A history of research on yeasts 4: cytology part II, 1950–1990
2002; Wiley; Volume: 19; Issue: 9 Linguagem: Inglês
10.1002/yea.875
ISSN1097-0061
AutoresJames A. Barnett, C. F. Robinow,
Tópico(s)Plant Disease Resistance and Genetics
ResumoThe first part of this review considered the history of early work on yeast cytology16. This, the second part, continues the story, dealing with developments that occurred after 1950. Vacuoles, nuclei, mitochondria and other structures were observed before 1950, but differences between the accounts of their characteristics and their alleged functions in the life of yeasts led to long-lasting controversies. After 1950, however, advances in microscopy, biochemistry, genetics and molecular biology made it practicable to establish the existence and characteristics of most organelles of yeasts. Masterly reviews of this more recent research have already been published: they include that of Matile and his fellow authors126 and those in volume 4 of the second edition of Rose and Harrison's The Yeasts176. Accordingly, the present article summarizes only selected aspects of yeast cytology and Table 1 lists chronologically some of the significant findings in yeast cytology that have been published between 1950 and 1990, not all of which are discussed below. No account is given of advances made since 1990, which involve such innovations as the green fluorescent protein reporter (GFP) system195, confocal microscopy45 and flow cytometric analysis48. Major contributors to the subject in the second half of the twentieth century include Breck Byers, Enrico Cabib, Byron F. Johnson, Philippe Matile, J. Murdoch Mitchison, Oldřich Nečas, Eva Streiblová, Kenji Tanaka and Don H. Williamson. Sadly, however, it is impracticable to pay proper tribute to the many skilful scientists ‘whose technical contributions …︁’ are, to use Fruton's words, ‘forgotten even by those who benefited from them …︁ The names of these highly skilled workers often appeared in print only in an acknowledgement at the end of a scientific article of which the leader of the research group was the principal author’ (72 pp. 31–32). Like most living cells, live yeasts are transparent to visible light, so techniques of staining were, in the past, of major importance for elucidating intracellular structures. But, as already described, alterations to the cellular structures caused by fixing and staining often made it exceedingly difficult to interpret what was seen16. Despite transparency, differences in refractive index exist (i) between unstained cells and the medium in which they are suspended, and (ii) between different parts of the cells. Phase contrast microscopy exploited these differences in refractive index, making them observable as differences in contrast in living cells, without having to use stained preparations of dead cells. Phase differences, invisible to the naked eye, are produced in light waves passing through objects which have differing refractive indexes. In the 1930s, Zernike3 developed an optical method of converting phase differences into observable differences of amplitude, so that normally transparent material becomes readily visible232-235. Variations in thickness or in refractive index of objects then appear as having differences of intensity.4 The firm of Carl Zeiss produced a prototype phase contrast microscope in 193686, but such instruments became widely available5 only after World War II, that is, from the late 1940s (27 p. 297). For any microscope using visible light, the maximum resolving power7 is about 0.2 µm. But another development in the 1930s was the production of the electron microscope with a resolving power of about 10 nm70; this made it possible to observe clearly the fine structure of yeasts and other cells. One of the first such electron microscopic studies of yeasts was of cell wall structure, published by Northcote and Horne in 1952149; and in 1957 Agar and Douglas described the ultrastructure of the yeast cell, using the transmission electron microscopy of thin sections fixed with KMnO41 (Figure 1). Electron micrograph of a section of S. cerevisiae. C.m., plasma membrane; C.w., cell wall; I.m., endoplasmic reticulum; M, mitochondrion; N, nucleus. Reproduced by permission from Agar and Douglas (1957)1 In the 1960s, the technique of freeze-etching, which had been applied successfully to biological materials by Steere in 1957197, greatly furthered the study of cell walls and other cytological features of yeasts. With an advanced version of this technique, Moor published many magnificent electron micrographs showing internal structures of yeast cells, including pores in the nuclear envelope (Figure 2). Yeast was (i) frozen to −100°C, (ii) put under high vacuum (2–3×10−6 mmHg), (iii) cut with an ultramicrotome supercooled to −196°C, (iv) freeze-dried to expose intracellular structures, and (v) shadowed with a carbon arc137, 139. This technique required a very high rate of cooling in glycerol to prevent formation of ice crystals in the cytoplasm, which destroy the organelles. Moor's diagram of his method is reproduced in Figure 3. Mühlethaler has published a brief history of freeze-etching141. The nucleus in a freeze-etched cell of S. cerevisiae, showing pores in the nuclear envelope: an electron micrograph published by Moor in 1966138. Reproduced from the Journal of Cell Biology, 1966, p. 155, by copyright permission ofRockefeller University Press Moor's diagram of the four processes of his method of freeze etching. (a) The yeast is frozen to −100°C; thereafter, under high vacuum, (b) the upper part, is cut off with a super-cooled knife; (c) ice is removed by sublimation; (d) the projecting material is shadowed with a carbon-arc (137 Abb. 1). Reproduced by permission from Moor H. 1964. Zeitschrift für Zellforschung und Mikroskopische Anatomie 62: 546–580.© 1964 Springer-Verlag In 1947, van Dorsten and his colleagues had used whole yeast cells for testing an experimental electron microscope and their Figure 8 indicates the presence of bud scars9216. Later, Northcote and Horne disintegrated baker's yeast mechanically and, after centrifuging, mounted the cell wall fraction in polyvinyl formal films.10 The walls proved to be stratified; after acid hydrolysis, chromatography showed that the outer layer was mainly mannan-protein; the walls contained 29% glucan, 31% mannan, 13% protein and 8.5% lipid149. Mannan in yeast cell walls had been reported previously96. The primary septum between a mother cell of Saccharomyces cerevisiae and its bud is composed mainly of chitin11136. In 1960, Mundkur boiled cellwalls of a strain of a Saccharomyces species in dilute acid to remove mannan and protein, and obtained a residue which he presumed to be chitin145 and which had already been found in various yeast species (61 p. 238). Six years later, Bacon and his colleagues treated pressed S. cerevisiae with NaOH, obtaining a residue ‘almost exclusively’ of bud scars (Figure 4). The infra-red spectrum showed ‘the presence of much chitin’ (about 1%) and some other polysaccharide (10 p. 37c). In 1971, Cabib and his colleagues found that chitin is restricted to rings around the scars in S. cerevisiae and S. pastorianus (carlsbergensis)37, 38. Electron micrograph by Bacon and his colleagues of bud scar chitin residues of S. cerevisiae, after dissolving away mannan and glucan and shadowing with nickel–palladium (10 Plate 1b). Reproduced by permission from Bacon JSD, Davidson ED, Jones D, Taylor IF. 1966. Biochemical Journal 101: 36c–38c.© 1966 the Biochemical Society Considerable effort by a number of workers was put into assessing the glucan and mannan content in baker's yeast (Table 2). In 1965, Edwards summarizes the techniques then used for isolating the polysaccharides from baker's yeast63. Glucan was prepared from crude extracts made by successive treatments of yeast with 6% or 3% NaOH and heating at 60°C or 80°C, the glycogen being removed by autoclaving at 135°C, first in 20 mM NaAc, pH 7, and then in water. For mannan, the yeast was autoclaved at 140°C, concentrated under reduced pressure, treated with acetic acid and the crude mannan precipitated with ethanol (Figure 5). Summary of methods involved in preparing mannan and glucans from baker's yeast. After Duffus et al.58 The walls of other species, too, were analysed in the 1960s and 1970s. SentheShanmuganathan and Nickerson found 81–91% carbohydrate, 2% hexosamine (presumably mostly chitin) and 0.6–1.4% phospholipid in Trigonopsis variabilis walls188; while Bush and his colleagues reported 74–82% glucan and 9–14% galactomannan in the walls of Schizosaccharomyces pombe33. From electron micrographs, made in 1964, of thin sections of S. cerevisiae, S. pastorianus, S. ludwigii and Sz. octosporus, Hagedorn estimated that the thickness of the walls varied between about 150 nm and 400 nm, varying not only between species, but also with different conditions of growth88. Some features brought to light in the 1970s and 1980s include the following. Glucans seem to determine the rigidity of the yeast cell walls and are protected from enzymic lysis by overlaying mannoproteins187; consequently, treating intact cells with a proteinase, which removed the dense outer layer completely, was found not to alter the cell shape237. The walls are negatively charged by phosphate groups which occur both in the mannan11 and in the protein108. Accordingly, invertase (β-fructofuranosidase), which Friis and Ottolenghi had shown in 1959 to be held in the cell walls of a S. cerevisiae/S. pastorianus hybrid, could be released by treating cells with 1,4-dithiothreitol71, as Kidby and Davies later found for Kluyveromyces marxianus (Saccharomyces fragilis) in 1970112, 113 and Sommer and Lewis for S. cerevisiae itself in 1971192. A consequent hypothetical structure of the cell wall proposed by Kidby and Davies is shown in Figure 6. Diagram of Kidby and Davies' (1970) hypothetical structure of the yeast cell wall. Phosphoric diester links are represented by —P—. Reproduced by permission from113 As long ago as 1950, Conway12 and Downey, working with centrifuged baker's yeast and with large molecules, such as inulin,13 found that the space between the cells constituted 23% of the total volume. By contrast, measurements with small molecules, such as lactose gave the larger volume of 33% total space. The difference was attributed to the penetration of the smaller molecules into the cell wall47. Later, several investigations have shown that mannoproteins of the cell wall limit its permeability, largely owing to the disulphide linkages, ionic interactions51, 52, 237 and the large mannan side-chains53; but porosity of the walls changes with the composition of the medium, the growth phase and the strain of yeast. Hence the cell wall is a dynamic structure39. De Nobel and Barnett have published a short account of the history of studying the passage of molecules through yeast cell walls50. Concanavalin A14 binds specifically to mannan and, in 1971, it was the first lectin15 used to label yeast walls211, 212, the fluorescein-conjugated lectin being located by fluorescence microscopy. Using mercury-labelling of concanavalin A, in order to make it visible by electron microscopy, Horisberger and his colleagues demonstrated mannan in bud scars of S. cerevisiae20, 104. They also used immunogold labelling and anti-mannoprotein antibodies to show that the outer part of the cell is rich in mannoproteins105. An electron microscopic study of Sz. pombe, published by Conti and Naylor in 1959, included an image of the septum formed during cell division. Such images showed that the septum was three-layered46 and this was confirmed by Tanaka in 1963208. From the electron microscopy of thin sections stained in lead acetate, in 1973, Johnson and his colleagues described the centripetal formation of this septum in some detail109 (Figure 7). Fission scars, formed when two cells separate, can be seen in a scanning electron micrograph (Figure 8). A hitherto unsuspected structure was amat of microfilaments involved in the cell division of Sz. pombe and described by Tanaka (personal communication). This can be seen in his striking electron micrograph showing a row of actin-associated vesicles on either side of a bundle of microfilaments16 at the site of an ingrowing septum (Figure 9). Such filaments were first seen by Girbardt79 and named a ‘microfilamentous septal belt’. Centripetal formation of the septum in cell division of Sz. pombe, described by Johnson and his colleagues in 1973. The primary septum is labelled AR (‘annular rudiment’) in (d) and (e); the secondary septum becomes the scar plug, which is the new end of the cell; in (f) FS is a fission scar and SP the scar plug. Reproduced by permission from Figure 1 of109 Sz. pombe: scanning electron micrograph showing fission scars. Courtesy of Teresa Niccoli Electron micrograph of part of a cell of Sz. pombe, prepared by freeze-substitution (see156); vesicles are arrayed on either side of a microfilament bundle associated with the ingrowing septum. Electron micrograph of K. Tanaka, reproduced from Figure 39c of171 In the 1950s, instruments available for disintegrating cell walls in order to prepare cell-free extracts, were exceedingly vigorous and damaged the organelles. Such, for example, were the Hughes press106 and the Nossal disintegrator150. Accordingly, the development of methods for preparing yeast protoplasts was most timely, and these methods soon proved valuable for studying organelles such as mitochondria (e.g.57), although yeast protoplasts then became a subject of study in themselves144, 147. In 1956, Nečas described the formation of some protoplasts or sphaeroplasts17 amongst spontaneously autolysing S. cerevisiae146. The next year, influenced by the observation of Giaja in the 1920s77 that the digestive juice of the edible snail Helix pomatia removed the yeast cell wall, Eddy and Williamson used such juice to obtain protoplasts of S. pastorianus (carlsbergensis)62. Subsequently, other lytic preparations were developed forpreparing yeast protoplasts, such as those derived from Streptomyces sp.76 or from Cellulosimicrobium cellulans (Arthrobacter luteus)59. Although bud scars were first described by Cagniard-Latour in 183640 (see13), they were not rediscovered until 1950 by Barton19. Subsequently, much work was done on them by Streiblová and others in the 1960s and 1970s26, and her introduction of fluorescence methods heralded later extensive researches on bud and birth scars for studying aspects of yeast population dynamics and ageing74, 75, 87. Electron microscopic studies on S. bayanus (uvarum) showed that bud scars never overlap each other, although they may overlap a birth scar22. In the 1970s and 1980s, the construction of newly synthesized cell walls and of septa between mother cell and bud was studied extensively by Cabib and his colleagues35, 36, 39. Chitin, associated with bud scars (as described above), is synthesized by chitin synthases18 bound to the plasmalemma39. Of these, Chs3 forms the chitin ring at budding; Chs2 repairs the septum when the bud separates and Chs1 is responsible for forming the primary septum236 (see Figure 10). Septum formation and bud and birth scars are easily observed, for chitin is readily stained by the fluorescent substances, primulin201, 202 or Calcofluor19110. Figure 11(A)-11(D) illustrates bud and birth scars of S. cerevisiae. Cabib's diagrammatic scheme of budding of S. cerevisiae, published in 198239. The insert, top left, depicts a tentative structure of the cell wall; magnified. A–G represent bud formation and separation. Chitin is shown in black and the plasmalemma as a dotted line. SP, septal primordia; PS, primary septum; SS, secondary septa; B Sc, bud scar From 39 Bud and birth scars of S. cerevisiae. First described by Cagniard-Latour in 183640, they were rediscovered by Barton in 1950. (A) One of Barton's photomicrographs: A is the birth scar, B is the first bud scar; photographed using mercury violet illumination, with an Ilford 601 filter. Reproduced by permission from19. Electron micrograph of bud scars. Reproduced by permission from Bartholomew and Mittwer (1953)18. Many bud scars shown using fluorescence microscopy of cells stained with primulin. Reproduced by permission from Streiblová200. Scanning electron micrograph showing birth scar (top left) and bud scar (below). Reproduced courtesy of Masako Osumi. The term ‘cell cycle’ refers to cellular processes occurring after the formation of a mother cell and before the subsequent complete formation of its daughter cell. For S. cerevisiae, the start of bud formation (‘initiation’) is the beginning of a new cell cycle. In a review article, Harold refers to the large number of genes of S. cerevisiae obtained which, when mutated, produce grossly misshapen cells; and for which the biochemical activity of each gene product is known in detail. Many of these were isolated as cdc20 mutants, that stopped the cell cycle at particular stages, causing ‘reproducibly abnormal morphology …︁’89 (see Table 3). Williamson and Scopes …︁ devised procedures for preparing synchronously dividing cultures ofSaccharomyces cerevisiae. Essentially, these processes involve subjecting a population of resting cells of uniform size to a series of cycles of alternate feeding and starvation. Conditions are arranged so that no new buds or cells are formed during the treatment, and the organisms thus remain in an effectively ‘resting’ state. However, on inoculation into a growth medium, they undergo several cycles of growth and division with a marked degree of synchrony (225 p. 759). Research on the cell cycles of S. cerevisiae and Sz. pombe has given information about the regulation of the size of the cells. Mitchison used interference microscopy, in 1957, to measure changes in the volume of individual cells of Sz. pombe. By measuring the cell dimensions from photographs, he calculated the volume of each cell from the formula for a cylinder with hemispherical ends.21 The volume increased slightly throughout much of the interphase, abruptly ceased changing about an hour before the cell split and increased markedly just after cleavage (Figure 12). Mitchison also found that the cells of Sz. pombe extended at one end only, usually the older one132. Subsequently, Button and Garver found that the size of cells of Candida (Torulopsis) utilis varies with the growth rate34. Mitchison also prepared synchronous cultures of Sz. pombe by sedimentation, writing that this method is not as good as the method of Williamson and Scopes ‘since the separation of small cells is incomplete …︁ It does, however, have many advantages. It is simple, and requires little in the way of apparatus …︁ and …︁ is relatively quick …︁ The whole procedure …︁ takes 20–80 min’133. Curves of the growth of one cell of Sz. pombe through one division to two cells. Results published by Mitchison in 1957. Reproduced by permission from Figure 2 of132 The order of events occurring during the cell division cycle of Saccharomyces cerevisiae, a budding yeast, have been rather well characterized and some studies on the fission yeast, Schizosaccharomyces pombe, have been reported. Most of these investigations were made possible by the availability of techniques for obtaining synchronous division in yeast (91 p. 377). There are two major events in eukaryotic cell division cycles: DNA replication (S phase) and nuclear division (M phase). The interval before DNA replication is called G1 and that before nuclear division G2; Figure 13 is Hartwell's diagram of this sequence of events in S. cerevisiae, which he published in 1974 and, for comparison, Figure 14 is a more recent version. In both S. cerevisiae and Sz. pombe, the initiation of DNA replication is precisely controlled, only occurring after nuclear division is complete. Many mutants of the cell division cycle (CDC) genes have been isolated, mutants that affect the start of DNA replication (for reviews, see151, 166). Hartwell's diagram of the sequence of events in the cell division cycle of S. cerevisiae, published in 197493. IDS, initiation of DNA synthesis; BE, bud emergence; DS, DNA synthesis; NM, nuclear migration; mND, nuclear division; IND, late stage of nuclear division; CK, cytokinesis (i.e. division of cytoplasm); CS, cell separation. Timings: G1, interval between previous cytokinesis and initiation of DNA synthesis; S, period of DNA synthesis; G2, time between DNA synthesis and onset of mitosis; M, period of mitosis.http://www.sciencemag.org. Readers may view, browse and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes. Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher. Reproduced with permission from Hartwell LH, Culotti J, Pringle JR, Reid BJ. 1974. Genetic control of the cell division cycle in yeast. Science 183: 46–51. © 1974 American Association for the Advancement of Science. Diagram of the mitotic cycle of S. cerevisiae, published by Williamson in 1991, illustrating major changes in nuclear cytology in relation to the stages of the cell cycle. Reproduced by permission from222 Hartwell published a series of papers describing over 100 cdc mutants of S. cerevisiae, having first, in 1967, recognized some among many temperature-sensitive lethal mutants90. Each of these mutants stopped division occurring at a specific stage of thecell cycle, irrespective of when during the cycle the cells were moved from a permissive to a restrictive temperature. In the mid-1970s, both Hartwell, working with S. cerevisiae and Nurse with Sz. pombe, showed that the events controlled by the various CDC genes were highly interdependent, any one event having to be completed before onset of the subsequent stage93, 154. Later, Hartwell called the control mechanisms which enforce this interdependency ‘checkpoints’94. Hartwell and Nurse have reviewed this work69, 92. …︁ sequenced, expressed in E. coli, the gene product purified and antibodies raised to provide tools which can be used to investigate the roles ofthe molecules during the cell cycle. In this waythe study can proceed initially from an abstract description of the problem based on identification of the genes involved, to a concrete molecular description of the components and processes making up the cell cycle. The strength of this approach is that nothing need be known about the nature of the molecules involved at thebeginning of the work, but this information will gradually emerge as the study proceeds (153 p. 239). The function of checkpoints in the cell cycle is toensure the completion of early events before late events begin. When checkpoints are eliminated by mutation …︁ infidelity of chromosome transmission, or increased susceptibility to environmental perturbations (like DNA damaging agents) result. …︁ when mitotic infidelity is rampant and reproducible as it is in many types of human tumours it may be fruitful to consider perturbations of the checkpoints that normally ensure mitotic fidelity as potential causes (94 p. 633). Cyclin was discovered by a group of students taking a summer laboratory course at Woods Hole, Massachusetts. They observed that the concentration of a hitherto unknown protein dropped precipitously at the end of mitosis, and increased during interphase (G1, S, and G2) – hence the name cyclin (203 p. 987). However, the final name on the paper reporting this discovery in 198365 was Tim Hunt; and, with Nurse and Hartwell, he too received the Nobel prize in 2001 for this work238. Under the ordinary microscope the nucleus of the living yeast cell is not readily distinguished from the cytoplasm. It becomes visible in the presence of 0.5% acetic acid (pH 3.4)…︁ The nucleus then appears clear and watery except for a peripheral cap or crescent of high refractility, the Kernkopf of Henneberg98. Townsend and Lindegren214 state that the acid-treated cells remain viable and we have confirmed this (173 p. 131). Müller24had described a method of observing nuclei in living yeast cells in 1956. He used phase contrast microscopy to observe cells suspended in media of high refractive index, such as 30% gelatin (n′≈1.38)25142, 143. Working at the Institut für Mikrobiologie und Experimentelle Therapie at Jena (Germany) in 1959, he spent a year using this method in making superb time-lapse films showing changes in nuclei, vacuoles and mitochondria during cell growth in various kinds of yeast.26In addition to studying fixed and stained preparations, Robinow and Marak also used phase contrast microscopy of budding yeast cells growing in 21% gelatin (plus nutrients) and noticed that the envelope of dividing nuclei seemed to remain intact173, thus confirming Guilliermond's conclusion, made in 191785, that the yeast nucleus divides by elongation followed by constriction.27Regrettably, Guilliermond follows this accurate generalization with an account and illustrations of meiosis (première mitose and seconde mitose) in Sz. pombe, to which his description does not apply. In the nuclei of the cells of most plants and animals, the nucleoli are spherical or ovoid and usually ‘float’ in the centre of the mass of chromatin that fills the rest of the nucleus. However, in cells of S. cerevisiae, the nucleolus does not lie in the centre of the nucleus and it has two faces: the outer face is tightly apposed to a large part of the inner surface of the nuclear envelope; the contours of the inner face are often irregular. The physiological significance of this peculiar and noteworthy arrangement is unknown. Its singular appearance has been an important factor in translucent, round vacuoles being mistaken for nuclei. Robinow and Marak's figures 24 and 25 showing fixed and stained cells, and reproduced here as Figure 15, revealed the nucleus as consisting of three materials: a dense, irregularly shaped crescent, deeply stainable with iron alum haematoxylin or acid fuchsin; a faintly stainable nucleoplasm (the chromatin); and some material readily stainable with acid fuchsin, within the chromatin area, which assumes the shape of a dot or that of a slender dumb-bell. That this material is composed of non-chromatinic components of the mitotic apparatus at early stages of anaphase is clear from various electron micrographs, such as that of S. pastorianus (carlsbergensis), made by Bjoern Afzelius and shown as figure 28 in reference126 and also from those of S. cerevisiae by Peterson and Ris159. Fixed and stained cells of S. cerevisiae from a paper published by Robinow and Marak in 1966. The fibre can be seen clearly in 24a and 25a, both cells stained with acid fuchsin; the cells in 24b and 25b were stained with Giemsa after hydrolysis173 From Robinow and Marak173 The nucleus of S. cerevisiae consists of an envelope, a nucleolus and chromatin. Within the space occupied by chromatin, a ‘fiber apparatus’ was discovered by Robinow and Marak173 which, depending on the yeast's stage in the cell cycle, may appear as a small dumb-bell or a straight, long fibre. High magnifications of thin sections have shown the dumb-bell phase, visible by light microscopy, represents the non-chromatinic components of nuclei at very early phases of anaphase of mitosis. Figure 16 reproduces figures 26–29 of Robinow and Marak: at advanced stages of nuclear division, the dot or dumb-bell apparatus becomes a long thinfibre with a spindle pole body at each end. These authors' electron micrographs of S. cerevisiae show that the fibre, or spindle, is composed of microtubules between two spindle pole bodies (Figure 17). The question of the relationship of the fibre to the 16 haploid or 32 diploid chromosomes of S. cerevisiae remains unknown. Cells of S. cerevisiae, Helly-fixed and acid fuchsin-stained, from Robinow and Marak173. The fibres inside the dividing nuclei are fully expanded; the crescentic nucleolus is deeply and the chromatin faintly stained From Robinow and Marak173 S. cerevisiae: electron micrograph of a dividing nucleus, showing microtubules of the spindle, attached (at the upper end of the photograph) to a spindle pole body. Figure 45 of Robinow and Marak173 The yeasts, whose cytology has been discussed so far, belong to the order Saccharomycetales of the Hemiascomycetes; but, among the basidiomycetes, there are fungi which, at some stage in their life cycle, occur as single cells. When budding, many look like the more familiar budding yeasts, but differ markedly from the latter in their mode of nuclear division. The nucleus appears to divide by a process of elongation and constriction during which roughly half of the nucleolus, along with the surrounding chromatin, passes into the bud. The nuclear membrane was found to persist during all stages of division (210 p. 127). In their budding phase, many of these yeasts do not look different from more familiar kinds of budding yeasts but they differ markedly from the latter in their mode of nuclear division (172 p. 85). In two papers, McCully and Robinow examined three of these weird organisms, as living cells, as stained preparations and also as McCully's brilliantly successful electron micrographs128, 129. Mitosis, here, strangely but regularly, takes place in the bud. The nuclear envelope does indeed not persist in its entirety, but nor does it ‘break down’. However, mitosis does involve, temporary, ‘large discontinuities’ of the nuclear envelope, giving rise to surreal configurations, evoking such comments as: ‘The nuclear envelope is present along the left side of the spindle but the right side of the spindle is open to the cytoplasm’. The cycle of mitosis is illustrated in figure 1 (reproduced here as Figure 18) and figure 4 of the first of these authors' papers128. A particularly clear example is provided by figure 4 of their second paper129, with a conventional anaphase image and emptiness of the protop
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