The poleward motion of a chromosome during mitosis or meiosis coincides with the shortening of its associated kinetochore fiber microtubules. Recent investigations on this motion have focused on determining the site(s) where kinetochore microtubule disassembly occurs, as well as on how the force for motion is generated. Two general models have arisen from these studies. In the “Pac-man” model, the kinetochore powers chromosome poleward motion, which occurs along kinetochore microtubules that shorten by subunit removal at the kinetochore (reviewed in Rieder and Salmon, 1994 ). In this model kinetochore-associated minus end-directed motors, such as cytoplasmic dynein, are envisioned to provide the force for chromosome movement, although it could also be generated by the disassembly of kinetochore microtubule plus ends within the kinetochore (Inoue and Salmon, 1995 ). Such a model is supported by the facts that dynein is present at kinetochores (Pfarr et al., 1990 ; Steurer et al., 1990 ; reviewed in Hoffman et al., 2001 ) and that its depletion attenuates the rate of poleward chromosome motion (Savoian et al., 2000 ; Sharp et al., 2000 ).

Alternatively, in the “traction fiber” model, the chromosome is dragged poleward by the poleward motion of its associated kinetochore microtubules that shorten by subunit removal at the pole (reviewed in Pickett-Heaps et al., 1996 ). In this model, force production is envisioned to occur, for example, as plus end-directed motors anchored within the spindle matrix interact with and push all spindle microtubules poleward (Mitchison and Sawin, 1990 ; Sawin and Mitchison, 1991 ). This model is supported by microinjection (Mitchison et al., 1986 ) and photoactivation studies (Mitchison, 1989 ), which reveal a “flux” of tubulin subunits that are constantly incorporated before anaphase into the plus ends of microtubules while being removed from their minus ends within the pole. The flux mechanism exerts a poleward force on the chromosome when subunit incorporation at the kinetochore ceases, as occurs at anaphase onset (Waters et al., 1998 ).

The relative contribution that each of these mechanisms makes to the poleward motion of a chromosome appears to depend on the system. The rate that kinetochore microtubules move poleward in spindles formed in Xenopus oocyte extracts is the same as the rate exhibited by the chromosomes at anaphase (Desai et al., 1998 ). This suggests that poleward motion in this in vitro system is powered entirely by flux. In contrast, in vertebrate somatic cells (Mitchison and Salmon, 1992 ; Zhai et al., 1995 ), both mechanisms appear to operate simultaneously, but the contribution made by flux is much less (~15–35%) than that made by the poleward movement of kinetochores.

In addition to those forces that act on kinetochores, the chromosome arms are also subjected to spindle-mediated forces throughout the division process. In vertebrate somatic cells, when the arm of a prometaphase chromosome positioned near a pole is severed from the kinetochore, it is ejected away from the pole (reviewed in Rieder and Salmon, 1994 ). The “polar wind” propelling that motion appears to be mediated by plus end-directed motors associated with the chromosome arms (i.e., chromokinesin; Antonio et al., 2000 ; Funabiki and Murray, 2000 ). In contrast, when a pole-directed arm of a metaphase chromosome during plant (Hemanthus) mitosis is similarly severed from its kinetochore, it is transported poleward at the same velocity exhibited by chromosomes during anaphase (Khodjakov et al., 1996 ). The force-producing mechanism behind this motion remains to be determined, but candidates include microtubule flux or chromosome-associated minus end-directed motors.

Insect spermatocytes have long been a popular system for studying the forces that move and position chromosomes. In crane fly spermatocytes, chromosome arms sometimes become aligned parallel to the spindle long axis during spindle formation, and maintain this alignment throughout anaphase (Adames and Forer, 1996 ). This suggests that in insect spermatocytes, as in plant mitosis, poleward forces act along the length of the chromosome independent of those acting on the kinetochore. To directly test this hypothesis we used laser microsurgery to sever chromosome fragments lacking kinetochores (i.e., acentric fragments) from pole-directed arms. As predicted, these fragments were invariably transported poleward at a velocity (~0.5 μm/min) similar to that exhibited by the kinetochore regions on anaphase chromosomes. We then investigated the mechanism responsible for this motion by repeating our experiments on cells treated with paclitaxel (taxol), a drug that inhibits microtubule flux (Derry et al., 1995 ; Waters et al., 1996 ) but not microtubule-dependent motor activity (Vale et al., 1985 ). In taxol, the poleward movement of acentric fragments was dramatically inhibited: <10% of the fragments generated during metaphase exhibited motion, and in those that did, velocity was greatly attenuated. From these findings we conclude that the poleward force that acts on the chromosome arms in these spermatocytes is generated by microtubule flux and not by molecular motors associated with the chromosome. Furthermore, because taxol treatment similarly attenuated the velocity of poleward chromosome motion during anaphase, it also is mediated largely by flux, as originally suggested by Wilson et al. (1994) .

Begg and Ellis, 1979

Janicke and LaFountain, 1986

Taxol (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at −20°C. For treatment of spermatocytes, the above-mentioned stock solution was diluted in TI buffer to obtain the desired concentration (100 nM-50 μM), and isolated testes were incubated in the various dilutions for 15 min to 1 h. During that incubation, taxol was taken up into the testicular fluid surrounding spermatocytes, as well as by spermatocytes that were suspended in that fluid. The concentration of taxol in the testicular fluid was not known; however, the effect of taxol on spermatocytes was evident in the taxol phenotype (see RESULTS) that was achieved. After incubation in taxol-TI buffer, testes were then ruptured under oil for microscopy. Under oil, spermatocytes remained suspended in testicular fluid that contained taxol. Typically, it took 0.5–2 h after cells were prepared for microscopy to find a cell suitable for either microsurgery or analysis of anaphase velocities. Thus, in some cases, results were obtained from cells that had spindles before taxol exposure, and they shortened during exposure. In other cells, nuclear envelope breakdown occurred during exposure to taxol and thus, short spindles were assembled in the presence of taxol. Microsurgical operations were performed on both types, and similar results were obtained

LaFountain et al., 1999

Oldenbourg and Mei (1995)

Figure 4 Taxol treatment induces the formation of short, broad spindles, but taxol does not inhibit anaphase onset or prevent the subsequent poleward motion of chromosomes. (A–D) Differential interference contrast micrographs of living DMSO (0.05%) (more ...)

Cole et al., 1995

Khodjakov et al., 1996

Digital images were captured on the laser LM workstation with the use of a Micromax charge-coupled device camera (RSP Princeton Instruments, Trenton, NJ), at 2 frames/min, and the illumination was shuttered between framing intervals. Time-lapse sequences were processed and stored as TIFF files on the hard drive with the use of ImagePro software (Media Cybernetics, Silver Springs, MD) running on a PC. They were then imported into Image J for movie making and further analyses.

Among males of N. suturalis, the karyotype includes three pairs of metacentric autosomes and two small telocentric sex chromosomes (X and Y). The autosomes pair into three bivalents for meiosis; sex chromosome behavior during meiosis is complicated. X and Y initially pair but then precociously separate into univalents for meiosis I; sex chromosomes behave normally as dyads during meiosis II. The spindle in spermatocytes is well defined, outlined by a sheath, or mantle, of aligned mitochondria (LaFountain, 1972 ).

Adames and Forer, 1996

Figure 1 Chromosome fragments lacking kinetochores are transported poleward. Selected frames from a time-lapse recording of a secondary (meiosis II) spermatocyte in which one of the noncoherent pole-directed arms of a dyad (B, large arrowhead) was severed (B) (more ...)

Figure 2 Chromosome fragments lacking kinetochores are transported poleward. (A–D) Selected frames from a time-lapse recording of a primary (meiosis I) spermatocyte in which a pole-directed arm (A, large arrowhead) was severed (B) from the kinetochore (more ...)

Table 1 Comparison of spindle organization, transport velocities, and the duration of meiosis 1 in control and taxol-treated spermatocytes

A thin linear ribbon of highly refractive material is generated in the cutting plane as the specimen is translated slowly through the focused laser beam (Figure 1B and 2B). These “sniglets” (Cole et al., 1995 ) can be formed at will, anywhere within the spindle or cell, and presumably consist of material denatured by the laser pulses. As chromosome arms were severed, conspicuous sniglets were formed, and these were also always transported poleward with a velocity similar to that exhibited by the adjacent acentric fragment (Figures 1, C–E, and 2, C and D, arrows; Table 1). When sniglets were formed by irradiating a region of the half-spindle that lacked chromosomes, they were also transported poleward with a velocity similar to that exhibited by acentric fragments and anaphase chromosomes (our unpublished data).

When acentric chromosome fragments were generated at metaphase in a fully formed spindle, they moved in a linear manner into the proximal pole (Figures 1, B–E, and 2, C and D). When generated in early- to mid-prometaphase cells, fragments frequently exhibited a gradual lateral displacement toward the sheath of mitochondria surrounding the spindle as they moved poleward (Figure 3). Thus, in addition to experiencing pole-directed forces, during spindle formation the chromosomes are also subjected to forces directed perpendicular to the spindle long axis that tend to eliminate them laterally from the central domain of the spindle. These so-called “transverse equilibrium forces” were originally described by Östergren (1945) , and likely represent the tendency of highly ordered dynamic microtubule arrays to sterically eliminate larger inclusions as they form (see examples in Tucker, 1977 ).

Figure 3 As the spindle forms during meiosis I and II, acentric fragments also experience a transverse force that eliminates them to the spindle periphery even as they are moving poleward. (A–D) Mid-prometaphase II cell in which a fragment, originally (more ...)

Snyder and Mullins, 1993

Waters et al., 1998

Table 2 Comparison of retardance magnitude in three spindle domains of control and taxol-treated spermatocytes

The effect of taxol on distribution of microtubules in the spindle was best resolved with polarized light (Figure 4, E–H). For the interpretation of images made with polarized light, white (maximal brightness) represents maximal retardance or birefringence; black represents no retardance or the absence of birefringence. Our images demonstrated that the taxol-induced broadening of the spindle poles correlated with greatly increased numbers and densities of microtubules that extended short distances (~2–3 μm) from the poles toward the equator. Microtubule densities in subpolar and equatorial domains after taxol treatment, however, did not appear to be different from those of controls. Spindle structure in those regions was especially important to this study. If taxol treatment had greatly altered the distribution of microtubules in those domains into which fragments were released after they had been severed from their chromosomes then any interpretation of fragment behavior would have to take those alterations into account. Because with the instrumentation we used the retardance magnitude within a given domain of the spindle is directly dependent on its microtubule number/density, we were able to quantify microtubules in those domains based on their retardance. We quantified retardance two ways: 1) within 0.55-μm² areas that were made within regions of interest (Figure 4, E and F), and 2) from line scans that were made along planes of interest (Figure 4, G and H). Taking those approaches, we found that retardance in central spindle domains (in the vicinities of chromosomes that could have been cut had we been performing operations) in taxol-treated spindles was not significantly different (Student's t test, p = 0.19; differences were regarded significant at p < .001) from those in control spindles (Table 2). Our quantitative analysis revealed that retardance at spindle poles was clearly increased after taxol treatment, and a taxol effect also was manifested in reduced retardance of kinetochore fibers (Table 2). The latter suggests there are fewer microtubules per kinetochore in taxol, an effect also apparent from the data presented by Wilson and Forer (1997) . The cause of this effect of taxol on kinetochore fibers is not known. One of the reviewers raised the possibility that the birefringence of kinetochore fibers in taxol is due to fewer than normal associated nonkinetochore microtubules in kinetochore fibers (Wise et al., 1991 ). Data needed to confirm that, however, will require serial section electron microscopic analysis, such as that performed on untreated and cold-treated spermatocytes by Scarcello et al. (1986) .

A final point regarding taxol effects on spermatocytes is that the doses of taxol that were effective in producing the taxol phenotype did not prevent entry into, or progression through, anaphase. For similar effects on spermatocytes from Drosophila, see Savoian et al. (2000) . This was true for both meiosis I and II, but to obtain quantitative data on these points, analysis was restricted to spermatocytes in meiosis I. In the 83 untreated cells that we monitored, it took ~83 min on average to reach anaphase I onset after the breakdown of the nuclear envelope (NEB) at the end of diakinesis (Table 1). Spermatocytes from testes that had been incubated in 5 or 10 μM taxol for 15 min (see MATERIALS AND METHODS) progressed through meiosis in taxol, and the duration between NEB to anaphase I onset lasted somewhat longer, averaging 112 min over a range between 78 and 166 min (Table 1), yet in all 167 cells analyzed, the onset of anaphase was not prevented. In the time between NEB and anaphase, events usually seen in untreated cells, including congression of autosomes to the equator and metakinetic movements of sex univalents, were also observed in taxol-treated cells. We analyzed anaphase I in taxol-treated cells and found that segregating half-bivalents exhibited very slow (average velocity = 0.1 μm/min; range 0.1–0.3 μm/min; n = 20) poleward motion (Table 1). This was true in the cases of spindles that existed before taxol exposure and then shortened during exposure, as well as spindles that were assembled in taxol. Because this velocity was significantly less (Student's t test, p < .0001) than in untreated spermatocytes (0.5 μm/min; see above), anaphase A in taxol-shortened spindles lasted 30–40 min compared with 15 min in the longer spindles of controls (Figure 4, A–D).

The ultimate outcome of anaphase in the presence of taxol varied. In some cells chromosome poleward motion (anaphase A) was followed by elongation of the previously shortened spindle; those cells usually initiated, and sometimes completed, cytokinesis. In contrast, in other cells, the spindle poles moved progressively closer to one another during anaphase A, and this gradual collapse of the spindle inhibited the initiation of cytokinesis. It is noteworthy that neither congression nor anaphase was inhibited even when testes were incubated in 50 μM taxol for >30 min before spreading under oil.

Figure 5 Acentric fragments, generated near the spindle equator in taxol-treated spermatocytes, are not transported poleward as they are in control spermatocytes. (A–H) Selected frames from a time-lapse sequence of a taxol-treated primary spermatocyte (more ...)

The original goal of our study was to test the hypothesis that spindles in crane fly spermatocytes exert a pole-directed force on chromosomes independent of kinetochores. To do this we used laser microsurgery to sever the arms from metaphase chromosomes, between their kinetochore and telomere regions. We found that the resultant acentric fragments invariably moved poleward with a uniform velocity similar to that exhibited by kinetochores during anaphase. We also found that ribbons or sniglets of denatured material, generated anywhere within a half-spindle by laser irradiation, also moved poleward with the same kinetics. From these data we conclude that the production of kinetochore-independent, poleward forces is a general feature of crane fly spermatocyte half-spindles.

Mitchison, 1989

Waterman-Storer et al., 1998

Taxol rapidly inhibits microtubule flux within spindles. It does not affect the activity of microtubule-based motors (Vale et al., 1985 ), and thus it provided the means for distinguishing between possible flux-based and motor-based mechanisms of arm fragment transport. At low concentrations taxol preferentially inhibits microtubule plus end dynamics in vitro and in vivo, whereas at higher concentrations both plus and minus ends are affected (Jordan et al., 1993 ; Derry et al., 1995 ). When vertebrate somatic cells are treated with 10 μM taxol during metaphase, microtubule subunit incorporation at the kinetochores is inhibited well before removal at the poles (Waters et al. 1996 ). Because of this differential inhibition, the kinetochore microtubules shorten as subunits are lost at the poles, and the spindle shortens as the poles hold on to shortening microtubules attached to the chromosomes (Waters et al., 1996 ; Derry et al., 1998 ).

We found that the spermatocytes obtained from testes treated with 5 or 10 μM taxol for 15 min contained significantly shortened spindles characteristic of the taxol phenotype (Table 2; Wilson and Forer, 1997 ). When we generated acentric chromosome fragments near the spindle equator in these cells, they failed to move poleward, or they displayed significantly attenuated poleward motion (~0.1 μm/min vs. ~0.5 μm/min in controls). The two fragments that exhibited this motion were likely generated in spindles in which the effects of taxol had not yet been fully reached.

Taxol promotes microtubule assembly (Schiff et al., 1979 ), and it was possible that an increase in microtubule density within each half-spindle impeded the poleward motion of acentric fragments. To evaluate this, we used quantitative polarization microscopy to determine the density of microtubules within those areas of taxol-treated spindles where fragments were released by our cutting operations. We found that taxol treatment did not significantly increase the density of microtubules in those regions, although it did enhance microtubule density near the spindle poles.

From these results, we conclude that the force for transporting acentric chromosome fragments and the other material poleward in crane fly spermatocytes is produced by microtubule flux. Chromosome arms must simply become trapped by the dense arrays of microtubules in the half-spindle (LaFountain 1974 , 1976 ; Scarcello et al., 1986 ) and then are directed poleward as their surfaces interact with fluxing microtubules. This conclusion provides a ready explanation for why areas of reduced birefringence, created on crane fly spermatocyte kinetochore fibers by UV irradiation, move poleward (Forer, 1966 ). It also reveals that the transport properties of crane fly spermtocyte spindles are similar to metaphase spindles in plant endosperm (Khodjakov et al., 1996 ) but differ from those of animal somatic cells in which a polar wind is generated by microtubule plus end-directed motors associated with chromosome arms (Rieder and Salmon, 1994 ).

Wilson et al. (1994)

Wilson and Forer, 1989

Although the rate at which kinetochores move poleward during anaphase in taxol-treated spermatocytes is reduced by 80% (from 0.5 μm/min to only ~0.1 μm/min), the chromosomes invariably completed this migration. Why and how this occurs is unclear. It is possible that our taxol treatment did not eliminate flux. That is, it did not completely inhibit the incorporation of microtubule subunits at kinetochores and their removal at the poles. However, our observation that the majority of acentric fragments generated in metaphase cells failed to exhibit poleward motion suggests that in most cases flux is shut down completely by the time of anaphase onset. The idea that subunit incorporation into kinetochore microtubules is inhibited, but that some residual removal at the pole continues, is not consistent with our finding that taxol-treated spindles reached an equilibrium length after which they no longer shortened. In their study on the sites of microtubule disassembly during anaphase in crane fly spermatocytes, Wilson et al. (1994) concluded that although 80% of kinetochore fiber shortening occurs by subunit removal at the pole, 20% can be attributed to subunit removal at the kinetochore. Thus, it is possible that the stability of kinetochore microtubule plus ends is suddenly modified at anaphase onset in taxol-treated cells by, for example, the rapid inactivation of the CDK1 kinase, which then allows them to shorten by subunit removal at the kinetochore.

Our conclusion that anaphase chromosome motion in crane fly spermatocytes is driven primarily by flux differs from Nicklas' (1989) finding that in grasshopper spermatocytes the force for poleward chromosome motion during anaphase is generated at or near the kinetochores and that during this motion microtubules shorten primarily by subunit removal at kinetochores. Recent work on zw10 and rod mutants also suggests that the force for anaphase motion in Drosophila spermatocytes is generated primarily at the kinetochore (Savoian et al., 2000 ; Sharp et al., 2000 ). Together, these studies imply that the relative contribution that each (redundant) force-producing mechanism contributes to moving chromosomes poleward during meiosis in insect spermatocytes varies between organisms.

Our findings add to the growing body of evidence in support of the conclusion that both kinetochore-based and flux-based mechanisms exist and that the mechanism that is emphasized depends on the particular system. Although flux is a contributor in animal somatic cells, forces produced by kinetochore-associated motors, or disassembling microtubule plus ends, appear to dominate (Mitchison and Salmon, 1992 ; Zhai et al., 1995 ). Here we show that each of the two opposing half-spindles in crane fly spermatocytes are “flux machines” that transport kinetochores, acentric chromosome fragments, and other inclusions poleward as they adhere to the surfaces or plus ends of microtubules. These spindles are therefore similar to those formed in Xenopus oocyte extracts (Murray et al., 1996 ; Desai et al., 1998 ) in that the force for poleward chromosome motion is also produced by microtubule flux as kinetochore microtubules shorten by subunit removal at the pole.

In such flux machines, “slippage” must occur between the plus ends of kinetochore microtubules and the kinetochores during metaphase, when poleward motion is prevented by the cohesion of homologs (or sister chromatids). As the machine continues to flux, this slippage, which in vertebrate somatic cells produces a “neutral” kinetochore state (Khodjakov and Rieder, 1996 ), could still maintain the tension on the kinetochores needed to stabilize attachment to the spindle (Nicklas, 1997 ). Then, when the chromosomes disjoin at anaphase onset, the sudden decrease in tension could release the clutch on the opposing kinetochores, engage the gears (i.e., stop slippage), and allow the force produced by flux to move the chromosome poleward.

Although the molecular basis for flux is unknown, it has been proposed that microtubule plus end-directed motors, anchored within the spindle matrix, could push the microtubule lattice toward the spindle pole (Sawin and Mitchison, 1991 ). An actin/myosin system located within the kinetochore fiber and spindle matrix could act in a similar manner (Waterman-Storer and Salmon, 1997 ; Silverman-Gavrila and Forer, 2000 ). Regardless of the mechanism, the flux-mediated production of forces for kinetochore poleward motion is compatible with traction fiber models (reviewed by Hays and Salmon, 1990 ) for chromosome positioning. In this view chromosomes become aligned on the spindle equator because the opposing poleward “pulling” forces, acting on sister kinetochores, are proportional to the length of the kinetochore fibers. Challenges for the future will be to determine whether chromosome congression in flux machines is indeed mediated by traction fibers and how poleward forces that act on the chromosome arm influence this process.

We gratefully acknowledge the contributions made to this study by A. Khodjakov, G. Rickards, S. Inoué, K. LaFountain, D. LaFountain, and A. Siegel. We also thank the reviewers who recommended revisions that improved the final version of this report. This research was supported by grants from the National Science Foundation (MCB-9808290 to J.L.) and National Institutes of Health (GM-40198 to C.R. and GM-49210 to R.O). Much of the work was completed in the Video LM Core Facility of the Wadsworth Center.