Eva Streiblová

Tubulin and actin topology during zygote formation of Saccharomyces cerevisiae.

The topology of tubulin and actin during mating of Saccharomyces cerevisiae was analysed by fluorescence microscopy with the monoclonal anti-tubulin antibody Tu01 and rhodamine-labelled phalloidin. Preconjugatory cells displayed an asymmetric distribution of the microtubule and actin cytoskeleton and an overall polarization of the cells preceding cell fusion. Prior to karyogamy, the haploid spindle pole bodies were associated with abundant cytoplasmic microtubules. Budding zygotes revealed the same tubulin and actin patterns as vegetative cells. Treatment of the mating mixture with the microtubule inhibitor nocodazole (10 micrograms ml-1) did not prevent polarization and fusion of haploids, zygote formation and emergence of the first zygotic bud. In marked contrast, the migration of the nucleus in preconjugatory cells as well as nuclear migration and fusion within the zygotes was unequivocally blocked by the action of the drug. It is suggested that the problem of the morphogenesis of mating should be approached by considering interactions at the cell periphery.

F-actin contractile rings in protoplasts of the yeast Schizosaccharomyces

By rhodamine-phalloidin fluorescence, distinct continuous F-actin rings were visualized in 18-20% of the protoplasts of Schizosaccharomyces pombe and S. japonicus var. versatilis, in addition to randomly distributed F-actin dots. Whereas the reversion of ring-lacking protoplasts coincided with the polarization of the dotted F-actin pattern, the ring-containing protoplasts became furrowed as the F-actin rings constricted. The furrowing was more conspicuous in S. japonicus var. versatilis than in S. pombe protoplasts and it was blocked when the reversion was inhibited by Novozyme 234 indicating that the cell wall formation is essential for the F-actin ring constriction.

Demonstration of yeast scars by fluorescence microscopy

D URING the multiplication of yeast cells scars develop at cell-wall sites after separation of the daughter cell, appearing in the optical microscope as an unevenness of the surface. Barton [4] was the first to study systematically the types of scars in living, plasmolysed and stained cells of Saccharomyces cerevisiae. Later the structure of the cell wall in whole cells [3], in isolated membranes [l, 6, 10) and in ultrathin sections [I, 2, 91 was studied with the aid of the electron microscope. Since one can determine the relative age of cells from the number of scars, methods designed to demonstrate yeast bud scars should be of interest to cytologists [3], but hitherto there has been no method for studying scars on intact cells [5]. In the course of cytological studies of yeasts in this laboratory with aid of the fluorescence technique, the fluorochrome primulin was used among other agents. This non-toxic vital dye was used for the differentiation of viable and non-viable yeast cells [7, 8, 121. It was found, however, that at higher concentrations primulin causes an intense greenish fluorescence of the scars on the cell wall. The species studied were grown in modified medium of Olson and Johnson [ll] for 24-48 hr at 28°C on a shaker. The centrifuged and washed cells were suspended in the primulin dye solution in concentrations ranging from 1: 1000 to 1: 5000. Specimens were prepared from the suspension and the coverglass embedded in Vaseline. They were stained for 5-30 min. The specimens were observed in the Nf microscope (Zeiss) with an illuminator 01-17 (USSR) fixed on the microscope tubus. The source of illumination was a highpressure mercury vapour lamp DRSH-250. The following filters were used: FS-12 mm, exciter filter with 85 per cent transmission at 380 my, and the S3S-7, S3S-14, BS-8 filters and the ZHS-18 suppression filters. The photographs were made with the Miflex (Zeiss) camera, Ilford HPS film was used. Fluorescence microscopy was used for studying the yeast cell surface with the scars developing during budding, fission and reproduction by the intermediate process. For this purpose 28 species of various genera of yeasts were used. Budding yeasts.-In the majority of the strains investigated two kinds of scars were found, birth scars and bud scars, which have been previously described [l, 4, 61. The birth scar (Fig. la) indicates the site of the separation of the new cell from the mother cell. No fluorescent substance was found to accumulate at its margin. The bud scars (Fig. 1 b and 5) are formed after the separation of the daughter cells. On their margin a circular fluorescent thickening was observed so that these scars resemble a crater. In Saccharomyces cerevisiae the maximum of 25 bud scars were counted at the end of the logarithmic phase of growth.

Types of multiplication scars in yeasts, demonstrated by fluorescence microscopy.

A new method permitting the study of scars on intact cells of different species of yeasts by means of secondary primulin fluorescence is described. The existence of two types of scars in budding yeasts was confirmed and their morphology was described in intact cells. In fission yeasts a number of division scars was found in individual cells and changes occurring in the lateral walls as a result of cytokinetic process were observed. In yeasts reproducing by bipolar budding, a new type of scar—the multiple scar—with an important morphogenetic function was discovered. The possibilities and prospects of the new method are discussed.AbstractОписЫвается нояій метод изучения шрамов на нена рушеннЫх клетках различнЫх видов дрожжей на основании вторичной флуоресценции примулина. БЫло подтверждено наличие у почкующихся дрожжей двух видов шрамов. ОписЫвается морфология шрамов ненарушеннЫх клеток. У делящихся дрожжей бЫло обнаружено большое количество шрамов от деления (division scars) на отделънЫх клетках; наблюдалисъ изменения боковЫх ктенок, развивающиеся в резулътате цитокинетического процесса. У дрожжей с бинолярнЫм способом бегетативнобо размножения. бЫл обнаружен новЫй тип шрамов—множественнЫе шрамЫ (multiple scars), связаннЫе с важной мовфогенетической функцией.—Обсуждаются возможности и перспективЫ нового метода.

Fractionation of a population ofSaccharomyces cerevisiae yeasts by centrifugation in a dextran gradient

A method of the fractionation of aSaccharomyces cerevisiae yeast population in dextran gradients is described. The elaboration of this method was based on the finding of a correlation between the size of individual cells and the number of bud scars on their surface and rapid indication of the scars by fluorescence microscopy. The basic conditions for fractionation (determined experimentally) were as follows: 2 ml. yeast suspension (100 mg. dry weight) was applied to the surface of a continuous dextran gradient of 9–16% concentration and was centrifuged at a relative centrifugal force of 200 G for 15 minutes. In fractionation of a whole population, the best fractionation was obtained in a linear gradient. Repeated separation of fractions obtained by centrifugation in a linear gradient in a concave gradient further separated cells without bud scars and accumulated cells with five scars and over. Three fractions were obtained by this technique. The first contained 90–98% cells without bud scars, the second 55–65% cells with 1–4 bud scars and the third 50% cells with five bud scars and over.AbstractОписан метод фракционирования популяции дрожжей Saccharomyces cerevisiae в декстрановых градиентах. Основной предпосылкой для разработки этого метода было обнаружение отношения между размерами отдельных клеток и количеством материнских шрамов на их поверхности, а также способ быстрой индикации шрамов с помощью флуоресцентной микроскопии. Экспериментально были установлены следующие основные условия для фракционирования: суспензию дрожжей (100 мг сухого вещества) в 2 мл вносили в горизонт декстрана с концентрацией, постепенно и непрерывно повышающейся от 9 до 16%, и центрифугировали 15 мин. при относительной центробежной силе 200 G. При центрифугировании всей популяции наиболее успешное фракционирование было получено в линейном градиенте. Путем дальнейшего разделения фракций, полученных в резулятате центрифугирования в линейном градиенте, с применением вогнутого градиента удалось отделить еще клетки без материнских шрамов и скопления клеток с 5 и более шрамами. Так были получены 3 фракции: первая содержала 90–98% клеток без материнских шрамов, вторая— 55–65% клеток c 1–4 материнскими шрамами, а третья—50% клеток с 5 и более материнскими шрамами.

Features of the cell periphery in the deformedras1− cells ofSchizosaccharomyces pombe

The rounding-up phenomenon in the sterileras1− mutant ofSchizosaccharomyces pombe was examined by fluorescence microscopy of Calcofluor- and rhodamine-phalloidin-labeled cells and by freeze-etching. In cells that entered the stationary phase, rapid and dramatic changes occurred in cell shape and isotropic cell expansion, with the cells becoming round and increasing in size. This was accompanied by a collapse of the actin-containing cytoskeleton and a transition of actin dot to transcellular filaments. A considerable part of the plasma membrane of the mutant was smooth and uninvaginated. Notable was a gradual delocalization of the cell wall deposition and a special kind of cell wall rearrangement which altered the preexisting cell wall architecture. This is the first report that describes the morphological consequences induced by the deletion of the singleras gene inS. pombe.

On the question of vegetative reproduction in apiculate yeasts

Fluorescence microscopy, carbon replicas, ultrathin sections and metal-shadowed walls were used for studying changes during cell division of yeasts. Some common features and differences between multipolar budding, fission and the so-called bipolar budding of apiculate yeasts are specified. The conclusion was reached that in apiculate yeasts we are not dealing with a kind of budding but with a type of reproduction that is synonymous with multipolar budding and fission.AbstractИзменения в промежуточных стадиях клеточного деления изучались с помощью флуоресцентннй микроскопии, угольных отпечатков, ультратонких срезов и напыления металлом клеточных оболочек дрожжей. Отмечаются общие черты и различия между почкованием, делением перегородками и т. н. биполярным почкованием у апикулятных дрожжей.—Делается вывод, что у апикулятных дрожжей имеет место такой способ размножения, который, по существу, равноценен с мультиполярным почкованием и перегородочным делением.

Ultrastructure of the surface of multiple scars inSaccharomycodes ludwigii

The use of induced primuline fluorescence led to the discovery of a new type of yeast scars (multiple scars) in the generaKloeckera, Saccharomycodes, Nadsonia andHanseniaspora. The structure and ultrastructure of their surface was studied by electron microscopy, using carbon replicas and isolated cell walls.AbstractУ родов Kloeckera, Saccharomycodes, Nadsonia, Hanseniaspora был на основании вторичной люминесценции примулина описан новый вид шрамов дрожжевого грибка—множес твенные шрамы. Строение и ультраструктура их поверхности изучались под электронным микроскопом на углеродных отпеча тках и на изолированных клеточных оболочках.

On the question of the taxonomy ofEndomycopsis javanensis (Klöcker) Dekker

The author studied the physiology, morphology, sexuality and cytology ofEndomycopsis javanensis. This microorganism does not bud, the mycelium breaks up into arthrospores, blastospores are not formed, sexual reproduction consists in somatogamous conjugation and the cells are mononuclear. Because of these properties,Endomycopsis javanensis cannot be included either in the genusEndomycopsis Dekker orEndomyces Reess. After comprehensive evaluation of all the findings, it was included in the genusSchizosaccharomyces Lindner, as the speciesSchizosaccharomyces javanensis (Klöcker) Streiblová.AbstractИзучались физиология, морфология, половой процесс и цитология Endomycopsis javanensis. У этого микроорганизма почкования не наблюдается; мицелий превращается в артроспоры; бластоспоры не образуются; половой процесс осуществляется путем соматогамической копуляции; клетки—одноядерные. По перечисленным признакам вид Endomycopsis javanensis не может быть отнесен ни к дрожжам рода Endomycopsis Dekker, ни к роду Endomyces Reess. На основе комплексной оценки всех данных было предложено отнести микроорганизм к роду Schizosaccharomyces Lindner с видовым названием Schizosaccharomyces javanensis (Klöcker) Streiblová.