The mechanism of anaphase spindle elongation

At anaphase chromosomes move to the spindle poles (anaphase A) and the spindle poles move apart (anaphase B). In vitro studies using isolated diatom spindles demonstrate that the primary mechanochemical event responsible for spindle elongation is the sliding apart of half-spindle microtubules. Further, these forces are generated within the zone of microtubule overlap in the spindle mid-zone.     

Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle

During mitosis a monooriented chromosome oscillates toward and away from its associated spindle pole and may be positioned many micrometers from the pole at the time of anaphase. We tested the hypothesis of Pickett-Heaps et al. (Pickett-Heaps, J. D., D. H. Tippit, and K. R. Porter, 1982, Cell, 29:729-744) that this behavior is generated by the sister kinetochores of a chromosome interacting with, and moving in opposite direction along, the same set of polar microtubules. When the sister chromatids of a monooriented chromosome split at the onset of anaphase in newt lung cells, the proximal chromatid remains stationary or moves closer to the pole, with the kinetochore leading. During this time the distal chromatid moves a variable distance radially away from the pole, with one or both chromatid arms leading. Subsequent electron microscopy of these cells revealed that the kinetochore on the distal chromatid is free of microtubules. These results suggest that the distal kinetochore is not involved in the positioning of a monooriented chromosome relative to the spindle pole or in its oscillatory movements. To test this conclusion we used laser microsurgery to create monooriented chromosomes containing one kinetochore. Correlative light and electron microscopy revealed that chromosomes containing one kinetochore continue to undergo normal oscillations. Additional observations on normal and laser-irradiated monooriented chromosomes indicated that the chromosome does not change shape, and that the kinetochore region is not deformed, during movement away from the pole. Thus movement away from the pole during an oscillation does not appear to arise from a push generated by the single pole-facing kinetochore fiber, as postulated (Bajer, A. S., 1982, J. Cell Biol., 93:33-48). When the chromatid arms of a monooriented chromosome are cut free of the kinetochore, they are immediately ejected radially outward from the spindle pole at a constant velocity of 2 micron/min. This ejection velocity is similar to that of the outward movement of an oscillating chromosome. We conclude that the oscillations of a monooriented chromosome and its position relative to the spindle pole result from an imbalance between poleward pulling forces acting at the proximal kinetochore and an ejection force acting along the chromosome, which is generated within the aster and half-spindle.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.     

Mitotic apparatus formation and cleavage induction by micromanipulation of the nucleus and centrosome: The centrosome forms a spindle together with only the chromosomes at a short distance

We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

Sites of microtubule assembly and disassembly in the mitotic spindle

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.     

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.     

Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.     

Micromanipulation of mitotic chromosomes in PTK2 cells using laser-induced optical forces ("optical tweezers")

To study the potential use of optical forces to manipulate chromosome movement, we have used a Nd:YAG laser at a wavelength of 1.06 microns focused into a phase contrast microscope. Metaphase and anaphase chromosomes were exposed while being monitored by video microscopy. The results indicated that when optical forces were applied to late-moving metaphase chromosomes on the side closest to the nearest spindle pole, the trapped chromosomes initiated movement to the metaphase plate. The chromosome velocities were two to eight times the normal rate depending on the chromosome size, geometry, and trapping site. At the initiation of anaphase, a pair of chromatids could be held by the optical trap and kept motionless throughout anaphase while the other pairs of chromatids separated and moved to opposite spindle poles. As a result, the trapped chromosome either was incorporated into one of the daughter cells or was lost in the cleavage furrow, or the two chromatids eventually separated and moved to their respective daughter cells. If the trap was removed at the beginning of anaphase B, the chromosome moved back to the poles. Our experiments demonstrate that the laser-induced optical force trap is a potential new technique to study noninvasively the mitotic spindle of living cells.     

Chromosome movement and spindle birefringence in locally heated cells: Interaction versus local control

A microheater was used to produce a temperature gradient within the mitotic spindle of living cells. The slope of the temperature gradient was estimated from thermal conductivity calculations and confirmed by measurements of spindle birefringence and by experiments on striated muscle. When the microheater was placed at one spindle pole or at one group of kinetochores, the gradient was steep enough to cause a large difference in birefringence between the two half-spindles, but the velocity of chromosome movement in anaphase was nearly the same in the warmer and cooler half-spindles. When the heater was shifted from the pole toward the interzone, the average velocity of chromosome movement increased approximately two-fold but was, again, nearly uniform in the two half-spindles. The rate of spindle elongation was especially sensitive to the site of heating, increasing ten-fold when the heater was shifted from the pole to the interzone. Regardless of heater position, the rate of chromosome movement was determined largely by the temperature of the coolest spindle region —chromosomes in the warmer half-spindle moved more slowly than expected from estimates of the temperature in that region. Since the microheater produces a substantial temperature gradient within the spindle, the near uniformity of chromosome velocity in both half-spindles must be due to some biological property of the spindle. Two very different explanations for the results are considered the most likely. According to one explanation, the near uniformity of velocity in both half-spindles is determined by the structure of the interpolar spindle, while changes in velocity involve force producers located both in the half-spindles and in the interzone. On the other explanation, the velocity is nearly the same in both half-spindles because the force producers are located exclusively in the interzone (Margolis et al., 1978).This paper is dedicated to Professor Sally Hughes-Schrader with admiration and affection     

Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase. The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase. Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole. The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis. After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers. There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form. In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosome displays many features of a standard anaphase and may be followed by a false cleavage. The process of polar separation during prometaphase occurs without any visible interzonal structures. Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement. The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.     

Temperature dependence of anaphase chromosome velocity and microtubule depolymerization

The time course of chromosome movement and decay of half-spindle birefringence retardation in anaphase have been precisely determined in the endosperm cell of a plant Tilia americana and in the egg of an animal Asterias forbesi. For each species, the anaphase retardation decay rate constant and chromosome velocity are similar exponential functions of temperature. Over the temperature range at which these cells can complete anaphase, chromosome velocity and retardation rate constant yield a positive linear relationship when plotted against each other. At the higher temperatures where the chromosomes move faster, the spindle retardation decays faster, even though the absolute spindle retardation is greater. Chromosome velocity thus parallels the anaphase spindle retardation decay rate, or rate of spindle microtubule depolymerization, rather than absolute spindle retardation, or the amount of microtubules in the spindle. These observations suggest that a common mechanism exists for mitosis in plant and animal cells. The rate of anaphase chromosome movement is associated with an apparent first-order process of spindle fiber disassembly. This process irreversibly prevents spindle fiber subunits from participating in the polymerization equilibrium and removes microtubular subunits from chromosomal spindle fibers.     

Cell division and the microtubular cytoskeleton]

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.     

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.     

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.     

Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.     

Chromosome micromanipulation

Two types of unusual motion within the spindle have heen studied in a grasshopper (Melanoplus differentialis) spermatocyte. The first is the motion of granules placed by micromanipulation within the normally granule-free spindle. The most specific motions are poleward, approximate the speed of the chromosomes in anaphase, and occur in the area between the kinetochores and the nearer pole during both metaphase and anaphase. Exactly the same transport properties were earlier observed by Bajer inHaemanthus endosperm spindles. The absence of significant motion in the interzone between the separating chromosomes at anaphase has been unequivocally demonstrated inMelanoplus spermatocytes. Thus very specific motion of non-kinetochoric materials is probably a general spindle capability which would much restrict admissible models of mitotic force production,if the same forces move both granules and chromosomes. The second unusual motion is seen following chromosome detachment from the spindle by micromanipulation during anaphase. These tend to move toNearer pole rather than to the pole the chromosome's kinetochoresFace. The latter preference was earlier demonstrated after detachment during prometaphase or metaphase and has been confirmed without exception in the present studies. The apparent preference for motion to the nearer pole in anaphase provides the first evidence for poleward forces within each half-spindle which cannot be entirely specified by the chromosomal spindle fibers. Almost certainly these would be the usual forces responsible for chromosome motion since they act specifically at the kinetochores of detached chromosomes. This evidence requires interpretation, however because additional factors influence chromosome motion following detachment at anaphase. On thesimplest interpretation, certain current models of mitosis clearly are not satisfactory and others are favored.     

Mitotic motors and chromosome segregation: the mechanism of anaphase B

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an 'on/off' switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned 'off', tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.     

Polar ejection forces are operative in crane-fly spermatocytes, but their action is limited to the spindle periphery.

Laser microsurgery was employed to reveal kinetochore-independent forces acting on chromosome arms in crane-fly spermatocytes. When a portion of an arm situated along the interpolar axis between the equator and a pole was cut off, the resultant acentric fragment was transported poleward and outward into the peripheral domain of the spindle. If the fragment was generated well in advance of the onset of anaphase, then at the spindle periphery, it changed direction and moved away from the pole and back toward the equator. That domain-specific movement-poleward in the central spindle and away from the pole at the spindle periphery-not only provides the first evidence for polar ejection forces acting on acentric fragments in a meiotic system, but it is the first example of kinetochore-independent forces in both directions at the same stage of division. Sniglets generated by laser pulses directed at specific sites in the spindle revealed that the mechanism underlying ejection forces was specific to chromosomes. At anaphase onset, polar ejection forces ceased, and pole-directed forces took over. At that time, chromosome fragments that had been ejected to the equator moved poleward again, providing clear evidence for kinetochore-independent forces on chromosome arms during anaphase.     

Reverse engineering of force integration during mitosis in the Drosophila embryo

The mitotic spindle is a complex macromolecular machine that coordinates accurate chromosome segregation. The spindle accomplishes its function using forces generated by microtubules (MTs) and multiple molecular motors, but how these forces are integrated remains unclear, since the temporal activation profiles and the mechanical characteristics of the relevant motors are largely unknown. Here, we developed a computational search algorithm that uses experimental measurements to ‘reverse engineer’ molecular mechanical machines. Our algorithm uses measurements of length time series for wild‐type and experimentally perturbed spindles to identify mechanistic models for coordination of the mitotic force generators in Drosophila embryo spindles. The search eliminated thousands of possible models and identified six distinct strategies for MT–motor integration that agree with available data. Many features of these six predicted strategies are conserved, including a persistent kinesin‐5‐driven sliding filament mechanism combined with the anaphase B‐specific inhibition of a kinesin‐13 MT depolymerase on spindle poles. Such conserved features allow predictions of force–velocity characteristics and activation–deactivation profiles of key mitotic motors. Identified differences among the six predicted strategies regarding the mechanisms of prometaphase and anaphase spindle elongation suggest future experiments.