The role of the cytoskeleton in positioning of the nucleus in premitotic

tobacco BY-2 cells

JUNKO KATSUTA, YUKI HASHIGUCHI and HIROH SHIBAOKA

Department of Biology, Faculty of Science, Osaka University, Toyonaka 560, Japan

Summary

In premitotic tobacco BY-2 cells, the nucleus movesfrom the periphery to the central region of the celland settles there. This premitotic migration of thenucleus occurs in the presence of aphidicolin, aninhibitor of DNA polymerase a, which suppressesthe formation of the preprophase band of micro-tubules (PPB). Thus, the formation of the PPB is nota prerequisite for this premitotic nuclear migration.Tethered by transvacuolar cytoplasmic strands,which contain both microtubules and actin fila-ments, the nucleus migrates to the site of futuredivision. This migration is not prevented by disrup-tion of actin filaments caused by treatment withcytochalasin, suggesting that microtubules play animportant role in the premitotic migration of thenucleus.

In the preprophase cell, microtubules were pres-ent in the PPB and in cytoplasmic strands, whichextended from the nucleus to the periphery of thecell near the PPB and near the poles, and whichwere associated with actin filaments. At the end ofprophase, microtubules in the cytoplasmic strandsand the PPB disappeared, leaving actin filaments in

the transvacuolar cytoplasmic strands. Thereafter,the position of the mitotic apparatus (or that of thecytokinetic phragmoplast) was maintained by cyto-plasmic strands that contained only actin filaments.The disruption of microtubules by treatment withpropyzamide prevented the formation of the PPB-like band of actin filaments, as well as that of thePPB itself. From these results, we propose thefollowing hypothesis. In premitotic tobacco BY-2cells, microtubules change their arrangement irres-pective of the presence of actin filaments. Theresultant premitotic network of microtubulesserves as a scaffold for building up the mitoticnetwork of actin filaments, which plays an import-ant role in maintaining the position of the mitoticapparatus and in controlling the direction of expan-sion of the phragmoplast.

Key words: actin filaments, microtubules, nuclearpositioning, preprophase band of microtubules, tobacco BY-2cells.

Introduction

There are many examples of the ways in which cytokin-esis determines the fate of daughter cells. Cytokinesisgiving rise to daughter cells of different sizes, i.e. unequalcell division, has been reported to be involved in thedifferentiation of plant cells; for example, in the forma-tion of stomatal guard mother cells, root hair cells, pollengrains and the rhizoids of Fucales (Wareing and Phillips,1981). The site of formation of the cell plate is predictedby the position of the nucleus at the premitotic phase. Inpremitotic plant cells, the nucleus migrates to the site offuture division and settles there. It has also been sugges-ted that the site of formation of the cell plate is deter-mined with the aid of the phragmosome and the prepro-phase band of microtubules (PPB) (Gunning, 1982).

In a previous report, we demonstrated that actinfilaments, but not microtubules, are responsible for thepositioning of the nucleus in tobacco BY-2 cells at

Journal of Cell Science 95, 413-422 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

interphase. We found that only actin filaments werepresent in the cytoplasmic strands that extend from thenucleus to the cell periphery and that the disruption ofactin filaments by cytochalasin caused the immediatedisplacement of the nucleus (Katsuta and Shibaoka,1988). However, in premitotic cells, microtubules seemto play an important role in the positioning of thenucleus. In pollen tetrads in Lilliaceae, the nucleusmoves to one end of the cell and is fixed there before theunequal division of the cells that leads to differentiationof the pollen. Studies using a microtubule-disruptingagent and electron microscopy revealed that the nucleusis tethered by microtubules (Tanaka and Ito, 1981;Dickinson and Sheldon, 1984). Mineyuki and Furuya(1986) also showed that it is the microtubules thatparticipate primarily in the positioning of the nucleus inpremitotic protonema tip cells of Adiantum. The premi-totic migration of nuclei in leaf explant cells of Nautilo-calyx appears to be suppressed by colchicine (Venverloo

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and Libbenga, 1987). The present study was undertakento ascertain whether the nucleus in premitotic tobaccoBY-2 cells is positioned by actin filaments, as in the caseof interphase tobacco cells, or by microtubules, as in thepremitotic cells mentioned above. We found that it wasthe microtubules that were involved, as in the latter case,and we attempted to clarify the way in which thecytoskeletal elements responsible for the positioning ofthe nucleus change from actin filaments to microtubulesduring the transition from interphase to premitoticphase. Incidentally, Traas et al. (1987) demonstratedthat in cultured carrot cells the nucleus is suspendedthroughout cell division by actin filaments.

The use of synchronized cultures of cells offers manyadvantages for the study of the intracellular structuresthat appear during only a limited period of the cell cycle.The cell cycle of tobacco BY-2 cells can be effectivelysynchronized by aphidicolin, a drug that inhibits DNApolymerase a (Nagata et al. 1982). Kakimoto and Shi-baoka (1988) used such synchronized tobacco BY-2 cellssuccessfully for the isolation of a preparation of phragmo-plasts with a high degree of purity. Mineyuki et al. (1988)showed, however, that aphidicolin did not prevent theformation of the PPB in root tip cells of Allium eventhough it arrested the progress of the nuclear cycle.Therefore, we first examined whether or not aphidicolinprevents premitotic cytoplasmic events, such as theformation of the PPB, so that timely treatment with thisdrug would result in the synchronized formation of PPBsin tobacco BY-2 cells. When it was confirmed thattreatment with aphidicolin over a defined period of timebrought about the synchronized formation of PPBs intobacco BY-2 cells, we examined the premitotic re-arrangement of the cytoskeletal array using tobacco BY-2cells in which formation of the PPB was synchronized bysuch treatment. We then investigated the role of thecytoskeleton in the premitotic positioning of the nucleus.

Materials and methods

Plant materialTobacco BY-2 cells were maintained in modified Linsmaier andSkoog's medium (LS medium) supplemented with 170mgl~KH2PO4, 0.2 mg I"1 2,4-dichlorophenoxyacetic acid and 3 %sucrose, and regularly subcultured at 7-day intervals (Nagata etal. 1981).

The cell cycle of tobacco BY-2 cells was synchronizedaccording to the method of Nagata et al. (1982). A 10 ml sampleof a 7-day-old suspension of cells (approximately 7xlO8 cells)was added to 95 ml of LS medium that contained 5 fig ml"aphidicolin (Wako Pure Chemical Industries, Ltd, Osaka,Japan). After the cells were cultured with the drug for 24 h, theywere washed five times with fresh LS medium, resuspended inthe same medium and cultured continuously at 27°C.

Mitotic index (MI) and percentage of PPB (%PPB)After treatment with aphidicolin, cells were harvested atappropriate intervals, and the cell walls were partially digestedfor 2.5-3 min with an enzyme solution that contained 0.5 %Cellulase Onozuka RS (Yakult Honsha Co., Ltd, Tokyo,Japan), 0.05% Pectolyase Y-23 (Seishin Pharmaceutical Co.,Ltd, Tokyo, Japan), 5 mM EGTA (Dojindo Laboratories,

Kumamoto, Japan), 2//M (p-amidinophenyl)-methanesulfonylfluoride hydrochloride (p-APMSF; Wako Pure ChemicalIndustries, Ltd, Osaka, Japan), 50//gml~ leupeptin, 0 .25Mmannitol, adjusted to pH 5.5. This brief treatment with enzymegreatly facilitated the penetration of antibodies into the cellswithout causing any appreciable change in cell shape. Thencells were fixed in 3 % formaldehyde in KP-Mg-EGTA buffer(SmM MgCl2, 25 mM KC1, 5 mM EGTA, 2fM p-APMSF,50jUgml~ leupeptin, in 50mM potassium phosphate buffer,pH6.8) that contained 0.06% Nonidet P-40 (NP-40), at roomtemperature for 1 h. Microtubules were stained and observed asdescribed previously (Katsuta and Shibaoka, 1988). DNA wasvisualized by staining with l/lgral"1 5',6'-diamidino-2-phen-ylindole (DAPI). The mitotic index (Ml) was assessed as thepercentage of cells with condensed chromatin, that is the sum ofthe percentages of cells in prophase, metaphase, anaphase andtelophase. The %PPB was assessed as the number of cells with aPPB, as a percentage of the total number of cells. For eachdetermination, more than 1000 cells were examined.

Triple staining of microtubules, actin filaments andDNAAbout 6h after the treatment with aphidicolin, cells wereharvested and cell walls were partially digested as describedabove. Cells were incubated in KP-Mg-EGTA buffer thatcontained 0.2mgml~ tropomyosin, 200 or 400 fM w-maleimi-dobenzoic acid Ar-hydroxysuccinimide ester (MBS, SigmaChemical Company, St Louis, MO, USA), 20/igml"1 taxol or2mM ethyleneglycolybis(succimidylsuccinate) (EGS, DojindoLaboratories, Kumamoto, Japan), 0.06% NP-40 and 10%dimethyl sulfoxide (DMSO), at room temperature for 30min,and fixed in KP-Mg-EGTA buffer that contained 3 % formal-dehyde for 1 h. Tropomyosin and MBS were used to stabilizeactin filaments (Kakimoto and Shibaoka 19876; Sonobe andShibaoka, 1989), and EGS or taxol was used to stabilizemicrotubules (Gorbsky et al. 1988; Mitchison and Kirschner,1985). MBS was dissolved in DMSO at 20mM and EGS inDMSO at 100 mM, and both were stored at -20°C. Tropomyo-sin was isolated from rabbit skeletal muscle according to themethod of Lehman and Szent-Gyoryi (1972), with somemodifications.

Cells were washed twice with KP-Mg-EGTA buffer andincubated with mouse monoclonal antibodies against tt-tubulinfrom chick brain (IgG) (Amersham International pic, Buck-inghamshire, England, UK) and 0.2/XM rhodamine-conjugatedphalloidin (Rh-ph, Molecular Probes, Inc, Eugene, OR, USA)for 1 h. Cells were washed twice with Tween 20-PBS (0.05%Tween 20, 0.05% NaN3, in 8mM PBS solution, pH7.0) andincubated with FITC-conjugated antibodies raised in rabbitagainst mouse IgG (ICN ImmunoBiologicals, Lisle, IL, USA)and 0.2^M Rh-ph for 1 h. They were washed twice with Tween20-PBS, incubated with 1 //gml~' DAPI and 0.2 iM Rh-ph for10 min, and washed once with Tween 20-PBS.

Stained cells were observed under an Olympus BHS-RFKfluorescence microscope with standard filter sets (BP 545 andEO 530 excitation filters and an O 590 barrier filter forrhodamine; BP 490 and EY 455 excitation filters and an O 515barrier filter for fluorescein; and a UG 1 excitation filter and anL 420 barrier filter for DAPI fluorescence).

Drug treatmentsCytochalasin D was purchased from Sigma Chemical Co. (StLouis, MO, USA) and dissolved in DMSO at 20mM. Propyza-mide (Akashi et al. 1988) was obtained from SumitomoChemical Co. (Takarazuka, Hyogo, Japan) and dissolved inDMSO at 200 mM. These solutions were stored at -20°C andwere diluted immediately before use to appropriate concen-

414 J. Katsuta et al.

trations as mentioned in Results. The final concentration ofDMSO was kept below 0.5 % (v/v) in all experiments.

Assessment of the position of the nucleusAt appropriate intervals, cells were harvested and fixed for 2hin KP-EGTA buffer (5 mM EGTA, 2fiu p-APMSF, in 50 mMpotassium phosphate buffer, pH6.8) that contained 2% for-maldehyde for 2h. They were washed twice with KP-EGTAbuffer and stained with l|Ugml~' DAPI at room temperaturefor lOmin. Cells were then washed once with KP-EGTAbuffer and observed under a fluorescence microscope equippedwith phase-contrast apparatus (Olympus BHS-RFK).

The cell with a central nucleus was taken as that cell in whichthe nucleus was located apart from the cell cortex, or that cellwith transvacuolar cytoplasmic strands between the nucleus andthe cortex. The number of cells with a central nucleus as apercentage of the total number of cells at interphase andprophase was estimated.

Results

Synchronization of formation of the PPBTo ascertain whether PPBs were formed synchronouslyin BY-2 cells after treatment of cells with aphidicolin for adefined period of time, the changes in MI and %PPBafter treatment with aphidicolin were examined (Fig. 1).The MI and the %PPB in aphidicolin-untreated cells inthe logarithmic phase of growth were 10.8 and 4.6,respectively. Treatment with aphidicolin for 24 h reducedboth the MI and the %PPB in cells in the logarithmicphase of growth; it reduced the MI and the %PPB to 1.7and 1.7, respectively. Since the MI and the %PPB wereextremely low in cells in stationary phase even beforetreatment with aphidicolin (0.1 and 0.2, respectively),the effects of aphidicolin were scarcely evident (after a 24-hour treatment, the MI and the %PPB were 0.0 and 0.2,respectively). However, the effects became evident afteraphidicolin-treated cells were washed and transferred toaphidicolin-free medium. The MI began to increase 5hafter the termination of treatment with aphidicolin andattained a maximum value (50-70) after 7h. The per-

2 3 4 5 6 7Time (h)

10 11

Fig. 1. Changes in %PPB (see text for definition) and themitotic index in tobacco BY-2 cells after the termination of a24-h treatment with aphidicolin. Thick line, %PPB; thin line,mitotic index; broken line, percentage of cells at prophase;dashed line, percentage of cells at metaphase; dotted line,percentage of cells at anaphase/telophase.

centage of cells in prophase, metaphase and anaphase/telophase reached a maximum after 6.5, 7 and 7.5 h,respectively. The %PPB began to rise 3.5 h after removalof aphidicolin and reached a maximum value (30-50)after 6 h (Fig. 2).

Changes in the cytoskeletal array in premitotic cellsTo investigate the relationships between microtubulesand actin filaments from interphase to metaphase, weemployed a method for the triple staining of the cytoskel-etal elements and DNA. As suggested by Kakimoto andShibaoka (19876) and Sonobe and Shibaoka (1989),tropomyosin and MBS were used to stabilize actinfilaments. BY-2 cells were treated with a combination oftropomyosin and MBS and were fixed. This procedureenabled us to stain the microtubules, actin filaments andDNA in the same cells and to examine their relationships.

Cells immediately after the termination of the 24-htreatment with aphidicolin seemed to fall into twocategories in terms of arrays of microtubules: cells with atypical interphase array of microtubules and cells with apremitotic array of microtubules. The former had micro-tubules in only the cell cortex (Fig. 3), while the latterhad microtubules not only in the cell cortex but also incytoplasmic strands that radiated from the nucleus(Fig. 4). In interphase-type cells, actin filaments werepresent in cytoplasmic strands and around the nucleus, aswell as in the cell cortex (Fig. 3). In premitotic-type cells,microtubules were present both in cytoplasmic strandsthat extended from the nucleus in a direction parallel tothe prospective spindle axis (nucleus-to-pole cytoplasmicstrands) and in strands that radiated from the nucleustoward the side walls (nucleus-to-side wall cytoplasmicstrands). Both microtubules in nucleus-to-pole cytoplas-mic strands and those in nucleus-to-side wall cytoplasmicstrands were associated with actin filaments. The per-centage of cells with a typical interphase array of micro-tubules decreased from 40-50% immediately after theremoval of aphidicolin to 5-10% during the subsequent4-6 h. The percentage of cells with a premitotic array ofmicrotubules increased from 50-60 % immediately aftertreatment with aphidicolin to more than 80% in 2h.However, the percentage of cells with a premitotic arrayof microtubules decreased during the next 2-4 h whilethat of cells with a PPB increased.

PPBs appeared more than 3 h after the termination oftreatment with aphidicolin (Fig. 5). Condensation ofchromatin was not seen in 30-80% of cells with a PPB.Such cells were considered to be preprophase cells.Microtubules were codistributed with actin filaments inthe PPB and in both nucleus-to-pole and nucleus-to-sidewall cytoplasmic strands. The width of the PPB-like bandof actin filaments was the same as that of the PPB in about30% of the cells with a PPB, but the former was largerthan the latter (by 18% on average and by 67% at themaximum) in about 70 % of such cells. In almost all cellswith a PPB, the meshwork of actin filaments was presentin the cortical region from which microtubules haddisappeared.

Premitotic nuclear positioning in plant cells 415

Fig. 2. Synchronized formation of PPBs. Cells sampled 6h after the termination of treatment with aphidicolin and stained formicrotubules. A. Phase-contrast micrograph. B. Immunofluorescence micrograph. Arrows, cells with PPB. Bar, 50/im.

Changes in the cytoskeletal array in prophase cellsAll prophase cells had PPBs, and the PPBs were narrowerthan those in preprophase cells, as has been reported incells from onion root tips (Wick and Duniec, 1983). AsPPBs became narrower, the number of nucleus-to-sidewall cytoplasmic strands became smaller and only thosein the region encircled by PPBs (in an equatorial plane)remained. Microtubules began to disappear first from thenucleus-to-pole cytoplasmic strands, leaving actin fila-ments in these strands. Concomitantly, microtubulesappeared around the nucleus, assuming a spindle-hkearrangement (Figs 6, 7). During the prophase-metaphase transition, either the microtubules in thenucleus-to-side wall cytoplasmic strands or those in PPBsalso disappeared. At the same time, PPB-like bands ofactin filaments also disappeared, but actin filaments innucleus-to-side wall cytoplasmic strands as well as thosein nucleus-to-pole cytoplasmic strands remained.

Cells with a PPB were also viewed from the pole of thecells (Figs 8-10). Through-focusing also showed thatnucleus-to-side wall cytoplasmic strands, which con-tained microtubules, were distributed over a wide rangeof depths in preprophase cells (Fig. 8) and over a limitedrange in prophase cells (Fig. 9).

Observations from the pole of the cells also showed thatactin filaments were codistributed with microtubules

both in PPBs and in nucleus-to-side wall cytoplasmicstrands in cells at preprophase or prophase (Figs 8, 9). Atlate prophase, actin filaments were not always associatedwith microtubules (Fig. 10), suggesting that the micro-tubules disappeared and left the actin filaments in thestrands. The accumulation of microtubules around thenucleus at late prophase was observed also during obser-vations from the pole (Fig. 10).

Effects of cytochalasin D (CD) and propyzamide on theposition of the premitotic nucleusThe close spatial relationship between microtubules andactin filaments suggested the possibility that the arrange-ment of the former was determined by the latter or viceversa. This possibility was examined. Three hours afterthe termination of the 24-h treatment with aphidicolin,when PPBs began to be formed, propyzamide or CDwere added to the suspension of cells to a final concen-tration of 100/iM or 50[MM, respectively. Cells werecultured in the presence of drug for 3 h, then fixed andstained.

All prophase cells had PPBs in untreated controlcultures, while neither PPB nor the PPB-like band ofactin filaments was formed in cells treated with 100/iMpropyzamide, which reduced the microtubules to frag-ments (Fig. 11). As has been reported in root tip cells of

416 jf. Katsuta et al.

Figs 3-5. Cells triple-stained for microtubules, actin filaments and DNA. A. Microtubules in cell cortex. B. Actin filaments inthe same plane as in A. C. Microtubules at the level of the nucleus. D. Actin filaments at the same level as in C. E. DNA.Fig. 3. A cell sampled immediately after termination of treatment with aphidicolin. DAPI staining shows that the cell was atinterphase. An array of microtubules typical of interphase is seen. Microtubules are seen only in the cortical region, while actinfilaments are seen in transvacuolar cytoplasmic strands as well as in the cell cortex. Most cortical microtubules are associatedwith actin filaments.Fig. 4. A cell sampled immediately after the termination of treatment with aphidicolin. DAPI staining shows that the cell wasat interphase. A premitotic array of microtubules is seen. Microtubules are seen not only in the cell cortex but also in thecytoplasmic strands that extend from the nucleus to the periphery of the cell. Most of them are associated with actin filaments.Fig. 5. A cell sampled S h after the termination of treatment with aphidicolin. DAPI staining shows that the cell was atinterphase. The PPB is seen. Actin filaments are present in the PPB. Microtubules are seen in nucleus-to-pole cytoplasmicstrands as well as in nucleus-to-side wall strands. Bar, 20,um.

onion (Palevitz, 1987), the disruption of microtubulesprevented the formation of the PPB-like band of actinfilaments, as well as that of the PPB.

CD at 50,UM caused most of the actin filaments to breakinto small fragments within 20 min. As has been found inroot tip cells of onion (Palevitz, 1987), PPBs were formed

in the presence of CD (Fig. 12). PPBs were formed 6hafter the termination of treatment with aphidicolin. A lotof short actin filaments were present in PPBs formed inthe presence of CD and they were closely associated withmicrotubules of the PPB (Fig. 12B, D).

The effects of anticytoskeletal drugs on premitotic

Premitotic nuclear positioning in plant cells 417

Figs 6, 7. Cells triple-stained for microtubules, actin filaments and DNA. A. Microtubules in the cell cortex. B. Actin filamentsin the same plane as in A. C. Microtubules at the level of the nucleus. D. Actin filaments at the same level as in C. E. DNA.Cells were sampled 6h after the termination of treatment with aphidicolin. DAPI staining shows that the cell in Fig. 6 was atmid-prophase and the cell in Fig. 7 was at late prophase. Microtubules in nucleus-to-pole cytoplasmic strands have disappeared,while actin filaments are still present in cytoplasmic strands as well as in the cell cortex. Microtubules have accumulated aroundthe nucleus, assuming a spindle-like arrangement. At late prophase (Fig. 7), microtubules begin to disappear from nucleus-to-side wall cytoplasmic strands and from the PPB. Although actin filaments in the PPB disappear at the same time as thedisappearance of PPB microtubules, they remain in cytoplasmic strands. Bar, 20 fim.

migration of the nucleus were examined in 7-day-oldsubcultured BY-2 cells. The nucleus was present in thecentral region in about 40% of these cells. Such suspen-sion cultures were divided into four groups and cells ingroup 1 were cultured in medium that contained5figml~' aphidicolin for 24 h and then in aphidicolin-free medium for 6h; cells in group 2 were cultured inmedium that contained aphidicolin and 100 fiM propyza-mide for 24 h and then in aphidicolin-free medium thatcontained propyzamide for 6h; cells in group 3 werecultured in medium that contained aphidicolin and 50 [iMCD for 24 h and then in aphidicolin-free medium thatcontained CD for 6h; cells in group 4 were cultured inmedium that contained aphidicolin, 100 fiM propyzamideand 50 fiM CD for 24 h and then in aphidicolin-freemedium that contained propyzamide and CD for 6h.After 2, 10, 24 and 30 h from the start of the firsttreatment, cells were sampled and the positions of thenuclei were determined (Fig. 13). During the treatmentwith aphidicolin, the percentage of cells with a centralnucleus increased gradually in the control cultures (group1), indicating that DNA synthesis is not a prerequisite forthe premitotic migration of the nucleus. The percentageof cells with a central nucleus continued to increase after

removal of aphidicolin (Fig. 13). In the presence of100/iM propyzamide (group 2), the percentage of cellswith a central nucleus decreased by about 15 % duringthe first 2 h and then increased and reached a value justbelow that for control cells within the subsequent 8h.However, after removal of aphidicolin, the percentagedid not increase at a rate similar to that in control cells(Fig. 13). Microtubules appear necessary for premitoticnuclear migration. Treatment with CD caused a rapiddecrease in the percentage of cells with a central nucleus;the percentage decreased from 40 % to 10 % within thefirst 2h but, thereafter, the percentage of cells with acentral nucleus increased at the same rate as in the controlcells. In CD-treated cells (group 3), the percentage ofcells with a central nucleus increased by 50 % during theperiod from 2 h to 30 h after treatment, while it increasedby 40 % in the control cells during the same period(Fig. 13). The premitotic migration of the nucleusseemed not to depend on actin filaments in BY-2 cells, asin the case of leaf explant cells from Nautilocalyx(Venverloo and Libbenga, 1987), although the involve-ment of fragmented residual actin filament in the move-ment of the nucleus cannot be excluded. In cells treatedwith both propyzamide and CD (group 4), the percent-

418 Jf. Katsuta et al.

Figs 8-10. Triple-stained cells with a PPB, viewed from the pole of the cells. A. Microtubules in the zone encircled by thePPB. B. Actin filaments in the same plane as in A. C. DNA.Fig. 8. A cell sampled 5 h after the termination of treatment with aphidicolin. DAPI staining shows that the cell was atinterphase. Many microtubules are seen in nucleus-to-side wall eytoplasmie strands. Most of them are codistributed with actinfilaments.Fig. 9. A cell sampled 6h after the termination of treatment with aphidicolin. DAPI staining shows that the cell was atprophase. Microtubules and actin filaments are seen between the nucleus and the PPB or the cell cortex.Fig. 10. A cell sampled 6 h after the termination of treatment with aphidicolin. DAPI staining shows that the cell was at lateprophase. Microtubules have been reduced in number in nucleus-to-side wall eytoplasmie strands and in the PPB. They haveaccumulated on the surface of the nucleus. Many actin filaments are seen in nucleus-to-side wall eytoplasmie strands. Bar,20 fan.

age of cells with a central nucleus fell below 1 % within2h and barely increased during the subsequent 28 h(Fig. 13).

The effects of propyzamide and CD on the position ofthe nucleus in cells pretreated with aphidicolin for 24 hwere also examined (Fig. 14). Seven-day-old subculturedBY-2 cells were treated with 5/igml~' aphidicolin for24 h. During the treatment with aphidicolin, the percent-age of cells with a central nucleus increased. Immediatelyafter the termination of treatment with aphidicolin, thenucleus was positioned in the central region in about 75 %of cells (Fig. 14). These aphidicolin-treated cells werethen treated with 100 [XM propyzamide and/or 50 fiMcytochalasin D. Treatment with propyzamide or CD for2 or 6h caused only a small decrease (5-15%) in thepercentage of cells with a central nucleus (Fig. 14).However, simultaneous treatment with propyzamide andCD caused displacement of the nucleus from the central

region to the periphery in more than 80 % of cells within2h(Fig . 14).

Discussion

Relationship between the nuclear cycle and formation ofthe preprophase band (PPB)Formation of the PPB in tobacco BY-2 cells wassynchronized by 24-h treatment with aphidicolin, aninhibitor of DNA polymerase a (Figs 1,2). Mineyuki etal. (1988) found that the %PPB was not reduced bytreatment with aphidicolin that decreased the mitoticindex six- to tenfold in root tip cells oiAllium. From thisresult they concluded that the eytoplasmie events ofcytokinesis were controlled in parallel to the nuclearcycle, rather than in an obligatorily coupled sequence.The present study demonstrates, however, that theformation of PPBs is closely related to the nuclear cycle in

Premitotic nuclear positioning in plant cells 419

Figs 11, 12. Cells treated with propyzamide or CD and triple-stained. A. Microtubules at the level of the surface of the cell.B. Actin filaments at the same level as in A. C. Microtubules at the level of the nucleus. D. Actin filaments in the same focalplane as in C. E. DNA.Fig. 11. A cell treated with aphidicolin for 24 h and then with 100 j.iM propyzamide for 3h before fixation. DAPI staining showsthat the cell was at prophase. Microtubules are broken and seen as small fragments or dots. Actin filaments seem normal, butthey show no PPB-like distribution.Fig. 12. A cell treated with aphidicolin for 24 h and then with SO (M CD for 3 h before fixation. DAPI staining shows that thecell was at prophase. Note that the PPB microtubules are associated with short actin filaments. Bar, 20 jum.

100i

50-

U

10Time (h) Time (h)

Fig. 13. Effects of CD and propyzamide on premitoticmigration of the nucleus in tobacco BY-2 cells. Seven-day-oldsubcultured cells were cultured in medium that containedaphidicolin (O), aphidicolin+lOOjUM propyzamide ( • ) ,aphidicolin+50/lM CD ( • ) or aphidicolin+100,UMpropyzamide+50;UM CD (A) for 24h and then in the samemedium but without aphidicolin. The shaded zone indicatesthe period of treatment with aphidicolin (APH).

Fig. 14. Effects of CD and propyzamide on the position ofthe nucleus in tobacco BY-2 cells that were pretreated withaphidicolin for 24 h. Aphidicolin-pretreated cells werecultured in the absence (O) or presence of 100 jUMpropyzamide ( • ) , 50[M CD ( • ) or 100/.M propyzamide+50,UM CD (A). At appropriate times, cells were sampled andthe position of the nucleus examined. The time is given inhours after removal of aphidicolin.

420 J. Katsnta et al.

tobacco BY-2 cells. The difference between the results ofMineyuki et al. and ours may be due to the difference inplant species or in the type of cell examined; Mineyuki etal. used cells of Allium tissue whereas we used suspen-sion-cultured tobacco cells. To clarify the discrepancies,the effects of aphidicolin on root tip cells of tobaccoplants or on cultured cells oi Allium should be examined.

Although one of the premitotic cytoplasmic events,namely the formation of the PPB, was found to be relatedto the nuclear cycle, another premitotic cytoplasmicevent, namely premitotic migration of the nucleus, ap-peared not to be coupled to the nuclear cycle in BY-2cells. Premitotic migration of the nucleus occurred inaphidicolin-treated cells that did not form a PPB, indi-cating that the PPB does not participate in premitoticmigration of the nucleus. This result is consistent withthe previous observation in epidermal cells in Nautiloca-lyx that both formation of the PPB and replication ofDNA were preceded by the premitotic migration of thenucleus (Venverloo and Libbenga, 1987). Our resultsalso indicate that the onset of the premitotic migration ofthe nucleus and that of formation of the PPB arecontrolled by different mechanisms.

The role of microtubules in premitotic positioning of thenucleusIn a previous paper (Katsuta and Shibaoka, 1988), weshowed that actin filaments, and not microtubules, areresponsible for positioning of the nucleus in tobacco BY-2cells at interphase. The cytoskeletal elements responsiblefor the positioning of the nucleus in premitotic cells seem,however, to be different from those in interphase cells,because (1) microtubules are present in cytoplasmicstrands that extend from the nucleus to the cell peripheryin premitotic cells (Fig. 4), while they are not present ininterphase cells; and (2) the nucleus was displaced aftertreatment with cytochalasin in interphase cells, while itwas not displaced in premitotic cells (Fig. 14). As hasbeen reported in several types of cell (Mineyuki andFuruya, 1986; Venverloo and Libbenga, 1987), micro-tubules seem to play an important role in premitoticpositioning of the nucleus in tobacco BY-2 cells. Lloydand Traas (1988) proposed a possible mechanism for thepremitotic migration of the nucleus. They suggested thatthe premitotic disappearance of cortical microtubulesallows the nucleus, whose position has been maintainedby cortical microtubules, to migrate to the site of futuredivision in a process that involves actin filaments. Ourobservation that the nucleus in premitotic cells was notdisplaced by disruption of microtubules alone, but wasdisplaced when both microtubules and actin filamentswere disrupted, also suggests the involvement of actinfilaments in the premitotic migration of the nucleus.However, the observations that microtubules emerge incytoplasmic strands that radiate from the nucleus onlyduring the premitotic stage (Fig. 4), and that the premi-totic migration of the nucleus is accomplished even in thepresence of CD (Fig. 13), lead us to suggest that micro-tubules are important in the premitotic migration of thenucleus. The result showing that fragmented actin fila-ments are present in CD-treated cells (Fig. 12 B, D)

suggests the possibility that residual fragmented actinfilaments might be responsible for bringing the nucleus tothe center of the cell. If this is the case, we may stillemphasize the importance of microtubules in thisphenomenon: the fragmented residual actin filaments canmove the nucleus only when they are associated withmicrotubules that radiate from the nucleus.

The role of microtubules in organizing the array ofmitotic actin filamentsAt preprophase, microtubules appear in transvacuolarcytoplasmic strands in which they are never observedduring interphase. The appearance of the PPB is also acharacteristic feature of preprophase cells. Microtubulescharacteristic of preprophase cells, namely microtubulesof the PPB and microtubules in cytoplasmic strands, arealways associated with actin filaments, suggesting that thearray of microtubules is determined by actin filaments orvice versa. The results of our experiments with variousdrugs indicate that it is the microtubules that determinethe arrangement of the actin filaments. Premitotic re-arrangement of microtubules (Figs 4, 5) takes place inthe presence of an inhibitor of actin filaments in whichmost of the actin filaments are broken into small frag-ments (Fig. 12), but the premitotic rearrangement ofactin filaments, i.e. formation of the PPB-like band ofactin filaments, did not occur in the presence of aninhibitor of microtubules (Fig. 11), as has been reportedin root tip cells from onion (Palevitz, 1987). Lloyd andTraas (1988) also demonstrated that the PPB-like band ofactin filaments is formed in the presence of CD in carrotcells.

Microtubules seem to be involved in positioning of thenucleus only from preprophase to prophase. At lateprophase, they begin to disappear from the cytoplasmicstrands that extend from the nucleus to the cell periph-ery. Thereafter, actin filaments only are responsible formaintaining the position of the dividing nucleus. Traas etal. (1987) have already demonstrated that the nucleus issuspended throughout cell division by actin filaments,Lloyd and Traas (1988) reported the presence of actinfilaments between the equatorial plane of the mitoticspindle and the cell cortex in carrot cells, and Kakimotoand Shibaoka (1987a) showed that actin filaments con-nect the margin of the phragmoplast with the cell cortexin tobacco BY-2 cells.

From our results, we suggest that the premitotic arrayof microtubules is set up as a scaffold for building up thearray of actin filaments that maintains the position of themitotic apparatus and the cytokinetic apparatus. Ourhypothesis will be validated only when we can demon-strate the presence in plant cells of microtubule-associ-ated proteins that can bind to actin filaments, similar tothose known to be present in animal cells (Griffith andPollard, 1978, 1982). Close association of microtubulesand actin filaments (or of thin filaments that resembleactin filaments) in plant cells, as revealed by double-staining fluorescence microscopy (Figs 3-10 and 12 inthis paper; Kakimoto and Shibaoka 19876; Palevitz,1987; Sonobe and Shibaoka, 1989) and by freeze-substi-tution electron microscopy (Lancelle et al. 1987; Nogu-

Premitotic nuclear positioning in plant cells 421

chi and Ueda, 1988; Tiwarie^a/. 1984), strongly suggeststhe presence of such proteins in plant cells. Studies toconfirm the presence of such proteins are now in pro-gress.

This work was supported in part by a Grant-in-Aid forScientific Research (no. 62480009) from the Ministry of Edu-cation, Science and Culture, Japan.

Taxol was kindly supplied by the Natural Products Branch,Division of Cancer Treatments, National Cancer Institute(Bethesda, Maryland).

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(Received 5 September 1989 - Accepted 22 November 1989)

Ml jf. Katsuta et al.