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J. Cell Sci. Suppl. 5, 229-241 (1986) Printed in Great Britain © The Company of Biologists Limited 1986 229 GROWTH POLARITY AND CYTOKINESIS IN FISSION YEAST: THE ROLE OF THE CYTOSKELETON JO H N M A R K S , I A I N M . H A G A N and JE R E M Y S. H Y A M S * D epartm ent o f Botany ¿2? Microbiology, University College London, Gower Street, London WC1E 6BT, U K S UMMA R Y The distribution of F-actin in the fission yeast Schizosaccharomyces pombe was investigated by fluorescence microscopy using rhodamine-conjugated phalloidin. Fluorescence was seen either at the ends of the cell or at the cell equator. End staining was predominantly in the form of dots whilst equatorial actin was resolved as a filamentous band. The different staining patterns showed a close correlation with the known pattern of cell wall deposition through the cell cycle. In small, newly divided cells actin was localized at the single growing cell end whilst initiation of bipolar cell growth was coincident with the appearance of actin at both ends of the cell. As cells ceased to grow and entered cell division, a ring of actin was seen to anticipate the deposition of the septum at cytokinesis. The relationship between actin and cell wall deposition was further confirmed in three temperature-sensitive cell division cycle (cdc) mutants; cdc 10, cdc 11 and cdc 13. Immunofluor escence microscopy of S. pombe with an anti-tubulin antibody revealed a system of cytoplasmic microtubules extending between the cell ends. The function of these was investigated in the coldsensitive, benomyl-resistant mutant ben A. In cold-grown cells actin was seen to form conspicuous filamentous rings around the nucleus. The origin of these and the possible role of microtubules in the cell-cycle-dependent rearrangements of F-actin are discussed. INTRODUCTION The use of simple model systems to study complex problems is a familiar strategy in biological research. Recent developments in molecular genetics have reinforced the value of organisms such as yeasts in the study of fundamental cellular processes. This is particularly true in the case of the cytoskeleton. Although the small size of yeast cells and the presence of a cell wall present obstacles to traditional approaches to the organization of cytoplasmic filament systems such as immunofluorescence microscopy, these have now been largely overcome (Kilmartin & Adams, 1984; Adams & Pringle, 1984). Coupled with the analysis of cloned actin and tubulin genes (Thomas et al. 1984; Yanagida et al. 1985) this opens up a combination of approaches to cytoskeleton structure and function that is possible in few other organisms. An additional attraction of yeasts as experimental systems is the availability of a large number and variety of mutants that affect the yeast cell division cycle (Pringle & Hartwell, 1981; Nurse, 1981). Thus, the relationship of the cytoskeleton to other cell cycle events is also open to direct study. Two yeasts in particular have been the focus of most attention, the budding yeast Saccharomyces cerevisiae and the fission •A u th or for correspondence. 230 J . Marks, I. M. Hagan andjf. S. Hyams yeast Schizosaccharomyces pombe. Of the two, 5. pombe may prove to be the better model, if only because its cell division cycle more closely resembles that of higher eukaryotes (Nurse, 1985). Fission yeast cells grow only at their ends (Johnson, 1965; Streiblova & Wolf, 1972; Mitchison & Nurse, 1985). In newly divided cells growth occurs solely at the old end, that is, the end that existed prior to septation. At a point in the Gz phase of the cell cycle termed N E T O (new-end take off) bipolar growth is initiated. This is maintained until the end of Gz, at which point growth ceases and the sequence of mitosis, cytokinesis and cell separation begins. In this paper we describe the relationship of the cytoskeleton to these changes in growth polarity. We also address the role of the cytoskeleton in establishing the division plane in these simple eukaryotes, both in wild-type cells and in various cell division cycle (cdc) mutants. MATERIALS AND METHODS Wild-type S. pombe strain 972h_ and the mutant strains cdclQ, 129h_ , cdc 11, 136h~ and cdc 13, 117h- (Nurse et al. 1976) were kindly supplied by D r Paul Nurse; strain ben A, D3 (Roy & Fantes, 1982) was kindly supplied by D r Peter Fantes. Cultures were grown as previously described (Marks & Hyams, 1985). Temperature-sensitive cdc strains were grown at 25°C to mid-log phase prior to arrest at 36°C for 5-7 h. The cold-sensitive mutant ben A was grown at 36°C to mid-log phase and blocked at 22-5°C for 24h. Intracellular F-actin distribution was determined by phalloidin staining (Wulf et al. 1979; Wieland & Govindan, 1974) according to Marks & Hyams (1985). Nuclear morphology, and thus the position of the cell in its mitotic cycle, was determined using DA PI (4'-6-diamidino-2-phenylindole; Williamson & Fennell, 1975). Calcofluor white was used to reveal both the cell wall and the septum (Darken, 1961). Tubulin staining of wild-type cells using the monoclonal antibody to yeast tubulin, Y O L 1/34 (Kilmartin et al. 1982), was performed essentially after the method of Kilmartin & Adams (1984). RESULTS A field of 5. pombe cells stained with rhodamine-conjugated phalloidin as a probe for F-actin is shown in Fig. IB. Fluorescence is seen either at one end of the cell, at both ends or at the cell equator. End staining is mainly in the form of dots whereas equatorial actin is resolved as a filamentous ring. The different staining patterns may be ordered with respect to the cell cycle by reference to Fig. 1A, which shows the same field of cells stained with the cell wall stain Calcofluor and the D NA probe D API. Newly divided 5. pombe cells grow only at the old end (Mitchison & Nurse, 1985). The two cell ends can be distinguished by their affinity for Calcofluor, the old (growing) end staining brightly whilst the new (non-growing) end appears as a dark, unstained hemisphere (Fig. 1A, cell 1 ). When new-end growth is initiated (N ETO ) this dark region is internalized and appears as a birth scar on the cell surface (Fig. 1A, cell 2 ). Comparison of the phalloidin staining patterns reveals that the distribution of actin coincides precisely with the polarity of cell growth; namely, actin resides at the single growing end before N ETO and at both growing ends after N E T O (compare cells 1 and 2 in Fig. IB). The transition from one-end to two-end staining also sees the transient appearance of fine filaments of F-actin possibly extending the length of the cell (cell J , Fig. IB). Fission yeast cytoskeleton 231 F ig. 1. T w o views of the same field of asynchronous wild-type S . pombe cells: A, stained with C alco flu or-D A P I; B, stained with rhodamine-conjugated phalloidin. N um bers designate the staining patterns referred to in the text. Bar, 10 j.lm. Double-end actin staining is maintained until the onset of mitosis (cell 4, Fig. 1A), which coincides with the completion of end growth. Actin now disappears from the poles but reappears as a ring at the cell equator where it overlies the dividing nucleus (cell 4, Fig. 1A ,B). T h e position of the actin ring anticipates the deposition of the septum , which stains intensely with Calcofluor (cell 5, Fig. 1A). As the septum grows centripetally the appearance of the equatorial actin changes from a filamentous ring to clusters of dots (cell 6, Fig. IB ). At the completion of septation the remnants of the equatorial actin lie at the new ends of the two daughter cells (cell 7, Fig. IB ). Since growth will be initiated at the opposite (old) end, actin must rapidly relocate to the other end of the cell before the next cell division cycle can begin. This transition is again accompanied by the transient appearance of fine actin fibres (not shown). T he complete sequence of actin distribution through the S. pombe cell cycle is summarized in Fig. 2. Further evidence for the relationship between actin and cell growth and division in fission yeast has been obtained from various temperature-sensitive cdc mutants. These become arrested at a specific point in the cell division cycle when grown at the restrictive temperature although their metabolic processes are maintained and they continue to elongate (Bonatti et al. 1972; Nurse et al. 1976). Fig. 3 shows Calcofluor and phalloidin images of cdc 10, which arrests in G\, i.e. prior to N E T O . Intense 232 J . Marks, I. M. Hagan andy. S. Hyams actin staining is seen at the growing old end although a few dots are also seen at the new end (Fig. 3B). Correspondingly, although Calcofluor staining indicates that growth is predominantly at the old end, a small amount of cell wall deposition at the new end is also detectable (Fig. 3A). None of the cells display equatorial actin nor do they form septa. Similar images of cdc 13 are shown in Fig. 4. This mutant arrests in mitosis although under certain conditions a proportion of the cells leak through the temperature block and form multiple, aberrant septa (Fig. 4A). Growing cell ends (as judged by Calcofluor staining) again reveal intense actin fluorescence in the form of both dots and fibres (cells 1, 2 and 3 in Fig. 4B). Actin dots also occur in the region of the multiple septa in cell 4 whilst cells 5 and 6 reveal the equatorial actin ring, which anticipates the septum. The contiguous nature of the ring is particularly clearly seen in these cells. In cell /, the equatorial actin is largely dispersed following the formation of a septum and intense staining is again seen at the cell poles. Fig. 5 shows the relationship of the equatorial actin ring to nuclear position in an early septation mutant cdc 11. These cells are unable to undergo cytokinesis at the restrictive temperature although nuclear division and cell elongation continue. At the first mitosis following the temperature block the daughter nuclei return to a point either side of the middle of the cell (cell 1, Fig. 5B). Reinitiation of end growth is indicated by the bipolarity of actin staining (cell 1, Fig. 5A). Although these cells do not form a septum, a pair of actin rings forms at the positions occupied by the two nuclei prior to mitosis (cell 2, Fig. 5A). The nuclei do not appear to be physically connected to their respective actin rings since they are able to move away from their original position at the second mitotic division (cell 2, Fig. 5A,B). At the next mitosis actin rings appear at the position occupied by each of the four daughter nuclei (not shown). Although not excluding other possibilities, we have begun to investigate whether the changing patterns of actin described above are dependent on the presence of cytoplasmic microtubules. S. pombe cells stained with anti-tubulin antibody are shown in Fig. 6. During interphase, groups of microtubules extend between the two ends of the cell. As the cell enters mitosis, these cytoplasmic microtubules disappear, to be replaced by an intranuclear spindle. The precise details of the cell cycle rearrangements of tubulin in S. pombe will be presented elsewhere (I. M. Hagan, P. Nurse & J. S. Hyams, unpublished data). Since anti-microtubule drugs have only a limited effect on fission yeast (Bums, 1973; Walker, 1982; our unpublished results) we have attempted to destabilize cytoplasmic microtubules by genetic means. The cold-sensitive, benomyl-resistant mutant benA is unable to undergo cell division at 20°C (Roy & Fantes, 1982). When cold-grown cells are stained with phalloidin, a dramatic rearrangement of actin is observed. Instead of multiple small dots at the ends of the cells, a single large dot is frequently present. Most obviously, however, much of the cellular F-actin is seen to be associated with the nucleus. Predominantly, this is in the form of a continuous ring, although sometimes this is twisted to form a figure eight and often bears a tail like a hangman’s noose (Fig. 7). These various perinuclear configurations are present in up to 90 % of the cells. 233 Fission yeast cytoskeleton (Os O O B -e- o $ H F ig. 2. Schem atic representation of the structural rearrangements of F-actin through the cell division cycle of 5 . pombe. T h e old end of the cell is at the top of the figure. T h e open circle in the centre of the cell is the nucleus, dots show the position of actin. Because of the asym m etric nature of cell growth in S . pombe, the cell wall material inherited by each daughter cell from its mother is different. The cell depicted in A has a ‘waistcoat’ of old cell wall material, which acts as a useful marker for the directionality of new wall deposition. Note that at the end of the sequence (cell-H) only one daughter inherits the waistcoat. T h e major transitions of actin distribution are seen in cell C, which is at N E T O , where actin filaments are also transiently observed; cell E , which shows the com pletion of end growth and the initiation of the actin ring (note that the shape of the nucleus has changed to signify that these events are coincident with the initiation of m ito sis); and cell H, where actin left at the cell equator by the breakdown of the actin ring m ust relocate to the opposite end of each daughter cell in order that new cell growth may be initiated. Fibres also appear transiently at this stage. (Redrawn from Marks & Hyams (1985).) F ig. 3. Tem perature-attested cells of cdc 10 double stained with: A, Calcofluor; and B , phalloidin. Note that as these cells block before N E T O both growth and the location of actin are restricted largely to the old ends. Bar, 10 fxm. 234 J . Marks, I. M. Hagan and J . S. Hyams £ ** 2 4A F ig. 4. Calcofluor (A) and phalloidin (B) images of temperature-arrested cdc 13 cells. N o te : the intense apical staining associated with the growing cell ends in cells 1 , 2 and 3 ; the diffuse arrays of actin dots associated with the multiple septa in cell 4 ; the actin ring preceding septum deposition in cells 5 and 6. Bar, 10 fxm. 235 Fission yeast cytoskeleton m *. 4% 5A F ig. 5. P h ase-D A P I (A) and phalloidin (B) images of two temperature-arrested cdc 11 cells. At the restrictive tem perature these cells undergo repeated nuclear divisions but do not divide. Following the first m itosis the daughter nuclei return to the middle of the cell and end growth is reinitiated (cell / ) . As the daughter nuclei synchronously enter the next mitotic division an actin ring appears at the site formerly occupied by the nuclei (cell 2 ) . T h e fact that cell 1 contains two interphase nuclei as opposed to a single mitotic nucleus is confirmed by the obvious difference in size between the nuclei in these two cells. Bar, lOjUm. DISCUSSION T h e precise manner of cell growth in S. pombe coupled with the availability of mutants affecting both the cytoskeleton and the cell division cycle make this a most attractive organism in which to investigate the structural rearrangements and inter actions of cytoskeletal proteins through the cell cycle and the mechanisms whereby these are integrated with other cellular events. In this paper we have shown that the two major growth transitions in the S. pombe cell cycle, that is, from monopolar to bipolar cell growth early in Gz (N E T O ), and the cessation of end growth and the initiation of cell division are accompanied by corresponding rearrangements of F-actin. T hese findings, which were originally established in wild-type cells (Marks & Hyam s, 1985), have been confirmed here by the use of cell division cycle mutants arrested at different points of the cell cycle by growth at the restrictive temperature (N urse et al. 1976). In cdc 10 both actin and cell growth were predominantly mono polar (consistent with the known execution point of this mutant before N E T O ). It was noticeable, however, that a small amount of actin staining, and a corresponding degree of Calcofluor staining, was always detectable at the opposite pole. A small amount of new-end growth prior to N E T O has been detected in wild-type cells although its extent has been difficult to assess, partly because it represents only a minor contribution to total cell growth and also because the methods of analysis used to date are relatively crude (Mitchison & Nurse, 1985). T he problem may be 236 J . Marks, I. M. Hagan and y . S. Hyams resolved when more sophisticated methods for detecting zones of cell expansion in yeasts are applied to 5. pombe (Staebell & Soli, 1985), or through the use of mutants such as cdc 10, which can be held prior to N E T O for extended periods. Preliminary observations of cd c\\ have shown that the normal relationship between nuclear division and the formation and disappearance of the equatorial actin ring is maintained even in the absence of septation. Mitchison & Nurse (1985) noted that cdc 11 showed pulses of cell growth interspersed with periods of quiescence. The fact that actin disappears from the cell ends with each cycle of ring formation provides an explanation for their observations. The relationship between the actin ring and septation is also clearly shown in cdc 13, where the ring cycle and nuclear cycle become uncoupled and multiple septa are laid down in the absence of nuclear division. At present we cannot distinguish whether the coincidence of actin and polarized cell growth is a cause or an effect relationship. In the case of septation, however, the situation is unambiguous, actin appearing at the cell equator prior to the F ig. 6. Wild-type cells: A, phase-contrast-D A P I; B, immunofluorescence microscopy using anti-tubulin antibody Y O L 1/34. D uring interphase groups of microtubules extend between the cell ends (e.g. cells 1 and 2 ) . At mitosis the cytoplasmic microtubules are replaced by a short intranuclear spindle (cell J ) , which elongates until the nuclei reach the ends of the cell (cell 4 ). Reinitiation of the interphase array occurs prior to spindle breakdown by the nucléation of microtubules at the cell equator (cells 4 and 5 ) . Bar, 10 fim . Fission yeast cytoskeleton 237 F ig. 7. Phalloidin staining of cold-treated benA cells. These contain fewer, larger actin dots and, in many cases, a conspicuous perinuclear ring. Bar, 10 fim. deposition of Calcofluor-staining material. Both end growth and septation involve the deposition of new cell wall macromolecules, albeit of different chemical com position (Bush et al. 1974; Horisberger & Rouvet-Vauthey, 1985). In fungi this requires the mobilization of vesicles containing wall precursors to the growing region (M cClure et al. 1968; Grove, 1978). Vesicles associated with the poles and septa of S.pombe cells have been reported (OuleveyeZ al. 1970; Johnson etal. 1973), and this suggests a possible explanation for the dot-like nature of actin staining. Although at present there is no evidence for the association of actin with cell wall vesicles in yeasts, vesicles coated with fine filaments of the approximate dimensions of F-actin have been observed in a filamentous fungus (Hoch & Howard, 1980). T he role of actin in wall deposition may not be finally clarified until cell wall vesicles are available for biochemical study. T he localization of myosin may also provide important clues (Watts et al. 1985) as will drugs that selectively interfere with cell wall morphology (Miyata et al. 1985, 1986), and experiments to these ends are in progress. Although the nature of the dot staining remains to be resolved, it is obviously a common feature of fungal cells, having been seen in three ascomycetous yeasts (S a . cerevisiae, Sa. uvarum and S. pombe) (Kilmartin & Adams, 1984; Adams & Pringle, 1984; M arks & Hyams, 1985) and a filamentous basidiomycete (Utvmycesphaseoli) (Hoch & Staples, 1983). T h is represents a broader sample than may initially be apparent, in view of the evolutionary divergence between budding and fission yeasts (Huysmans 238 J . Marks, I. M. Hagan andy. S. Hyams et al. 1983) and the fact that their mode of division is quite different. Cytokinesis in budding yeast is assymmetric and involves a chitin ring, whereas fission yeasts divide symmetrically and lack chitin (Bush et al. 1974). The nature of actin staining as well as its position undergoes a marked change at the initiation of mitosis. Dot staining at the cell ends is replaced by a filamentous ring, which presumably occupies the thin layer of cytoplasm between the nucleus and the cell membrane (Streiblova & Girbardt, 1980). Our finding that a ring of actin anticipates the formation of the septum in S. pombe is consistent with earlier studies of cytokinesis in fungi, which have provided ultrastructural evidence for the presence of such a structure (Girbardt, 1979). The intimate relationship between the nucleus and the actin ring is most clearly demonstrated by cdc 11, which can go through multiple nuclear divisions at the restrictive temperature without undergoing cyto kinesis (Nurse et al. 1976). An actin ring forms in association with each daughter nucleus at the start of mitosis; however, as the nuclei divide this spatial relationship is lost. The fact that cdc 11 forms apparently normal actin rings and yet fails to lay down any detectable septal material may well be of value in establishing the coupling of these two events. The relationship between the nucleus and the actin ring may also be addressed by our findings with ben A. This mutant has the classic phenotype of a tubulin gene mutation, namely, it is benomyl-resistant and cold-sensitive (Roy & Fantes, 1982). Genetic studies have shown, however, that the ben A gene is distinct from the known tubulin genes of S. pombe (P. Fantes, personal communication; see Yanagida et al. 1985). The exciting possibility therefore exists that ben A codes for a protein that interacts with microtubules, i.e. a microtubule-associated protein (MAP). This is supported by immunofluorescence staining of ben A cells with anti-tubulin anti body, which reveals an apparently normal array of cytoplasmic microtubules (our unpublished results). The most conspicuous cytological feature of cold-treated ben A cells is the presence of a perinuclear F-actin ring, reminiscent of the rings of intermediate filaments that form around the nuclei of cultured cells treated with colchicine (Goldman, 1971). A possible explanation for the origin of these structures is that, like the /3-tubulin mutant nda3 (Hiraoka et al. 1984), cold-treated ben A cells arrest at mitotic prophase. Since this is the time at which the equatorial actin ring appears, the perinuclear rings in ben A may be the equatorial ring displaced from its normal location. Although Roy & Fantes reported that ben A does not exhibit classical cell cycle arrest, we have used a different temperature from that used in their study and this can have a profound effect on the phenotype (Hiraoka et al. 1984). Clearly, examination of the distribution of actin in known tubulin mutants of. S. pombe will be of value in further establishing the nature of the ben A mutation as well as clarifying the relationship between actin and tubulin in this organism. Investigations of the role of microtubules in 5. pombe will also be considerably aided by the introduction of the immunofluorescence techniques described here. Spindle microtubules have previously been demonstrated in this way (Hiraoka et al. 1984) but this is the first report of the visualization of cytoplasmic micro tubules. These are much more abundant than has previously been appreciated from Fission yeast cytoskeleton 239 electron microscopy (Hereward, 1974; Streiblova & Girbardt, 1980; King & Hyams, 1982a,b\ Tanaka & Kanbe, 1986) and extend from one end of the cell to the other. Cytoplasmic microtubules are clearly involved in the establishment and maintenance of cell morphology since treatment of fission yeast with anti-micro tubule drugs results in a variety of morphological changes (Walker, 1982), as does the disruption of microtubules by means of tubulin gene mutations (Toda et al. 1983; Hiraoka et al. 1984). Whether microtubules are actively involved in the transport of cell wall precursors or merely establish general cell polarity is at present unknown. However, the fact that bud expansion in S. cerevisiae proceeds in the absence of microtubules (Pringle et al. 1984) encourages us to favour the latter alternative. Irrespective of this, our results have clearly shown that the distribution of F-actin in fission yeast is controlled in a cell cycle site-specific manner. Whether understanding of this regulation will emerge through the wider study of cell cycle controls (Hayles & Nurse, 1986) or, more specifically, through the use of cloned actin genes and the identification and characterization of the actin binding proteins remains to be seen. We thank Professor T h. Wieland and D r John Kilmartin for the generous gifts of phalloidin and tubulin antibody, respectively. This work was supported by Action Research for the Crippled Child and the Science and Engineering Research Council. REFERENCES E. M. & P r i n g l e , J. R. (1984). Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic mutant Saccharomyces cerevisiae. J . Cell Biol. 98, 934-945. B o n a t t i , S . , S i m i l i , M. & A b b o n d a n d o l o , A . (1972). Isolation of temperature sensitive mutants of Schizosaccharomyces pom be.J. B a d . 109, 484-491. B u r n s , R. G . (1973). 3H-Colchicine binding: Failure to detect any binding tosoluble proteins from various lower organisms. E xpl Cell Res. 81, 285-292. B u s h , D . A ., H o risberg er , M ., H orman , I. & W u r sc h , P. (1974). The wall structure of Schizosaccharomyces pombe. jf. gen. Microbiol. 81, 199-206. D a r k e n , M. A. (1961). Applications of fluorescent brighteners in biological techniques. Science 133, 1704-1705. G irbard t , M. (1979). A microfilamentous septal belt (F SB ) during induction of cytokinesis in Trametes versicolor (L . ex Fr). E xplM ycol. 3, 215-228. G o l d m a n , R. D . (1971). The role of the three cytoplasmic fibres in BHK-21 cell motility. 1. Microtubules and the effect of colchicine. J . Cell Biol. 51, 752-762. G rove , S. N. (1978). The cytology of hyphal tip growth. In The Filamentous Fungi, vol. 3 (ed. J . E. Smith & D . R. Berry), pp. 28-50. New York: John Wiley & Sons. H a y l e s , J . & N u r s e , P. (1986). Cell cycle regulation in yeast. J . Cell Sei. Suppl. 4, 155-170. H e r e w a r d , F. V. (1974). Cytoplasmic microtubules in a yeast. Planta 117, 355-360. H iraoka , Y ., T o da , T . & Y an a gida , M. (1984). The ND A3 gene of fission yeast encodes ßtubulin. A cold sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39, 349-358. H och , H . C. & H ow ard , R . J . (1980). Ultrastructure of freeze-substituted hyphae of the basidiomycete Laetisaria arvalis. Protoplasma 103, 281-297. H o c h , H . C. & S t a p l e s , R. C. (1983). Visualization of actin in situ by rhodamine-conjugated phalloin in the fungus Uromyces phaseoli. Eur. J . Cell Biol. 32, 52-58. H o r i s b e r g e r , M. & R o u v e t -V a u t h e y , M. (1985). Cell wall architecture of the fission yeast Schizosaccharomyces pombe. Experientia 41, 748-750. A dams A . 240 J . Marks, I. M. Hagan a n d j. S. Hyams E ., D a m s , E ., V a n d e r b e r g h e , A. & d e W a t c h e r , R. (1983). The nucleotide sequences of the 5 S rRNAs of four mushrooms and their use in studying the phylogenetic position of basidiomycetes among the eukaryotes. Nucl. Acids Res. 11, 2871-2880. J o h n s o n , B. F. (1965). Autoradiographic analysis of regional cell wall growth of yeasts. E xpl Cell Res. 39, 613-624. J o h n s o n , B. F ., Y o o , B. Y . & C a l l e j a , G. B. (1973). Cell division in yeasts: Movement of organelles associated with cell plate growth of Schizosaccharomyces pombe. J . B a d . 115, 358-366. H u y sm an s, K ilm a r tin , J . V. & A dam s, A. E . M . (1984). Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J . Cell Biol. 98, 922-933. J . V., W r i g h t , B. & M i l s t e i n , C. (1982). Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J . Cell Biol. 93, 576-582. K i n g , S. M. & H y a m s , J. S. (1982a). Synchronization of mitosis in a cdc mutant of Schizosaccharomyces pombe released from temperature arrest. C a n .J. Microbiol. 28, 261-264. K in g , S. M . & H yam s, J. S. (19826). Interdependence of cell cycle events in Schizosaccharomyces pombe. Term inal phenotypes of cdc mutants arrested during D N A synthesis and cell division. Protoplasma 110, 54 -6 2 . M a r k s , J. & H y a m s , J. S. (1985). Localization of F-actin through the cell division cycle of Schizosaccharomyces pombe. E u r.J . Cell Biol. 39, 27-32. M c C lu r e , W. D ., P a r k , D . & R o b in so n , P. M . (1968). Apical organization in the somatic hyphae of fungi, jf. gen. Microbiol. 50, 177-182. M i t c h i s o n , J . M . & N u r s e , P. (1985). Growth in cell length in the fission yeast Schizo saccharomyces pombe. J . Cell Sci. 75, 357-376. M i y a t a , M ., K a n b e , T . & T a n a k a , K . (1985). Morphological alterations in the fission yeast Schizosaccharomyces pombe in the presence of aculeacin A: Spherical wall formation. J . gen. Microbiol. 131, 611—621. M i y a t a , M . , M i y a t a , H. & J o h n s o n , B. F. (1986). Assymetric location of the septum in physiologically altered cells of the fission yeast Schizosaccharomyces pombe. J . gen. Microbiol. 132, 883-891. N u r s e , P. (1981). Genetic analysis of the cell cycle. In Genetics as a Tool in Microbiology. SG M Sym p. 31, pp. 291-315. Cambridge University Press. N u r s e , P. (1985). Cell cycle control genes in yeast. Trends Genet. 2, 51-55. N u r s e , P., T h u r i a u x , P. & N a s m y t h , K . (1976). Genetic control of the cell division cycle of the fission yeast Schizosaccharomyces pombe. Molec. gen. Genet. 146, 167-178. O u l e v e y , N ., D e s h u s s e s , J . & T u r i a n , G. (1970). Étude de la zone septale de Schizo saccharomyces pombe en division a ses etapes succesives. Protoplasma 70, 217-224. P r in g l e , J . R ., C o lem a n , K ., A d a m s , A ., L il l ie , S ., H aarer , B ., J a cobs , C ., R o bin so n , J . & E v a n s , C . (1984). Cellular morphogenesis in the yeast cell cycle. In Molecular Biology o f the Cytoskeleton (ed. G . G . Borisy, D . W. Cleveland & D . B. M urphy). New Y ork: Cold Sprin g K ilm a r tin , H arbor Laboratory Press. J . R. & H a r t w e l l , L . H. (1981). The Saccharomyces cerevisiae cell cycle. In The M olecular Biology o f the Yeast Saccharomyces (ed. J. N. Strathern, E. W. Jones & J . R. Broach). New York: Cold Spring Harbor Laboratory Press. R o y , D . & F a n t e s , P. A. (1982). Benomyl resistant mutants of Schizosaccharomyces pombe coldsensitive for mitosis. Curr. Genet. 6, 195-201. STAEBELL, M. & S o l l , D . R. (1985). Temporal and spatial differences in cell wall expansion during bud and mycelial formation in Candida albicans. J . gen. Microbiol. 131, 1467-1480. S t r e i b l o v a , E. & G i r b a r d t , M. (1980). Microfilaments and cytoplasmic microtubules in cell division cycle mutants of Schizosaccharomyces pombe. C an.J. Microbiol. 26, 250-254. S t r e i b l o v a , E. & W o l f , A. (1972). Cell wall growth during the cell cycle of Schizosaccharomyces pombe. Z . Allg. Mikrobiol. 12, 673—684. T a n a k a , K . & K a n b e , T . (1986). Mitosis in fission yeast Schizosaccharomyces pombe as revealed by freeze-substitution electron microscopy. J . Cell Sci. 80, 253-268. P r in g l e , Fission yeast cytoskeleton 241 J . H ., N o v i c k , P. & B o t s t e i n , D . (1984). Genetics of the yeast cytoskeleton. In M olecular Biology o f the Cytoskeleton (ed. G. G. Borisy, D. W . Cleveland & D. B . Murphy). New York: Cold Spring Harbor Laboratory Press. T o d a , T . , U m e s o n o , K . & H ir a o k a , A. (1983). Cold-sensitive nuclear division arrest mutants of the fission yeast Schizosaccharomyces pom be.jf. molec. Biol. 168, 251-270. W a l k e r , G . M. (1982). Cell cycle specificity of certain antimicrotubular drugs in Schizo saccharomyces pom be.jf. gen. Microbiol. 128, 61-71. W a t t s , F . Z . , M i l l e r , D . M . & O r r , E . (1985). Identification of myosin heavy chain in Saccharomyces cerevisiae. Nature, Lond. 316, 83-85. W i e l a n d , T h . & G o v i n d a n , V. M . (1974). Phallotoxins bind to actins. F E B S L e tt. 46, 351-353. W i l l i a m s o n , D . H. & F e n n e l l , D. J. (1975). The use of fluorescent D N A binding agent for detecting and separating yeast mitochondrial DN A. Meth. Cell Biol. 12, 335-351. W u l f , E . , D e b o b e n , A ., B a u t z , F . A ., F a u l t i s c h , H . & W i e l a n d , T h . (1979). Fluorescent phallotoxin, a tool for the visualisation of cellular actin. Proc. natn. Acad. Sei. U .S A . 76, 4498-4502. Y a n a g i d a , M . , H i r a o k a , Y ., U e m u r a , T ., M i y a k e , S. & H i r a n o , T . (1985). Control mechanisms of chromosome movement in mitosis of fission yeast. In Yeast Cell Biology, UCLA Sym p. vol. 33 (ed. J . Hicks). New York: Alan R. Liss. T h o m a s,