relationship between preprophase band organization f-acti,n and … · 2005-08-26 · and basal...

Relationship between preprophase band organization, F-actin and the

division site in Allium

Fluorescence and morphometric studies on cytochalasin-treated cells

Y. MINEYUKI* and B. A. PALEVITZj

Department of Botany, University of Georgia, Athens, GA 30602, USA

* Present address: Botanical Institute, Faculty of Science, Hiroshima University, Nakaku, Hiroshima 730, Japant Author for correspondence

Summary

The preprophase band (PPB) of microtubules (Mts),which appears in the G2 phase of the cell cycle inhigher plants but disappears well before the end ofkaryokinesis, is implicated in the determination ofthe division plane because its location marks the siteat which the phragmoplast/cell plate will fuse withthe parental plasmalemma during cytokinesis. ThePPB first appears as a rather wide array, whichprogressively narrows before or during prophase.Actin-containing microfilaments (Mfs) have recentlybeen reported in the PPB, but the role of theseelements in PPB organization and/or functionremains unclear. The present study employed fluor-escence and pharmacological methods in symmetri-cally and asymmetrically dividing epidermal cells ofAllium to probe this problem. Our results show thatPPBs in cells treated with 2-200/iM cytochalasin D(CD) are still transversely aligned but remain two tothree times wider than mature bands in control cells.Treatment for 0.5 h at 20 ;iM is sufficient to make thePPBs abnormally widel Premitotic nuclear mi-

gration in asymmetrically dividing cells is alsoinhibited by CD, as is the positioning of the mitoticapparatus and the new cell plate. The plate is stilltransverse, however. Band-like arrays of cortical Mfsbecome evident in most interphase cells by prophase.The band remains quite wide compared to the finaldimensions of the Mt PPB, and clearly encompassesit. Levels of CD as high as 200/IM decrease thenumber of cells with transverse actin bands,although a majority still retain them. Other F-actinarrays are disrupted by the drug. Thus, while CDdoes not inhibit the formation of an initial, broad,transverse PPB in most cells, it does prevent thenarrowing process that defines the precise divisionsite. The role of actin in this effect is discussed.

Key words: actin, Allium, asymmetrical division, cytochalasin,cytoskeleton, division plane, microfilament, microtubule,phragmoplast, preprophase band, stomate.

Introduction

Because plant cells are surrounded by relatively rigidwalls, they are not free to migrate during growth anddevelopment. Instead, the position and orientation of theplane of division in most cases establishes spatialrelationships between cells that are critical in morphogen-esis and differentiation (Sinnott, 1960). The preprophaseband (PPB) of microtubules (Mts) replaces the interphasecytoskeletal array in the cortex of G2 somatic cells andmarks the position at which the phragmoplast/cell platecomplex meets the parental plasmalemma during cytokin-esis (Wada et al. 1980; Gunning, 1982; Gunning and Wick,1985; Palevitz, 1986; Mineyuki etal. 19886). The PPBitself disappears before karyokinesis. While the phragmo-plast clearly interacts with the cortical division siteduring cytokinesis (Ota, 1961; Gunning and Wick, 1985;Palevitz, 1986), whether the PPB actually influences thissite (and if so, how) remains to be determined. Likewise,the manner in which the PPB is organized is not wellunderstood. The band first appears as a relatively broadarray, which then narrows and consolidates (Wick and

Journal of Cell Science 97, 283-295 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

Duniec, 1983; Mineyuki etal. 1989). Evidence indicatesthat in at least some cells the PPB is first formed byrearrangement of the preceding interphase Mt array (e.g.see Pickett-Heaps, 19696; Wick and Duniec, 1983;Mineyuki et al. 1989; Mullinax and Palevitz, 1989). Otherobservations employing rhodamine-phalloidin and anti-body labeling have shown that the PPB and phragmoplastalso contain F-actin (Kakimoto and Shibaoka, 1987;Palevitz, 1987a,6; Traas et al. 1987; McCurdy et al. 1988;McCurdy and Gunning, 1990). Because actin-specificcytochalasins (Cooper, 1987) interfere with the correctalignment of the cell plate as well as other aspects of cellpolarity (Quatrano, 1973; Schnepf and von Traitteur, 1973;Palevitz and Hepler, 19746; Palevitz, 1980, 1986; Brawleyand Robinson, 1985; Gunning and Wick, 1985; Venverlooand Libbenga, 1987), it is appropriate to enquire into therole of actin in the organization and function of the PPBand the phragmoplast.

The stomatal apparatus provides excellent material forthe study of division plane regulation and its role in plantdevelopment. Highly asymmetrical transverse divisionsin the epidermis are responsible for cutting off stomatal

283

guard mother cells (GMCs) in monocots (Bunning, 1958;Stebbins and Jain, 1960; Stebbins and Shah, 1960;Palevitz and Hepler, 1974a). Accessory subsidiary cells arealso created by asymmetrical divisions in many species(e.g. see Stebbins and Shah, 1960; Pickett-Heaps, 1969a,6;Galatis etal. 1983). On the other hand, symmetricaltransverse divisions in the vicinity are proliferative innature in that they lead to the duplication of nonstomatalepidermal cells. GMC division, though symmetrical, ischaracterized by a cell plate oriented 90° to most or allother division planes in the epidermis, and the resultingdaughter cells then differentiate into a highly specializedguard cell pair (Palevitz and Hepler, 1974a,6, 1976;Galatis, 1980, 1982; Palevitz, 1986; Cleary and Hardham,1989; Marc et al. 1989a,6; Palevitz and Mullinax, 1989).All these divisions are characterized by appropriatelypositioned PPBs (Pickett-Heaps and Northcote, 1966;Palevitz and Hepler, 1974a; Galatis, 1982; Galatis etal.1983; Cho and Wick, 1989; Cleary and Hardham, 1989;Mullinax and Palevitz, 1989). In the present paper, weinvestigate the relationship between PPB organization, F-actin and the division plane in symmetrically andasymmetrically dividing epidermal cells in the cotyledonsof Allium seedlings using fluorescence localizations,quantitative cytology and cytochalasin D (CD) treatments.

Materials and methods

Plant materialSeeds of Allium cepa L. cv White Portugal (Harris Moran SeedCo., Rochester, NY) were sown in moist vermiculite andmaintained at 25°C under a 12h/l2h, light/dark, regime usingcool-white fluorescent lamps. Two- or 3-day-old seedlings wereused in these experiments.

Drug treatmentsStock solutions of CD (Sigma Chemical Co., St Louis, MO) wereprepared in dimethylsulfoxide (DMSO) and then diluted to2-200 ^M with deionized water. DMSO concentrations neverexceeded 1 %, a level that does not appear to affect thecytoskeleton in plant cells (e.g. see Palevitz and Hepler, 19746;Palevitz, 1980). For short-term exposures (0.5 to several h), 3-day-old seedlings were transferred to 1.3 cm (diameter) glass vialscontaining 1 ml of solution. For longer exposures (19-24 h), 2-day-old seedlings were placed in filter paper soaked in 3 ml of solutionin 9 cm Petri dishes. Control seedlings were treated withdeionized water or concentrations of DMSO matching those usedfor the drug exposures (see figure legends). To assess the effects ofMt disruption on microfilaments (Mfs), seedlings were treatedwith 1.0 or 10.0 mM colchicine (Sigma) in deionized water for 2h.

Fluorescence localizationsFor Mt localizations, that portion of the cotyledon between thebase and the hook was excised, sliced in half and fixed overnightin a solution containing 4 % paraformaldehyde, 0.2 % glutaralde-hyde, 5 mM EGTA, 1 mM MgSO4, 1 % glycerol and 50 mM Pipesbuffer, pH6.8. The immunocytochemical localization of Mts wasthen performed according to the procedures of Mineyuki et al.(1988a, 1989), Marc and Hackett (1989) and Marc et al. (1989a,6).For Mf staining, seedlings were preincubated in 50 ^M m-maleimidobenzoyl-Af-hydroxysuccinimide ester (MBS; Sonobeand Shibaoka, 1989) (Pierce Chemical Co., Rockford, IL) dissolvedin 20 mM Pipes bufer, pH 7.5, for 20 min. The cotyledons were thentransferred to staining medium and thin slices excised andincubated for 1 h. The medium was modified from those of Traasetal. (1987) and Kakimoto and Shibaoka (1987) and contained0.1 ji(M rhodamine-phalloidin (Molecular Probes, Eugene, OR),5 mM EGTA, 1 mM MgS04, 0.01 % Nonidet P-40,1 % DMSO, 0 . 1 Mmannitol, 1% bovine serum albumin, 3 HIM dithiothreitol, 0.1%

re-propyl gallate, protease inhibitors (1 mM phenylmethylsulfonylfluoride, 40iugmn1 leupeptin, 25,ugml~1 chymostatin, and5^gml~x each of antipain, aprotinin, alpha-2-macroglobulin andpepstatin), 20^gmr f Hoechst 33258 and 50mM Pipes buffer,pH6.8.

Epidermal cells were examined with a Universal microscope(Carl Zeiss, Thornwood, NY) equipped for epifluorescence anddifferential interference contrast (DIC) viewing. Photographswere taken on a Nikon UFX-II exposure system (NikonInstruments, Garden City, NY) using Tri-X film (Eastman Kodak,Rochester, NY) and developed in HC-110 (Kodak).

Quantitative analysesQuantitative methods were used to assess various aspects of PPBorganization, cell morphology and drug effects. For each point inindividual experiments, six half-cylinders were obtained from thecotyledons (again, the portion between the base and hook) of threeseedlings and processed according to the procedures describedabove. From these six samples, 30-60 prophase cells and 30-60cells at later stages in division could usually be found. For somemeasurements as many as 180 cells were employed.

PPB width as well as nuclear and cell length were measureddirectly on the microscope in midoptical sections using a 40 xobjective and an eyepiece micrometer. In order to designate theposition of the PPB in a standard manner from experiment toexperiment, the center of the PPB (as determined from its width)was specified in terms of the percentage of cell length from theapical end (Fig. 1). Bands located in the apical and basal 20 % ofcell length are designated apical and basal, while those located inthe mid 40-60% are considered central. All others are subapicaland subcentral. Apical, relative to the cotyledon axis, is defined asthe position closest to the seed coat and away from the root; basalis the reverse. For the nucleus and the mitotic apparatus, asomewhat different procedure was used. Because of variations inthe size of nuclei and spindles between cells, the position of theircenters is not necessarily an accurate indicator of the degree ofdivision asymmetry. Therefore, a nucleus or mitotic spindle isconsidered apically or basally positioned if it appears to be incontact with the apical or basal ends of the cell, respectively, whileit is considered centrally located if its center is positioned between40 and 60 % of the length of the cell from the apical end. Thosenuclei and spindles not located at the cell center or at the apicaland basal walls are defined as subapical or subcentral. While

Apical end

PPB

Basal end

Fig. 1. Diagram illustrating the method by which the positionof the PPB is defined in epidermal cells of the Alliumcotyledon. In the cell drawn here, the center (X) of the PPB ispositioned between 20 and 30 % of the length of the cell fromthe apical end. The center (x) of the nucleus (N) is also shown;for convenience it is positioned 70 % from the apical end of thecell.

284 Y. Mineyuki and B. A. Palevitz

these procedures were difficult in small cells (e.g. near the apicalhook region), they were not a problem for most of the cells usedhere, which were at least twice as long as their width.

Results

PPB development during symmetrical and asymmetricaldivision in untreated tissuePPB formation during asymmetrical transverse divisionswas compared with that attending similarly orientedsymmetrical divisions in nearby cells. At early stages inseedling growth (2-4 days), the basal portion of the Alliumcotyledon contains a mixture of the two division types.Moreover, prophase nuclei and mitotic spindles occurfrequently and are relatively easy to locate using Hoechstfluorescence. Most of the Mts in interphase epidermal cellsare cortical; relatively few are seen deeper in thecytoplasm or around the nuclei (Fig. 2A). The orientationof the cortical Mts varies from longitudinal to transverse,and different orientations including criss-crossed patternscan be discerned (Fig. 2A).

Two different types of PPB are easily distinguishable inthe epidermis of 3-day-old seedlings. One is located in thecentral part of the epidermal cells (Fig. 2A), while theother is positioned apically (Fig. 2B). The former appearsto be the PPB of symmetrically dividing cells, while thelatter corresponds to that of asymmetrical divisions. Mostof these PPBs are transversely aligned, but in asymmetri-cally dividing cells with an oblique apical end wall, thePPB is often oblique as well (bottom cell in Fig. 2B). Asimilar arrangement has been seen in the epidermis ofgrasses (Cho and Wick, 1989).

The first sign of PPB formation occurs in late inter-phase. The cortical Mt array appears to be rearranged intoa broad band (i.e. with a band width greater than 50 % ofnuclear length; see below) of transverse elements locatednear the center or apical end of the cell (Fig. 2C,D). A fewobliquely oriented Mts can traverse the band (Fig. 2C). Inthe case of centrally located PPBs, the transverse Mts atboth edges of the band at this stage gradually interminglewith more steeply aligned cortical elements extending

towards either end of the cell (Fig. 2C). A gradation in Mtorientation is usually seen only at the basal edge of theapically located bands, since the apical edge often lies tooclose to the adjacent transverse wall to allow adequatevisualization of detail (Fig. 2D). These early, broad PPBsare always accompanied by perinuclear Mts (data notshown). While single, broad bands are most common atthis time, some cells contain double PPBs similar to thoseseen in roots (data not shown; see Wick and Duniec, 1983).Double bands are most commonly seen in early prophase,indicating that they represent a transition stage betweenbroad and narrower bands.

By prophase, all cells have PPBs, and most of these arerelatively narrow (see below), although a few cells withbroad or double bands can still be detected as well.Typically, Mts link the PPB with those ensheathing thenucleus (Fig. 2E-G). Narrow PPBs can also be seen ininterphase cells, but these are relatively rare.

Analysis of PPB width, cell length and nuclear length insymmetrical and asymmetrical prophase cellsIn order to characterize symmetrical and asymmetricalprophase cells better in terms of their PPBs, cell length,nuclear length and nuclear position, a more quantitativeapproach was taken. Because epidermal cells between thebase and hook of the cotyledon vary greatly in size, wedecided to normalize PPB position to cell length. Fig. 3Bshows that, as surmised from the general inspection of thecells above, PPB positions fall into two distinct groups.One group, belonging to asymmetrically dividing cells, issituated in the apical 30 % of the cell (mainly between theapical 10 and 20% of cell length), while the other iscentrally disposed and corresponds to cells about toundergo symmetrical division. The two groups are clearlyseparated in histogram presentations of PPB position(Fig. 3C,D). PPBs do not abut against the apical trans-verse wall, nor are they seen in the basal 20 % of the cell.

Next, we examined PPB width, cell length and nuclearlength in prophase cells (Table 1). Distinct differences incell and nuclear length characterize the two cell popu-lations. Symmetrically dividing cells are generally longer

Table 1. Effect of CD on cell length, nuclear length and PPB width in prophase cells of asymmetrical andsymmetrical division in the epidermis of Allium cotyledons

Asymmetrical division

Treatment

WaterDMSO (0.5 %)CD(2/JM)CD(20/IM)

Symmetrical division

Treatment

Cell length(/im)

38.2±1.940.1±2.636.7±3.135.5±2.4

Cell length((im)

Nuclear length(^m)

19.0±0.718.9±0.719.8±0.918.2±0.5

Nuclear length(/im)

PPB width(fan)

3.9±0.23.9±0.38.3±0.6**

12.2±2.0**

PPB widthQun)

PPB width/nuclear length

0.21±0.010.21±0.020.43±0.05**0.68±0.12**

PPB width/nuclear length

n"

15216

13

WaterDMSO (0.5 %)CD (2 IM)CD(20HM)

54.4±2.0##

56.5±4.2##

47.1±2.2*'#

45.4±2.0**'**

24.8±0.8##

23.8+0.8**19.7±0.6**18.4±0.6"

6.3+0.5**5.4±0.5##

12.9±0.7**'#

15.7±1.2»

0.25±0.030.23±0.020.67±0.04**'*0.87±0.06**

302028b

30

The lengths of cells and nuclei and the widths of PPBs in the epidermis of the cotyledons of 3-day-old Alliuni seedlings incubated in 2 or 20 /IM CDfor 1 day were compared with values from distilled water and 0.5% DMSO controls. All values represent the mean±the standard error of the mean(S.E.M.). Student's t-test was used to compare differences in the averages of cell length, nuclear length and PPB width in each treatment to thedistilled water control (*•**). Symmetrical divisions were also compared to asymmetrical divisions (*'**). * Significantly different from the deionizedwater control (P<0.05). ** Significantly different from the deionized water control (/><0.005). # Significantly different from the same value forasymmetrical divisions (P<0.05). ** Significantly different from the same value for asymmetrical division tP<0.005).

"Number of cells counted.b As there was a cell with a double PPB among these 28 cells, the mean width of the PPB was calculated on the basis of values from 27 cells.

PPB organization and the division plane in plants 285

In all micrographs, the longitudinal axis of the cotyledon is verticallyoriented, and the apical end of the organ is positioned toward the topof the page.Fig. 2. (A,B) Tubulin immunofluorescence images of two differentepidermal layers from 3-day-old cotyledons of Allium viewed at theinner cell surface (A) and at mid-depth (B). A prophase cell in A (O)contains a perinuclear spindle and is girdled by a narrow,symmetrically placed PPB. Three cells in B are dividingasymmetrically; narrow PPBs are present in two of these (*), while thethird (O) contains a broader band. All three cells contain perinuclearMts. One of the interphase cells in A also has a broad PPB(arrowhead). Note that some of the interphase epidermal cells in Acontain nontransverse cortical Mts. x890. (C,D) Tubulinimmunofluorescence images of broad, early PPBs in symmetrical (A)and asymmetrical (B) epidermal cell divisions. Note the divergent Mtorientations at the edge of the PPBs. x 1850. (E-G) Serial opticalsections through an asymmetrically dividing epidermal cell containinga migrating prophase nucleus. The section in E is close to the cellsurface, while that in G is near midlevel. Note Mts that appear to linkthe perinuclear spindle with the narrow PPB. Small squares in Gmark the dimensions of the cell, x 1850.

(30-100 /an; see Fig. 3 and Table 1) than asymmetricallydividing cells (20-60 /im). Cells between 30 and 60 (im candivide in either manner. Prophase nuclei in symmetricallydividing cells are also longer (Table 1).


While symmetrically dividing prophase cells are rela-tively long, their PPBs are also significantly wider(average 6 jxra; Table 1) than those in asymmetricaldivisions (average 4^m). However, when PPB width is

03OHOH

Base 50Cell length (,um)

100 0 10 20 0 10 20Number of PPBs

Fig. 3. Graphical representation of the position of PPBs in prophase cells in the cotyledons of 3-day-old Alliuin seedlings. Forillustrative and definition purposes, a PPB with its center marked by a dot and its limits marked by a bar is showndiagrammatically in A. In B, the positions of PPBs of various dimensions in a population of epidermal cells are displayed as afunction of absolute cell length (abscissa) and % of cell length from the apical end (ordinate). The PPB marked by the combinedcontinuous and dotted line is a double PPB. (C) Histogram representation of the frequency of PPBs in B at any given positionalong an epidermal cell (expressed as % of length from the apical end). (D) Histogram representation of the distribution of PPBcenters in B. Arrows in C and D indicate 30 % of cell length from the apical end.

normalized to nuclear length, no significant differencebetween the two division types is seen. Most prophasePPBs are relatively narrow, with widths that are 10—30 %of nuclear length. A few PPBs in symmetrically dividingcells are still relatively broad, however, with widthsgreater than 50 % of nuclear length, and some of these aredouble PPBs. These are rare in asymmetrical prophasecells, however.

PPB actinBecause F-actin-containing Mfs are labile structures,their preservation for fluorescence and electron mi-croscopy has been problematic, and various fixation,permeabilization and staining procedures have been usedto visualize them in plant cells (e.g. see Parthasarathyetal. 1985; Kakimoto and Shibaoka, 1987; Palevitz,1987a,6, 1988; Traas etal. 1987; Sonobe and Shibaoka,1989; McCurdy and Gunning, 1990). In our case, initialexperiments indicated that while thick actin cables canusually be found, thin cortical Mfs, and especially thoseassociated with the PPB, are not detectable in many cellswhen epidermal slices are treated by the method wenormally use to observe live cells (i.e. slices incubated inwater for l h to allow cytoplasmic streaming to recover)before staining with rhodamine-phalloidin. However,actin bands (Fig. 4) encompassing the PPB are detectablein most prophase cells (about 97 %) when cotyledons aretreated with MBS cross-linking agent, sliced directly inthe staining medium and the slices then mounted in thesame solution.

Prophase epidermal cells contain broad arrays oftransversely aligned cortical Mfs that stain with rhod-amine-phalloidin. In symmetrically dividing cells, thesebroad bands can occupy a third of the cell surface (Fig. 4A).The bands are typically wider relative to cell size inasymmetrically dividing cells, occupying greater than50 % of the cell surface, and as expected are concentratedin the apical half of the cell (Fig. 4C). The borders of the

bands are often difficult to define, since the orientation ofthe Mfs gradually shifts from transverse to randomelsewhere in the cortex (Fig. 4A,C). In addition, Mfdensity gradually decreases beyond the band. Despite thisdifficulty, certain band characteristics are obvious. Actinbands vary in width, from somewhat narrower arrays tothose covering nearly the entire cell surface. The lattercondition is not typical, however, and in most cells thetransverse Mfs are band-like. In no case is the Mf band asnarrow as the Mt PPB, although it clearly encompasses it.For example, the width of most prophase Mt PPBs is lessthan half of the length of the nucleus (Table 1), while theMf bands are wider than half of the nuclear length in 77 %of asymmetrically dividing prophase cells and 91% ofsymmetrically dividing cells. The band remains broadeven in later stages of prophase, when the Mt PPB isnarrowest. Indeed, 33 % of metaphase cells still have broadactin bands.

Transverse actin bands are also detectable in someinterphase cells, though they may be dimmer than thosefound in prophase. Such bands are not found in mostinterphase cells, although thick Mf cables runningthroughout the cytoplasm and around the nucleus(Fig. 5C), as well as a weakly staining cortical meshwork,can be detected (Fig. 5A,B). Cytoplasmic and perinuclearactin remains in place throughout prophase, includingbefore and after nuclear migration in asymmetricallydividing cells. Actin arrays specifically linking thenucleus to the PPB site (Lloyd and Traas, 1988) have notbeen seen in these epidermal cells, although Mts arepresent in this location (Fig. 2E-G).

Effect of CD on the PPBAllium seedlings were treated with 2-20 ftM CD forvarious times and the size, position and orientation of thePPB in symmetrical and asymmetrical prophase cellswere compared with the same parameters seen in controltissue exposed to water or dilute DMSO solutions. Table 1


Table 2. Effect of high concentrations of CD on thearrangement of microtubules in the cortex of prophase

epidermal cells

Fig. 4. Broad, transversely aligned cortical Mfs insymmetrically (A) and asymmetrically (C) dividing prophaseepidermal cells stained with rhodamine—phalloidin. In B, thenucleus of the cell seen in A is visualized with Hoechstfluorescence. X1850.

shows that PPB width dramatically increases in bothasymmetrical and symmetrical prophase cells exposed toCD. In the former, average PPB width increases twofold ata concentration of 2[IM compared to controls, while athreefold increase is seen when the drug concentration iselevated to 20/iM (Table 1; Fig. 6A). Similar increases areseen for symmetrical prophases (Table 1; Fig. 6B). Almostall prophase epidermal cells have broad PPBs aftertreatment with 20 fas. CD (Table 2), and more than 90 % ofthe PPBs in symmetrical cells are broad after thistreatment. No significant difference in PPB width is foundbetween asymmetrical and symmetrical cells at this CDconcentration, although such a difference is seen at 2 [IM(Table 1). The effect on PPB width is seen at CDconcentrations up to 200 ^M (Table 2).

Treatment

WaterDMSO (1 %)CD (20/(M)CD (100 JIM)CD(200/(M)

Cortical microtubule

Transverse PPB

Narrow

86+278±21±100

Broadb

14±322±299±199+1••.mm

arrangement

Other"

000

1±14±2

Two-day-old seedlings were incubated for 19 h with deionized water,1 % DMSO or various concentrations of CD, and the percentage ofprophase cells with narrow PPBs, broad PPBs or other types of corticalMt arrays was determined. The concentration of DMSO in 20, 100 or200 ^M CD was 0.1, 0.5 and 1 %, respectively. All values represent themean±s.E.M. obtained from three independent experiments. About 45cells were scored for each percentage value.

a Cortical MTs are located throughout the cell surface and/or invarious orientations and no distinct PPB is detectable.

b A broad PPB is denned as a band whose width is more than half ofnuclear length.

These results indicate that CD induces widening of theprophase PPB. This conclusion is reinforced when theratios of PPB width to nuclear length are compared indrug-treated and control cells. This ratio increases two- tothreefold with CD (Table 1). Cell and nuclear length arereduced after CD treatment in symmetrical cells, while nosignificant effect is found in asymmetrical cells (Table 1).

In order to ascertain how rapidly the drug exerts itseffects on the PPB, seedlings were exposed to 20 /.IM CD forvarious periods of time (Fig. 7). The results show that adramatic widening can be seen within a few min insymmetrical cells and reaches a maximum in 0.5 h(Fig. 7B). Asymmetrical cells seem to respond moreslowly; the increase in PPB width reaches a maximumafter 1 h of treatment (Fig. 7A).

We also determined whether CD affects the location ofthe PPB. Fig. 8 shows histogram presentations of thedistribution of the center of the PPB along the epidermalcell axis in water (Fig. 8A), DMSO (Fig. 8B), and CDsolutions (Fig. 8C,D). The data clearly show a decline inthe number of apically positioned bands relative to thoselocated centrally and subapically. No PPBs were found inthe apical-most segment of epidermal cells after CD, incontrast to the condition seen in controls. However, the CDeffect was not so strong as to produce PPBs in the basal-most regions of the cells. These data indicate that in apopulation of CD-treated epidermal cells, the center of thePPB is shifted to a more middle position along the lengthof the cell. However, many cells are still asymmetrical,even though the PPB is not as highly polarized (Fig. 6A).

Examination of CD-treated epidermal cells shows thatthe normal orientation of the PPB is unaffected by thedrug at concentrations of 2 or 20jUM (Fig. 6). However, wewere curious as to whether elevated levels would alter thisparameter. Although the PPB is abnormally broad afterCD treatment, it still consists of transverse MTs in mostcells, even at concentrations as high as 200 f<M (Table 2).While Mt alignment does change at the band periphery,similar deviations are seen in control cells. In a few cells,transverse to oblique Mts are distributed more uniformlyalong the cortex (Fig. 9A).

Effect of CD on the division planeAsymmetrical division in the epidermis of Allium cotyle-


Fig. 5. Mfs in an interphase epidermal cell viewed in three focal planes, n, nucleus, x 1850.

dons produces long epidermal cells and apically positionedGMCs (Biinning, 1958; Palevitz and Hepler, 1974a;Mineyuki etal. 1989). The GMCs then grow into arectangular shape before they divide. Approximately 15 %of the cells in the portion of the cotyledon used here areGMCs, when both water and DMSO-treated controlseedlings are examined. In the presence of 20 ^M CD,however, the disparity between small and large cellsdeclines (Table 3). The percentage of GMC-like cells in theepidermis is reduced, and the short cells that remain arelargely restricted to a small part of the cotyledon near thehook (which is also the oldest part of this section of theorgan and therefore probably contains cells producedbefore treatment). In other words, new walls appear to beshifted to more central regions of the cell under theinfluence of CD. The walls remain transverse, however.

To probe this effect further, we ascertained whether theposition of the mitotic apparatus is affected by CD. Themajority of prometaphase, metaphase and anaphasechromosomes in the epidermis of water and DMSO-treatedcotyledons are located in either the central or apicalsegments of the cell, thus reflecting the dichotomybetween symmetrical and asymmetrical divisions

Table 3. Effect of CD on the formation of GMC-likecells in the epidermis o/"Allium cotyledons

Treatment GMC-like cells (%)

WaterDMSO(0.5%)CD(20UM)

15±115+35±1

Two-day-old seedlings were incubated for 19 h with distilled water,DMSO or CD and the percentage of GMC-like cells in the cotyledonepidermis was determined. A GMC-like cell is denned as a smalltrapezoid to rectangular cell whose height is less than its width, andwhich is accompanied by a large, basal epidermal cell. Each % is basedon the examination of 500-1100 cells in five slices. All values representthe mean±s.E.M. Student's (-test was used to compare differences inaverage % between CD and the deionized water control. ** Significantlydifferent from water control (P-cO.005).

(Fig. 10A,B). A few spindles are found in subapical andsubcentral locations. No cell divisions are seen in thebasal-most regions. As the concentration of CD increases,the population of highly polarized spindles decreases,while the percentage of cells with subapical spindlesincreases (it doubles in Fig. 10C,D). These data show that


15

Fig. 6. PPBs in asymmetrical (A) and symmetrical (B)divisions in 3-day-old cotyledons treated with 20 (M CD for19 h. Hoechst-stained images in C and D are complementary toA and B, respectively. Note that the PPBs are still broad,despite the fact that the cells are in prophase, as judged fromHoechst staining. X1850.

CD affects the position of the mitotic apparatus duringasymmetrical division.

To probe this effect still further, we asked whetherpremitotic nuclear migration is disrupted by CD. In waterand DMSO controls (Fig. 11A,B), half the prophase nucleiin the epidermal cell population are centrally located,while the other half are asymmetrically placed. Of thelatter, a quarter are apical and another quarter aresubapical. Very few are subcentral and none are basal.Most prophase nuclei in subapical regions and a few ofthose in the central part of the cell are accompanied by anapically placed, narrow PPB, indicating that nuclearmigration was in progress during prophase in these cases.

CD

DMSO

H,0

CD

DMSO __..—-jji

I I H2O

Time (h)

Fig. 7. Time course of the effect of CD on widening of the PPBin asymmetrical (A) and symmetrical (B) prophase epidermalcells. CD, 20 (JM; controls, deionized water and 0.5 % DMSO.

Mts link the perinuclear region and the PPB in such cells(Fig. 2E-G). In 2 or 20 (IM CD, most of the prophase nucleiare located in the central part of the cell, and thepercentage of apically placed nuclei is dramaticallyreduced (Fig. 11C,D). Thus, nuclear migration precedingasymmetrical division is inhibited by CD.

Effect of CD on MfsIn order to assess the effect of CD on actin-containing Mfs,drug-treated epidermal cells were stained with rhoda-mine-phalloidin. Mfs that ramify throughout the cyto-plasm are fragmented by 2 jUM CD, and many of these seemto cluster at the spindle poles (Fig. 9B). However, broadband-like arrays of transversely oriented Mfs remain inthe cortex of prophase cells in the presence of the drug.Most are still present in 20 (IM CD as well (Table 4), andmore than half of the prophase cells have transverse actinbands even at 200 ^M (Fig. 9C; Table 4).

Mt disruption and the Mf bandThe effect of Mt disruption on the Mf band was alsoascertained. In treatments with colchicine at 1 to 10 mMfor 2 h, exposures that are sufficient to destroy most or allepidermal cell Mts, respectively, most of the prophase cellshave no detectable Mf band (data not shown). Only 16 % ofthe cells have Mf bands at 1 mM, while 8 % have them at10 mM.

Discussion

Our results show that CD has a dramatic effect on the PPBin dividing epidermal cells. Whereas the band starts out asa broad Mt array that progressively narrows duringprophase in control material, it remains broad in thepresence of the drug. The effect is seen in both symmetri-cally and asymmetrically dividing cells. In the latter,


S3 5ft-

T

B

T

T_

c 0

Position along cell

Fig. 8. Positions of PPBs in epidermalcells of cotyledons treated withdeionized water (A), 0.5% DMSO (B),2;IM CD (C) and 20;IM CD (D) for 19 h.The position of the center of the PPBin dividing cells was ascertainedaccording to the criteria set forth inMaterials and methods and Fig. 1. Theordinate shows the average percentageof cells obtained from threeindependent experiments. Positionalong the cell is displayed on theabscissa. For reference sake, b refers tothe basal end, c the center and a theapical end of the cell. The arrow refersto the position 30 % down the length ofthe cell from the apical end. Thelongitudinal bars show standard errorsof the mean.

however, the position of the center of the band is alsoaltered, perhaps as a consequence of the fact that beingasymmetrically disposed, it takes up more room in thebasal direction as it widens. In addition to its effect on thePPB, CD also alters the position of new cell walls producedin the epidermis. The frequency of apical cell plates

declines in favor of those more centrally located, withfewer GMC-like cells produced as a result.

A wealth of data have implicated the PPB in denning ordetermining the site at which the cell plate fuses with theparental plasmalemma during plant cytokinesis (Wadaetal. 1980; Gunning, 1982; Gunning and Wick, 1985;

Fig. 9. (A) Aberrant cortical Mts in aprophase epidermal cell of a 3-day-oldseedling treated with 200 [IM CD for19 h. It is difficult to recognize adistinct, normal PPB. X1850.(B,C) Rhodamine-phalloidin staining ofsymmetrically dividing epidermal cellsin 3-day-old cotyledons treated with2 HM (B) or 200 /m (C) CD for 19 h. Thecells are in prophase as determined bycoordinate Hoechst staining (notshown). The cells still have intactcortical transverse Mfs even at highCD levels (C), while Mfs deeper in thecell are fragmented (B). Many of thefragments accumulate at the spindlepoles, n, nucleus. X1850.


100 r A

50

lOOi- B

50

0

lOOrC

50

b

100r D

50

0a b c a

Position along cell

Fig. 10. The effect of CD on the position of the mitoticapparatus in epidermal cell populations was determinedaccording to the criteria set forth in Materials and methods. Inthis case, however, spindles were grouped into 5 categories:basal (b), subcentral, central (c), subapical and apical (a). Thedata were obtained from three experiments. The longitudinalbars show standard errors of the mean. (A) Deionized water;(B) 0.5% DMSO; (C) 2;<M CD; (D) 20;JM CD.

100 r A

50

2? 0

100 r B

50

0

& 100rC

50

b

100 rD

50

a bPosition along cell

Fig. 11. Effect of CD on the position of prophase nuclei inepidermal cell populations. Details as in Fig. 10.

Palevitz, 1986). The present results support and extendthis concept. Our data show that while the PPB remainsbroad under the influence of CD, its normal transverseorientation is unaltered. However, the final position of thecell plate along the parental cell is abnormal. Preliminaryresults of observations on GMCs, in which the divisionplane is normally orientated 90° to those elsewhere in theepidermis, are also consistent with these findings. Specifi-cally, although a broad PPB is maintained in CD-treatedcells, it still has the expected longitudinal alignment (Y.Mineyuki and B. A. Palevitz, unpublished results). Thesedata support the hypothesis (Mineyuki etal. 1989) that

Table 4. Effect of high concentrations of CD on thepresence of cortical transverse Mfs in prophase epidermal

cells of Allium cotyledonsTransverse actin filaments

Treatment Detectable Not detectable

WaterDMSO (1 %)CD (20 tat)CD (100/uw)CD (200 MM)

97±3100

80±266+1859+12

3±30

20±234±1842±12

Two-day-old seedlings were incubated for 19 h with deionized water,1 % DMSO or various concentrations of CD, and the percentage ofprophase cells with a cortical transverse Mf band was determined. Theconcentration of DMSO in 20, 100 and 200//M CD was 0.1, 0.5 and 1%,respectively. All values represent the meants.E.M. obtained from threeindependent experiments. About 30 cells were examined for eachtreatment in each independent experiment.

division plane determination consists of two elementaryprocesses: (1) formation of a broad PPB associated withfixation of division plane orientation, and (2) narrowing ofthe PPB, which determines the exact position at which thecell plate will fuse with the parental wall. Cytochalasin-sensitive elements do not seem to be involved in the firstprocess, but do appear to regulate the second.

Cytochalasin D modifies asymmetrical divisions inanimals (Strome and Wood, 1983) as well as Allium (theseresults). Moreover, effects of cytochalasins on otheraspects of asymmetry and cell polarity in plants have beenreported (e.g. see Quatrano, 1973; Brawley and Robinson,1985). While cytochalasins have been found to alter cellplate formation in several plant cell types (Palevitz andHepler, 19746; Palevitz, 1980, 1986; Gunning and Wick,1985; Y. Mineyuki and B. E. S. Gunning, unpublisheddata), the effects vary from those seen here. In particular,the other anomalies are only inconsistently observed, withcytokinesis quite normal in some cells while incomplete orabnormally curved plates are found in others. In thesecases, instead of blocking PPB maturation, the drugs maybe influencing the actin-containing phragmoplast or itsinteraction with the PPB site during cytokinesis.

It is now known that the PPB contains F-actin(Kakimoto and Shibaoka, 1987; Palevitz, 1987a; Traasetal. 1987). While original studies in our laboratoryindicated that actin could be seen in the PPBs of 70 % ofAllium root cells (Palevitz, 1987a), the present resultsusing improved fixation and staining methods show that100 % of cotyledon epidermal cells have actin bands byprophase (Table 4). In addition, Mf bands are seen in athird of the metaphase cells. Mf bands at metaphase havealso been reported in carrot suspension cells (Traas et al.1987). Although these early studies reported Mfs in thePPB, two recent papers maintain that while Mfs parallelto the PPB appear just before division, they become spreadout along the entire length of the cell (McCurdy et al. 1988;McCurdy and Gunning, 1990). On the basis of micrographsprovided here, a prophase distribution between theseextremes seems likely: that is, the Mfs are distinctly band-like in arrangement, although the band is broader thanthe confines of the prophase PPB Mts.

Our results clearly implicate actin-containing Mfs inPPB organization and/or function. Such a role would notbe surprising, since Mf-Mt interactions are known orsurmised in a variety of cells (e.g. see Menzel and Schliwa,1986; Forscher and Smith, 1988; Kobayashi et al. 1988). Inthe flagellated stage of Physarum, a Mf-Mt complex


exhibits ATP-induced sliding (Uyeda and Furuya, 1987).On the basis of our own data, it is unlikely that the actinband is responsible for the formation of the initial, broadMt PPB. Indeed, the reverse may be true, since resultswith anti-Mt agents reported here and elsewhere (Pale-vitz, 1987a) seem to show that Mts are required for theorganization of the PPB Mfs. Our results do indicate thatactin plays an active role in the subsequent narrowing ofthe PPB. It may also maintain the PPB in the narrowstate, since widening of the band is quite rapid (it isdetectable only several minutes after the onset of CDtreatment and reaches a maximum at 0.5 h in symmetri-cally dividing cells), indicating that previously narrowedbands rewiden.

Unfortunately, interpretation of the CD effects relativeto the role of actin in PPB organization is made moredifficult by the fact that the actin band is not visiblyaltered by low levels of the drug in most Allium epidermalcells, although the frequency of actin bands markedlydecreases as the concentration increases. The percentageof cells with actin bands declines to 59 % at 200 JAM, whilethe fraction of cells with a broad PPB remains at morethan 95 %. Nevertheless, it is noteworthy that the majorityof prophase cells retain cortical actin bands in the presenceof CD. Resistance of cortical actin bands to CD has alsobeen reported in carrot suspension cells by Lloyd andTraas (1988). It is possible that CD can inhibit the functionof the Mf band (and affect PPB width) at low concen-trations without altering its organization. Disruption ofthe actin band may be forestalled by enhanced stabilityimposed through close Mf-Mt associations in the PPBregion, perhaps mediated by bridging moieties such as Mt-associated proteins (Griffith and Pollard, 1978; Sattilaroet al. 1981; Cyr and Palevitz, 1989). It is also possible thatthe Mf band exerts an effect on Mts via short, CD-sensitiveactin chains (Morris and Lasek, 1984; Fath and Lasek,1988) that are difficult to visualize in control cells.Alternatively, the actin band may be unimportant in PPBorganization, and attention should be directed insteadtoward CD-sensitive elements elsewhere in the cell, suchas those associated with the nucleus.

Further complications in the interpretation of drugeffects arise from discrepancies between various reports.The present results and those of Lloyd and Traas (1988)indicate that PPB actin filaments remain after cytochal-asin treatment, while Palevitz (1987a) and McCurdy andGunning (1990) maintain that they disappear. Our data(Palevitz, 1987a, and present results) show that thecortical Mfs are removed by colchicine treatment, butMcCurdy and Gunning (1990) report that they remain inthe presence of another Mt drug, oryzalin. It is impossibleto account for these and other reported differences in actinlocalization and behavior at this point. They could be dueto the use of different probes (rhodamine-phalloidinversus antibodies), fixation/permeabilization techniquesor cell types. For example, various plant actins may varyat their phalloidin binding sites (Meagher, 1990). Alterna-tively, rhodamine—phalloidin and anti-actins may differin their ability to stain F-actin after cytochalasintreatment (Tang et al. 1989). Recent experiments haveemployed rhodamine-phalloidin staining of nonfixed,permeabilized cells (Traas etal. 1987; Lloyd and Traas,1988), while other studies made use of various fixationprotocols (e.g. see Parthasarathy et al. 1985; Kakimoto andShibaoka, 1987; Palevitz, 1987a,6, 1988; Sonobe andShibaoka, 1989; McCurdy and Gunning, 1990). The workdescribed here used MBS (Sonobe and Shibaoka, 1989)

cross-linking in conjunction with permeabilization indetergent and DMSO. It is possible that cells treated inthese various ways respond differently to rhodamine-phalloidin. For example, when introduced into unfixedcells, the agent may induce artifactual actin polymeriz-ation or aggregation. Spurious formation of Mf bundles isseen after long exposure to low doses microinjected intoendosperm cells (Schmit and Lambert, 1990).

Migration of nuclei to specific division sites is a commonphenomenon in plant cells (Sinnott and Bloch, 1941;Bunning, 1958; Mineyuki and Furuya, 1980). In theepidermis of Allium, data obtained here and elsewhere(Bunning and Biegert, 1953) suggest that this processtakes place during prophase. The fact that CD decreasesthe number of distally located prophase nuclei and mitoticspindles in favor of those more centrally positioned showsthat nuclear migration is inhibited by this agent. Earlierobservations indicated that cytochalasin B also has suchan effect (B. A. Palevitz, unpublished results). Further-more, nuclear migrations in other systems are inhibitedby cytochalasins as well (e.g. see Schnepf and vonTraitteur, 1973). The mechanisms responsible for nuclearmotion remain unknown, but the involvement of an actin-based system seems likely. For example, actin has beenimplicated in positioning of the nucleus in tobaccosuspension cells (Katsuta and Shibaoka, 1988). A commonfeature of many of the actin localizations to date is thepresence of Mfs ensheathing the nucleus and extendinginto the cytoplasm (Traas etal. 1987; Kakimoto andShibaoka, 1987; Palevitz, 19876, 1988). Such nucleus-Mfassociations are seen in Allium epidermal cells throughprophase, when a layer of Mts also surrounds the nucleus.It would thus appear that Mfs are in the right position toimpel nuclear migration. Alternatively, cytoskeletal el-ements linking the nucleus to the PPB may be important.In this case, the inhibition of nuclear migration in theAllium epidermis might be related to the inhibition of PPBnarrowing. We routinely observe Mts extending betweenthe PPB and nucleus in prophase (Fig. 2), and Mfs havebeen reported in this location in another system (althoughwe have not seen them in epidermal cells; Lloyd andTraas, 1987). A role for the PPB in nuclear migration wasquestioned a long time ago because movement in somecases can occur before the PPB appears (Pickett-Heaps,1969a). However, the electron-microscopic proceduresused at the time could have missed early stages when theband is quite broad.

It is possible that the effects of CD on the PPB andnuclear migration are independent processes. Gunninget al. (1978) have argued that polarization of the cell is thefundamental process behind division plane determination,of which the PPB is just one manifestation. Whether thePPB appears before or after nuclear migration would be oflittle consequence. Further, it is this earlier, basic processthat may be sensitive to CD, and both nuclear migrationand PPB narrowing would be altered as a result. Evidencein favor of this hypothesis comes from experiments withalgal eggs, in which cytochalasins block changes in thepattern of ion movements leading to cell polarization andasymmetrical division (Brawley and Robinson, 1985; alsosee Kropf et al. 1989). Hopefully, further experiments willclarify some of these issues in the near future.

We thank Ms Christine Brushwood for printing the micro-graphs and Ms Carol Hahn for preparing the final versions ofPigs 1 and 11. We also gratefully thank Drs Jan Marc and RichardJ. Cyr for many helpful discussions and suggestions. This


research was supported by funds from the National ScienceFoundation (DCB-8703292) and the University of GeorgiaResearch Foundation Inc.

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(Received 20 February 1990 - Accepted, in revised form, 28 June 1990)


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