Metabolic Inhibitors Block Anaphase A In Vivo

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells o f Tradescantia virginiana consists almost entirely o f chromosome-topole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome mot ion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol t reatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and /o r electrical gradients generated by cellular membranes. W rHILE no one doubts that chromosome separation during mitosis requires energy, there has been an ongoing controversy over the last 30 years about when this energy is made available, in what form, and for which processes it is needed. Early studies showing that various metabolic inhibitors have little or no effect after the onset of prophase led to the concept that cells become preloaded with a sufficient energy reservoir to carry them through the rest of mitosis and cytokinesis. Mazia (29), in summing up this work in 1961, referred to "points of no return" during mitosis as stages after which the cell is committed to progressing through the mitotic cycle. Subsequent work by Epel (14) and Amoore (1, 2), however, indicated that the "energy reservoir" and "points of no return" concepts are not tenable (see also reference 30). For example, when sea urchin eggs are cultured in the presence of carbon monoxide they can be stopped at any stage of mitosis (14). Inhibition occurs when the ATP supply drops to 50% or less of the normal level. All stages of mitosis in pea roots including anaphase are sensitive to cyanide and oxygen deprivation. Amoore (1) reports, however, that inhibition is not due to reduced ATP levels but rather to an effect on a nonrespiratory ferrous complex. Regardless of the details, the conclusion that emerges from these studies is that there is no energy reservoir that is capable of carrying a cell through division. In light of these studies, we were not surprised to find, during experiments on division plane determination in plants several years ago (36, 37), that anaphase motion is rapidly and reversibly inhibited by 2,4-dinitrophenol (DNP) ~ and sodium azide. These results were communicated in abstract form (34). Since that report, however, the possibility that anaphase uses stored energy has res~urfaced. Using a permeabilized PtKl cells, Cande (8, 9) has reported that anaphase A (movement of chromosomes to the poles; 19) but not anaphase B (separation of the poles) is insensitive to the absence of ATP or the presence of ATPase inhibitors. It can thus be argued that anaphase A uses stored energy. The localization of creatine kinase in the spindle (21) and the movement of chromosomes in permeabilized cells in the presence of phosphoryl creatine (10) add credence to this conclusion. Further support comes from the work of Pickett-Heaps and Spurck (39) who found that in diatoms, metabolic inhibitors cause prometaphase chromosomes to quickly move to one pole or the other before stopping altogether. The concept that anaphase A is driven by stored energy has received additional impetus from studies on pigment granule migration in chromatophores. Based on observations that the outward movement of granules requires energy whereas movement towards the cell center does not, Luby and Porter (24) have suggested that the organization of the cytoplasmic matrix (microtrabecular lattice) is a source of energy which, when liberated via the contraction of the matrix, moves the particles inward. Both Mclntosh (31) and Cande (8) have used these findings in comparing anaphase A to the inward moveI. Abbreviations used in this paper: CCCP, carbonyl cyanide m-chlorophenyl hydrazone; d'H20, deionized water; DNP, 2,4 dinitrophenol; GMC, guard mother cell; MT, microtubule; SHC, stamen hair cell. © The Rockefeller University Press, 0021-9525/86/06/1995/11 $1.00 The Journal of Cell Biology, Volume 102, June 1985 1995-2005 1995 on Jauary 6, 2018 jcb.rress.org D ow nladed fom ment of pigment granules. In addition, Pickett-Heaps et al. (40) have proposed that the collar material in dividing diatoms is an elastic matrix similar to the microtrabecular lattice that can move chromosomes poleward. These ideas are attractive and may help resolve vexing questions concerning chromosome motion. However, it should be noted that considerable disagreement still exists concerning the role of the cytoplasmic matrix in the inward and outward movement of pigment granules (e.g., 12, 45). Furthermore, to the extent that any mitotic mechanism relies on the stored energy concept, we submit that it cannot be generally applied. We base this conclusion on the work reported here which shows that in two species of higher plants and in two cell types, anaphase A can be quickly and, depending on the inhibitor, reversibly blocked. Thus, anaphase A requires a continuous supply of energy in these cells. Part of this work appeared in the aforementioned abstract (34). Materials and Methods Two mitotic systems have been used in this study: (a) guard mother cells (GMCs) ofAlliurn cepa L. (onion) and (b) stamen hair cells (SHCs) of Tradescantia virginiana L. (spiderwort). To prepare GMCs for analysis in the light microscope, paradermal slices from the cotyledons of 5-d-old seedlings were excised with a No. 11 scalpel blade and placed on microscope slides in deionized water (d'H20) or 0.01-0.05 M potassium phosphate buffer, pH 7.0, as previously described (36). The GMC, being the product of a highly asymmetric division in the epidermis, is easily recognized by its small size. Under simple culture conditions these cells divide longitudinally to form an immature guard cell pair which then differentiates into the stomatal complex (35). Mitotic events can be examined directly in GMCs using Nomarski differential interference contrast optics. While monitoring mitosis directly or with the aid of a video camera, respiratory inhibitors including DNP, sodium azide (both obtained from Sigma Chemical Co., St. Louis, MO), and carbonyl cyanide mchlorophenyl hydrazone (CCCP) (Calbiochem-Behring Corp., La Jolla, CA; stock solution prepared in absolute ethanol) diluted in d'H20 or phosphate buffer were perfused under the coverslip and subsequent chromosome motion was observed. After various times, the inhibitors were washed out by perfusion with water or buffer and the ability of the cells to resume anaphase was ascertained. In some experiments, inhibitor was replaced with the same solution containing colchicine or with colchicine alone (in d'H20 or phosphate buffer). Observations were performed on a Zetopan microscope (C. Reichert, Vienna) and photographs taken with an automatic exposure system using PanatomicX film. When a newvicon camera (model 65, Dage/MTI, Michigan City, IN) was used to facilitate observations, exposures were made directly from a video monitor using a 35-ram camera. In addition, video recordings were made on a 3/4 inch time-lapse cassette unit (model VC-7505, Nippon Electric Co., Elk Grove, IL). Tradescantia stamen hairs were handled in much the same way as the Allium epidermal slices. However, because the hairs have a thin cuticle that impedes diffusion of drugs through the plasmalemma, they were pretreated for ~ l h at 20"C in a cutinase solution (0.1 mg/ml in 0.02 M Hepes-0.02 M KCI, pH 8.0) before mounting in 0.02 M Hepes-KCl, pH 7.0, in a glass slide chamber. The hairs were then examined on Reichert Zetopan or Photomicroscope III (Carl Zeiss, Inc., Oberkochen, Wuerttenberg, FRG) microscopes equipped with differential interference contrast optics. Again cells were allowed to enter anaphase under normal conditions. Thereafter, respiratory inhibitors diluted in 0.02 M Hepes-KCl, pH 7.0, were perfused through the chamber and the effect on anaphase movement was determined. Observations were again aided with a video camera and tape recordings were used to ascertain rates of anaphase motion. Ultrastructural analyses were performed on control and treated GMCs of Allium. Whole seedlings or paradermal slices were treated with inhibitors for various periods of time and then fixed and fiat-embedded for electron microscopy as previously described (36). Slices embedded in thin plastic wafers were examined in the light microscope and dividing GMCs identified. The cells were excised, thin sectioned, poststained in uranyl acetate and lead citrate, and viewed in an EM 10A electron microscope (Carl Zeiss, Inc.) at 60 kV. Results Anaphase Consists o f Chromosome-to-Pole Motion, Or Anaphase A Allium GMCs maintained in d 'H20 or dilute phosphate buffer readily continue division when observations are begun in late prophase or prometaphase. Cells remain in rnetaphase for a variable period of time before entering anaphase, which lasts for 10-20 rain. Because of constraints imposed by the relatively small size of the cell (10-20 zm), the spindle occupies the maximum volume available by aligning more or less diagonally across the GMC (Fig. I A). However, the future plane of the new cell wall is longitudinal, and to achieve this alignment, the daughter nuclei and forming cell plate undergo reorientation movements in late anaphase-early telophase (36, 37). While measurements of pole position and separation are difficult because the poles are initially placed near opposite comers of the cell and often in different planes of focus, it is nevertheless evident that the spindle during anaphase exhibits mostly chromosome-to-pole movement and comparatively little anaphase B. Indeed, significant pole separation would be difficult if not impossible because by metaphase or the beginning of anaphase the poles are usually abutted against the wails at opposite comers of the cell and are thus separated by nearly the maximum distance available. Subsequently during the initiation of spindle reorientation in late anaphaseearly telophase, there may be a small reduction in the poleto-pole distance as the daughter nuclei migrate along the side walls, but there is no increase. We conclude from these observations that anaphase in Allium GMCs is composed almost entirely of anaphase A. The mitotic apparatus is usually not as confined in Tradescantia SHCs as it is in Alliurn GMCs (Figs. 1 B and 2). The pole-to-pole separation may be 30-35 zm, with the end walls 50-60 mm apart. Nevertheless the motion displayed by the chromosomes is >90% anaphase A (Fig. 2). Despite the fact that the dimensions of the cell often permit a significant amount of pole separation, it does not occur. These observations show that the process we are examining in both cell types is anaphase A. Respiratory Inhibitors Block Anaphase A DNP and azide rapidly and reversibly inhibit anaphase motion. Cells were identified at metaphase or earlier and monitored until the onset of anaphase. The appropriate inhibitor was then perfused under the coverslip. A total of 31 cells were treated in this manner (16 GMCs and 15 SHCs). With GMCs, 1-3 × 10 -4 M DNP stops anaphase in 5-10 min (Fig. 3), while 2 × 10 -3 M sodium azide acts more rapidly (1-3 min; Fig. 4). Anaphase can be halted early, soon after the chromosomes separate, or later as they approach the poles. With both agents, the chromosomes seem to "freeze" in place and become more refractile in appearance (Fig. 3B). Cells can also be arrested in prometaphase (or metaphase), but unlike the results reported for diatoms and animal cells (39, 40), the chromosomes do not move to the poles during treatment at this stage. Both DNP and azide also stop telophase reorientation and cytoplasmic streaming. The results with Tradescantia SHCs are essentially the same. Under conditions in which the cuticle has been etched The Journal of Cell Biology, Volume 102, 1986 1996 on Jauary 6, 2018 jcb.rress.org D ow nladed fom