Localization of C 2 CF-Tubulin to Single Microtubules

In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 #m/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150/xM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between ,075 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase. I N recent years, the availability of molecular probes to study microtubule dynamics has provided new insights into microtubule behavior in the mitotic spindle. Photobleaching experiments have shown that microtubules turn over rapidly in the spindle (Cassimeris et al., 1988; Salmon et al., 1984a; Saxton et al., 1984; Wadsworth and Salmon, 1986), explaining in part its rapid response to changes in levels of microtubule polymerization (Inoue and Sato, 1967; Salmon et al., 1984b). Kinetochore microtubules as a class are more stable than most spindle microtubules, both in terms of turnover and resistance to depolymerizing conditions (Gorbsky and Borisy, 1989; Mitchison et al., 1986; Rieder, 1981). Different rates of microtubule turnover in the context of a relatively stable, differentiated structure such as the spindle suggest that regulation of microtubule dynamics is likely to be intimately involved in spindle morphogenesis and function (Inoue and Sato, 1967; Kirschner and Mitchison, 1986; Mitchison, 1988). By studying more carefully the behavior of microtubules in the spindle we hope to gain insights into how microtubule dynamics may be involved in spindle morphogenesis and chromosome segregation in mitosis. It was originally demonstrated in vitro that microtubule plus ends captured by chromosomes can incorporate exogenous labeled tubulin while maintaining attachment at the kinetochore (Mitchison and Kirschner, 1985), and subsequent experiments in vivo showed that microinjected biotinlabeled tubulin incorporates into microtubule ends attached to kinetochores at metaphase (Mitchison et al., 1986). At increasing times after microinjection, kinetochore microtubules became progressively labeled from their plus ends polewards towards their minus ends. To maintain the metaphase spindle at steady-state, the progressive addition of new tubulin subunits at microtubule plus ends requires the concomitant depolymerization of microtubules at minus ends. This would result in the steady poleward movement of tubulin subunits in the microtubule lattice towards spindle poles, as was postulated by Margolis and Wilson on the basis of experiments in vitro (Margolis and Wilson, 1981). This inferred movement, which we have termed poleward microtubule flux, was recently demonstrated directly in living tissue culture cells using a novel photoactivatable fluorochrome, bis-caged fluorescein (C2CF), covalently coupled to tubulin (Mitchison, 1989). After microinjection of unactivated (nonfluorescent) C2CF-tubulin into metaphase tissue culture cells and incorporation of the tagged tubulin into spindles, fluorescence was locally activated in the spindle by UV light and followed by low-light videomicroscopy. Activated fluorescence moved polewards at a rate of 0.4-0.6/~m/min, indicating movement of the microtubule lattice. Poleward flux appeared to be specific to kinetochore microtubules; other spindle microtubules lost fluorescence rapidly, presumably because of their high rate of turnover. The results obtained from photoactivation of fluorescence have agreed with those obtained by photobleaching methods, with respect to the rapid turnover of most spindle microtubules. However, photobleaching experiments have not detected any significant poleward movement of microtubules in © The Rockefeller University Press, 0021-9525/91/03/941 / 14 $2.00 The Journal of Cell Biology, Volume 112, Number 5, March 1991 941-954 941 spindles (see references above), with one possible exception (Hamaguchi et al., 1987), and the reasons for this are not yet clear (see Mitchison and Sawin, 1990 and references therein). In the preceding paper we described different assembly pathways for spindles reconstituted in cell-free extracts of Xenopus eggs (Lohka and Mailer, 1985; Murray and Kirschner, 1989; Sawin and Mitchison, 1990); in this paper we report the identification of poleward microtubule flux in spindles assembled in vitro, extending our previous observations of poleward microtubule flux. Spindles assembled in vitro provide a powerful model system for studying microtubule dynamics: they are among the largest described in higher eukaryotes (40-60 #m, pole-to-pole) and are therefore easily marked and followed by light microscopy. In addition, because they are not surrounded by a plasma membrane, spindles assembled in vitro can be subjected to a number of experimental conditions without the difficulties introduced by microinjection. The ability to manipulate this reconstituted system should allow us to pursue the mechanism of poleward flux at a biochemical level. Materials and Methods Extracts, Sperm, and Tubulin The methods used for the preparation of Xenopus egg extracts arrested in mitosis ("mitotic extracts") or induced to enter interphase and arrested in the following mitosis ("interphase-to-mitotic extracts"), for the isolation and fluorescent labeling of Xenopus sperm nuclei, and for tubulin labeling have been described in the preceding paper (Sawin and Mithison, 1990). In some experiments sperm nuclei were covalently labeled with the carboxymethylindocyanine dye CY5.18, using the dye N-hydroxysuccinimide-ester, CY5.18-OSu (Southwick et al., 1990) and the procedure described in the previous paper. CY5.18-OSu was the generous gift of Swati Mujumdar and Alan Waggoner (Carnegie-Mellon University, Pittsburgh, PA). Bovine brain tubulin was purified and labeled with X-rhodamine and C2CE using the dye N-hydroxysuccinimideand N-hydroxy-sulfo-succinimide esters, respectively (Hyman et al., 1990). X-rhodamine tubulin was used in place of tetramethylrhodamine tubulin (Sawin and Mitchison, 1990) for better spectral separation from the activated C2CF (i.e., fluorescein) signal. The synthesis and properties of C2CF and C2CF-labeled tubulin have been described previously (Mitchison, 1989).