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During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin [1, 2]. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors [3–5]. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin.
Centrosomes, kinetochores, and chromatin each provide distinct microtubule-organizing sites or signals that contribute to mitotic spindle assembly [6–8]. Of these, only the chromatin-mediated signal is sufficient for spindle formation . This signal is thought to consist of at least two molecular activities, a RanGTP signal generated by the chromatin-bound Ran-GEF RCC1, and an Aurora B signal generated by localization of Aurora B kinase as part of the chromosomal passenger complex (CPC). The function of the RanGTP signal is best understood; relatively high concentrations of RanGTP near chromatin DNA locally release spindle assembly factors (SAFs) from sequestration by importins [3–5]. Current models hypothesize that a RanGTP gradient provides an essential spatial cue for biasing microtubule polymerization to the vicinity of chromatin. While the CPC has been shown to contribute to anastral spindle assembly [10, 11], the molecular function of chromatin-bound CPC in this process is less well characterized than the RanGTP system. It is hypothesized that Aurora B stimulates microtubule assembly around chromosomes by locally inhibiting factors that promote microtubule catastrophes [11, 12].
The role of specific chromatin domains for signaling microtubule assembly is poorly understood. Specifically, the relative contribution, at a molecular level, of bulk chromatin versus centromeric chromatin to locally support microtubule polymerization is unclear. RCC1 localizes throughout chromatin  while the CPC, which also localizes along chromosome arms, is concentrated at the inner centromere . Kinetochores have been shown to locally promote microtubule nucleation and stabilization, and appear to play a key role in spindle assembly in some systems [15, 16].
To quantify the role of centrosomes and kinetochores in spindle assembly dynamics, we performed time-lapse microscopy of spindles assembling around either chromatin-coated beads or sperm nuclei in Xenopus egg extracts. (Figure 1A; and Supplemental Data, Movie 1–Movie 3). Chromatin bead spindles lack centrosomes and kinetochores, while sperm nuclei that have replicated their DNA contain kinetochores and centrosomes. In sperm spindles, the centrosomal array rapidly disassembled concomitant with nuclear envelope breakdown (NEBD) and often dissociated from assembling structures (Supplemental Data, Movie 3), suggesting that centrosomes are not required for normal sperm spindle assembly.
Although detailed assembly kinetics differed from spindle to spindle, certain generalities emerged, shown in the representative curves (Figure 1B, C). A pronounced lag phase was observed before the detectable onset of microtubule polymerization around both chromatin beads and sperm nuclei. Once assembly started, the rate of increase in microtubule density, and the time taken to reach a plateau, was faster for sperm nuclei, independent of the presence of obvious centrosomes. We do not believe that this discrepancy is due to significant differences between the chromatin content of sperm versus beads since the protocol used in this study yields beads with ~0.3 pg of DNA/bead  or 1/10th the amount of DNA as a single sperm nucleus. Since every bead cluster imaged consisted of at least 10 beads there may actually be higher amounts of DNA present in the bead spindle reactions. Thus, we conclude that sperm nuclei contain molecular activities that accelerate microtubule polymerization relative to chromatin beads. While we cannot rule out centrosomes as contributing to the faster kinetics, the fact that nuclei without associated centrosomes accumulated polymer faster than bead clusters makes us favor the interpretation that chromatin-associated activities on the sperm are largely responsible for the faster kinetics.
RanGTP is required for spindle assembly around both sperm nuclei and DNA beads in egg extracts , but it is not clear if its activity must be spatially regulated to localize spindle assembly. To test this, we experimentally flattened the RanGTP gradient for each type of assembly reaction while providing a constant background concentration of RanGTP by adding two different Ran mutants to spindle assembly reactions. RanT24N mimics the nucleotide-free state of Ran and binds with high affinity to RCC1, inhibiting local production of RanGTP by chromatin [17, 18]. RanQ69L, a hydrolysis defective mutant, is constitutively GTP-bound . On its own, Q69L promotes formation of asters throughout the extract, independent of chromatin [19–21]. We added excess T24N to completely inhibit assembly (30 µM). ~80% of sperm nuclei either had no associated microtubules or asters while >90% of bead clumps had no associated microtubules at this concentration (Figures 2A, B and not shown). We then titrated Q69L and looked for rescue of assembly (Q/T Reaction). When Q69L was added at ~15 µM, we observed rescue of spindle assembly around ~80 % of sperm nuclei. (Figures 2A, B and Supplemental Data, Figure S1). Under these same conditions, chromatin beads failed to assemble microtubule structures (Figure 2A). Increasing the concentration of Q69L did not rescue assembly around beads and at higher levels promoted assembly of microtubule asters (not shown). By imaging with two different FRET reporters , we found that the mixture of T24N and Q69L completely flattened the spatial gradients of RanGTP and liberated SAFs around chromatin, replacing them with a uniform concentration of each that was higher than in normal extracts (Figure 2C and Supplemental Data, Figure S2). Thus, sperm nuclei can assemble bipolar spindles in the absence of a localized gradient of RanGTP, while chromatin beads cannot, suggesting that sperm nuclei provide additional spatial signals that promote microtubule polymerization.
The molecular function of Ran-regulated SAFs in directing microtubule polymerization around chromatin remains poorly characterized. Interestingly, SAF activation by Q69L in T24N-treated reactions could be bypassed by the addition of three-fold excess End-binding 1 (EB1), a treatment that globally promotes microtubule assembly in the extract (Figures 2D, E). Identical to the Q/T reactions, this treatment failed to rescue bead spindle assembly (Figure 2D). EB1 promotes microtubule polymerization in Xenopus extract by increasing the rescue frequency and decreasing the catastrophe frequency of microtubules independent of RanGTP . Thus, under conditions of enhanced microtubule polymerization, spindle assembly not only occurs without a RanGTP gradient, but also without the activation of SAFs by RanGTP. Since EB1 is not known to nucleate microtubules but rather promotes microtubule elongation, this data suggests that SAF activities need not directly nucleate microtubules to achieve spindle assembly around sperm nuclei. Further, this provides additional evidence that sperm chromatin contains other RanGTP-independent signals to specifically localize bipolar spindle assembly.
A second spatial signal from chromatin is proposed to come from the chromosomal passenger complex (CPC), consisting of Aurora B, INCENP, Survivin and Borealin\DasraA [11, 23, 24]. We depleted INCENP, and observed spindle assembly defects in both sperm and bead reactions. In both cases bipolar spindles were much smaller, and nuclei and bead clusters lacking microtubules were observed (Figures 3A–C). The two types of reaction were similarly perturbed, showing that normal spindle assembly in both reactions depends on CPC activity. Interestingly, Q/T reactions no longer supported localized microtubule polymerization around sperm nuclei following immunodepletion of INCENP (Figures 3A, B). Small molecule inhibition of Aurora B with VX-680 had the same effect as INCENP depletion in all experiments (not shown).
INCENP depletion effects do not distinguish if the CPC acts locally, near chromatin, or globally, to promote microtubule assembly. To observe possible local effects, we inhibited the catastrophe-promoting kinesin-13 MCAK using a function perturbing antibody that has the effect of stabilizing microtubules. Aurora B is thought to stimulate local microtubule assembly in part by inhibiting this kinesin . MCAK inhibition greatly increased the size of the microtubule assemblies around sperm nuclei in INCENP deleted extracts (Figure 3D). As previously noted , this double inhibited reaction retains spatial control, as assembly occurs near chromatin. However, spatial control was lost when the mixture of T24N and Q69L was added to MCAK inhibited reactions (Figure 3D), suggesting that the RanGTP gradient provides a major microtubule localizing cue when INCENP is missing. When both the RanGTP/SAF and Aurora B/MCAK systems were made spatially uniform, microtubule assembly became completely delocalized from chromatin. These results are in agreement with previous findings that suggest that the RanGTP and CPC pathways function independently [11, 24].
The RanGTP gradient is essential for DNA beads to promote localized microtubule polymerization but it is not essential for sperm nuclei to do so. Since INCENP is required for sperm nuclei to support RanGTP gradient-independent spindle assembly, we next examined whether artificial localization of the CPC could confer gradient-free spindle assembly activity to DNA beads. CPC-localizing beads were made by coating them with α-INCENP IgG. These were mixed with chromatin coated beads, and the mixed clusters were tested for their ability to promote spindle assembly in untreated extract. Both anti-INCENP and control IgG mixed clusters supported spindle assembly (Figure 4A). In extracts supplemented with T24N and Q69L, control hybrids failed to support spindle assembly, while anti-INCENP hybrids supported assembly of ~4X as many bead-associated microtubule arrays and >10X the number of bipolar spindles compared to controls (Figures 4B, C). Anti-INCENP beads alone supported assembly of microtubule arrays in Q/T-treated extract but failed to assemble bipolar structures (not shown).
The difference between beads and sperm cannot be attributed to differences between CPC levels on each chromatin source because sperm and beads had comparable levels of associated Aurora B and INCENP (Figure 4D). Thus, the centromere may provide a local environment that promotes Aurora B activity at a molecular level by localizing additional Aurora B activators and/or at a structural level by spatially organizing the CPC in a conformation that most efficiently activates Aurora B. The hybrid beads had significantly higher levels of Aurora B and INCENP and Aurora B appeared to be hyper-phosphorylated since multiple higher molecular-weight Aurora B bands were detectable by western blot (Figure 4D). This data shows that the sperm-mediated regulation of CPC, which is lacking on DNA beads can be artificially provided by anti-INCENP coated beads. The ability of hybrids to support spindle assembly may be due to the significant enrichment of CPC and/or hyper-activation of Aurora B although the latter is most likely a consequence of the former. We conclude that locally concentrated CPC activity is able to spatially promote spindle assembly even when RanGTP levels are made uniform.
Our results provide significant insight into how microtubules are preferentially assembled around chromosomes during cell division. Release of SAFs by RanGTP most likely activates a gradient of microtubule stabilization. However, we have found that spatial localization of RanGTP per se is not absolutely required since localization of CPC is sufficient to target assembly to the vicinity of chromatin. Conversely, centromere-enriched CPC activity is not required for spindle assembly in egg extracts as spindles form around generic chromatin beads without centromeric DNA. Since clustering of INCENP has been shown to promote autoactivation of Aurora B , centromeric clustering of CPC likely provides a threshold of Aurora B activity that is necessary for RanGTP gradient-independent spindle assembly.
While we cannot entirely discount a contribution of centrosomes to gradient-free spindle assembly in the extract we favor the centromere-centric model for three reasons. First, the addition of purified centrosomes to Q/T treated bead spindles did not induce spindle assembly, suggesting centrosomes alone are not sufficient (data not shown). Secondly, nuclei that lack clearly associated centrosomes go on to build spindles. Third, the Q/T sperm spindle assembly mechanism is absolutely dependent upon INCENP, which is a centromere-enriched protein and not a centrosome-associated factor. Indeed, recent work has shown that the centromere/kinetochore region of chromosomes provides a preferential site for microtubule assembly, and both CPC and the RanGTP system were implicated in this preference [25, 26]. While chromatin beads are sufficient to direct assembly of a bipolar array, they cannot entirely recapitulate chromatin-controlled microtubule assembly mechanisms since they lack the protein assemblies that normally concentrate at centromeres and kinetochores. Adaptation of the chromatin bead system by targeting the assembly of specific microtubule promoting activities to mimic particular “chromosomal” domains will be a valuable tool in the future.
Time-lapse fluorescence imaging of bead spindle assembly.
Time-lapse fluorescence imaging of sperm spindle assembly in the absence of centrosomes.
Time-lapse fluorescence imaging of sperm spindle assembly in the presence of centrosomes.
We acknowledge all members of the Salmon and Mitchison labs as well as previous cell division group participants Chris Field, Sophie Dumont, Dan Needleman and Martin Wühr. We are also grateful to Rebecca Heald, Petr Kalab, and Karsten Weis for the FRET sensors as well as Alex Kelly and Hiro Funabiki for INCENP antibody. This work was supported by the American Cancer Society (grant PF0711401 to T.J. Maresca), the National Cancer Institute (grant CA078048-09 to T.J. Mitchison) and the National Institutes of Health (grant F32GM080049 to J.C. Gatlin and grant GM24364 to E.D. Salmon).
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