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 A ril 9, 2017 D ow nladed fom Published June 1, 1986