Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.

Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.

https://www.cell.com/cell/pdf/0092-8674(88)90060-8.pdf

cdc2 is a component of the M phase-specific histone H1 kinase: Evidence for identity with MPF

We have characterized the changes in microtubule organization that occur through the cell division cycle of the fission yeast Schizosaccharomyces pombe by indirect immunofluorescence microscopy. During interphase, groups of cytoplasmic microtubules, independent of the spindle pole body (SPB), form an array extending between the cell tips. These microtubules are involved in positioning the nucleus at the cell equator and in the establishment of cell polarity. At mitosis, the interphase array disappears and is replaced by an intranuclear spindle extending between the now duplicated SPBs. Elongation of the spindle sees the appearance of astral microtubules emanating from the cytoplasmic face of the SPBs. These persist until the end of anaphase whereupon the spindle microtubules depolymerize and two microtubule organizing centres (MTOCs) at the cell equator re-establish the interphase array. We have used the unique properties of various cell division cycle mutants to investigate further the function of these different microtubule arrays and their temporal and positional control.

The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe

The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast.

https://rupress.org/jcb/article-pdf/129/6/1601/1264411/1601.pdf

Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle.

Centrioles are present in oogonia and oocytes of hamsters, mice, rats, gerbils and man up to the pachytene stage. In subsequent meiotic stages, centrioles are absent. At the time of germinal vesicle breakdown in the mouse several foci composed of electron-dense fibrillar material from which microtubules radiate appear near the nucleus. These fibrillar aggregates, referred to as microtubule foci, form small asters and collectively perform the role of the spindle pole during the two meiotic divisions. There are multiple centres at each pole and their number may vary. Thus, centrioles are not obligatory organelles of the spindle apparatus.

Absence of Centrioles in the First and Second Meiotic Spindles of Mouse Oocytes

Mitosis in Schizosaccharomyces pombe has been followed in living cells by phase-contrast microscopy and studied in fixed and suitably stained preparations by light microscopy. Successful preservation of nuclear fine structure in this yeast, not previously achieved, has allowed us to confirm and extend the observations made with light microscopy. Without first arranging themselves on a metaphase plate, mitotic chromosomes become grouped in 2 clusters radiating, finger-like, from 2 points of attachment at opposite poles of an elongating nucleus. At these 2 sites electron microscopy reveals the presence of disk-shaped electron-dense organelles which we have called kinetochore equivalents (KCE). At mitosis the KCEs are connected across the nucleus by a narrow bundle of parallel microtubules which we refer to as the spindle. Integration of our observations has led us to propose that at mitosis the separation of the KCEs and their attached chromosomes is initiated by a differential expansion of the nuclear envelope restricted to the region between recently divided KCEs and that expansion of the nuclear envelope later becomes general, resulting in a marked elongation of the nucleus. Displacement of the nuclear contents to the ends of the elongated nucleus gives it the shape of a dumbbell. The elongation of the microtubule bundle keeps in step with the elongation of the nucleus but does not appear to be the cause of it. It may have the function of keeping the separated KCEs rigidly apart. During mitosis the nucleolus persists and stretches out within the unbroken envelope of the nucleus as it elongates. Towards the end of division equal amounts of nucleolar material are found in the rounded ends of the dumbbell-shaped nucleus. The break up of the dumbbell shape into daughter nuclei seems to involve the breaking of its tenuous middle part and a pivoting of its 2 ends in opposite directions. In the course of our work on mitosis we have become aware of features in the cytoplasm of growing S. pombe cells which are described here for the first time. The cells invariably contain several prominent vacuoles containing an extremely electron-dense material which stains metachromatically with toluidine blue and may be polyphosphate. The mitochondria are of special interest for 2 reasons. First, because they have unique mesosome-like membrane invaginations and secondly, because a mitochondrion is regularly associated with the single KCE by the side of the interphase nucleus, as well as with each one of the 2 KCEs that occupy opposite ends of the intranuclear spindle during mitosis.

Mitosis in the Fission Yeast Schizosaccharomyces Pombe: A Comparative Study with Light and Electron Microscopy

Leucosporidium scottii , for many years considered to be an asporogenous yeast in the genus Candida , is now known to have a heterobasidiomycetous life-cycle. Although morphologically similar to ascomycetous yeasts such as Saccharomyces cerevisiae , budding cells of L. scottii have a strikingly different type of mitosis. In L. scottii , mitosis is not intranuclear as it is in S. cerevisiae . Neither does the nucleus constrict and separate into daughter nuclei in the neck region between mother cell and bud. In L. scottii the chromatin-containing portion of the nucleus moves into the bud before division. There is partial breakdown of the nuclear envelope and the chromatin divides along a spindle apparatus formed inside the bud within remnants of nuclear envelope. A portion of the nucleus containing the nucleolus is left behind in the mother cell and disintegrates there when the envelope breaks down. After division, the nuclear envelope reforms around daughter nuclei and one daughter moves back to the mother cell. These events have been established on the basis of 3 types of observation: on living cells with phase-contrast microscopy; on cells stained for chromatin and studied with light microscopy; and on glutaraldehyde-fixed cells studied with electron microscopy. As in L. scottii , breakdown of nuclear envelope during division has been reported in several mycelial basidiomycetes. The sequence of chromatin movement into the bud of L. Scottii division inside the bud, and return of one daughter nucleus to the mother cell, is closely comparable to the movements of chromatin in and out of clamp connexions known to occur in dikaryotic hyphae of Polystictus versicolor . These mitotic similarities are in accord with the taxonomic relationship of L. scottii to the basidiomycetes. Observations on a cytoplasmic organelle located outside interphase nuclei of L. scottii have convinced us of its considerable importance during mitosis. This structure consists of 2 globular electron-dense ends joined by a bridge-like middle piece. It accompanies the advancing tips of nuclei which enter the bud. After breakdown of the nuclear envelope, the 2 spherical components of this organelle are seen at opposite poles of the mitotic spindle. Therefore we refer to it as the ‘microtubule organizing centre’ (MTOC). We have also speculated about a possible role for the organelle in the regulation of nuclear envelope growth, breakdown, and reformation.

Mitosis in Heterobasidiomycetous Yeasts: I. Leucosporidium Scottii (Candida Scottii)

Mitotic events similar to those in Leucosporidium scottii also take place in 2 other heterobasidiomycetous yeasts -- Rhodosporidium sp. and Aessosporon salmonicolor , which were formerly known as Rhodotorula glutinis and Sporobolomyces salmonicolor . Therefore it iS proposed that a mitosis which takes place within the bud may be a distinctive characteristic of heterobasidiomycetous yeasts which can be used as a taxonomic indicator of basidiomycete relationships in asporogenous yeasts. Mitosis in A. salmonicolor seems to be identical to that which we have previously described in L. scottii but a few differences have been found in Rhodosporidium sp. These are now described in detail.

Mitosis in heterobasidiomycetous yeasts. II. Rhodosporidium sp. (Rhodotorula glutinis) and Aessosporon salmonicolor (Sporobolomyces salmonicolor).

Clusters of cytoplasmic vesicles resembling those in growing hyphal apices of mycelial fungi are found near the tips of buds in three heterobasidiomycetous yeasts, Rhodotorula glutinis, Candida scottii, and Sporobolomyces salmonicolor.

E. Kathleen McCULLY

Papers

Mitosis in the Fission Yeast Schizosaccharomyces Pombe: A Comparative Study with Light and Electron Microscopy

Mitosis in Heterobasidiomycetous Yeasts: I. Leucosporidium Scottii (Candida Scottii)

Mitosis in heterobasidiomycetous yeasts. II. Rhodosporidium sp. (Rhodotorula glutinis) and Aessosporon salmonicolor (Sporobolomyces salmonicolor).

Apical Vesicles in Growing Bud Cells of Heterobasidiomycetous Yeasts