The Motor for Poleward Chromosome Movement in Anaphase Is in or near the Kinetochore

I have tested two contending views of chromosome-to-pole movement in anaphase. Chromosomes might be pulled poleward by a traction fiber consisting of the kinetochore microtubules and associated motors, or they might propel themselves by a motor in the kinetochore. I cut through the spindle of demembranated grasshopper spermatocytes between the chromosomes and one pole and swept the polar region away, removing a portion of the would-be traction fiber. Chromosome movement continued, and in the best examples, chromosomes moved to within 1 #m of the cut edge. There is nothing beyond the edge to support movement, and a push from the rear is unlikely because cuts in the interzone behind the separating chromosomes did not stop movement. Therefore, I conclude that the motor must be in the kinetochore or within 1 /zm of it. Less conclusive evidence points to the kinetochore itself as the motor. The alternative is an external motor pulling on the kinetochore microtubules or directly on the kinetochore. A pulling motor would move kinetochore microtubules along with the chromosome, so that in a cut half-spindle, the microtubules should protrude from the cut edge as chromosomes move toward it. No protrusion was seen; however, the possibility that microtubules depolymerize as they are extruded, though unlikely, is not ruled out. What is certain is that the motor for poleward chromosome movement in anaphase must be in the kinetochore or very close to it. T HE beautiful, precise movements of chromosomes in mitosis have been known for over a century. Four years after the discovery, Van Beneden (1883) proposed that chromosomes in anaphase are pulled to the poles by traction fibers. In current terms, traction forces pull poleward on the microtubules attached at the kinetochore of each chromosome. This ancient theory naturally suffered some reverses in the course of a century, but by 1960 the traction fiber idea was so well-established that it seemed pretentious to call it a theory. The traction fiber seemed an elementary fact, and the center of speculation shifted to the molecular mechanism of traction (for review, see Inou6, 1981; Nicklas, 1988). But now the traction fiber idea is once again a theory, a theory that may be wrong. The new alternative is a kinetochore that participates actively in its own movement, rather than being passively dragged toward a pole by traction fibers. In vitro, microtubules appear to be pulled toward kinetochores as microtubules disassemble (Koshland et al., 1988), the opposite of a microtubule pulling on a chromosome. However, the relevance of the in vitro microtubule movement to chromosome movement in living cells is not certain. Because the movement in vitro has not yet been studied in real time, a detailed comparison with movement in living cells is precluded. Also, the structure of the kinetochore-microtubule junction in vitro is not yet known, so it is uncertain whether the movement involves the precise structural arrangement seen in cells. Strong evidence from living cells against traction fibers and in favor of an active kinetochore comes from experiments of Gorbsky et al. (1987, 1988). They reasoned that a traction fiber would move poleward in anaphase along with the chromosome it pulls, so a marker on the kinetochore microtubules should move poleward along with the chromosomes. They made spindle microtubules fluorescent by injecting ceils with fluorescent subunits. Then the spindle was irradiated with a band of light to bleach the fluorescence, so that many microtubules were marked by a nonfluorescent band. They found that as chromosomes moved toward the poles, the bleached band did not move much relative to the chromosomes. Particularly striking are instances in which chromosomes caught up with a band and moved through it (Gorbsky et al., 1988). Gorbsky and co-workers concluded that kinetochore microtubules are not pulled poleward and do not pull chromosomes poleward as in a traction fiber model. Instead, kinetochore microtubules are stationary mils on which chromosomes glide, propelled poleward by a motor in the kinetochore (Gorbsky et al., 1987, 1988). While the evidence is impressive, it is not absolutely conclusive. The authors admit that the movement of "one or a very few kinetochore microtubules" cannot be ruled out (Gorbsky et al., 1988). Major concerns are first that some microtubules remain unbleached (of. Fig. 6 in Gorbsky et al., 1987) and hence are not marked; any movement they undergo is undetectable. Second, the fluorescence increases in the bleached band as chromosomes move toward it (Gorbsky et al., 1988); this © The Rockefeller University Press, 0021-9525/89/11/2245/11 $2.00 The Journal of Cell Biology, Volume 109, November 1989 2245-2255 2245 on July 0, 2017 jcb.rress.org D ow nladed fom might be due to microtubules that move poleward, so that their unbleached fluorescent parts intrude into the bleached region. Additional concerns have also been raised (Forer, 1988; Vigerset al., 1988; Wolniak, 1988). For me, however, these elegant in vitro and in vivo experiments are more noteworthy for the novel view they offer of mitosis and the kinetochore than for any shortcomings (Nicldas, 1988). Certainly the recent experiments raise serious doubts about traction fibers, even if they are not a compelling reason to discard a theory that has worked so well for so long (Forer, 1988; Wolniak, 1988). A novel approach to testing a role of kinetochore microtubules as traction fibers is to cut them off; do chromosomes continue to move when a large part of the putative traction fiber is simply not there? Surprisingly, such direct experiments are possible. Certain insect spermatocytes with large spindles can be demembranated mechanically, allowing free access to the spindle (Nicklas, 1977). The spindle can be cut as desired merely by pressing it against the coverslip with a needle (Nicklas et al., 1989). Remarkably, anaphase chromosome movement continues in cut spindles, even when a large part of the spindle is not present. The characteristics of that movement and the implications for the mechanism of chromosome movement in anaphase are the subjects of this report. The pioneers in cutting the spindle are Hiramoto and Nakano (1988) and Hiramoto and Shoji (1982). They reported continued chromosome movement, but the extent of movement was not clear in their reports, nor was it established that the spindle was severed cleanly and completely. Materials and Methods Only certain cells can be demembranated mechanically and yield functional spindles (Nicklas et al., 1989). Chromosome movement has been studied mainly in the exceptionally large spindles of spermatocytes from several grasshoppers of the subfamily Oedipodinae: Arphia sulphurea (Fabricius), A. xanthoptera (Burmeister), Chortophaga viridifasciata, (DeGeer), and Dissosteira carolina (L.). Those species were collected as seasonably available from local natural populations. Chromosome-to-pole movement in anaphase is very similar in rate and extent in spermatocytes of all these species, so the results have been pooled. However, for the spindles illustrated, the species are as follows: Figs. I and 8, Dissosteira; Figs. 2, 3, and 4, Chortophaga; Figs. 5 and 6, Arphia xanthoptera. Cricket (Acheta domestica L.) spermatocytes were used for immunochemical studies (Fig. 7). Acheta is available year-round from a commercial supplier (H. O. Brewer, Durham, NC). Spermatocyte culture, demembranation, micromanipulation, and timelapse movie recording and analysis were carried out as previously described (Nicklas et al., 1989 and references therein). Culture temperature was usually 25-26"C (range: 23-26"C). Spermatocytes are covered with an inert oil and viewed on an inverted microscope; the micromanipulation needle is placed in the oil and lowered down onto the ceils. Spindles were observed by phase contrast, polarized light microscopy, and anti-tubulin immunochemistry. Standard immunochemical procedures were used with mouse monoclonal anti-ct-tubulin and anti-~-tubulin (Amersham Corp., Arlington Heights, IL) as described in Nicklas et al. (1989). Zeiss phase contrast optics were used: an inverted "Plankton" microscope was equipped with a 100x/1.3 NA (numerical aperture) Neofluar objective and an 0.9 NA long working distance condenser. For polarized light observations, a Nikon "M-stand" inverted microscope was used, equipped with a Nikon rectified 40x/0.65 NA achromatic objective and a rectified 0.52 NA long working distance condenser. The polarizers, light source, and heat and green filters were as described earlier (Nicklas, 1979). Unenhanced images were recorded on Kodak Tech Pan 35-mm film exposed at ASA 40 and processed in Kodak HC410 developer, dilution B, for 12 min at 75"E Some spindles were observed by video-enhanced polarized light microscopy, using the procedures for microscope andvideo adjustment, monitor photography, etc., so well described by Inou6 (1986). A series 70 video camera (Dage-MTl, Wabash, MI) with a 1-inch Newvicon tube was used with an Image I-AT image processor (Universal Imaging Corp., Media, PA). Video images were processed as follows: 8 or 16 frames were summed to reduce noise, an out-of-focus background image was subtracted to cancel inhomogeneities in illumination, and contrast was optimized. Processed images were recorded on an optical memory disk recorder (model TQ-2021FCB; Panasonic Video Systems, Secaucus, N J). The only spindles of interest are those in which both the pole of the uncut half-spindle and the cut edge in the other half were clearly visible. In such spindles, measurements of distance and position were reproducible to within 0.25-0.5 ttm. Cutting spindles removes some of the usual bench marks for measurements. When one pole was cut away, everything was measured and plotted relative to the only available marker, the pole of the uncut halfspindle (defined by centrioles or the convergence of spindle birefringence). Images of focal levels in which the pole was not visible (e.g., Fig. 1, upper row) were aligned to an image showing the pole at nearly the same time (within a minute). The two images (one at the level of the pole and one at another focal level) were aligned using either the cut edge or other markers, if the spindle had not moved. Measuring and plotting everything relative to the centrioles of the uncut half-spindle has a peculiar effect when spindle elongation is present. Only the chromosomes in the cut half-spindle appear to be moved by spindle elongation (e.g., Fig. 2). Actually, of course, elongation moves the chromosomes in the two spindle halves an equal distance away from the center of the spindle. This plotting artifact is easily recognized and corrected and has no effect on the measurements reported below. As is conventional, distances in the graphs are marked off relative to the equator (e.g., Fig. 2), with the pole at a constant distance from the equator. The pole-to-equator distance was measured soon after the spindle was cut; the equator was defined as a line midway between the separating kinetochores.