Topology of microtubules and actin in the life cycle of Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The morphology of budding and conjugating cells and associated changes in microtubules and actin distribution were studied in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by phase-contrast and fluorescence microscopy. The non-budding interphase cell showed a nucleus situated in the central position and bundles of cytoplasmic microtubules either stretching parallel to the longitudinal cell axis or randomly distributed in the cell; none of these, however, had a character of astral microtubules. During mitosis, the nucleus divided in the daughter cell, cytoplasmic microtubules disappeared and were replaced by a spindle. The cytoplasmic microtubules reappeared after mitosis had finished. Actin patches were present both in the bud and the mother cell. Cells were induced to mate by transfer to ribitol- containing medium without nitrogen. Partner cells fused by conjugation projections where actin patches had been accumulated. Cell fusion resulted in a zygote that produced a basidium with parallel bundles of microtubules extended along its axis and with actin patches concentrated at the apex. The fused nucleus moved towards the tip of the basidium. During this movement, nuclear division was taking place; the nuclei were eventually distributed to basidiospores. Mitochondria appeared as vesicles of various sizes; their large amounts were found, often lying adjacent to microtubules, in the subcortical cytoplasm of both vegetative cells and zygotes.

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.

Response of yeast protoplasts to their mating partners.

The mating process between two protoplasts or between a protoplast and a cell in the yeastSaccharomyces cerevisiœ was manifested by a specific morphological response of only the cell partner. The cells produced projections, up to 5 μm long, to meet their protoplast partners. The protoplasts responded, after a period of nonspecific hernia-like growth, by ceasing to grow and assuming oval or spherical shapes. They never formed mating projections, apparently due to the absence of complete cell walls. Similarly to the cells, nuclear division in protoplasts was arrested and the nucleus migrated towards the plasma membrane at the site of protoplast-cell contact. Cytoplasmic microtubules were directed to this site, indicating the position of the spindle pole body (SPB) on the nucleus adjacent to the plasma membrane. Actin patches accumulated also in this region. These cytological features of the protoplasts were reminiscent of the reorganization of the cytoskeleton and nucleus characteristic of mating cells. This implies that the ability of protoplasts to produce and receive mating signals was unaffected by protoplasting. Fusion, however, was not initiated due to the absence of the complete cell wall in one of the partners. Thus, the cell wall appeared to be necessary for the expression of polarized growth during mating and for cell fusion.