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J. Cell Sci. 46, 341-352 (1980)Printed in Great Britain © Company of Biologists Limited 1080

THE INFLUENCE OF THE MICROTUBULE

INHIBITOR, METHYL BENZIMIDAZOL-2-YL-

CARBAMATE (MBC) ON NUCLEAR DIVISION

AND THE CELL CYCLE IN

SACCHAROMYCES CEREVISIAE

R. A. QUINLAN, C. I. POGSON* AND K. GULLBiological Laboratory, University of Kent, Canterbury, Kent, CTz 7NJ, U.K.

SUMMARY

Methyl benzimidazol-2-yl-carbamate (MBC), at a concentration of 100 fiM, has a pronouncedeffect on the growth of Saccharomyces cerevisiae, resulting in the accumulation of cells as largedoublets. We have determined a specific execution point for the effect of MBC on the yeast cellcycle, and have shown that this execution point is between the cell cycle events of spindle polebody duplication and spindle pole body separation. An ultrastructural examination of theMBC-treated cells revealed the absence of cytoplasmic and spindle microtubules. MBCtreatment also produced an altered spindle pole body morphology, causing the disappearance ofthe outer component. Nuclear size was also markedly increased in the MBC-induced doubletcells, although septa were completely absent from these doublet cells.

It is proposed that MBC inhibits microtubule polymerization, rather than causing thedepolymerization of stable microtubules.

INTRODUCTION

The benzimidazole carbamate group of anti-microtubule agents have becomeimportant fungicides and antihelmintics (Clemons & Sisler, 1971; Borgers et al. 1975).Benomyl is a much used commercial fungicide, the active component being methyl-2-benzimidazole carbamate (MBC). Hammerslag & Sisler (1973) have shown that MBCaffects overall DNA, RNA and protein synthesis in Ustilago maydis and Saccharomycescerevisiae. However, these effects on macromolecular synthesis were secondary to aprimary inhibition of nuclear division and cytokinesis. Davidse & Flach (1977) haveused Aspergillus nidulans to show that fungi possess an MBC-binding protein whichhas many characteristics expected of tubulin - the main constituent protein ofmicrotubules. Mutants of A. nidulans resistant to MBC have been isolated and wereshown to have altered tubulins (Shier-Neiss, Lai & Morris, 1978; Morris, Lai &Oakley, 1978).

Microtubules can be purified from mammalian cells and tissues by an assembly/disassembly protocol (Weisenberg, 1972). This in vitro assembly of microtubules is

• Present address: Department of Biochemistry, University of Manchester, Manchester,M13 9PL, U.K.

Address for correspondence: Dr K. Gull, Biological Laboratory, University of Kent,Canterbury, Kent, CTz 7NJ, U.K.

342 R. A. Quinlan, C. I. Pogson and K. Gull

inhibited by the benzimidazole carbamates (Ireland, Gull, Gutteridge & Pogson,1979). Also a number of the benzimidazole carbamates have been shown to bind topurified tubulin (Hoebeke, Van Nijen & De Brabander, 1976; Laclette, Guerra &Zetina, 1980). Studies of microtubule-mediated processes in lower eukaryotes haveoften been hindered by the fact that lower eukaryotic tubulin appears to have a loweraffinity for colchicine than that from mammalian cells (Burns, 1973). The benzimida-zole carbamates offer a better prospect for an antimicrotubule agent for use withlower eukaryotes.

In S. cerevisiae microtubules play a central role in nuclear division. The spindle polebody (SPB) is embedded in the nuclear membrane and mitosis is intranuclear(Robinow& Marak, 1966). In the Gx state, the SPB has attached microtubules whichradiate into the intranuclear space (Byers & Goetsch, 1975). These microtubules aremaintained in SPB duplication, and are approximately 100-250 nm long. They arenot yet functional in connecting the 2 SPBs but both have an associated group ofintranuclear microtubules. These microtubules then elongate to 500 nm, and someto 1 /£m (Peterson & Ris, 1976). It is the longer microtubules which form the spindle,but as shown by Peterson & Ris (1976), they are not functional in SPB separation.The SPBs move apart on the nuclear periphery, and reorientate so that the respectivebundles of microtubules are parallel, and at this point, the spindle is formed. At thisstage the spindle is slightly shorter than the average diameter of the nucleus, the SPBslying within cytoplasmic indentations of the nuclear envelope (Moens & Rapport,1971). During the first stage of nuclear division, and coinciding with a bud of similarsize to the mother cell, the spindle elongates rapidly to 6-8/tm (Matile, Moor &Robinow, 1969). It is speculated that this rapid growth in the spindle provides theforce for the final stage of nuclear elongation (Byers & Goetsch, 1974), although thishas yet to be proved. The nucleus divides and cytokinesis is completed by the deposi-tion of a septum.

Functioning of the SPB has been implicated as an important control in the cellcycle (Byers & Goetsch, 1974). The cytoplasmic microtubules which radiate from theSPB complex after duplication are oriented towards the site of bud emergence (Byers& Goetsch, 1975). In this study we have used MBC to inhibit microtubule-orientedprocesses in S. cerevisiae, with a view to examining the importance of these organellesin control of the cell cycle.

MATERIALS AND METHODS

Organism used

Saccharomyces cerevisiae, strain 20B-12, a mutant deficient in proteases A, B and C (Jones,1977)-

Growth conditions

S. cerevisiae was grown in a fully defined medium, using (NH1),SO1 as the nitrogen source(Clayton, Pogson & Gull, 1979). Cultures were incubated at 26 °C on an orbital shaker, rotatingat 100 rev/min. Drug-treated cultures were incubated with 100 fiM methyl benzimidazol-2-yl-carbamate (MBC), using dimethyl formamide at a final concentration of 2 % v/v as an organic

MBC and Saccharomyces cell cycle 343

carrier. Control cultures were grown in the presence of the same final concentration of dimethyl-formamide.

Light scattering and cell number data

Light scattering was monitored using a KJett Summerson photoelectric colorimeter, readingagainst a medium blank.

Cell numbers were determined using a Coulter Counter, model FN fitted with a 7o-/tmorifice probe. Cells were diluted into sterilized 0-9 % w/v NaCl solution. These samples weresonicated to break cell clumps for 15 s at maximum power, using a MSE 150-W ultrasonicdisintegrator (Mk. 2), fitted with a o/s-mm-diameter probe.

Light microscopy

Light micrographs were taken on a Zeiss Universal Light Microscope, using phase contrast.For time-lapse photomicroscopy, cells from an actively growing culture were placed on theabove defined medium containing 2 % w/v agar and 100 fiM MBC and observed over a 23-hperiod.

Electron microscopy

Cells were harvested and prefixed for 45 min in 25 % v/v glutaraldehyde. The cells werethen pretreated for 30 min with o-i M /?-mercaptoethanol, prior to cell wall digestion with theenzyme, Glusulase (Endo Laboratories). After 2 h digestion, the cells were fixed in 2-5 % v/vglutaraldehyde for 30 min, and then postfixed in 1 % w/v osmium tetroxide for 1 h. Dehydra-tion of the cells was in a graded ethanol series, after which they were embedded in Spurr'sresin (Spurr, 1969).

Thin sections were cut on a Reichert OMU3 ultramicrotome, and then poststained withuranyl acetate and lead citrate, before being viewed in an AEi 801A electron microscope at anaccelerating voltage of 60 kV.

RESULTS

Effect of MBC on growth

As cells at different stages of growth may show different sensitivities to MBC, weused inocula taken at 2 different stages of the batch growth curve - mid-exponentialand early stationary cells. The effect of MBC was monitored by changes in cell numberand light scattering (Klett units). Using mid-exponential phase cells as an inoculum,MBC severely inhibits growth as seen from the cell number data (Fig. 1). MBCrestricts growth to just over one doubling in cell number. This represents a 15-foldinhibition by MBC of the final population achieved in control cultures (Fig. 1). Asimilar inhibition by MBC of the growth of an early stationary phase inoculum wasalso seen (data not shown). The light-scattering data for both inocula were much thesame as the cell-number data, although the cell-number data indicated a more pro-nounced inhibition by MBC. This is reflected by the final population achieved in thepresence of MBC. The cell-number data indicated a 15-fold inhibition by MBC of thefinal population achieved, whereas light-scattering data revealed only a 5-fold inhibi-tion by MBC.

One likely explanation of the discrepancies between the light-scattering and cell-number data is that in the presence of MBC there is a progressive increase in cellvolume. In order to verify this, light micrographs of both control and MBC-treatedcultures were taken after 6h (Fig. 2 A, C) and 24 h (Fig. 2B, D). After 6 h in the

344 R- A- Quinlan, C. I. Pogson and K. Gull

presence of MBC (Fig. 2C), the culture consists mainly of cells blocked in the cellcycle at some stage before cytokinesis. The bud cell has nearly reached the samedimensions as the mother cell, and this morphology will be referred to as a ' doubletcell' (see Fig. 2C, D). After 24 h the doublet cells have increased greatly in size (Fig.2 D), and clearly this increase in volume, and the presence of the doublet cells, can

50 1-

oX

3

Fig. 1. Growth of mid-exponential inoculum with no inhibitor ( • ) , and 100 /JM MBC(A), as expressed in terms of cell number.

account for the increase in light scattering. Single cells are observed in the MBC-treated culture after 24 h, but comparison with the control culture reveals an increasein cell volume. Size analyses of these 24-h populations using a Coulter Counter, showaverage cell sizes in control cultures of 120 /<m3, compared with 265 /4m3 for MBC-treated cultures.

Effect of MBC on the yeast cell cycle

The morphology of the doublet cells in the MBC-treated culture is very similar tothe morphology of certain cell division cycle (cdc) mutants grown at their restrictivetemperature, e.g. cdc 13 (Hartwell, 1974). There are 2 groups of cdc mutants whichare characterized by the number of cycles completed at the restrictive temperature,cdc mutants that on commencing the cell cycle at the restrictive temperature areinhibited in the first cycle, are said to exhibit 'first cycle arrest' (Hartwell, 1974).


It is then possible to determine an execution point for these particular mutants, usingtime-lapse photomicroscopy.

MBC treatment of a culture results in just over one doubling in cell number. Afterprolonged MBC treatment, sonication does result in breakage of some of the doubletcells, and as this would artificially raise the cell number, the execution point for theaction of MBC on the cell cycle was determined by time-lapse photomicroscopy.Fig. 3 shows a group of cells photographed over a period of 23 h, after being spreadon to agar containing 100 fiM MBC. From this figure it can be seen that single cellsincrease in size, bud normally, but then become blocked, adopting a doublet cellmorphology. Cells with very small buds also terminate at this same diagnosticmorphology. It is not until a slightly larger bud size is reached, that it is possible forthe cell to complete 1 cycle. However, each of the resulting daughters block in thesecond cycle. Clearly, as with the cdc mutants, the diagnostic terminal morphologydoes not always coincide with the execution point (Hartwell, 1974). Fig. 4 summarizesthese data and indicates the position of the execution point for MBC inhibition of theyeast cell cycle. At the execution point the bud diameter/cell diameter ratio isapproximately 0-25.

Fig. 2. Samples of cultures grown with no inhibitor at 6 h (A) and 24 h (B), and with100 /iM MBC at 6 h (c) and 24 h (D). dc, doublet cell, x 800.

23 CEL 46


Fig. 3. Time-lapse photomicroscopy of a group of cells inoculated onto 2 % w/v agarin minimal medium, containing 100 fiM MBC. Photomicrographs were taken at o h (A) ;1-25 h (B); 2 0 h (c); 3-0 h (D); 4-0 h (E); 5-0 h (F); 5-7 h (G); I I -O h (H) ; 230 h (1).


The effect of MBC on the intracellular morphlogy of SPBs and microtubules

On comparison of the morphology of the SPBs found in control cells with those inMBC-treated cells, 2 differences are immediately apparent. First, in MBC-treatedcells there are no intranuclear SPB-associated microtubules (compare Fig. 5 A-D withFig. SE-H). Secondly, control cells possess a 2-component SPB (Fig. 5 A-D); however,the outer component on the cytoplasmic face of the SPB is missing in MBC-treated

Fig. 4. Diagrammatic representation of the effect of MBC upon the cell cycle ofSaccharomyces cerevisiae. ex, execution point.

cells (Fig. 5E-H). Also the SPBs of MBC-treated cells were slightly larger (average0-13 fim) than those of control cells (average o-i /im). Extranuclear microtubules areknown to be oriented from the outer component towards the site of bud emergence,and a possible role of these microtubules in bud emergence has been suggested(Byers & Goetsch, 1975).

Towards the end of the G2 phase of the yeast cell cycle the 2 SPBs are separated byabout 1 /tm, and are positioned near the neck of the bud, as shown in Fig. 51. Rapidelongation of the spindle microtubules then occurs, with subsequent nuclear division.In MBC-treated cells, the 2 separated SPBs are directly opposed to each other,approximately 0-5 /tm apart, near the neck of the mother and bud cells (Fig. 5J, K).There are no microtubules connecting the 2 SPBs and again it can be clearly seen thatthe SPBs lack the outer component. The positioning of the 2 SPBs across a smallportion of the nucleus gives it a highly lobed appearance. In some electron micro-graphs, it is clear that the nuclear membrane has increased dramatically in area,causing the formation of large loops (Fig. 5 L). In these MBC-treated cells this large

23-2


5 A B

F > :t. X' IT


lobed nucleus is often distributed throughout the doublet cell (Fig. 5M). These largeMBC-induced doublet cells never possess septa.

DISCUSSION

It is now well established that the benzimidazole carbamate groups of compoundscan disrupt microtubule function in vitro and in vivo (Dustin, 1978; Ireland et al.1979). MBC is the active antimicrotubule agent in the commercial fungicide, Benomyl(Clemons & Sisler, 1969). A large amount of evidence exists suggesting that there is anMBC-binding site on the tubulin of Aspergillus nidulans (Davidse & Flach, 1977;Sheir-Neiss, Lai & Morris, 1978). One of the main consequences of this site of actionof MBC is an inhibition of nuclear division in A. nidulans hyphae (Davidse, 1973).

Our results with S. cerevisiae show that it is possible to determine a specific execu-tion point for MBC in the yeast cell cycle. When treated with MBC, unbudded cells,or cells with small buds (up to a bud: mother cell diameter of about 0-25:1) block as alarger doublet cell. Cells with larger buds are able to complete their division; howeverthe daughter cells then block in the subsequent cell cycle.

We have observed that in MBC-treated cells the SPB has been duplicated. Afterseparation, the SPBs then adopt a position on the nuclear membrane reminiscent ofearly spindle morphology in control cells, that is, they are oriented opposite to eachother, separated by about 0-5 /tm. Hammerslag & Sisler (1973) have previously shownthat DNA synthesis can continue during treatment of S. cerevisiae cells with MBC.The duplication of the SPB occurs at approximately the same time as DNA replicationand bud emergence. It has been postulated that this SPB duplication event couldserve to integrate the cell cycle by triggering the initiation of both DNA synthesis andbud emergence (Byers & Goetsch, 1974). In MBC-treated cells, although SPBduplication does occur, the SPBs have an altered morphology, lacking the outercomponent. There is also a complete absence of cytoplasmic and spindle microtubulesin these MBC-treated cells. Clearly these differences are not sufficient to preventinitiation of DNA synthesis or bud emergence. Therefore the SPB duplication which

Fig. 5. The effect of MBC upon the ultrastructure of the spindle pole body (SPB),spindle microtubules and the nucleus in S. cerevisiae. In each of the electron micro-graphs A-H, the nucleoplasm is at the bottom of the electron micrograph and the cyto-plasm is at the top.

A-D. Morphology of the SPB and associated microtubules in control cells. The longarrows mark the outer component of the SPB. The SPB-associated microtubulescan be seen between the short arrows, A, B, C, X 58000; D, X 80000.

E-H. Morphology of the SPB in MBC-treated cells. Note the absence of theassociated microtubules. x 65 000.

1. Control cell spindle, x 58000.j . MBC-treated cell. Two separated SPBs with no microtubules. x 93 300.K. MBC-treated cell. Two separated SPBs with no microtubules. x 60000.L. Nuclear morphology in MBC-treated cells. A region of cytoplasm (c) is enclosed

by a loop of the nucleus (n). One SPB is arrowed, x 45 800.M. MBC-induced doublet cell. Note the nucleus distributed throughout the doublet

cell and the absence of a septum, x 9200.


does occur in MBC-treated cells is sufficient to allow further cell cycle events such asDNA synthesis and bud emergence. However, the formation of a septum in the MBC-treated cells is inhibited, emphasizing the dependence of this cell cycle event upon thecompletion of prior events in the cell cycle such as nuclear migration and nucleardivision.

Peterson & Ris (1976) observed that after SPB duplication, the SPB-associatedintranuclear microtubules elongate to form 2 groups of microtubules, one consistingof shorter microtubules (average length 0-5 fim) the other consisting of longermicrotubules (average length 1 /tm). These longer microtubules will constitute theinterpolar microtubules of the spindle, with no additional increase in length required.Spindle formation occurs when the SPBs have been oriented directly opposite eachother across the nucleus, the length of this early spindle being approximately 1 /tm.With MBC treatment, SPB separation does occur, and when both SPBs are visiblein the section, they are approximately 0-5 /tm apart, facing each other across thenucleus. This observation supports the proposal of Peterson & Ris (1976) that theseparation of the SPBs is nuclear membrane-mediated. However, as the SPBs of theMBC-treated cells are separated by a slightly smaller distance than in control cells it ispossible that the complete process of SPB separation does involve a late event whichis microtubule mediated.

The cell cycle data reveal an execution point just after bud emergence and yetprior to SPB separation. Microtubules are, however, present throughout most, if notall, of the yeast cell cycle. Therefore we have to explain why MBC appears to affectonly the short spindle in the early part of the cell cycle.

There is evidence that it is microtubule polymerization rather than the preformedmicrotubule which is affected by the benzimidazole carbamates (Hoebeke et al. 1976).Perhaps the presence of possible microtubule-associated proteins on the yeast micro-tubules alters the binding capacity for MBC or the microtubules are stabilized in someother way. In other organisms, these well characterized microtubule-associatedproteins are known to competitively inhibit colchicine binding (Nunez, Fellows,Francon & Lennon, 1979). Colchicine binding to mammalian tubulin is competitivelyinhibited by the benzimidazole carbamates, oncodazole and mebendazole, suggestingsimilar binding sites (Hoebeke et al. 1976; Ireland et al. 1979; Laclette et al. 1980).Colchicine does not bind to intact microtubules, as shown by Wilson & Meza (1973).However, the depolymerization of intact microtubules in vitro by colchicine can beexplained, if microtubules are considered to be a dynamic equilibrium systemconsisting of constant disassembly at one end and constant assembly at the other(Inoue & Sato, 1967). Colchicine can be regarded as inhibiting the assembly portionof the equilibrium system so producing a net disassembly. However, highly cross-linked structures such as cilia, centrioles and basal bodies are not sensitive to colchi-cine depolymerization. Cross-bridges are a frequent observation in spindles from avariety of sources (Fuge, 1974) and, of course, the spindle microtubules are oftenattached at both ends, either to the SPBs, or kinetochores. Moreover, Byers, Shriver &Goetsch (1978) have reported that in Saccharomyces cerevisiae the spindle micro-tubules at the site of attachment to the SPB have closed ends. Taken together with


our observations this indicates that MBC might inhibit microtubule assembly ratherthan cause disassembly of spindle microtubules. This would explain the executionpoint in the cell cycle as it is at this time that the spindle microtubules are beingpolymerized.

At concentrations which do not fully inhibit growth, the effect of partial inhibitionof tubulin polymerization into spindle microtubules might be to produce spindleswithout the full complement of microtubules. This would explain why the benzimida-zole carbamates, particularly MBC, cause chromosome non-disjunction (Kappas,Georgopoulos&Hastie, 1974; Kappas, 1978).

We would like to thank Mr R. Newsam for his assistance with the electron microscopy.R. A. Quinlan is in receipt of an S.R.C. studentship. K. Gull thanks the Royal Society for fundstowards the sectioning equipment used in this study.

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(Received 1 May 1980)

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