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...

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Growth cones are intimately involved in determining the direction and extent of neurite elongation during development. They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP-43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha-actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A-CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.

Optimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271-284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0-50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2-3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature. When highly coupled preparations were reactivated at 21 degrees C, double reciprocal plots of beat frequency vs. [MgATP] were biphasic with extrapolated Fmax values of 22 and 44.8 Hz. However, when reactivated at 10 degrees C and 30 degrees C, linear plots were generated with Fmax values of 18.3 and 48.9 Hz, respectively. The beat frequencies of cultured cells and reactivated axonemes also varied biphasically with temperature. Our data suggest that newt lung respiratory cilia possess an intra-axonemal activation mechanism involving a temperature- and MgATP-induced transition between two distinct states whose maximum beat frequencies differ by 200-300%.

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