Capture and incorporation of preformed microtubule bundles into the monopolar spindle in monastrol-treated cells
We used near-simultaneous three-dimensional (3-D) GFP fluorescence/two-dimensional (2-D) differential interference contrast (DIC) multimode microscopy to follow the behavior of both the microtubules and chromosomes in live monastrol-treated PtK2 cells that express α-tubulin/GFP (PtK-αT). Typically, we began observation immediately after NEB and then followed the cell for 2 h, recording one DIC image and a corresponding 3-D fluorescence stack every minute. In some experiments (e.g., )
, we followed cells over shorter periods (10–30 min) but at higher temporal resolution (10–30-s intervals).
Figure 1. Formation, looping, and incorporation of microtubule bundles in monastrol-induced monopolar spindles in PtK-αT cells. Selected frames are shown from a near-simultaneous 3-D fluorescence/DIC time-lapse microscopy experiment. The top half of each (more ...)
Our 2-h time-lapse recordings of 20 cells revealed that within 5–10 min after NEB, all chromosomes in monastrol-treated cells became mono-oriented and assumed a star-like configuration, with their centromere regions oriented toward the centrosomes and the arms pointing outwards (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200208143/DC1
). At the moment of NEB in the presence of monastrol, the centrosomes were often already spatially separated (). This has also been observed in untreated PtK cells (Roos, 1976
). In the presence of monastrol, centrosomes generated two radial microtubule arrays that coalesce within a few minutes after NEB (). Initially, the chromosomes may be positioned only on one side of the centrosomes, but, over time, they gradually rearranged into a spherical array encircling the centrosomes. These aspects of monopolar spindle formation were largely expected from previous fixed cell analyses of monastrol-treated cultures (Mayer et al., 1999
, Kapoor et al., 2000
One unexpected feature conspicuous in our time-lapse recordings was that in all cells imaged, we observed prominent bundles of microtubules extending from the chromosomes toward the cell periphery. These bundles appeared in the vicinity of the centromere regions of chromosomes and then rapidly grew outwards (). Temporal resolution in most of our time-lapse records was not sufficient to determine precise elongation rates of the bundles that can often reach up to 10–12 μm within 3 min. To document these events in greater detail, we used a spinning-disk confocal microscope, which acquired images at a higher temporal resolution, sampling the cellular volume every 15 s. Under these conditions, we observed elongation rates of ~3–4 μm/min (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200208143/DC1
After reaching ~10 μm in length, the bundles usually underwent a rapid bending, and their ends distal to the chromosome moved back toward the center of the spindle (). As a result, the bundle formed a transient microtubule “loop” as its distal end moved inwards to the pole while the proximal end remained relatively stationary. This configuration was transient, and in ~2–5 min, the bundle made a complete 180° turn so that its distal end incorporated into the spindle. Overall, this behavior is suggestive of the distal end of the microtubule bundle being suddenly captured and experiencing a force directed toward the center of the spindle.
The phenomenon of formation, capture, and incorporation of microtubule bundles was very common in monastrol-induced monopolar mitoses. On average, we observed ~10–12 such events in a cell during a 2-h observation period (range 8–20; 19 events in Video 1). Once a bundle of microtubules formed (reached ~10-μm length), it was typically captured within 5 min. Infrequently, some bundles remained extended for up to 15–20 min before incorporating into spindles.
This microtubule formation, capture, and incorporation phenomenon is not limited to the PtK-αT cells. We also observed similar behavior of microtubule bundles in other cell types, including LLC-PK and CV-1 (both constitutively expressing α-tubulin/GFP; unpublished data).
Microtubule loops result in the formation of syntelic-oriented chromosomes in monastrol-treated cells
To determine the structural organization of microtubule loops, we followed a cell by 3-D fluorescence/ 2-D DIC microscopy and then fixed it during a microtubule looping event. The fixed cells were subsequently processed for immunofluorescence analysis. This analysis revealed that the proximal end of the microtubule bundle (one that remains stationary during looping) was always associated with the primary constriction of a chromosome ()
. As a result, upon incorporation of the distal end of the looping microtubule bundle into the spindle, the chromosome becomes syntelic, i.e., its primary constriction connected to the spindle pole by two bundles of microtubules (). This configuration implies that before the distal microtubule bundle was captured and looped toward the spindle pole, it extended from the primary constriction toward the cell periphery. To confirm this, we analyzed a population of monastrol (100 μM)-induced monopolar mitoses in PtK-αT cells after fixation and immunostaining for microtubules, kinetochores, and chromosomes. Our analysis revealed that ~10% of monopolar mitoses contain conspicuous bundles of microtubules that emanate directly from distal kinetochores and extend for several micrometers away from the spindle pole and toward the cell's periphery (
Figure 2. Small aggregates of NuMA are associated with the parts of the microtubule loop that move poleward. Same cell as in . (A–D) Maximal projections of 3-D fluorescence volumes representing microtubules (anti–α-tubulin; (more ...)
Figure 3. Some of the distal kinetochores in monastrol-induced monopolar spindles are associated with well-developed bundles of microtubules (K-fibers). (A–D) A PtK-αT cell stained for microtubules (anti-α-tubulin; A), kinetochores (more ...)
To investigate the structural organization of these distal K-fibers at a greater resolution, we analyzed two monopolar mitoses in a population of PtK cells treated with 100 μM monastrol by serial section EM. The cells were selected without bias, and spindle structures were not evaluated by immunofluorescence before processing for EM. Nevertheless, in these two cells, we found three unambiguous cases of well-developed K-fibers emanating from distal kinetochores toward the cell periphery (, E–K). Each individual kinetochore plate was associated with several microtubules (up to 12). It is important to emphasize that in all three cases, the distal kinetochore faced directly away from the spindle pole and was shielded from the astral microtubules by the chromosome mass (, E–K). Thus, our serial section EM data confirmed the existence of K-fibers not directly oriented to the spindle pole.
Inhibition of NuMA prevents microtubule fiber looping
Thus far, our data revealed that microtubule loops form when the free ends of preformed K-fibers are captured and actively transported toward the spindle pole. To examine the molecular mechanism of this poleward sliding of spindle microtubules, we examined the localization of NuMA, a protein responsible for maintaining microtubule ends focused at spindle poles (Gaglio et al., 1995
; Merdes et al., 1996
; Gordon et al., 2001
). We found NuMA to be present at the leading end of the looping microtubule bundle in all cells analyzed (n
= 4) ( F, arrow). Because NuMA has been shown to interact with the dynein/dynactin complex (Merdes et al., 1996
), this observation is consistent with the capture and incorporation of microtubule bundles being driven by dynein motility.
To test whether NuMA activity is required for microtubule looping, we microinjected cells with a NuMA-specific antibody (Gaglio et al., 1996
). We previously demonstrated that injection of this antibody into cultured cells aggregates NuMA and prevents it from interacting appropriately with spindle microtubules (Gaglio et al., 1996
; Gordon et al., 2001
). For these experiments, we used human CFPAC-1 cells, as available anti-NuMA antibodies do not react sufficiently with marsupial NuMA to inhibit its function in PtK cells. Inhibition of Eg5 function in human CFPAC-1 cells through either injection of Eg5-specific antibodies (unpublished data) or monastrol treatment prevented centrosome separation and led to the formation of monopolar spindles (
A). The microtubule distribution in these monopolar spindles was indistinguishable from that observed in PtK-αT cells, with only a few microtubule bundles extending toward the cell periphery (on average one bundle in every other cell; data from 16 cells analyzed by 3-D microscopy). In contrast, upon simultaneous perturbation of Eg5 (by either treatment with monastrol [unpublished data] or injection of Eg5-specific antibodies) and NuMA (by antibody injection), numerous straight microtubule bundles were seen to extend from the chromosomes in an orientation opposite that of the pole defined by the two unseparated centrosomes ( B; on average five to six bundles per cell; data from 17 cells analyzed by 3-D microscopy). If monastrol was removed from cells injected with NuMA antibodies and treated with monastrol, then we observed centrosome separation, but K-fibers failed to recruit appropriately toward the centrosomes (unpublished data), resulting in disorganized spindles with splayed spindle poles analogous to those observed after perturbation of NuMA alone (Gaglio et al., 1996
; Gordon et al., 2001
). These changes in microtubule distribution are consistent with the idea that NuMA is functionally responsible for the capture and incorporation of preformed K-fibers. Upon inhibition of NuMA, the fibers that would normally loop back to the single pole remained extended and accumulated over time.
Figure 4. NuMA is required for K-fiber orientation in monopolar spindles formed in cells lacking Eg5 activity. Human CFPAC-1 cells treated with 100 μM monastrol (A) or injected with both Eg5- and NuMA-specific antibodies (B) were fixed in mitosis. Mitotic (more ...)
Capture of preformed microtubule bundles occurs during spindle bipolarization after monastrol washout
The mitotic arrest due to monastrol is completely reversible, and monopolar spindles rapidly rearrange into normal bipolar mitoses upon monastrol washout (Kapoor et al., 2000
). To investigate whether the capture and looping of preformed microtubule bundles occurs during the transformation of monopolar structures into bipolar spindles, we examined microtubule behavior in cells released from monastrol arrest. Our initial attempts to follow these transformations revealed that the redistribution of microtubules can often be too complex to be followed by wide-field fluorescence microscopy. Therefore, we employed near-simultaneous 3-D confocal fluorescence/2-D DIC time-lapse microscopy for these experiments. The use of a spinning-disk confocal microscope allowed us to track individual microtubule bundles within complex arrays with greater precision than conventional wide-field fluorescence microscopy. Scanning depth was set to match the parameters of our wide-field time-lapse recordings used to examine cells in the presence of monastrol. Images sampling the cell volume were acquired at 30-s intervals.
Our recordings revealed that bipolarization of the spindle began immediately upon monastrol removal, and cells consistently initiated anaphase ~75 min after washout. The bipolarization began with the separation of centrosomes, which often detached from the rest of the spindle ( and )
. Intriguingly, the orientation of the axis of centrosome separation was not related to the original orientation of the K-fibers within the monopolar spindle, and the centrosomes often separated in a direction perpendicular to the majority of the K-fibers (). As the centrosomes separated, they remained associated with prominent arrays of astral microtubules. These microtubules overlap and appear to interact, forming a structure very similar to the “chromosome-free spindles” described by Faruki et al. (2002)
in PtK-αT polykaryons. Detachment of centrosomes did not immediately affect the organization of the monopolar spindle. K-fibers remained focused at a single spindle pole that now lacked astral microtubules ( B). These K-fibers exhibited rapid changes in length corresponding to oscillations of attached chromosomes (see Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200208143/DC1
Figure 5. The “capture” of stable K-fiber minus ends contributes to bipolar spindle formation and chromosome alignment in PtKαT cells released from a monastrol arrest. Selected frames from a near-simultaneous 3-D confocal fluorescence/2-D (more ...)
Figure 6. Looping and capture of microtubules contributes to spindle morphogenesis in PtKαT cells released from a monastrol arrest. PtKαT cells with monopolar spindles formed in the presence of 100 μM monastrol were placed in monastrol-free (more ...)
Importantly, during bipolarization of the spindle, the K-fibers continued to exhibit capture and incorporation of their minus ends into the spindle. However, in contrast to monopolar spindles where K-fibers looped around chromosomes and became incorporated into the single spindle pole, during bipolarization of the spindle, each bundle exhibited one of two types of motion. First, those K-fibers that emanated from the side of chromosomes that faced the centrosomes exhibited direct translocations toward one of the two separating centrosomes. As a result, each chromosome became either syntelic (when minus ends of both K-fibers were captured by the same centrosome) or properly bioriented (). Second, those K-fibers that emanated from chromosomes toward the cell's periphery behaved exactly as the distal K-fibers in monopolar spindles. The K-fibers bended and looped around chromosomes, with their minus ends sliding toward one of the two separating centrosomes ().
Capture and incorporation of K-fiber minus ends was a common phenomenon we observed in every cell released from a monastrol arrest. Looping of K-fibers distal to the centrosome was often seen during the initial stages of spindle bipolarization with an average frequency of seven loops per cell (range 1–24; n = 15). The high density of microtubules on the side proximal to the centrosomes precluded accurate quantification of direct translocations of K-fiber minus ends toward the separated centrosomes. However, this phenomenon was at least as common as the “looping” of distal K-fibers during the initial stages of spindle bipolarization and predominant during later stages (Videos 2 and 3).
Our observations reveal that spindle morphogenesis in vertebrates does not only depend on the microtubule plus-end search and capture mechanism but also includes the capture and incorporation of preformed K-fibers at their minus ends.
NuMA is consistently associated with the minus ends of K-fibers during spindle bipolarization
Our data have shown that minus ends of distal K-fibers incorporating into monopolar spindles were always associated with NuMA (see above). To determine if this was the case for the persistent K-fibers that faced centrosomes and exhibited more direct translocation toward a centrosome, we examined the distribution of NuMA in cells released from a monastrol arrest (
, A–E). In all cells examined, NuMA was distributed in numerous small patches spread over the region between the separating centrosomes. The strongest NuMA staining corresponded to the ends of K-fibers ( E; 2.5× magnification of three-color overlay and line scan). This is consistent with our data that NuMA function is required for the capture and incorporation of the preformed K-fibers into the mitotic spindle.
Figure 7. In cells establishing bipolar spindles after release from a monastrol arrest, kinetochore microtubule minus ends are associated with interpolar microtubules and NuMA but not centrosomes. (A–D) PtK2 cells arrested in the presence of monastrol (100 (more ...)
The fact that NuMA was consistently spread over a large area between the separating centrosomes raised a formal possibility that centrosomal material was similarly fragmented in cells released from monastrol. To evaluate this, we examined localization of γ-tubulin, a protein that has been shown to delineate the boundaries of centrosomes (Khodjakov and Rieder, 1999
). This analysis revealed that in contrast to the NuMA distribution, the centrosomal material remained focused at the spindle poles, and thus the centrosomes were not fragmented under these conditions (, F–I). Overall, these observations suggest that common mechanisms contribute to the capture and incorporation of K-fiber minus ends in monopolar spindles and during spindle bipolarization.
Capture of preformed microtubule bundles occurs during mitotic spindle formation in control cells
Thus far, our data revealed that capture and incorporation of preformed K-fibers contributes to spindle morphogenesis in cells treated with monastrol. The question remained whether this phenomenon also occurs during normal bipolar spindle formation in unperturbed cells. We reviewed a library of time-lapse recordings of spindle formation in control PtK-αT cells (~20 cells) and found two examples of clear incorporation of preformed K-fibers into the forming spindle.
illustrates one such event (see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200208143/DC1.
In both cases, the fibers were incorporated into the correct half spindle, resulting in accurate biorientation of the chromosome.
Figure 8. Capture and incorporation of preformed K-fibers into the spindle occurs during spindle formation in control PtK cells. Selected frames from a fluorescence time-lapse recording of a PtK-αT cell also stained with Hoechst 33342 to visualize chromosomes. (more ...)