Taxol-induced anaphase reversal: evidence that elongating microtubules can exert a pushing force in living cells.

Abstract

The effects of taxol on mitosis in Haemanthus endosperm were studied. Immuno-gold staining was used to visualize microtubules; observations on microtubule arrangements were correlated with studies in vivo. Mitosis is slowed down, but not arrested, by taxol over a wide range of concentrations. Taxol promotes the formation ofabundant new microtubules and lateral association within and between microtubule arrays (spindle fibers). This leads to a pronounced reorganization of the spindle, especially at the polar regions. Chromosome arms may be pushed toward the equator in metaphase. Anaphase chromosomes, with their kinetochores still pointing to the poles, move backward before resuming their poleward migration. During anaphase, the interzone is depleted of microtubules and trailing chromosome arms are stretched and often torn apart by rapidly elongating polar microtubules. Fragments are transported away from the poles, apparently "riding" on the tips of microtubules. This provides evidence of "pushing" by elongating microtubules. The desynchronization of anaphase, often observed as one of the first effects of taxol, indicates that the anchorage of different kinetochore fibers varies. The data draw attention to modifications of spindle structure due to increased microtubule lateral associations and to the role of this process in spindle integrity and chromosome movement.Taxol, a low molecular weight alkaloid from the eastern yew (Taxus brevifolia) promotes the assembly of microtubules in vitro by lowering the critical concentration of tubulin required for polymerization (1)(2)(3). Experiments with taxol carried out on living systems (4-8) are in general agreement with studies in vitro. In a variety of cells, taxol induces the formation of microtubules that are resistant to disassembly. Most studies have been done with animal cells and describe the long-term effects ofthis drug. It has been reported that taxol blocks mitosis either in the G2 or the M phase. The data on immediate short-term effects on mitosis are either fragmentary (9) or do not include direct observations in vivo (10).We have studied the effects of taxol on mitosis in living cells ofa higher plant, Haemanthus endosperm, and correlated these experiments with immuno-gold staining (IGS) (11,12). The IGS technique allows us to examine spindle microtubule organization at high resolution in the light microscope on a large number ofcells under precisely the same experimental conditions. This overcomes the limited sampling inherent in studies in vivo or with an electron microscope. It also makes the variation between cells, which normally increases the difficulty of interpretation of small samples, an advantage in identifying the essential features of the effects studied because extensive comparison of average and extreme cases can be made.The absence of centriolar asters in Haemanthus is a further advantage for this type of study. Centrioles (centrosomes) induce microtubule nucleation, a process that is enhanced by taxol. This introduces additional c...

Model of anaphase chromosome movement based on polymer-guided diffusion.

“…It has indeed been observed that in anaphase, all the chromosomes move at a velocity independent of size, indicating that their displacement is not limited by their hydrodynamic properties. Also, this displacement is inhibited if the depolymerization is blocked by taxol (17). This is consistent with our model in which slowing down depolymerization results in slowing particle displacement.…”

Independently Regulated Neocentromere Activity of Two Classes of Tandem Repeat Arrays

“…The primary effects included aberrant spindle formation and extra microtubule arrays in prometaphase, a heavier packing of microtubules (visualized as a more uniform staining of tubulin), and a widening of the spindle midzone and premature clearing of microtubules from the future cell plate at anaphase (as if a phragmoplast were beginning to form at anaphase). These also were the primary effects of taxol noted by Bajer and colleagues in Haemanthus (Bajer et al, 1982;Molè-Bajer and Bajer, 1983). A noteworthy difference is that we observed more pronounced effects at prometaphase than did the previous authors.…”

“…Taxol treatments sufficient to induce gross alterations in spindle architecture caused no apparent reduction in the extent or number of neocentromeres ( Figures 5B and 5E, Table 1), suggesting that microtubule dynamics contribute little to neocentromere motility. A slight enhancing effect of taxol on neocentromere activity (Tables 1 and 2) can be explained if taxol impairs kinetochore-mediated movement (Bajer et al, 1982;Waters et al, 1996) but has no effect on neocentromere movement. The fact that neocentromeres are pronounced on the most disturbed spindles supports the view espoused previously (Yu et al, 1997) that Ab10 provides or recruits proteins to neocentromeres that generate their movement de novo.…”

“…We anticipated that if the differential poleward motility were a function of flux, taxol would cause a measurable reduction in neocentromere activity. Taxol is not expected to have any effect on motor activity per se, but it could generally slow the anaphase movement of the chromosomes (which may be driven in part by flux [Bajer et al, 1982;Waters et al, 1996]). The neocentromere-mediated stretching of chromosome arms could be increased by taxol if kinetochore-mediated movement is slowed but neocentromere activity is not.…”

Relationships between the structures of taxol and baccatine III derivatives and their in vitro action on the disassembly of mammalian brain and Physarum amoebal microtubules.

“…Among the numerous microtubular poisons, taxol (25,26) possesses the unique property of stabilizing microtubules both in vivo (27,28) and in vitro (29). The effects of taxol are widespread among eukaryotic cells, including mammalian cells (27,28,30,31), Xenopus eggs (32), sea urchin eggs (33), Haemanthus endosperm (34), Trypanosoma (35), Plasmodium (unpublished results), and Physarum amoebae (36,37). In order to compare the interaction of taxol on tubulin from two distinct evolutionary lines of eukaryotic cells, we have studied the effects of various taxol derivatives, in particular baccatine III ( Fig.…”