The Effect of Cytochalasin D on Preprophase Band Organization in Root Tip Cells of Allium

Journal of Cell Science 103, 989-998 (1992) 989 Printed in Great Britain © The Company of Biologists Limited 1992

The effect of cytochalasin D on preprophase band organization in root tip cells of Allium

E. P. ELEFTHERIOU* and B. A. PALEVITZ†

Department of Botany, University of Georgia, Athens, GA 30602, USA *Permanent address: Department of Botany, School of Biology, University of Thessaloniki, GR-54006 Thessaloniki, Greece †Author for correspondence

Summary

The relationship between microfilaments (Mfs) and tion dependent, and the effect of 10 M CD is near max- microtubules (Mts) in the organization of the pre- imal only 15 min after application of the drug. This prophase band (PPB) was investigated in Allium root tip rapid response suggests that a rebroadening of already cells subjected to treatment with cytochalasin D (CD). condensed PPBs takes place. After as little as 15 min in Mts and Mfs were visualized by indirect immunofluo- CD, Mfs are replaced by many small specks and rods. rescence and various parameters such as PPB width Dual localizations of both Mts and Mfs show that were analyzed quantitatively. In control samples, the prophase cells contain broad PPBs without Mfs. The PPB first appears as a wide Mt band that progressively rapid disorganization of Mfs, by CD, therefore coincides narrows to an average width of 4 m in mid-prophase. with the rebroadening of PPBs. CD-treated cells in Randomly oriented Mfs are present throughout the metaphase, anaphase and telophase contain larger actin cytoplasm of most interphase control cells. Preprophase aggregates at the poles, as previously reported. The and prophase cells, however, contain cortical Mfs results indicate that Mfs play an important role in the arranged parallel to the PPB. The Mfs initially occupy narrowing of the PPB, which in turn is essential for much of the cortex but in most cells they progressively determination of the exact position of the plane of divi- become restricted to an area wider than the PPB. In the sion. They also indicate that movement of intact Mts is presence of CD, the PPB fails to narrow and remains important in PPB organization. at least two-fold wider than in control cells. PPB width expressed as a percentage of nuclear or cell length also Key words: Allium, actin, cytochalasin, division plane, increases compared to controls. Widening is concentra- microtubule, mitosis, preprophase band.

Introduction PPB site. In highly vacuolate cells, this plane is often occupied by a distinct plate of cytoplasm, termed the phragmo- Determination of the division plane is of critical importance some, which contains additional Mts and Mfs (Lloyd, in growth, differentiation and morphogenesis in plants 1991). Furthermore, Mts link the PPB to the new bipolar (Gunning, 1982). However, despite considerable progress spindle that ensheaths the nucleus in prophase (Mineyuki in identifying cell structures that may participate in divi- et al. 1991; Wick, 1991). While the long axis of the sion plane control, our understanding of the process remains spindle is usually aligned perpendicular to the PPB plane, incomplete. The presence of a specific cortical division site it can also assume more oblique configurations, a con- at which the phragmoplast/cell plate meets the parental dition that is corrected in anaphase or telophase via plasma membrane (PM) at telophase is made manifest in reorientation movements (Palevitz, 1986; Mineyuki et al. most tissue cells before division begins in the form of the 1988a,b). preprophase band (PPB), an array of microtubules (Mts), Definition of the division site appears to involve at least microfilaments (Mfs) and associated proteins that appears two phases (Mineyuki and Palevitz, 1990). First, the appear- in the G2 phase of the cell cycle, reaches its greatest promi- ance of an initial, broad PPB establishes the orientation of nence in prophase, and gradually erodes in late prophase- the future cell plate and its approximate location in the early prometaphase (Gunning, 1982; Wick, 1991; also see cortex. In phase two, progressive narrowing of the band Eleftheriou, 1985; Gunning and Sammut, 1990). The exact precisely marks the position at which the phragmoplast will timing of these events may vary between species and cell meet the cortex/PM in telophase. Various proposals have type. In most situations, the premitotic nucleus or spindle been put forward to explain how the phragmoplast “sees” eventually comes to lie in the plane circumscribed by the and “pursues” the cortical zone once occupied by the PPB. 990 E. P. Eleftheriou and B. A. Palevitz

These include interactions between phragmoplast Mts and Because the potential link between PPB organization, residual elements in the PPB zone (Palevitz, 1986) and actin and the division plane is so important, we have rein- guidance by Mfs extending from the phragmoplast to the vestigated the effect of CD on Allium root cells. Our goal PM in the plane of the phragmosome (Lloyd, 1991). was to assess whether CD induces widening of the PPB in Recently, another hypothesis was put forward: the PPB gov- this material as it does in cotyledons, and if so, whether erns the deposition of factors in the parental wall which sta- Mfs remain in the PPB in the presence of the drug. Instead bilize or harden the new cell plate (Mineyuki and Gunning, of using rhodamine phalloidin to detect Mfs, as was done 1990). in our original paper, antiactin immunocytochemistry was Cytochalasins are useful tools for probing the role of Mfs employed. A separate paper documents in greater detail the in eukaryotic cells (Cooper, 1987; Staiger and Schliwa, behavior of cortical Mfs during mitosis (Liu and Palevitz, 1987; Seagull, 1989), and treatment with these agents leads 1992). to a number of effects relevant to division plane determination in plants. For example, cytochalasins B and D (CB, CD) inhibit or retard certain nuclear migrations, including those involved in asymmetrical divisions that generate Materials and methods guard mother cells (GMCs) in the cotyledons of Allium (Mineyuki and Palevitz, 1990). When those same GMCs Drug treatments divide, the drugs inhibit telophase reorientation of the ini- Seeds of Allium cepa L. cv White Portugal (Joseph Harris Seed tially oblique phragmoplast (Palevitz and Hepler, 1974; Co., Rochester, NY) were sown in moist vermiculite at room tem- Palevitz, 1986; Cho and Wick, 1990). In the former case, perature (RT; 21-23°C). Intact seedlings 5-6 days old were trans- the cell plate formed under drug treatment is more proxi- ferred to glass vials containing 2 ml of control or CD solution. mally positioned in the parental protodermal cell than Only the roots were immersed in the solutions. Cytochalasin D normal, and the resulting GMC is unusually long (Mineyuki was obtained from Sigma Chemical Co. (St. Louis, MO) and and Palevitz, 1990). While broad, properly oriented PPBs working solutions were prepared by dilution with deionized water are formed in the presence of CD, they fail to narrow. More- of a 2 mM stock prepared in 100% DMSO. All working solutions over, previously narrowed bands appear to rewiden during contained 0.5% DMSO, a level that does not appear to affect the treatment. This effect on the PPB is not limited to asym- cytoskeleton of plant cells (e.g. Mineyuki and Palevitz, 1990). Nevertheless, drug-treated seedlings were compared to controls metrically dividing protodermal cells; it also occurs during incubated in either deionized water or 0.5% DMSO. equal divisions. Two sets of experiments were conducted. In one, seedlings were These results directly tie PPB organization to the final exposed to a series of concentrations of CD (1, 2, 4 or 10 mM) position of the future cell plate and appear to implicate F- for 2 h at RT in the dark. The second set constituted a time-course actin in the control of PPB development. The link seems experiment in which seedlings were treated with 10 mM CD for even firmer, given that in many cells the PPB contains and 0.25, 0.5, 1, 2 or 4 h under the same conditions. is encompassed by parallel cortical Mfs (Kakimoto and Shibaoka, 1987; Palevitz, 1987; Traas et al. 1987; Ding et Immunofluorescence localizations al. 1991; but also see Cho and Wick, 1990). Nevertheless, Microtubules the conclusions remain tentative because of conflicting Root tips, 1 mm in length, were excised in a drop of fixative using observations in the literature. For example, Mineyuki and a No. 11 scalpel blade and then transferred to vials containing Palevitz (1990) found that although the number of Allium fresh fixative. Fixation continued for 1.5 h at RT. The fixative cotyledon cells with cortical Mfs was reduced with CD, consisted of 4% formaldehyde (freshly prepared from most seemed to retain them. This finding is in accord with paraformaldehyde) in a Mt stabilization buffer (MSB) containing the results of Lloyd and Traas (1988), who maintained that 50 mM PIPES, pH 6.8, 1 mM MgSO4 and 5 mM EGTA. The PPB actin is resistant to CD treatment in cultured carrot root tips were then rinsed in MSB for at least 45 min (3-4 cells (although for somewhat different results, see Traas et changes), digested in 1% Cellulysin (Calbiochem-Behring, La al. 1987). Such resistance would not be surprising, if sta- Jolla, CA) in MSB for 15 min and rinsed again in MSB for 45 bilizing interactions occur between Mfs and Mts. However, min. The tips were then squashed between a 22-mm square cov- the original report on PPB actin (Palevitz, 1987) maintained erslip and a microscope slide subbed with chrom-alum and gelatin, large tissue pieces were removed, and the dispersed cells were air- that CD leads to the loss of these same Mfs in Allium root dried for several hours at RT or, for longer periods, in a cold tip cells, a finding that has since been confirmed in Triticum room. The cells were permeabilized in 0.5% Triton X-100 in MSB roots by McCurdy and Gunning (1990). Katsuta et al. for 15 min and then immersed in cold methanol (- 20oC) for 10 (1990) took an intermediate position based on experiments min. The slides were rinsed in phosphate-buffered saline (PBS) with tobacco suspension cells: they reported that short Mfs for at least 15 min at RT, after which the cells were incubated for are present in the PPB after CD treatment. Thus, the results 1 h at RT with a rabbit polyclonal antibody raised against Hibis - are mixed: two reports indicate that CD leads to the loss of cus tubulin (courtesy of Richard J. Cyr) diluted 800-fold in PBS PPB Mfs, two others maintain that it has little effect, and containing 1% bovine serum albumin (BSA: Sigma). Following a a fifth is less certain. Only Mineyuki and Palevitz (1990) 15 min rinse in PBS, the cells were incubated in a fluorescein mentioned any effect of cytochalasin on the final dimen- isothiocyanate (FITC)-conjugated goat anti-rabbit second antibody (Sigma) diluted 50-fold in PBS plus BSA for 1 h. After a 15-min sions of the PPB. Interestingly, the seemingly conflicting wash, the cells were finally mounted in a medium consisting of reports of Palevitz (1987) and Mineyuki and Palevitz (1990) 0.1 M Tris buffer, pH 9.2, containing 50% glycerol, 0.1 mg/ml were obtained with the same species, Allium cepa, albeit Hoechst 33258 (Sigma) to stain DNA and 1 mg/ml of the antifade with different organs. agent p-phenylenediamine (Sigma). Effect of cytochalasin D on preprophase band organization 991

Actin Results For actin localizations or actin/tubulin dual localizations, a pro- cedure similar to that described above for MTs was applied, with PPB development in control samples the following modifications. The fixative consisted of 3% In order to describe adequately the effects of CD, it is nec- paraformaldehyde, 0.1% glutaraldehyde, 2% glycerol and 2% essary to review PPB organization and development in con- DMSO prepared in MSB, pH 6.8. Fixation lasted for 1 h at RT. trol cells. The treatment here will be brief, however, Enzyme digestion was carried out in a medium consisting of 1% Cellulysin and 1% BSA in MSB, with the addition of protease because more extended coverage can be found elsewhere inhibitors (80 mg/ml phenylmethylsulphonyl fluoride (PMSF), 40 (Gunning, 1982; McCurdy and Gunning, 1990; Mineyuki mg/ml leupeptin, 5 mg/ml aprotinin and 5 mg/ml pepstatin). and Palevitz, 1990; Wick, 1991; Liu and Palevitz, 1992). Extended exposures to Triton of up to 12 h were employed in The majority of interphase cells contain a dense array of some cases. After methanol treatment and before incubation in cortical Mts oriented transverse to the long axis of the cell primary antibody, the cells were treated for up to 45 min at and spread over the entire cell periphery (Fig. 1). By pre- RT with 0.1 M glycine in 0.5% BSA and 0.1% Triton X-100. prophase, marked by the first signs of chromatin conden- Alternatively, slides were incubated in 1% NaBH4 in PBS for 10 sation in the nucleus (Fig. 2C), the cortical array appears or 20 min. The primary antibody consisted of C4 monoclonal to be rearranged into a broad band that encircles the antiactin (Lessard, 1988; ICN, Costa Mesa, CA) diluted 400-fold nucleus. In mid-optical sections, the PPB often occupies in PBS plus BSA. The secondary antibody was Texas Red- conjugated sheep anti-mouse IgG (Amersham, Arlington Heights, much of the lateral walls (Fig. 2A). At this stage, the fib- IL) diluted 100-fold in the same medium. For actin/tubulin rillar nature of the bands is clearly evident in tangential sur- dual localizations, the primary antibody solution also contained face views (Fig. 2B). Mts are also evident outside the con- anti-tubulin (final dilution 1200ϫ), while FITC goat anti-rabbit fines of the band, while weak anti-tubulin fluorescence can was added to the second antibody solution at a dilution of be detected at the nuclear periphery (Fig. 2A,B). 100ϫ. The PPB gradually narrows by prophase, until it loses its fibrillar appearance due to the close packing of individual Microscopy Mts (Fig. 3A,B). Moreover, cortical fluorescence is now All preparations were examined with a Zeiss Axioskop micro- restricted to the PPB, as seen near mid-depth planes of focus scope (Carl Zeiss, Thornwood, NY) equipped for epifluorescence (Fig. 3A). Perinuclear Mt fluorescence is much brighter at illumination using Neofluar objectives. Photomicrographs were this stage (Fig. 3A), apparently due to increased numbers taken on a Nikon UFX-II exposure system (Nikon Instruments, of perinuclear Mts, many of which show bipolar alignment Garden City, NY) using Tri-X film (Eastman Kodak, Rochester, (Fig. 3B). Hoechst fluorescence shows that chromatin con- NY) and developed in HC-110 (Kodak). densation is advanced (compare Figs 1C, 2C and 3C). Quantitative cytology In order to assess the effects of increasing CD concentration and Distribution of Mfs in the cortex exposure time on PPB organization and cell morphology, a quan- In order to study the distribution of Mfs, actin was local- titative cytological analysis was undertaken. PPB width, cell ized immunocytochemically in fixed root tip cells using the length and nuclear length were measured directly on the micro- C4 monoclonal antibody. Both individual localizations of scope in mid-optical sections using the 100ϫ objective and an actin, and dual localizations of actin and tubulin were per- eyepiece micrometer (Mineyuki and Palevitz, 1990). The position of the PPB was specified by measuring the distance between one formed. Only results from the latter are presented because end wall and the center of PPB (as determined from its width). they afforded direct comparisons between Mts and Mfs. All measurements were carried out in the direction perpendicular However, Mf distributions were cross-checked in cells sub- to the plane of the PPB. In order to standardize our observations, jected to single localizations in which stages of the cell only cells at prophase were counted. Organization of the PPB cycle were again determined with Hoechst fluorescence. reaches its zenith (i.e. brightest signal and narrowest dimensions) Most interphase root cells that contain cortical arrays of at this stage (refer to Fig. 3A,B). Prophase was defined by two Mts also contain a network of fine Mfs or Mf bundles (Fig. principal criteria: the presence of bright perinuclear anti-tubulin 4A,B) similar to those reported elsewhere (e.g. Clayton and fluorescence and the existence of distinct chromosomes within the Lloyd, 1985; Parthasarathy et al. 1985; Palevitz, 1988; Cho nucleus, as judged by Hoechst staining (see Figs 3, 5). In addition, and Wick, 1990, 1991; McCurdy and Gunning, 1990). The the nucleus was often spindle shaped. Because the squashing tech- nique used in this experiment releases a great number of meris- Mfs traverse all regions of the cytoplasm from the cell tematic cells, which vary considerably in size and shape, care was periphery to the nuclear surface, where they form a exercised to measure as many cells as possible from each prepa- more concentrated layer. In many elongated cells, the ration. The mean value for each parameter and the standard devi- Mfs are also arrayed in thicker, bright bundles primarily ation of the mean were calculated. Student’s t-test was used to oriented parallel to the long axis of the cell (data not compare differences in the averages of PPB width, cell length, shown). nuclear length, and the percentages of PPB width/cell length and Cells with relatively broad PPBs (Fig. 5A) contain Mfs PPB width/nuclear length between CD treatments and the con- arranged parallel to the Mts and occupying much of the trols. cortex (Fig. 5B). As the PPB narrows (Figs 3, 6), the dimen- Other parameters, such as the number of cells with split PPBs sions of the Mf array varies. In some cells, the area cov- and those with PPBs of nonuniform or uneven width were also examined. Nonuniform PPBs included those whose width in two ered by Mfs is still broad (Fig. 7), while in most the actin optical sections differed by more than 1.5 mm. Depending on their array has narrowed, although it never matches the width of shape, all cells were characterized as isodiametric or elongate, and the Mt band (Fig. 6; see also Liu and Palevitz, 1992). In the nuclei as oval or round. cells with a very narrow PPB, the Mfs begin to bracket the 992 E. P. Eleftheriou and B. A. Palevitz

Figs 1-3. Tubulin immunofluorescence images of control root tip cells of Allium cepa treated with 0.5% DMSO. Fig. 1. Mid-focal plane image (A) of an interphase cell showing Mt fluorescence at the cell periphery. In tangential optical section (B), the dense array of cortical Mts is oriented mainly transverse to the long axis of the cell. Mts occupy the entire cortex. Note that varying degrees of particulate staining with anti-tubulin, deeper in the cytoplasm, were encountered, in part depending on the antibody in use. Hoechst fluoresence of the nucleus is seen in C. nu, nucleolus. A and B, ´ 1300; C, ´ 1450. Fig. 2. A preprophase cell at mid-plane focus (A). The PPB occupies much of the lateral walls, while weak fluorescence can be seen at the nuclear periphery. (B) Tangential focus to visualize the fibrillar nature of the broad PPB. Additional Mts are seen away from the main band. Hoechst staining in C shows an early stage of chromatin condensation. ´ 1300. Fig. 3. A prophase cell with a mature PPB. A mid-optical section in A shows strong cortical fluorescence restricted to the PPB, and bright perinuclear fluorescence. A tangential view of the same cell in B shows the narrow, evenly bright PPB and numerous perinuclear Mts, many of which display bipolar alignment. The oval prophase nucleus with condensed chromatin is seen in C. ´ 1300.

band, with no Mfs actually coincident with it (Fig. 6B). normal for this stage. In addition, when focusing on their Few if any Mfs are encountered in the inner cytoplasm by tangential surface, the PPBs are also distinctly fibrillar (Figs this stage (data not shown). 9B, 10B) compared to the compact organization seen in control prophase cells (Fig. 3B), and Mts at both edges of Effects of CD on PPB organization the bands intermingle with other elements (Figs 9B, 10B). Qualitative observations Although the PPBs are at first glance similar to those seen The effects of CD on PPB organization are presented in at an earlier stage of organization in control cells, close Figs 8-10. Prophase cells treated for as little as 15 min with examination reveals that perinuclear fluorescence is strong 10 mM CD contain PPBs encompassing the centrally (compare Figs 2A and 8A) and chromatin condensation is located nuclei (Fig. 8). However, the PPBs are broader than advanced (compare Figs 2C and 8B). These characteristics Effect of cytochalasin D on preprophase band organization 993

Figs 4-7. Dual localizations of tubulin (4A, 5A, 6A) and actin (4B, 5B, 6B) in control cells. Corresponding nuclei stained with Hoechst are seen in 4C and 5C. Fig. 4. Interphase cell with dense, cortical array of Mts spread over the entire cortex (A; part of the cortex is out of focus). Actin Mfs in the cortex are randomly oriented (B). ϫ1350. Fig. 5. A preprophase-prophase cell with a broad PPB (A). Fine Mfs occur at the cell periphery (B). Their orientation largely matches that of the PPB. ϫ1350. Fig. 6. A prophase cell with a narrow PPB (A). Cortical Mfs (B) have a similar orientation. However, they appear to bracket the band with no Mfs actually coincident with it. ϫ1350. Fig. 7. Cortical actin in a prophase cell that has a PPB (not shown) in a stage of development similar to that in Fig. 6A. Note the abundance of Mfs in the cortex. ϫ1350.

are typical of prophase. Similar images are seen in root cells play about the same degree of perinuclear ﬂuorescence, treated for 0.5 h (Fig. 9), 1h (data not shown), 2h (Fig. 10) while their nuclei are similar in shape and in the degree of and 4h (data not shown). Moreover, the effects on the PPB chromatin condensation. However, the PPB of the CD- are independent of cell shape; they are seen in nearly iso- treated cell (Fig. 10) is at least two-fold broader and dis- diametric (i.e. square; Fig. 9) and more elongate (Fig. 10) tinctly ﬁbrillar in appearance compared to the control band cells. Occasionally, split PPBs or those of unequal width (Fig. 3). (Fig. 9A) are observed. The effects of CD on the PPB are particularly evident Quantitative analysis when comparing Figs 3 and 10, which are nearly identical In order to demonstrate more precisely the effects of CD in certain respects. Both cells have the same shape and dis- on the PPB, quantitative data were collected. These are pro- 994 E. P. Eleftheriou and B. A. Palevitz

Table 1. Effects of increasing CD concentration and time course of drug treatment on PPB and cell parameters in root tips of Allium cepa PPB Cell Nuclear PPB width/ PPB width/ Cells with Nuclei with width length length cell length nuclear length unequal PPB oval outline (mm) (mm) (mm) (%) (%) width (%) (%) n Controls (2 h treatment) Water 4.2±1.0 35.9±6.0 22.9±3.5 11.7±1.4 18.3±1.6 8.3 73.5 34 0.5% DMSO 3.8±0.9 32.9±7.1 20.1±3.7 11.5±1.1 18.9±1.3 0.0 54.8 31 Effect of CD concentration (2 h treatment) 1 mM 5.2±1.5** 33.1±7.5 20.6±3.5 15.7±1.3** 25.2±1.7** 7.7 53.8 26 2 mM 7.3±1.8** 40.3±7.4** 24.4±4.0 18.1±1.3** 29.9±1.5** 15.8 73.7 38 4 mM 7.9±2.4** 35.1±7.8 22.6±4.3 22.5±1.4** 35.0±1.6** 29.0 54.8 31 10 mM 8.2±2.4** 38.6±8.6* 24.2±4.4 21.6±1.3** 33.9±1.6** 30.0 52.5 40 Time course of effect of CD (10 mM) 0.25 h 7.5±1.9** 33.7±6.6 21.5±4.4 22.3±1.3** 34.9±1.2** 46.7 36.7 30 0.5 h 8.0±2.1** 34.4±9.1 21.9±3.6 23.3±1.3** 36.5±1.6** 27.0 46.4 37 1 h 7.5±2.8** 37.7±7.0 22.6±3.5 19.9±2.0** 33.9±2.4** 40.0 54.3 35 2 h 7.6±2.7** 37.8±7.9* 22.9±4.0 20.1±1.7** 33.2±2.0** 44.4 70.4 27 4 h 7.6±1.9** 34.8±6.8 21.1±3.2 21.8±1.3** 36.0±1.6** 36.7 50.0 30

*Statistically significant difference from DMSO control (PϽ0.05). **Statistically significant difference from both controls (PϽ0.05). n=Number of cells counted. vided in Tables 1 and 2. As shown in Table 1, column 1, examined in Table 2, trends very similar to those in Table the increase in PPB width is dependent on CD concentra- 1 are seen. tion. As CD rises, there is a progressive increase in PPB We also attempted to ascertain how quickly root cells width, which levels off around 4 mM. While the effect of can respond to CD. For this purpose, we chose a concen- 1 mM is smaller, the difference compared with the water tration of 10 mM and assayed roots at 0.25, 0.5, 1, 2 and 4 and DMSO controls is statistically significant. The effect is h. As shown in Tables 1 and 2, the maximum effect was specific for the PPB, in that cell length, nuclear length and seen at the earliest sample time, 0.25 h, and no increase in nuclear shape are unaltered by the drug. Thus, PPB width PPB width was noted with longer exposures. A similar trend also increases when normalized against cell and nuclear is seen when PPB width is normalized to cell and nuclear length. length (Tables 1 and 2). The reader may note that the two The measurements in Table 1 were taken from cells of values for PPB width at 2 h using 10 mM CD vary in column various sizes and shapes. In an attempt to make more uni- 1 of Tables 1 and 2. It is important to realize, however, that form comparisons, we selected a subset of the data from the two data sets represent separate experiments performed cells of similar shape. Specifically, we chose more isodia- on different occasions. In any event, the values are not sta- metric (square) cells for this purpose. When such data are tistically different.

Table 2. Effects of increasing CD concentration and time course of drug treatment on PPB and cell parameters in isodiametric cells of Allium cepa root tips PPB Cell Nuclear PPB width/ PPB width/ Cells with Nuclei with width length length cell length nuclear length unequal PPB oval outline (mm) (mm) (mm) (%) (%) (%) (%) n Controls (2 h treatment) Water 3.8±0.7 31.1±3.6 20.3±1.6 12.2±1.6 18.7±2.3 7.7 18.2 11 0.5% DMSO 3.6±0.9 28.5±4.9 18.5±2.4 12.6±1.4 19.5±1.9 0.0 7.1 14 Effect of CD concentration (2 h treatment) 1 mM 5.1±1.4** 28.8±6.5 18.4±2.3 17.7±1.2** 27.7±2.2** 7.1 21.4 14 2 mM 6.5±1.3** 32.8±3.4* 20.0±1.9 19.8±1.9** 32.5±2.1** 8.3 25.0 12 4 mM 7.0±1.6** 29.9±4.0 19.6±2.1 23.4±1.7** 35.7±2.1** 17.6 17.6 17 10 mM 8.2±1.8** 32.6±3.5* 20.9±1.9 25.1±2.0** 39.2±2.4** 33.3 16.7 18 Time course of effect of CD (10 mM) 0.25 h 7.8±1.5** 31.2±2.7 19.8±1.4 25.0±2.2** 39.4±2.7** 44.4 11.1 18 0.5 h 7.6±1.8** 27.4±3.5 19.6±1.4 27.7±1.8** 38.8±3.3** 33.3 16.7 18 1 h 6.8±2.1** 32.8±4.2* 20.3±2.0 20.7±2.4** 33.5±3.1** 53.3 26.7 15 2 h 6.9±1.4** 32.7±5.8 20.3±3.1 21.1±1.1** 34.0±1.3** 22.2 22.2 9 4 h 7.5±1.8** 29.5±5.2 18.7±2.5 25.4±1.4** 40.1±1.8** 50.0 8.3 12

*Statistically signiﬁcant difference from DMSO control (PϽ0.05). **Statistically signiﬁcant difference from both controls (PϽ0.05). n=Number of cells counted. Effect of cytochalasin D on preprophase band organization 995

Figs 8-10. Effects of 10 mM CD for various time periods on PPB organization. Fig. 8. A prophase cell treated for 0.25 h. The PPB is relatively broad for this stage (A); note the clear perinuclear staining. Hoechst fluorescence of the nucleus is seen in B. ϫ1300. Fig. 9. 0.5 h treatment. This cell has a broad PPB of unequal width (A). In addition, the PPB is still distinctly fibrillar (B), while Mts at the band edges are randomly aligned. ϫ1470. Fig. 10. 2 h treatment. A cell at nearly the same stage of development as that shown in Fig. 3, as judged by perinuclear fluorescence, nuclear shape and chromatin condensation (not shown). The PPB is about two-fold wider than in controls at this stage, while perinuclear fluorescence is sharp (A). The PPB is distinctly fibrillar (B). ϫ1300.

In the great majority of control cells, the width of each is the appearance of large actin aggregates at the spindle PPB was uniform across the cell. In CD-treated roots, how- poles at metaphase through telophase (Palevitz, 1988; ever, the frequency of cells with split or unevenly wide McCurdy et al. 1991). These aggregates were seen in the PPBs increases. Once again, these abnormalities were con- CD-treated material examined here (Figs 14, 15). Little or centration but not time dependent (i.e. beyond an exposure no anti-actin signal was detected in the phragmoplast (Fig. of 0.25 h; Tables 1 and 2, column 6). 15), which is known to contain F-actin in control root cells (e.g. Palevitz, 1988). Effect of CD on cortical Mfs Because PPB organization responds quickly to CD, we wished to ascertain whether the drug has an effect on cor- Discussion tical Mfs in the same time period. Indeed, we found that effects on Mfs can be seen in many root cells in as little Our results show a dramatic, rapid effect of CD on the PPB as 0.25 h, although the thick bundles characteristic of of Allium root cells. In prophase, when the PPB normally elongate cells respond more slowly. Actin filaments are reaches its narrowest, most compact configuration, CD replaced by fine specks and rods in both interphase (Fig. induces at least a two-fold increase in band width. More- 11) and dividing cells (Figs 12-15). Fig. 12 illustrates a over, the PPB is distinctly fibrillar after CD treatment, in prophase cell of a root exposed to 10 mM CD for 0.25 h. contrast to the condition seen in controls where individual This cell displays a broad PPB, but the actin Mfs found in Mts are difficult to discern. This change is accompanied by control cells are entirely absent. Instead, only very fine loss of the cortical Mfs that encompass the PPB, which are specks can be detected in the cortical cytoplasm of pre- replaced by numerous fine specks similar to those reported prophase or prophase cells (Fig. 12B). Similar images were elsewhere (Palevitz, 1988; McCurdy and Gunning, 1990; observed in cells treated for 0.5 h (Fig. 13). In some cases, McCurdy et al. 1991). It seems likely that the effect of CD the actin specks seem to be ordered in lines parallel to the on F-actin is related to the changes in PPB morphology. PPB (Fig. 13A,B). Hoechst fluorescence confirms that the Our data extend and amplify those of Mineyuki and Pale- cells in Figs 12 and 13 are in prophase (Fig. 13C). vitz (1990), who reported on the effects of CD on the PPB A reliable indicator that CD enters and affects onion roots and the division plane in Allium cotyledon cells. The change 996 E. P. Eleftheriou and B. A. Palevitz

Figs 11-15. Dual localization of tubulin and actin in CD-treated root tip cells of Allium. Fig. 11. An interphase cell treated for 0.5 h with 10 ␮M CD. Fine actin specks are present in the cortex. ϫ1500. Fig. 12. A prophase cell treated for 0.25 h with 10 ␮M CD before fixation. A broad PPB is seen in surface view in A. Note its distinctly fibrillar nature. Actin staining reveals only fine specks in the cortex (B), although a hint of transverse order can be seen. ϫ1500. Fig. 13. A prophase cell treated for 0.5 h with 10 ␮M CD before fixation. The broad, fibrillar PPB (A) is accompanied by fine actin specks in the cortex (B). Hoechst staining of the nucleus is shown in C. ϫ1500. Fig. 14. An anaphase cell exposed to 10 ␮M CD for 0.5 h contains actin aggregates at one of the spindle poles. Aggregates at the opposite pole are out of focus. The anaphase chromosomes are seen with Hoechst staining in B. ϫ1500. Fig. 15. A telophase cell treated for 0.5 h. Actin aggregates are located at the polar regions. The phragmoplast region between daughter nuclei is unstained. ϫ1500. in PPB width reported here is very similar to those results; study reported here, we sampled cells of roots in direct con- however, while Mineyuki and Palevitz failed to detect sig- tact with the drug. Similar considerations of drug access nificant alterations in PPB-associated Mfs, rendering con- could account for the differences seen in the effects of clusions on the relationship between the PPB and actin cytochalasin in other cell types (see below). somewhat tentative, the effects reported here are far less Although CD appears to prevent normal narrowing of the ambiguous. We cannot offer a firm explanation for the dif- PPB, the very rapid nature of the response (near saturation ference, other than the fact that the earlier study examined in only 15 min at 10 mM) implies that the drug also leads cotyledons. Since the cotyledons are invested with a cuticle, to rebroadening of previously narrowed bands. Thus, cor- the final concentration of CD in the target cells may not tical Mfs probably play a key role in PPB maturation by have been sufficient to disrupt Mfs but could have been progressively confining PPB Mts to a restricted zone in the adequate to affect their function in PPB organization. In the cortex and maintaining the Mts in this zone once the band Effect of cytochalasin D on preprophase band organization 997 has narrowed. Additional observations reinforce our con- the formation of the PPB. Most interphase cells in Allium clusions. For example, rather than maintaining a compact roots contain only randomly oriented Mfs that permeate the arrangement in which individual Mts are hard to discern, entire cytoplasm. McCurdy and Gunning (1990) came to CD-treated prophase PPBs return to a distinctly fibrillar the same conclusion using wheat root tips. However, as appearance. In addition, the increased percentage of split or shown in our laboratory, parallel Mfs appear among the uneven bands in the presence of CD supports the view that cortical Mts before chromatin condensation is obvious in a Mfs act to contain the PPB. Recent results using fern pro- small population of cells. At the same time, aligned Mfs tonemata are also of direct relevance. Kadota and Wada tend to disappear from internal regions of the cytoplasm, a (1992) have shown that a band of cortical Mts behind the characteristic maintained through later stages of division advancing apex in Adiantum is disorganized following dis- (Liu and Palevitz, 1992). Thus, formation of a uni- ruption of a coincidental array of cortical Mfs by CB. These formly distributed Mf system in the cortex precedes the authors proposed that the Mfs are crucial for maintaining PPB. the integrity of the Mt band. Finally, Mfs have been impli- While our results indicate that Mfs play an important role cated in the organization of cortical Mts in other plant cells in PPB organization, feedback between Mts and Mfs cannot (Fukuda and Kobayashi, 1989; Seagull, 1990) and interest- be ruled out. Indeed, prior results with colchicine indicat- ing examples of Mt-Mf colocalizations have been reported ing that elimination of PPB Mts leads to the loss of corti- (e.g. Pierson et al. 1989; Kengen and de Graaf, 1991). cal Mfs support such interactions (Palevitz, 1987; Mineyuki The manner in which Mfs restrict PPB Mts to a pro- and Palevitz, 1990). On the other hand, similar experiments gressively narrower zone and then maintain that arrange- reported elsewhere offer a different perspective. McCurdy ment is unknown. The mechanism could involve direct and Gunning (1990) maintained that oryzalin-induced loss interactions between the two classes of cytoskeletal ele- of PPB Mts is not accompanied by disappearance of ments mediated by accessory proteins (Griffith and Pollard, coaligned cortical Mfs. Lloyd and Traas (1988) reported 1978). Such interactions are indicated by the close physi- that PPB actin is not lost in the presence of CIPC. In cal relationship between Mts and Mfs in the PPB (Ding et addition, colchicine-induced disruption of the subapical Mt al. 1991). The mechanism could also operate more indi- band in Adiantum protonemata does not lead to the disap- rectly through an effect on the PM. Elucidation of the mech- pearance of the coincident F-actin array (Kadota and Wada, anism may in part depend on an adequate picture of the 1992). Further experiments are clearly needed to clarify this distribution of cortical Mfs during PPB development. aspect of the “cross talk” between Mts and Mfs during PPB Unfortunately, our information in this regard, once again, formation. is mixed. While published micrographs generally show that Our results raise an additional important question: the Mf array is superimposed on the PPB (Kakimoto and namely, in the transition between one Mt array and another, Shibaoka, 1987; Palevitz, 1987; Traas et al. 1987), its do all the Mts of the new array represent new polymer, or arrangement changes as the PPB develops. McCurdy and are at least some of them incorporated intact from the old Gunning (1990) reported that, in root cells of Triticum, par- array (for review see Palevitz, 1991)? Ideas on the forma- allel Mfs appear in the PPB after it has narrowed signifi- tion of Mt arrays have over the past several years concen- cantly. Coverage of the cortex by transverse Mfs then trated on the role of dynamic instability and the selective expands until the entire cell surface is occupied. Later, as spatial stabilization of growing Mts (Kirschner and Mitchi- the PPB degrades, the Mfs disappear as well. On the other son, 1986). However, past speculation on the construction hand, work performed in our laboratory indicates that par- of the PPB has also invoked the reutilization of intact Mts allel Mfs cover much of the cortex very early in PPB organ- of the previous interphase cortical array (e.g. Wick and ization and are then progressively restricted to the midzone Duniec, 1983; see Wick, 1991). Such a mechanism could as the PPB narrows (Mineyuki and Palevitz, 1990; Liu and operate together with or instead of an assembly-disassem- Palevitz, 1992). The width of the Mf band always exceeds bly process. Recent observations on the guard mother cells the area covered by Mts, however. Although Ding et al. of grasses (Cho and Wick, 1989; Cleary and Hardham, (1991) maintained that Mfs permeate narrow PPBs in 1989; Mullinax and Palevitz, 1989) can be interpreted in tobacco root cells, our results show that in cells with a very terms of construction of the PPB in part from preexisting narrow, compact PPB, the cortical Mfs re-expand toward Mts (Mullinax and Palevitz, 1989). In addition, Pickett- the ends of the cell, leaving the midzone free of aligned fil- Heaps (1969) proposed that Mts of the PPB are incorpo- aments (Fig. 6A,B; also see Liu and Palevitz, 1992). A pat- rated into the spindle apparatus, an idea fueled by promi- tern in which aligned Mfs cover the entire cortex early, after nent Mts linking the two arrays (for recent images see which the array progressively narrows but always remains Mineyuki et al. 1991). The effect of CD in dramatically wider than the Mt band, is consistent with (but does not yet preventing PPB narrowing supports the movement of intact prove) a mechanism in which the Mfs control PPB mor- polymer in the cortex, as does rewidening of previously phology. However, this model does not account for the narrowed bands. The extent to which such movement reported lack of Mfs in the PPBs of certain stomatal cells occurs in the organization of other Mt arrays, as well a (Cho and Wick, 1990). determination of Mt dynamics in plants, deserves intensi- It is also noteworthy that, contrary to reports that a net- fied scrutiny. work of cortical Mfs parallels the interphase Mt array (e.g. Seagull et al. 1987; Traas et al. 1987; Sonobe and Shibaoka, We thank Bo Liu for advice during the course of our experi- 1989), results from our laboratory indicate that such an ments. Supported by funds from the National Science Foundation arrangement in root cells represents a very early stage in to B.A.P. (DCB-9019285). E.P.E. was sponsored by the Univer- 998 E. P. Eleftheriou and B. A. Palevitz sity of Thessaloniki during a sabbatical leave at the University of comparative immunocytochemical study. Cell Motil. Cytoskel. 18, 107- Georgia. 112. Mineyuki, Y. and Gunning, B. E. S. (1990). A role for preprophase bands of microtubules in maturation of new cell walls, and a general proposal on the function of preprophase band sites in cell division in higher plants. J. References CellSci. 97, 527-537. Mineyuki, Y., Marc, J. and Palevitz, B. A. (1988a). Formation of the Cho, S.-O., and Wick, S. M. (1989). Microtubule orientation during oblique spindle in dividing guard mother cells of Allium. Protoplasma stomatal differentiation in grasses. J. Cell Sci. 92, 581-594. 147, 200-203. Cho, S.-O. and Wick, S. M. (1990). 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