Our goal here was to observe and quantitate the relative activity of a subset of MT motors on spindle pole positioning during the assembly and elongation of bipolar mitotic spindles in Drosophila embryos. To this end, we have used time-lapse laser scanning confocal microscopy on embryos containing fluorescently labeled tubulin to measure the extent of spindle pole separation as a function of time in the presence and absence of inhibitors of three mitotic motors, namely the bipolar kinesin KLP61F, the C-terminal kinesin Ncd, and cytoplasmic dynein.
Quantitative Analysis of Mitotic Spindle Pole Positioning in the Drosophila Syncytial Blastoderm
Because the cell cycles in Drosophila
early embryos become progressively longer as they near cellularization (Foe and Alberts, 1983
), it is important to compare spindle pole movements that occur within the same cycle. For technical reasons (see MATERIALS AND METHODS, Embryo Microinjections), in this study we focus on the spindle pole movements that occur in one of the final two cycles before cellularization during three distinct stages in the pathway of mitotic spindle formation and function: 1) interphase–prophase of cycle 13 (the last cycle before cellularization) when duplicated spindle poles migrate to nearly opposite sides of the nuclear envelope; 2) prometaphase–metaphase (between NEB and the onset of anaphase A) of cycle 12; and 3) anaphase B (spindle elongation) of cycle 12. In the quantitative studies presented below, each line plot was derived from the analysis of 10 mitotic spindles from two different embryos (see MATERIALS AND METHODS, Quantitative Image Analysis).
Figure A shows plots of spindle pole separation as a function of time during these three stages of mitosis. The top panel plots the positions of pairs of spindle poles, which separate around the nuclear envelope during interphase and prophase and come to lie ~7 μm (arc length) or ~120° apart (Figure B). The middle panel (Figure A) shows a second phase of spindle pole movements that occur during prometaphase as the spindle elongates from ~7 to ~10 μm (Figure , C and D, respectively). Finally, Figure A, bottom panel, shows a plot of spindle pole movements that occur during anaphase B when the spindle elongates from ~10 to ~14 μm (Figure E). Although it is clear that there is a general trend for the spindle poles to separate throughout mitosis, spindle pole separation does not occur at a linear rate. Instead, the rate of pole separation as reflected in the slopes of the curves in Figure changes in a complex manner with stops, starts, and rate changes.
During the first 300 s of spindle pole migration in interphase–prophase of cycle 13 (Figure A, top panel) spindle poles appear to separate in a roughly hyperbolic manner. The initial rate of this separation is ~0.11 μm/s, which gradually slows down to a plateau at ~175–180 s when the spindle poles are ~6 μm apart. After this hyperbolic phase there is a slower, roughly linear rate of spindle pole separation (~0.01 μm/s) during the ensuing 150 s that pushes apart the poles until they lie 7–8 μm apart just before NEB. After NEB in cycle 12, the length of the spindle remains constant (at ~7 μm) for ~25 s and then displays a nearly linear rate of elongation (~0.06 μm/s) driving the poles to a separation length of ~10 μm during metaphase (Figure A, middle panel). Finally, as anaphase begins there is another nearly linear phase of spindle elongation at a rate of ~0.09 μm/s driving the spindle to reach a peak length of ~14 μm (Figure A, bottom panel). Spindle length then decreases slightly during telophase. (A video showing spindle formation during these mitoses can be viewed with Figures , B–E.)
A plausible explanation for this complex behavior is that the rate of spindle pole separation remains constant when the net force acting on the poles is constant, whereas any change in the rate reflects a corresponding change in this net force. Specifically, an increase in the rate reflects an enhancement in the net force serving to separate the poles, whereas a decrease in the rate reflects either the decrease in this force or the addition of an antagonistic force that slows spindle pole separation down.
Antagonistic Microtubule Motors Involved in Spindle Pole Migration during Interphase and Prophase
Two motors that might provide force to drive spindle pole separation during the early phases of mitosis are cytoplasmic dynein (Vaisberg et al., 1993
; Robinson et al., 1999
) and the bipolar kinesin KLP61F (Heck et al., 1993
). Our previous studies suggest that KLP61F does not act until the later stages of mitosis (Sharp et al., 1999a
), and this was supported by our current analyses, which indicate a rate for interphase–prophase spindle pole separation after the injection of anti-KLP61F antibodies that is indistinguishable from controls (our unpublished results). Thus, we assessed the role of cytoplasmic dynein in the initial separation of spindle poles. For this, cytoplasmic dynein activity was inhibited in Drosophila
embryos by two separate methods. In one set of studies we disrupted dynein activity by injecting human p50 dynamitin into Drosophila
embryos. p50 is a component of the dynein “activator” dynactin (Gill et al., 1991
; Schroer and Sheetz, 1991
) and has been shown to specifically inhibit cytoplasmic dynein when overexpressed (Echeverri et al., 1996
). In a second set of studies, we injected a mAb that specifically recognizes the dynein heavy chain (anti-DHC; Figure A) into embryos. Immunofluorescence using anti-DHC shows a cortical staining pattern, very similar to the actin-rich “caps” known to surround each nuclear domain (Warn et al., 1984
; Karr and Alberts, 1986
; Kellogg et al., 1988
), into which the ends of astral MTs extend (Figure B). Diffuse staining is also seen on the central spindle but not at the poles after NEB (Figure C). For unknown reasons, this localization for the dynein heavy chain in Drosophila
early embryos is different than that reported previously (Hays et al., 1994
), although similar cortical staining has been observed in vertebrate epithelial cells (Busson et al., 1998
). Possible explanations for this observation include that anti-DHC is specific for a distinct dynein isoform or recognizes a site on the same dynein isoform that is masked unless the motor is bound to specific cellular targets such as the cortex.
Figure 2 Specificity of the anti-DHC mAb and the immunolocalization of dynein in Drosophila early embryos. (A) Immunoblots of purified MAP fractions from Drosophila embryonic high-speed supernatant probed with the anti-DHC monoclonal antibody (lane 1) and similar (more ...)
When microinjected into Drosophila embryos, both p50 dynamitin and anti-DHC substantially reduce the rate and extent of spindle pole migration during interphase–prophase of cycle 13 (Figure , top panel). The initial rapid phase of spindle pole separation is almost completely eliminated, and spindle poles separate to a distance of ~4 and 3 μm in p50- and anti-DHC-injected embryos, respectively, compared with 7 μm in controls. In some cases, the inhibition of dynein also results in a prophase arrest in the affected spindles (see Figure , top panel, for video). These data suggest that cytoplasmic dynein located on the cortical actin caps (cortical dynein) exerts pulling forces on astral MTs to provide the major force for spindle pole separation during early phases of mitosis. The inhibition of dynein was also observed to result in the formation of abnormally large nuclei with four associated spindle poles, suggesting defects in karyokinesis (our unpublished results). Such nuclei were never included in our quantitative analyses of spindle pole migration.
Figure 7 Spindle defects induced by inhibiting either dynein or KLP61F are reversed by double knockouts with Ncd. Confocal micrographs from live wild-type embryos coinjected with fluorescent tubulin and anti-DHC (top left) or anti-KLP61F (top right) are shown. (more ...)
Previous studies suggested that the C-terminal kinesin Ncd provides a force that antagonizes the pole-separating activity of the bipolar kinesin KLP61F at stages subsequent to NEB (Sharp et al., 1999b
). To determine whether Ncd performs a similar counterbalancing function to cortical dynein in earlier phases of mitosis, we exploited the Ncd null mutant claret-nondisjunctional (cand
; see MATERIALS AND METHODS, Drosophila
Stocks and Embryo Collections) (Sturtevant, 1929
; Lewis and Gencarella, 1952
). Strikingly, the overall rate and extent of spindle pole separation in cand
embryos is much greater than in wild-type embryos (Figure , center panel). Closer analysis reveals that in the absence of Ncd activity the early fast phase of spindle pole separation occurs at roughly the same rate as in wild-type embryos (~0.19 vs. 0.11 μm/s) but overshoots. This overshoot causes the spindle poles to separate nearly completely within the first 100 s of this phase and also results in an overall decrease in the length of each mitotic cycle, in general as illustrated in Figure (see associated video). Based on these observations, we propose that Ncd serves as a brake during the initial migration of spindle poles, limiting its rate and length and preventing the premature separation of spindle poles. This activity may result from the putative capacity of Ncd to cross-link antiparallel microtubules and generate minus-end–directed forces, which would serve to oppose spindle pole separation. In the absence of this control, spindle poles from adjacent nuclei may form aberrant contacts, which could, in turn, result in the formation of microtubule “spurs” often observed between spindles lacking normal Ncd activity (see Figure , top right panel, arrow) (Endow and Komma, 1996
). The resulting structural instability of these spindles may ultimately decrease the fidelity of chromosome segregation (Endow et al., 1990
In anti-DHC-microinjected cand embryos, we observed a complete rescue to the wild-type rate of spindle pole migration (Figure , bottom panel; see Figure , bottom left panel, for video). The plots of spindle pole migration versus time for the wild-type embryos and anti-DHC-injected cand embryos after the perturbation of cortical dynein activity are essentially identical. This strongly supports the notion that cytoplasmic dynein and Ncd generate antagonistic forces during the initial separation of spindle poles. Moreover (as discussed below), this observation suggests the existence of an underlying mechanism for spindle pole migration that is independent of cortical dynein and Ncd.
Antagonistic Microtubule Motors Involved in Spindle Pole Separation during Prometaphase and Metaphase
During prometaphase and metaphase of cycle 12, our observations suggest that KLP61F and dynein cooperate to drive the separation of spindle poles, whereas Ncd continues to antagonize this activity by pulling them together. Figure , top left panel, shows the temporal sequence of events occurring in wild-type embryos injected with anti-KLP61F antibodies. As previously reported (Sharp et al., 1999b
), spindles collapse to form MT monoasters under these conditions. However, our current analyses show that these spindles do not begin to collapse immediately after NEB and maintain a constant spacing of ~7 μm for 25–30 s (similar to controls) before the spindle poles begin to slide together at a rate of ~0.06 μm/s (see Figure , top right panel, for video). Figure , top right panel, shows the effects of anti-DHC injections during the same stage in spindle formation. Although these spindles do not collapse, the rate and extent of spindle elongation are greatly reduced, with spindles reaching a length at metaphase of only ~8 vs. ~10 μm in controls. Similar results were obtained after the injection of p50 dynamitin (our unpublished results). These observations are consistent with the hypothesis that KLP61F and cortical dynein work in concert to elongate the spindle during prometaphase. Finally, in cand
embryos, the temporal plot of prometaphase–metaphase spindle pole separation appears nearly identical to wild type under control conditions (our unpublished results), but the absence of Ncd activity ameliorates the effects resulting from the injection of anti-KLP61F or anti-DHC antibodies (Figure , bottom panels; see Figure , bottom right panel, for video). This indicates that Ncd has a role in this process that is antagonistic to both KLP61F and dynein.
Figure 5 KLP61F and dynein function in concert to separate spindle poles during prometaphase–metaphase and are antagonized by Ncd. Shown is a comparison of spindle pole separation versus time during prometaphase and metaphase in control injected wild-type (more ...)
Antagonistic Microtubule Motors Involved in Spindle Elongation during Anaphase B
As in prometaphase–metaphase, the activity of both dynein and KLP61F appears to be required for the proper separation of spindle poles during anaphase spindle elongation. Figure , top left panel, shows the effects of anti-DHC injection on anaphase B in wild-type embryos. Although spindles are abnormally short in anti-DHC-injected embryos at the onset of anaphase (resulting from an abnormal prometaphase), they elongate at an initial rate that is nearly identical to that observed in controls. However, later anaphase B movements (from 25 to 55 s) are severely hampered, and the spindles shorten significantly, suggesting that dynein is involved in late but not early anaphase B. The mechanical basis for this observation is unclear but may result because, early in anaphase B, spindles are too short to allow extensive contacts to form between astral microtubules and cortical dynein. An entirely similar inhibition of anaphase B was observed in p50-injected embryos, as well (our unpublished results). Figure , top right panel, shows the effects of anti-KLP61F injection on anaphase B. Because spindles collapse during prometaphase when KLP61F is inhibited in wild-type embryos, it was necessary to perform this set of experiments in cand embryos. Overall, under these conditions, both the early and later phases of anaphase B are greatly diminished (although elongation does occur in some spindles), supporting the notion that KLP61F actively drives the apparently dynein-independent early movements in anaphase B. Ncd on its own, however, appears to have little or no influence on anaphase B because, as shown in Figure , bottom two panels, the temporal plots of anaphase B in the presence or absence of Ncd activity appear nearly identical in both control and anti-DHC-injected embryos (Figure , bottom right and bottom left panels, respectively). Thus, it is possible that anaphase B is triggered by the down-regulation of Ncd, allowing first KLP61F alone and then KLP61F in concert with cortical dynein to drive the poles apart. Further experimentation will be required to test the merits of this hypothesis.
Figure 6 Dynein and KLP61F drive anaphase B spindle elongation. Comparison of spindle pole separation versus time during anaphase B is shown. Top left panel, Control-injected wild-type embryos versus anti-DHC-injected wild-type embryos. Bottom left panel, anti-DHC-injected (more ...)
Simultaneous Functional Inhibition of Pairs of Antagonistic Motors Uncovers an Underlying “Backup” Mechanism for Mitosis
One striking observation that should be noted is that the inactivation of pairs of counterbalancing MT motors at appropriate stages of mitosis leads to a rescue of successful mitotic spindle assembly and function (Figure ). For example, the coinhibition of Ncd with dynein (left panels) or Ncd with KLP61F (right panels) results in a nearly complete restoration of normal spindle pole positioning and bipolar spindle assembly during interphase–prophase or prometaphase–metaphase, respectively. Thus, these “double knockouts” may have uncovered underlying mechanisms for bipolar spindle assembly and maintenance before anaphase. Although the identity of these backup mechanisms is unknown, possibilities include: 1) a low level of residual KLP61F or dynein motor activity that is sufficient to drive spindle formation in the absence of the antagonistic forces generated by Ncd; 2) redundant sets of MT motors whose activities are normally masked by dynein, KLP61F, and Ncd; 3) the force derived from MT dynamics; 4) interactions between MTs and the dynamic actin network that surrounds the spindle; and 5) a novel, unidentified mechanical system that contributes to mitosis.