Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

INTRODUCTION

Chromosome segregation is the essential biological processes in which chromosomes are partitioned into the two daughter cells during cell division. In eukaryotes, chromosome segregation is carried out by the spindle, which is predominately composed of microtubules. Chromosomes first align in the middle of the spindle and are then segregated in anaphase. It is often the case that chromosome segregation is accompanied by an elongation of the spindle. In many systems, the speed of spindle elongation is slower than chromosome segregation, such that chromosomes move closer to spindle poles over the course of anaphase. The movement of the chromosomes toward spindle poles is referred to as anaphase A; the elongation of the spindle (i.e., the separation of spindle poles) is referred to as anaphase B. The motion of anaphase A and anaphase B has been thought to depend on different behaviors of microtubules at different parts of the spindle (Scholey et al., 2016; Asbury, 2017).

The movement and turnover of spindle microtubules in anaphase have been measured with a variety of techniques, including photobleaching (Mallavarapu et al., 1999; Khodjakov et al., 2004; Redemann et al., 2017), photoconversion/photoactivation (Mitchison and Salmon, 1992; Ferenz et al., 2010; Vukusic et al., 2017), and fluorescence speckle microscopy (Brust-Mascher et al., 2004; Cameron et al., 2006; Pereira et al., 2016). Many previous studies have focused on characterizing kinetochore microtubule dynamics, providing great insights into the mechanisms of spindle assembly (Mitchison, 1989; Maiato et al., 2005; Cameron et al., 2006; Ma et al., 2010; Redemann et al., 2017) and the poleward chromosome motion in anaphase A and anaphase B (Mitchison and Salmon, 1992; Brust-Mascher et al., 2004; LaFountain et al., 2004). The behaviors of microtubules in the central-spindle, between segregating chromosomes, have been less thoroughly characterized than kinetochore microtubules. It has been shown that microtubules of the central-spindle slide apart while spindles elongate in diatoms, yeasts, flies, and human cells (Saxton and McIntosh, 1987; Masuda et al., 1988; Mallavarapu et al., 1999; Scholey et al., 2016; Vukusic et al., 2017). However, we are aware of few studies that simultaneously measure the speeds of central-spindle microtubules and chromosomes (Vukusic et al., 2017) during anaphase A and anaphase B. Such data are potentially interesting, as they might provide further insight into the role of central-spindle microtubules in chromosome segregation. Formally, there are three possibilities: central-spindle microtubules might move slower, faster, or at the same speed as segregating chromosomes (Figure 1).

While simultaneous observation of microtubule and chromosome dynamics can reveal whether their motions are correlated, perturbing spindle microtubules provides a means to test the extent to which their motions are coupled to the motion of chromosomes. A number of groups have used laser ablation to cut selected populations of microtubules and concluded that, in some anaphase spindles, central-spindle microtubules resist pulling forces from astral microtubules (Aist and Berns, 1981; Aist and Bayles, 1991; Aist et al., 1993; Kronebusch and Borisy, 1981; Grill et al., 2001); in some anaphase spindles, central-spindle microtubules exert pushing forces that contribute to anaphase chromosome motion (Khodjakov et al., 2004; Nahaboo et al., 2015; Laband et al., 2017; Vukusic et al., 2017). However, perturbation experiments must always be interpreted with care. One complication is the potential for redundant mechanisms. Thus, the observation that chromosome segregation can proceed without a connection to spindle poles in many systems (Nicklas, 1989; Khodjakov et al., 2004; Elting et al., 2014; Nahaboo et al., 2015; Vukusic et al., 2017) is equally consistent with two different explanations: that a loss of connection to the pole can be compensated by alternative processes, or that pole connections minimally contribute to chromosome motion. Another concern with any perturbation experiment is the possibility of collateral damage/off-target effects. These concerns can be minimized (but not eliminated) by focusing on the immediate response to a rapid perturbation using a technique with a highly localized impact. In this regard, laser ablation with femtosecond pulses is advantageous, as cuts can be performed in seconds with ∼300 nm resolution with no discernible impact outside of the ablated region (Konig et al., 1999; Schaffer et al., 2001a,b; Watanabe et al., 2004; Shen et al., 2005; Supatto et al., 2005; Vogel et al., 2005; Chung et al., 2006; Gattass and Mazur, 2008; Vitek et al., 2010), unlike ablation with other lasers which can produce a significant amount of denatured protein (Brenner et al., 1980; Khodjakov et al., 1997, 2000).

Light microscopy has insufficient resolution to resolve individual microtubules in spindles. Thus, a detailed study of spindle architecture requires alternative techniques, such as electron microscopy (Muller-Reichert et al., 2018). The structure of central-spindle microtubules has been studied using electron microscopy in a number of spindles, which has revealed different structures in different spindles. In yeast and diatom spindles, central-spindle microtubules span from the middle of the spindle to the poles (McDonald et al., 1977, 1979; Ding et al., 1993; Winey et al., 1995, 2005). In contrast, in PtK1 mitotic spindles, many central-spindle microtubules have both ends between chromosomes, while other central-spindle microtubules extend from the interchromosomal region and terminate around kinetochore fibers (Mastronarde et al., 1993). In Caenorhabditis elegans female meiotic spindles, partial electron microscopy reconstruction revealed that many central-spindle microtubules have both ends between chromosomes, some of which appear to make end-on contacts to chromosomes (Laband et al., 2017; Redemann et al., 2018). These different structures make it unclear to what extent central-spindle microtubules are coupled to chromosomes in different spindles.

In this work, we investigated the relationship between central-spindle microtubules and chromosomes during anaphase in human mitotic spindles and C. elegans mitotic and female meiotic spindles. We used the same laser ablation and electron microscopy techniques on all of these spindles to avoid the potential complications of interpretation that can arise when different techniques are applied to different systems. We found that central-microtubules slide apart at the same speed as chromosomes, even when chromosomes move closer to poles. Damaging the central-spindle microtubules by laser ablation caused immediate and complete cessation of chromosome motion, even when anaphase A and anaphase B occur simultaneously, suggesting a strong coupling between central-spindle microtubules and chromosomes. Our electron tomographic reconstructions further reveal that these anaphase spindles all contain microtubules with both ends between segregating sister chromosomes and other central-spindle microtubules that terminate on either chromosomes or kinetochore fibers. Taken together, this work suggests that central-spindle microtubules are strongly coupled to chromosomes in anaphase spindles.

RESULTS

Central-spindle microtubules slide apart at the speed of chromosome motion in anaphase human mitotic spindles

We set out to simultaneously measure the relative motion of central-spindle microtubules and chromosomes by generating a stable human cell line expressing mEOS3.2::tubulin and GFP::CENP-A. This cell line allowed us to photoconvert a subset of labeled microtubules between chromosomes while simultaneously tracking the motion of kinetochores and poles. We focused on studying chromosome motion 30–60 s after the onset of anaphase. Tracking the motions of kinetochores and spindle poles reveals that, at this time, chromosomes move away from the center of the spindle with a speed of 2.7 ± 0.2 µm/min, while poles move away from the spindle center at a speed of 1.5 ± 0.1 µm/min (n = 8, p < 10^-4 compared with chromosome-to-spindle center moving speed), so the chromosome-to-pole distance decreases at a speed of 1.2 ± 0.2 µm/min (Figure 2A). Thus, our experiments probe a time window in which the speed of chromosome-to-pole motion is similar to the speed of pole-to-spindle center motion (p = 0.19), that is, anaphase A and anaphase B were both underway at this time. Next, a line of fluorescence-labeled microtubules across the spindle was photoconverted 30–60 s after the onset of anaphase. The single line of photoconverted tubulin split apart over time, demonstrating the sliding of central-spindle microtubules (Figure 2B, magenta; Supplemental Movie S1). The total fluorescence intensity of the photoconverted tubulin was tracked over time and was fitted well by a single exponential with a characteristic decay time of τ = 21 ± 0.3 s. Kymographs revealed that the speed of central-spindle microtubule sliding was similar to the speed of chromosome motion and substantially faster than pole motion (Figure 2C). To quantify the speed of the central-spindle microtubules, we took line profiles and tracked the motion of photoactivated regions (Figure 2D), in addition to chromosomes and poles (see Materials and Methods). We found that the photoconverted microtubules slide at a speed of 2.8 ± 0.1 µm/min (n = 46), which is indistinguishable from the speed of chromosome motion, 2.7 ± 0.1 µm/min, (n = 46, p = 0.85) and substantially greater than the speed of pole motion, 1.5 ± 0.1 µm/min (n = 46, p < 10^-11) (Figure 2D, right). This result shows that central-spindle microtubules and chromosomes move at the same speed in human mitotic anaphase spindles, even at times when anaphase A and anaphase B occur simultaneously, suggesting a coupling between them.

Laser-ablating central-spindle microtubules immediately stops anaphase chromosome motion in human mitotic spindles

We next sought to investigate to what extent central-spindle microtubules are coupled to chromosome motion in human mitotic spindles. We built a custom laser ablation system utilizing an ultrafast femtosecond laser, which enables subdiffraction-limited cuts (Vogel et al., 2005) in nearly arbitrary three-dimensional patterns. These cuts are performed within a few seconds and generate minimal collateral damage outside of the ablated region (see Materials and Methods). We performed a rectangular plane-cut, 12 µm in length by 6 µm in depth, perpendicular to the spindle axis between separating chromosomes 30–60 s after the onset of anaphase (Figure 3A, left; Supplemental Movie S2). This ablation led to an immediate cessation of chromosome motion, reducing its speed to 0.1 ± 0.3 µm/min (n = 6; indistinguishable from 0, p = 0.83). When the motion of chromosomes stopped in response to disrupting the central-spindle, the chromosomes-to-pole distance continued to decrease at a speed of 0.7 ± 0.2 µm/min (n = 14), indistinguishable from the speed of 0.8 ± 0.2 µm/min (n = 9) in uncut spindles (p = 0.71) (Supplemental Figure S1). After ∼20 s, chromosome segregation resumed with a speed similar to that of controls, presumably due to the microtubule regrowth, replacing the damaged population of microtubules (Figure 3A, right). In contrast, chromosome segregation continued (at a reduced rate) after ablating a region between chromosomes and poles (Supplemental Figure S2), while ablating an area in the cytoplasm did not impact the rate of chromosome motion (Supplemental Figure S3), which strongly argues that the observed pause in chromosome segregation after ablating the central-spindle is not a generic stress response to cellular damage from the laser. The finding that damaging the central-spindle in anaphase leads to the immediate cessation of chromosome motion further argues that central-spindle microtubules are strongly coupled to chromosome motion, even at times when anaphase A and anaphase B occur simultaneously.

Electron tomography reveals the presence of interchromosomal microtubules in human mitotic spindles

To further investigate how the central-spindle microtubules are coupled with chromosomes, we next studied the structure of human mitotic spindles in anaphase using large-scale electron tomographic reconstructions (Figure 3B, green). This work revealed the presence of interchromosomal microtubules: some of which had two free ends between chromosomes (Figure 3B, red), others of which had one end near the center of the spindle, with the other end contacting chromosomes (Figure 3B, orange). Other microtubules extended from the interchromosomal region, passed chromosomes, reaching a micron or so beyond them (Figure 3B, yellow). We found no microtubules, which extended all the way from the pole to the region between chromosomes, and no microtubules, which passed across the entire interchromosomal region, directly bridging microtubules between the two poles. The three classes of microtubules we observed are often tightly associated into bundles, which appear to connect interchromosomal microtubules to kinetochore microtubules, near chromosomes (Figure 3C). The microtubules from a single interchromosomal bundle spread out and associate with multiple kinetochore fibers, showing that there is not a simple one-to-one correspondence between interchromosomal bundles and kinetochore fibers. This structure illustrates how interchromosomal microtubules in the central-spindle region may cross-link to kinetochore microtubules, which thus would couple the motion of central-spindle microtubules to the motion of chromosomes. This structure of central-spindle microtubules is reminiscent of what was previously seen in PtK1 cells (Mastronarde et al., 1993).

Central-spindle microtubules slide apart at the speed of chromosome motion in C. elegans anaphase mitotic spindles

Having characterized dynamics, function, and structure of central-spindle microtubules in human mitotic spindles, we next explored whether central-spindle microtubules in other spindles have similar behavior and organization. We first set out to measure the relative motion of central-spindle microtubules and chromosomes in anaphase C. elegans mitotic spindles expressing GFP::tubulin and mCherry::histone. We were unable to reliably photobleach central-spindle microtubules in C. elegans mitotic spindles due to the rapid oscillations of these spindles in anaphase (Supplemental Movie S3). To better maintain the spindle in the focal plane, we knocked down GPR-1/2 using RNA interference (RNAi), which greatly reduces the cortically pulling forces (Supplemental Movie S4) (Grill et al., 2003; Srinivasan et al., 2003; Park and Rose, 2008). To understand the effect of gpr-1/2(RNAi) on chromosome segregation, we acquired time-lapse images of spindles for the first 3 min of anaphase in the first mitotic division of wild-type and gpr-1/2(RNAi) C. elegans embryos (Figure 4A; Supplemental Movies S3 and S4) and averaged the traces of pole motion, chromosome motion, and chromosome-to-pole motion (Figure 4B). In the wild-type embryos, the chromosome-to-pole distance stayed almost constant during this time (Figure 4B, top, orange) (Oegema et al., 2001), that is, the mean speed of chromosomes, 1.1 ± 0.02 µm/min, was indistinguishable from the mean speed of poles, 1.1 ± 0.05 µm/min (Figure 4C, top, n = 5, p = 0.93). In contrast, time-lapse microscopy of gpr-1/2(RNAi) embryos showed that while pole motion was significantly reduced, the speed and extent of chromosome motion remained very similar to that in controls (compare Figure 4A, top and bottom; Supplemental Movies S3 and S4). We averaged the traces of pole motion, chromosome motion, and chromosome-to-pole motion and found that the distance between chromosomes and poles continuously decreased over the course of anaphase (Figure 4B, bottom, orange), as the mean speed of poles, 0.45 ± 0.04 µm/min was significantly less than the mean speed of chromosomes, 0.90 ± 0.03 µm/min (Figure 4C, bottom, n = 8, p < 10^-6). Consistent with previous results (Nahaboo et al., 2015), this experiment demonstrates that cortically pulling forces are not required for chromosome segregation in C. elegans mitosis. Since GPR-1/2 knockdown greatly reduced spindle movement and only had a minor impact on chromosome motion, we used this RNAi condition to investigate the relative motion of chromosomes and central-spindle microtubules.

We conducted photobleaching experiments in gpr-1/2(RNAi) embryos 40–60 s after the onset of anaphase, when the chromosome-to-pole speed was 0.5 ± 0.07 µm/min (n = 7) and the pole-to-spindle center speed was 0.3 ± 0.05 µm/min (n = 7). Thus, at this time, the chromosome-to-pole distance shrank at a faster rate than the poles separate (i.e., anaphase A and anaphase B were both underway at this time). We photobleached GFP-labeled tubulin to create two parallel fiducial marks on microtubules near the spindle center (Figure 4D, left). Kymographs showed that these fiducial marks moved apart at a speed similar to the speed of chromosomes separation (Figure 4D, right). The movement of the photobleached marks demonstrates that microtubules between chromosomes slide apart in anaphase. To further quantify this motion, we took line ­profiles of the bleached region (Figure 4E), tracked their motion and the motion of chromosomes (see Materials and Methods), and found that the central-spindle microtubules move at a speed of 0.8 ± 0.09 µm/min (n = 7), which is indistinguishable from the speed of chromosome movement (0.8 ± 0.04 µm/min, n = 7, p = 0.53) and substantially greater than the speed of pole movement (0.3 ± 0.05 µm/min, n = 7, p < 0.001). Therefore, our results reveal that central-spindle microtubules slide apart at the same speed as chromosomes, even at times when anaphase A and anaphase B occur simultaneously. Similar to the results in the human mitotic spindles, these results argue for a coupling between the central-spindle microtubules and chromosomes in C. elegans mitotic spindles.

Laser-ablating central-spindle microtubules immediately stops anaphase chromosome motion in C. elegans mitotic spindles

We next sought to investigate the coupling between the central-spindle microtubules and the chromosomes in C. elegans mitotic spindles using our custom laser ablation system. We first cut a rectangular plane, 4 µm in length by 6 µm in depth, perpendicular to the spindle axis halfway-through between separating chromosomes in gpr-1/2(RNAi) embryos (Figure 5A; Supplemental Movie S5). Cuts were performed 30–60 s after the onset of anaphase when chromosomes and poles move away from the spindle center at a speed of 1.2 ± 0.1 and 0.5 ± 0.1 µm/min, respectively, so that chromosomes move toward poles at a speed of 0.7 ± 0.1 µm/min. Thus, at this time, the chromosome-to-pole distance shrinks at a faster rate than the poles separate (i.e., anaphase A and anaphase B were both underway at this time). The ablation led to an immediate cessation of chromosome motion, reducing its speed to 0.1 ± 0.2 µm/min (n = 7; indistinguishable from 0, p = 0.73). After approximately ∼20 s, chromosome segregation resumed with a speed similar to that of the uncut controls, presumably due to microtubule regrowth and replacement of the damaged microtubule population (Figure 5A, right). Thus, ablating microtubules between chromosomes completely stopped chromosome motion, while, at the time of these cuts, chromosome-to-pole distance was decreasing and spindle poles were separating. The result further argues that the central-spindle microtubules are strongly coupled to chromosome motion in gpr-1/2(RNAi) C. elegans embryos.

This strong coupling in the embryos with GPR-1/2 depleted might be due to an activation of compensatory processes not present in wild-type embryos. To test the coupling between central-spindle microtubules and chromosomes in the wild-type embryos, we ablated two rectangular regions, 8 µm in length by 6 µm in depth, between chromosomes and spindle poles on both sides of the spindle (Figure 5B, left) while the sister chromosomes were moving apart. After the cuts, the spindle poles detached from the central body of the spindle (Figure 5B, middle, green) and rapidly moved apart at a speed of 60.8 ± 2.2 µm/min (n = 13). In the subsequent ∼15 s the poles underwent irregular movements while the central body of the spindle showed no correlated displacement or rotation, further arguing that the cut disconnected the central body of the spindle from the poles (Supplemental Movie S6). Chromosome segregation continued after the cuts (Figure 5B, middle, magenta). The cuts did not pause chromosome motion, but reduced their speed from 9.2 ± 0.3 µm/min (n = 7) in controls to 5.2 ± 0.1 µm/min (n = 13, p < 10^-10) (Figure 5B, right). Thus, while previous work showed that C. elegans mitotic spindles can segregate chromosomes if the poles are destroyed before the onset of anaphase (Nahaboo et al., 2015), our new results demonstrate that chromosomes can continue segregating even immediately after severing the connection between chromosomes and poles in anaphase. The continued motion of chromosomes after sudden detachment from both spindle poles argues that, even in wild-type C. elegans mitotic embryos, the central-spindle microtubules are coupled to chromosomes in a manner that is not entirely dependent on connections through spindle poles.

The anaphase C. elegans mitotic spindle contains interchromosomal microtubules

To further investigate how the central-spindle microtubules couple to chromosomes, we performed serial-section electron tomography to reconstruct the C. elegans anaphase mitotic spindles (Figure 5C, left). Our reconstructions revealed the presence of interchromosomal microtubules: some of which had two free ends between chromosomes (Figure 5C, red), others of which had one end near the center of the spindle, with the other end contacting chromosomes (Figure 5C, orange). In addition, there are also microtubules with one end extending from the interchromosomal region passing around chromosomes to contact kinetochore microtubules (Figure 5C, yellow). Again, we also found no micro­tubules, which extended all the way from the pole to the region between chromosomes, and no microtubules, which passed across the entire interchromosomal region directly bridging microtubules between the two poles. Therefore, electron tomography demonstrates that interchromosomal microtubules also exist in anaphase C. elegans mitotic spindles, similar to those in human anaphase spindles. This structure suggests that either the coupling from central-spindle microtubules to chromosomes could be through direct contacts at the inner surface of chromosomes (Figure 5C, orange microtubules) or, alternatively, the connection to chromosomes could be indirect, by microtubules with one end extending from the interchromosomal region passing around chromosomes to contact kinetochore microtubules (Figure 5C, yellow). The lack of microtubules connecting the central-spindle region directly to the poles may explain the continued motion of chromosomes immediately after severing the connections between poles and chromosomes in anaphase.

Electron tomography confirms the presence of interchromosomal microtubules in C. elegans female meiosis

Having characterized the dynamics, function, and structure of central-spindle microtubules in human and C. elegans mitotic spindles, we next sought to apply the same approach to study a spindle with very different morphology and dynamics: acentrosomal C. elegans female meiotic spindles. We first generated electron tomographic reconstructions of all microtubules in C. elegans female meiotic spindles at various stages of anaphase (Figure 6A, left). Nearly all microtubules in the spindle lie between chromosomes at these stages of anaphase (Figure 6A, left). The organization of interchromosomal microtubules in C. elegans female meiotic spindles is highly reminiscent of those found in C. elegans mitotic spindles: some microtubules have both their ends between the chromosomes, without touching the chromosomes (Figure 6A, center), while some microtubules make end-on contact with chromosomes, with their other end terminating between the chromosomes (Figure 6A, right). Our observation of numerous interchromosomal microtubules in anaphase of C. elegans female meiotic spindles is consistent with previous partial electron tomography reconstructions (Laband et al., 2017).

Laser-ablating central-spindle microtubules immediately stops anaphase chromosome motion in C. elegans meiotic spindles

To test the coupling of these interchromosomal microtubules to chromosomes, we used laser ablation to cut them during anaphase (Figure 6B; Supplemental Movie S7). We found that, as in mitotic spindles, this damage caused an immediate cessation of chromosome motion, with their speed reducing to 0.01 ± 0.01 µm/min (n = 6; indistinguishable from 0, p = 0.36) from a speed of 0.7 ± 0.1 µm/min (n = 5) in controls (Figure 6C). Similar to mitotic spindles, chromosome motion resumed at a speed similar to that of controls after ∼20 s, presumably due to the spontaneous repair of the damaged population of microtubules. Thus, this result demonstrates that interchromosomal microtubules are strongly coupled to chromosomes in C. elegans female meiotic spindles at the stages we studied. A previous laser ablation study also found that cutting interchromosomal microtubules halted chromosome motion in C. elegans meiotic spindles (Laband et al., 2017), although in that study, severed spindles never recovered, likely due to severe collateral damage caused by using a UV laser for ablation.

Interchromosomal microtubules slide apart at the speed of chromosome motion in C. elegans female meiotic spindles

We next investigated the relative motion of interchromosomal microtubules and chromosomes in C. elegans female meiotic spindles (Figure 1). We photobleached GFP-labeled tubulin to create a fiducial mark on interchromosomal microtubules adjacent to a set of sister chromosomes during anaphase and found that they moved at a similar speed as chromosomes moved (Figure 6D). To better quantify this motion, we made line profiles of the photobleached regions and tracked their motion, along with the motion of chromosomes (Figure 6E) (see Materials and Methods). Averaging measurements over multiple spindles gave a mean speed of microtubule sliding away from the spindle center of 0.5 ± 0.2 µm/min (n = 6), indistinguishable from the mean speed of chromosome movement, 0.3 ± 0.1 µm/min (n = 6, p = 0.3). These results further argue that central-spindle microtubules are strongly coupled to anaphase chromosomes in C. elegans female meiotic spindles.

DISCUSSION

We used optical microscopy, laser ablation, and electron tomography to characterize central-spindle microtubules in anaphase in human mitotic spindles and C. elegans mitotic and female meiotic spindles. We found that central-spindle microtubules slide apart at the same speed as chromosomes, even at times when anaphase A and anaphase B occur simultaneously. Laser ablating central-spindle microtubules leads to an immediate cessation of chromosome motion, even at times when anaphase A and anaphase B occur simultaneously. These results argue that central-spindle microtubules are tightly coupled to chromosomes in anaphase. Because of technical limitations, our most detailed characterization of C. elegans mitotic spindles were performed when pulling forces were inhibited by gpr-1/2(RNAi), a perturbation that only minimally impacted chromosome motion (while greatly reducing pole oscillations and movements). Our laser ablation experiments demonstrate that chromosomes continue to separate even immediately after severing the connection between chromosomes and poles, suggesting that a tight coupling between central-spindle microtubules and chromosomes is also present in wild-type C. elegans mitotic spindles.

It has previously been reported that damaging the central-­spindle results in faster chromosome segregation in some systems, including in C. elegans mitotic spindles and in some vertebrate spindles (Aist and Berns, 1981; Aist and Bayles, 1991; Aist et al., 1993; Sakai et al., 1982; Bayles et al., 1993; Grill et al., 2001). In other systems, it has been reported that damaging the central-spindle impairs anaphase chromosome motion, including in human tissue culture cells and C. elegans meiotic spindles (Hiramoto and Nakano, 1988; Khodjakov et al., 2004; Laband et al., 2017; Vukusic et al., 2017). It is possible that the central-spindle was only partially damaged in some of the previous studies that observed impaired, but continued, motion of chromosomes, and more complete damage might have halted chromosomes. Our interpretation of these diverse results is that the extent of chromosome motion after damaging the central-spindle is determined by the extent of the external pulling forces from outside of the spindle in different organisms (irrespective of whether those external pulling forces drive chromosome motion in undamaged spindles). We believe that the three types of wild-type spindles studied in our article are normally subject to different strengths of pulling forces, and thus, consistent with previous literature, respond differently to central-spindle damage. However, our results argue that central-spindle microtubules are strongly coupled to chromosomes in both anaphase A and anaphase B in all of these spindles.

To gain further insight into the manner by which central-spindle microtubules are coupled to chromosomes in anaphase, we also performed large-scale electron tomography reconstructions of these microtubules in human mitotic spindles and in C. elegans mitotic and female meiotic spindles (Supplemental Table S1). We found that all of these spindles contained microtubules with both ends between segregating chromosomes, some of which appeared to directly contact chromosomes. Human and C. elegans mitotic spindles also both contained microtubules with one end extending from the interchromosomal to contact kinetochore microtubules. In all the spindles we studied, we found no microtubules that extended all the way from the pole to the region between chromosomes and no microtubules that passed across the entire interchromosomal region, directly bridging kinetochore-microtubules from the opposite two poles. Thus, our data argue that central-spindle micro­tubules could be connected directly to chromosomes, or the coupling could be indirect through their mutual connection to kinetochore microtubules (or both). It is an exciting challenge for future research to disentangle these possibilities.

MATERIALS AND METHODS

C. elegans strains

Strain SA250 (tjIs54 [pie-1p::GFP::tbb-2 + pie-1p::2xmCherry::tbg-1 + unc-119(+)]; tjIs57 [pie-1p::mCherry::his-48 + unc-119(+)]) and a strain expressing GFP::tubulin and mCherry::histone (a gift from Marie Delattre’s lab, Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, France) were used for experiments of fluorescence imaging, laser ablation, and fluoresce recovery after photobleaching. Wild-type (N2) C. elegans embryos and oocytes were used for the preparation of electron tomography. All strains were cultured at 24°C and fed on OP50 bacteria on nematode growth medium plates.

Human cell lines and culture

Human bone osteosarcoma epithelial (U2OS) cells were engineered to stably express fluorescence-labeled proteins by retroviral transfections. Three stable fluorescence U2OS lines were used: one expresses GFP::CENP-A and mCherry::tubulin; another expresses GFP::centrin, GFP::CENP-A, and mCherry::tubulin; the other expresses GFP::CENP-A; and mEOS3.2::tubulin. U2OS cell lines were maintained in DMEM (Life Technologies), supplemented with 10% fetal bovine serum (Life Technologies) and 50 IU/ml penicillin and 50 μg/ml streptomycin (Life Technologies) at 37°C in a humidified atmosphere with 5% CO₂.

C. elegans imaging preparation and RNAi

For imaging of mitotic spindles, gravid C. elegans hermaphrodites were cut in half, and the released embryos were transferred onto a 4% agarose pad between a slide and a coverslip (Walston and Hardin, 2010). Meiotic spindles in oocytes were observed in uterus, as adult hermaphrodites were mounted between a coverslip and a thin 4% agarose pad on a slide. Polystyrene microspheres (microspheres 0.10 μm; Polysciences) in solution were added to help immobilize worms.

RNAi was carried out following the RNAi feeding protocol from the Ahringer lab (Kamath et al., 2001). Plasmid pMD082 (a gift from the Marie Delattre lab) containing the sequence of gpr-1/2 was cloned into the L4440 plasmid, which was transformed into HT115 bacteria. L2 hermaphrodites were transferred to gpr-1/2 (RNAi) plates and fed on the RNAi bacterial lawn for 36–48 h at 24°C.

Human cell imaging preparation

In preparation for imaging, cells were grown on a 25-mm-diameter, #1.5-thickness, round coverglass coated with poly-d-lysine (GG-25-1.5-pdl, neuVitro) to ∼80–90% confluency. The cells were incubated in imaging media, which is FluoroBrite DMEM (Life Technologies) supplemented with 4 mM l-glutamine (Life Technologies) and 10 mM HEPES, for ∼15–30 min before imaging. The coverglass was mounted on a custom-built temperature-controlled microscope chamber at 37°C, while covered with 1.5 ml of imaging media and 2 ml of white mineral oil. An objective heater (Bioptech) was used to maintain the objective at 37°C.

Sample preparation for electron tomography

Wild-type (N2) C. elegans embryos and oocytes collected in cellulose capillary tubes (Pelletier et al., 2006; Redemann et al., 2018) and HeLa Kyoto cells immobilized on sapphire disks (Guizetti et al., 2011) were high-pressure frozen as described using either an EM PACT2+RTS or an EM ICE high-pressure freezer (Leica Microsystems, Vienna, Austria) (Muller-Reichert et al., 2007). Freeze substitution was performed over 2–3 d at –90°C in anhydrous acetone containing 1% OsO₄ and 0.1% uranyl acetate. Samples were embedded in Epon/Araldite and polymerized for 1–3 d at 60°C. Serial semithick sections (300 nm) were cut using an Ultracut UCT Microtome (Leica Microsystems, Vienna, Austria) collected on Formvar-coated copper slot grids and poststained with 2% uranyl acetate in 70% methanol followed by Reynold’s lead citrate. For dual-axis electron tomography (Mastronarde, 1997), a series of tilted views were recorded using a TECNAI F30 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) operated at 300 kV and equipped with a Gatan US1000 CCD camera (2k x 2k). Images were captured every 1.0° over a ±60° range. The IMOD software package (http://bio3d.colourado.edu/imod) was used for the calculation of electron tomograms (Kremer et al., 1996). The Amira software package with an extension to the filament editor was used for the segmentation, automatic tracing, stitching, and 3D visualization of microtubules and for data analysis (Stalling et al., 2005; Weber et al., 2012, 2014).

Spinning disk confocal fluorescence imaging

Live imaging was performed using a spinning disk confocal microscope (Nikon Ti2000, Yokugawa CSU-X1) equipped with 488- and 561-nm diode lasers, an EMCCD camera (Hamamatsu), and a 60× water-immersion objective (CFI Plan Apo VC 60X WI, NA 1.2, Nikon). Acquisition parameters were controlled by a home-developed LabVIEW program (LabVIEW, National Instruments). For C. elegans mitotic and female meiotic spindles, images were acquired every 2 or 4 s with a single z-plane. For human mitotic spindles, images were acquired every 2 or 4 s with three z-sections every 1 µm, and the middle planes were presented.

Laser ablation

The laser ablation system was constructed on the above-mentioned spinning disk confocal microscope. Femtosecond-pulsed near-infrared lasers with either 80-MHz or 16-kHz repetition rate were adapted to perform laser ablation. Femtosecond pulses (80 MHz) with 0.3-nJ pulse energy and 800-nm center wavelength came directly from a Ti:sapphire pulsed laser (Mai-Tai; Spectra-Physics, Mountain View, CA). A 16-kHz femtosecond pulse train with ∼6-nJ pulse energy was produced by selecting pulses from the above Ti:sapphire pulsed laser using a pulse picker (Eclipse Pulse Picker; KMLabs). The ablation laser was focused through the same objective for imaging, and laser ablation was performed by moving the sample on a piezo-stage (P-545 PInano XYZ; Physik Instrumente) in three dimensions controlled by a home-developed LabVIEW program (National Instruments). Scanning line cuts with z-steps were created by moving the stage perpendicular to the spindle long axis back and forth on the focal plane while lowering the stage in the z direction. The parameter for ablation in length by depth was 8 × 6 µm for C. elegans mitotic spindles, 12 × 6 µm for human mitotic spindles, and 6 × 2 µm for C. elegans meiotic spindles. The moving speed of the stage was 50 µm/s.

Photobleaching experiments

Photobleaching experiments used the same setup and software control as the laser ablation, except that a 80-MHz Ti:sapphire pulsed laser was used (800 nm wavelength, ∼70 fs pulse width, ∼0.1-nJ pulse energy; Mai-Tai; Spectra-Physics, Mountain View, CA). The parameter for photobleaching in length by depth was 7 × 6 µm in C. elegans mitotic spindles and 6 × 2 µm in C. elegans meiotic spindles. The moving speed of the stage was 50 µm/s.

Photoconversion experiments

Photoconversion experiments used the same setup and software control as the laser ablation, except that a 405-nm continuous-wave diode laser was used (Thorlabs). The parameter for photoconversion in length was 12 µm in human mitotic spindles. The moving speed of the stage was 50 µm/s.

Quantitative analysis in C. elegans mitotic spindles

Quantitative analysis of the chromosome distance with a subpixel resolution was achieved using a home-written MATLAB (MathWorks, Natick, MA) program. The center positions of two centrosomes in each frame of the time-lapsed images were either manually selected in spindles expressing GFP-labeled tubulin or automatically located in spindles expressing mCherry-labeled γ-tubulin based on a public MATLAB program for particle tracking (https://site.physics.georgetown.edu/matlab/index.html). The center positions of the two centrosomes in each frame determines the spindle long axis as well as the spindle length and were used to generate line scans of overlaid images of mCherry-labeled histones (corresponding to chromosomes). Averaged mCherry fluorescence intensities from histones along these line scans were extracted, and the fluorescence profile around histone-enriched regions were Gaussian-like shapes. A double-peak Gaussian function was used for fitting the line scans to locate the centers of two groups of histones, presumably reflecting the central positions of sister chromosomes. The distance of these two fitted center positions was defined as the chromosome-to-chromosome distance. A straight line was fitted to the change of chromosome distance versus time to extract the velocity of chromosome separation before and after laser ablation. In controls, the velocity was computed before and after the chromosome distance was 2.4 µm, the chromosome distance around which most laser-ablation experiments were performed.

Movement and recovery of photobleaching makers were also tracked using a program written in MATLAB. Line scans of GFP-labeled tubulin between chromosomes along the spindle axis were extracted over the course of anaphase. Each line scan at each time point can be divided into two halves by the middle plane of the spindle. The half of the line profile with the bleaching mark was normalized to the other half of the profile by a reflection of symmetry around the middle plane of the spindle. This profile normalization was used to remove spatial variations in the background fluorescence, a valid procedure assuming mirror symmetry of the spindle around its middle plane. A Gaussian function was used to fit the normalized profile to locate the center of the bleached mark, and thus the position of the bleached mark versus time was extracted. A straight line was fitted to the position of the bleached mark versus time to retrieve the velocity of the bleached mark.

Quantitative analysis in C. elegans meiotic spindles

Chromosome distance in C. elegans meiotic spindles was computed with a combination of Fiji (Schindelin et al., 2012) and a MATLAB program. Time-lapse images of spindle expressing mCherry::histone (corresponding to chromosomes) were realigned in a routine for matching, rotation and translation using Rigid Body of Fiji’s StackReg plug-in, so that the random displacement of the spindle due to the spontaneous motion of the worm was corrected. On the realigned time-lapse stack, a straight line passing through sister chromosomes was manually drawn in line with the spindle axis, and the kymograph of mCherry:histone intensities along this line was generated. On a MATLAB program, each line scan of mCherry:histone was fitted to a double-peak Gaussian function for computing the center positions of sister chromosomes and thus the chromosome distance. A straight line was fitted to the data of chromosome distance versus time to extract the velocity of chromosome separation before and after laser ablation. In controls, separation velocity was computed before and after the chromosome distance was 3.3 µm, the chromosome distance around which most laser-ablation experiments were performed.

Movement of photobleaching makers was also tracked using a program written in MATLAB. Line scans of GFP-labeled tubulin between chromosomes along the spindle axis were extracted over the course of anaphase. A Gaussian function was used to fit the line-scanning profile to locate the center of the bleached mark, and thus the position of the bleached mark versus time was extracted. A straight line was fitted to the position of the bleached mark versus time to retrieve the velocity of the bleached mark.

Quantitative analysis in human mitotic spindles

Distance information of interest was extracted from spindles expressing GFP::CENP-A (corresponding to kinetochores) and GFP::centrins (corresponding to spindle poles) in time-lapse z-stacks based on the following steps. First, using approaches from a particle tracking algorithm (Pelletier et al., 2009), a GUI MATLAB program was developed to retrieve the three-dimensional coordinates of the center positions of each kinetochores and the two poles. Next, a support vector machine algorithm was adapted to find the best plane to separate kinetochores of one half-spindle from those of the other half-spindle. This best plan is the one with the largest margin between two sides of kinetochores and corresponds to the middle plane of the spindle. A unit vector perpendicular to this plane was thus in line with the direction of spindle long axis. All positions of kinetochores and spindle poles in space were projected onto this unit vector and thus converted to one-dimensional information of projection lengths. Finally, chromosome-to-chromosome distance was computed as the length difference between the averaged projection lengths of kinetochores at one side of the half-spindle and those at the other side. Similarly, the chromosome-to-pole distance was computed as the length difference between averaged projection lengths of kinetochores and the projection length of the pole at the same side of the half-spindle. The above procedure was performed for each time point of a z-stack, so that the rotation and translation of spindles in space over the course of anaphase was corrected. A straight line was fitted to the data of chromosome-to-chromosome distance, and chromosome-to-pole distance versus time to compute the separation velocity of the chromosomes, and the velocity between chromosomes and poles before and after laser ablation, individually. In controls, the separation velocity of chromosomes was computed before and after the chromosome distance was at 6.1 µm.

Movement of photoconverted mEOS3.2-labeled microtubules were also tracked using a program written in MATLAB. Line scans of photoconverted tubulin between chromosomes along the spindle axis were extracted over the course of anaphase. A Gaussian function was used to fit the profile to locate the center of the photoconverted microtubules, and thus the position of the photoconverted microtubules versus time was extracted. A straight line was fitted to the position of the photoconverted microtubules versus time to retrieve the velocity of the photoconverted microtubules.

Statistical analysis

Statistics are presented as mean ± SEM, and p values were calculated by the “ttest2” function in MATLAB.

FOOTNOTES

ACKNOWLEDGMENTS

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