Spindle fusion requires dynein-mediated sliding of oppositely oriented microtubules

Abstract

Background: Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts.

Results: When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole.

Conclusions: Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.

Figure 1. Parallel spindles with overlapped poles merge by sliding

Meiosis II arrested bipolar spindles were positioned using microneedles with their interpolar axes parallel and with overlapped proximal poles. Subsequent alignment and fusion was monitored using polarization optics and fluorescence microscopy (A). Directly labeled anti-NuMA antibodies were added to the extracts to mark the poles and DAPI was used to label the chromosomes at the metaphase plate. In this starting configuration, the majority of spindles fused by sliding parallel to the interpolar axis (A,B). In C, the position of the distal poles is plotted versus time. For all time courses, the zero time point represents the time the first image was acquired following spindle positioning. This typically occurred within a few seconds after moving the microneedles away from the spindles. The average velocity of alignment (the slope) is 1.2 ± 0.4 μm/min (n = 9 spindle pairs). For D, the same data as in C was used to determine the velocity as a function of percent overlap (i.e. 100 × overlap/ average initial spindle length). Values were obtained by averaging the slopes of linear regressions for sliding that occurred during the fraction of overlap indicated. Analysis using a pairwise Student's t-test indicated no statistically significant differences between the velocities (all p > 0.05). All scale bars represent 25 μm.

Figure 2. Perpendicular spindles merge by “jackknifing”

Spindles positioned with perpendicular interpolar axes were monitored as in Figure 1. “T-boned” spindles also align, but in the absence of antiparallel microtubule overlap, the proximal poles come together as the spindles rotate, ultimately creating a single, fusiform bipole (A,B). Graph in B shows the fate of the proximal poles plotted by the initial spacing between them. In almost all cases, the two nearest poles moved together and fused. All scale bars are 25μm.

Figure 3. Peripheral microtubules facilitate spindle alignment and fusion through a distance

High resolution confocal imaging of rhodamine-tubulin labeled spindles reveals numerous peripheral microtubules extending beyond the circumference of the main spindle body, A. In B, complementary studies using fluorescently labeled EB-1 (125 nM) added to pre-assembled spindles to visualize the dynamics of growing microtubule ends also show peripheral microtubule extending beyond the margins of the spindle. The image shown in B represents a single frame from a time-lapse series spanning ~ 2 minutes (result is typical of the n = 20 spindles observed). The centers of overlayed circles mark the most distal positions of EB1 trajectories observed during imaging whereas the arrows indicate the direction of EB1 comet trajectories. Colors indicate from which pole the growing microtubule likely emanated from. This was determined by assuming a linear trajectory for each EB1 comet and finding the point where it intersected a line running through both spindle poles (i.e. the extended interpolar axis). The pole closest to this point was assumed to be the originating pole. In some cases, the comet trajectories did not intersect with the extended interpolar axis, so we assumed the comet originated from the nearest pole. The distances from the poles to each EB1 comet were plotted against the angle of the comet trajectory to the horizontal, with 0° oriented on the horizontal extending behind the pole and 180° on the horizontal toward the metaphase plate (C). To test whether spindles could merge through a distance, spindle pairs were positioned on the same axis with separation between their proximal poles (D). In most cases, the spindles first came together, fused at their proximal poles, and then pivoted or “jackknifed” around the shared pole (D, E). Scale bars = 25μm.

Figure 4. Dynein is required for bipolar spindle alignment and fusion

Polarization images of spindles pairs positioned with their proximal poles overlapped and their interpolar axes parallel to one another. Shown in A are images of bipolar fusion from a time-lapse series following addition of 25 μM vanadate. In B, antibodies to dynein intermediate chain (final concentration ~1.0 mgml^-1) were added 5-10 minutes prior to the start of imaging. Notice that spindle alignment and fusion fail under both experimental conditions. To confirm the requirement for dynein, the ability of micromanipulated spindles to fuse was assayed following assembly in immunodepleted extracts containing less than 2.5% of the endogenous protein (C; see Supplemental Data, Figure S2). In these assays, pairs of bipolar microtubule arrays, which lacked focused poles as expected, were positioned initially with their long axes parallel to one another and with overlapped birefringence. All perturbations of cytoplasmic dynein function completely blocked spindle fusion. In contrast, spindles assembled in XCTK2-depleted extracts still fused (D). Scale bars represent 25 μm.

Figure 5. The fusion of Eg5-inhibited monopoles is mediated by dynein-dependent sliding of antiparallel microtubules

Following spindle assembly, Xenopus egg extracts were treated with the Eg5-specific inhibitors STLC (25μM) or monastrol (100μM). Poles were labeled with anti-NuMA antibodies conjugated to AlexaFluor 488 to facilitate automated pole tracking. Fusion was monitored using polarization optics and fluorescence microscopy. A, three examples of Eg5-inhibited monopole fusion demonstrating varying degrees of increased interpolar birefringence observed during fusion (total n > 20 pairs). B, addition of 25μM vanadate (~5-10min prior to imaging) inhibited monaster fusion. For each experimental condition, the interpolar distance between anti-NuMA labeled poles was plotted as a function of time (C). In these plots, the initial separations between monopole pairs are equal to the interpolar distance values at time t = 0. D, growing microtubule plus ends were visualized by addition of 125 nM AlexaFluor 488-labeled EB1 and recorded using confocal microscopy. The image is a single frame selected from a ~2min time lapse recording overlayed with EB1 tracks (n = 1396 tracks with a minimum lifetime of 4 consecutive frames). The histogram shows the distribution of radial distances from the monopole center (see Materials and Methods) to the distal end of each EB1 track. In E, Eg5-inhibited monopoles were spun down on coverslips following fixation as described in [48]. The monopoles were then processed for immunofluorescence microscopy using antibodies against tubulin (green) and the 70.1/74 kDa intermediate chain of dynein (red). The extent and dynamics of microtubule plus ends during monopole fusion were assessed by labeling growing microtubule ends by adding 125nM GFP-EB1 to assembled monopoles (F and G; see Movie 2). The images in F (shown in inverted contrast) are from a time-lapse series of two monopoles, marked by asterisks, at the initial stages of interaction (a third monopole is out of the field towards the lower right). Microtubule density increases in a region half-way between the poles. G, a kymograph of the linear region between the two arrowheads in F is used to show movements of EB1-labeled ends toward the interpolar axis (asterisks mark the approximate position of the poles). All scale bars represent a length of 25μm, except for the scale bar in panel G, which is equivalent to 10μm.

Figure 6. A model for bipolar spindle fusion

A, a cartoon schematic showing ways in which dynein-dependent sliding of antiparallel, peripheral microtubules might occur. These two models differ in that the first relies on plus end growth to license the dynein motor, permitting crosslinking only near growing plus ends. Whereas in the second, dynein crosslinking is independent of plus end growth and can occur anywhere along the length of the microtubule. B, dynein-dependent sliding of oppositely oriented, peripheral microtubules extending from each pole can account for both modes of bipolar spindle merging. For the sliding mode, we predict that the sum of forces generated between the outer pole of one spindle and inner pole of the other is greater than the attractive force between the two inner poles. Eg5-dependent sliding in regions of antiparallel overlap between spindle microtubules (highlighted in yellow) might antagonize the dynein-mediated pulling forces, contributing to proximal pole separation. The jackknifing mode is more complex as it requires the generation of torque. In each of our “T-bone” experiments, the two nearest poles moved together first, likely due to F_proximal being greater than F_distal. However, this imbalance would not be expected to generate the torque required for the observed “jackknifing”. We propose that the initial geometry creates a mechanical advantage (i.e. longer lever arm) for F_distal-outer thereby generating a torque that favors a rotation in the counter-clockwise direction as drawn.