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 occu- pied 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 determi- nation 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.