NuSAP Is Enriched at Meiotic Spindles in Xenopus Oocytes
NuSAP was discovered as a microtubule-interacting protein that is conserved in vertebrates and essential for progression through mitosis in cultured mammalian HeLa cells (Raemaekers et al., 2003
). It was shown that NuSAP localizes specifically to spindle microtubules that are in proximity to chromatin throughout metaphase and anaphase. This particular distribution has not been observed for other microtubule-binding proteins, and it prompted us to study NuSAP and its mechanism of function in detail. As a first step, we asked whether NuSAP is also detectable on meiotic spindles using Xenopus
oocytes as an example. We cloned and expressed full-length NuSAP from Xenopus
, generated antibodies, and analyzed the distribution of NuSAP on meiotic spindles in intact X. laevis
oocytes by indirect immunofluorescence (). Indeed, the NuSAP protein was detectable on the entire length of the metaphase spindle microtubules. As in HeLa cells, the NuSAP protein was enriched at microtubules in the immediate vicinity of chromatin in the oocytes (). The localization of NuSAP was similar in spindles assembled with DNA beads and sperm chromatin in meiotic Xenopus
egg extracts (our unpublished data).
Figure 1. NuSAP localizes to microtubules and is enriched around chromatin of the meiotic spindle of Xenopus oocytes. Stage 6 oocytes were treated with 5 μg/ml progesterone to induce maturation. The oocytes were fixed and stained with a rabbit anti-Xenopus (more ...)
NuSAP Function Is Critical for Spindle Formation in Xenopus Egg Extract
To analyze the function of NuSAP in meiotic spindle formation, we prepared extracts from Xenopus
eggs arrested in metaphase of meiosis II (CSF-arrested extracts). NuSAP was immunodepleted from the extracts by affinity-purified antibodies, and spindle assembly was allowed to proceed around DNA beads for 60 min at 20°C (Heald et al., 1996
; Desai et al., 1999
). DNA beads were used in this experiment instead of purified Xenopus
sperm chromatin (Lohka and Masui, 1984
) to ensure that NuSAP, which is a nuclear protein, was not added to the depleted extracts with the sperm nuclei.
At depletion levels of ~90% (A), microtubules were still produced around DNA beads (C). However, we observed different defects in spindle organization. In some cases, microtubules formed distorted bipolar arrays that contained many buckled microtubules and had inconsistent pole-to-chromatin distances (C, third panel). Moreover, although the spindle microtubules were usually still focused at the poles, they often appeared poorly organized around chromatin (C, third panel). The microtubule density around chromatin often appeared to be reduced (C, third panel). In extreme cases, spindles were impaired in establishing or maintaining bipolarity, and spindle poles were not located on opposite sides of the chromatin (C, right panel). To quantify the defects we collectively categorized all spindles that were not of compact bipolar shape as abnormal and determined their percentage in four different experiments. The percentage of abnormal spindles was significantly increased in NuSAP-depleted extracts (more than 80% compared with 40% in mock-depleted extracts; B). Thus, NuSAP appears to be essential for the proper assembly of meiotic spindles.
Figure 2. NuSAP function is critical for spindle formation in Xenopus egg extract. (A) NuSAP protein was immunodepleted from meiotic Xenopus egg extract with an affinity-purified α-NuSAP antibody. Western blot analysis shows that this resulted in the reduction (more ...)
Two problems complicated the interpretation of this experiment. First, it was exceedingly difficult to deplete NuSAP to more than 90% from the extracts (A). The residual NuSAP may be sufficient to provide partial function and lead to an underestimation of the effects of NuSAP depletion. Second, add-back of recombinant NuSAP to depleted extracts restored microtubule bundling but did not produce well-organized spindles. The chromatin-associated microtubules were strongly bundled (our unpublished data; but see B), but other defects, such as impaired positioning of the spindle poles, were not rescued by recombinant NuSAP alone. Thus, either the recombinant protein was not fully functional or factors that codepleted with NuSAP contribute to the production of the defects. We are currently addressing this problem. In summary, it appears that NuSAP activity is required to establish and/or maintain spindle integrity. The observed defects point toward a function of NuSAP in organizing chromosome-proximal microtubules. However, from these experiments we are unable to conclude whether this effect is direct or indirect.
Figure 3. NuSAP efficiently bundles spindle microtubules in egg extract. (A) Recombinant full length Xenopus NuSAP was expressed in Escherichia coli from pQE80 and purified via nickel-agarose and subsequent gel filtration. The marker (left lane) shows that recombinant (more ...)
NuSAP Efficiently Bundles Spindle Microtubules in Meiotic Egg Extract
In the next step, we studied the effect of excess NuSAP on spindle assembly around sperm chromatin in Xenopus egg extracts (B). When surplus NuSAP was added to the extract before the onset of spindle assembly, the spindle reactions were highly inefficient, and only a few chromatin samples acquired microtubules. It appeared that the presence of excess NuSAP blocked spindle formation. As an alternative approach, we therefore tested the effect of surplus NuSAP on already existing spindle structures. Isolated sperm chromatin was added to egg extract, and spindle assembly was allowed to proceed for either 15 or 45 min. Thereafter, the spindle reaction was supplemented with 0.2 μM recombinant NuSAP (4–10 times the endogenous NuSAP concentration) and further incubated for 10 min. The addition of surplus NuSAP had two reproducible effects on the majority (>90%) of the spindle structures (B). First, the spindle microtubules became strongly bundled into prominent fibers, and second, they often appeared to grow longer than the microtubules in control spindles.
The strength of the effect on spindle intermediates depended on the concentration of recombinant NuSAP added. At concentrations below 0.2 μM (0.05 and 0.1 μM were tested), the bundling of microtubules was still detectable, but less prominent, and fewer spindle structures were affected. In contrast, at concentrations above 0.5 μM, the spindles structures became strongly distorted and often lost their bipolar configuration.
The function of NuSAP was also probed in the context of chromatin-free spindle assembly mediated by RanQ69L. RanQ69L is a mutant of Ran that is locked in the active, GTP-bound state (Bischoff et al., 1994
). Like chromatin, RanQ69L activates a set of spindle assembly factors such that microtubule asters and bipolar spindle-like structures emerge (see Introduction
When recombinant NuSAP alone was added to the egg extract, no detectable microtubule structures were formed (our unpublished data). This means that NuSAP activity is either inhibited or requires additional factors to efficiently produce microtubules in the extract. In contrast, the addition of 15 μM RanQ69L resulted in the formation of microtubules and their organization into asters, which over time evolved into bipolar spindle-like structures (C). To analyze the effect of surplus NuSAP in chromatin-free spindle organization, the reaction was initiated with 15 μM RanQ69L. After 30 min, when Ran-spindles had reached the bipolar state, 0.2 μM recombinant NuSAP was added. The spindle reaction was allowed to proceed for a further 10 min before the samples were fixed (C).
To monitor the position of spindle poles in chromatin-free spindles, a fluorescently labeled antibody of the spindle pole marker NuMA was added to the reaction (Merdes et al., 1996
; Mitchison et al., 2005
). The addition of surplus NuSAP to bipolar structures resulted in a strong bundling and extension of microtubules. Interestingly, the tips of the spindle poles were strongly curled. Moreover, NuMA became spread along the spindle axis instead of being tightly focused at the poles. Furthermore, the symmetric bipolar configuration of the spindles often appeared distorted. Together, these observations indicate that in the presence of surplus NuSAP, microtubules within spindle-like structures are excessively bundled, leading to aberrant spindle morphology.
NuSAP Converts Growing Microtubules into Aster-like Structures and Thick Fibers
Next, we probed the effect of recombinant NuSAP on purified tubulin. Recombinant NuSAP (1 μM) was added to different concentrations of soluble tubulin from 3 to 24 μM, and the reaction was incubated at 37°C for 15 min (A). At 3 μM tubulin, NuSAP produced mainly small, aster-like structures with a dense tubulin core and short microtubules emanating from the center. At higher tubulin concentrations (6–24 μM; A), NuSAP produced not only asters but also prominent microtubule fibers. The NuSAP-generated microtubule fibers became longer and more abundant with increasing tubulin concentrations (A). In control samples, both the aster-like structures and the prominent microtubule fibers as were detectable with 6, 12, or 24 μM tubulin were absent. Thus, in the presence of NuSAP, net microtubule polymerization was significantly increased compared with the buffer control.
Figure 4. Recombinant NuSAP efficiently bundles and stabilizes pure microtubules. (A) Recombinant NuSAP (1 μM) was incubated with different concentrations of tubulin at 37°C for 10 min. The reaction was performed in BrB80 buffer with 1 mM GTP, and (more ...)
NuSAP is a highly basic protein with an isoelectric point of 9.9 and might cause nonspecific aggregation of tubulin. To test this possibility, we analyzed the effect of other highly basic proteins (a mixture of core histones) on pure tubulin (Supplemental ). While the NuSAP-generated structures contained microtubules and thus reflected some degree of organization, histones appeared to induce formation of disorganized aggregates of tubulin (Supplemental ). This indicates that the formation of NuSAP-asters is not due to nonspecific aggregation but the result of specific interaction.
The coexistence of two morphologically different structures is at first sight puzzling and may have two different explanations: First, the asters may represent early intermediates that evolve into microtubule fibers. Second, asters and fibers may result from two independent functions of NuSAP. We will return to this point later.
We used electron microscopy to obtain further insight into the organization of NuSAP and microtubules in the previously examined conditions. Thick bundles with closely adjacent microtubules were detected (B). The interconnected microtubules displayed a strong tendency for parallel alignment. NuSAP itself formed irregularly shaped structures along the outside walls of the microtubules, without any detectable preference for the microtubule ends. NuSAP strongly accumulated on the microtubule bundles and was largely absent from microtubule-free regions, suggesting that its local concentration at the bundles is due to specific interaction with the microtubules. NuSAP alone gave rise to a small number of aggregates (B). At the bundles, NuSAP appeared to fill the space between individual microtubules. Many microtubules were packed into bundles (B), whereas others were cross-linked via their ends to form chains (B, top left).
Importantly, the NuSAP–microtubule bundles not only contained intact microtubules but also prominent sheets consisting of long protofilaments (B, top left, arrowheads; and enlargement, top right). Such prominent sheets were rarely detected in samples without NuSAP. This suggests that NuSAP efficiently promotes either the assembly or the maintenance of protofilament tubulin sheets.
These experiments revealed two properties of NuSAP. First, it appears to strongly promote the net production of microtubules and protofilaments. Second, it can efficiently cross-link microtubules into asters and thick fibers.
Recombinant NuSAP Can Efficiently Cross-Link Pure Microtubules and Tubulin Sheets and Stabilize Them against Depolymerization
NuSAP could in principle promote the production of microtubules and tubulin sheets by two different mechanisms. It may catalyze the assembly of tubulin dimers into protofilaments and microtubules. Alternatively, it may stabilize spontaneously formed microtubules and protofilaments against depolymerization.
We designed an assay to directly test whether NuSAP can stabilize microtubules, i.e., prevent the disassembly of dynamically unstable microtubules. Microtubules were first assembled at 50 μM, a concentration well above the critical tubulin concentration for polymerization (15 μM). Then, the microtubules were diluted to 5 μM, a concentration at which unstabilized microtubules rapidly disassemble. The dilution buffer contained GTP alone, or GTP plus recombinant NuSAP at different concentrations. In the absence of NuSAP, microtubules disassembled rapidly and none were detectable by fluorescent microscopy after 5 min (A). In contrast, when NuSAP was present, large networks of microtubules were detectable.
The microtubules detected in the presence of NuSAP may be due to stabilization of the provided microtubules. Alternatively, they may have been newly generated by NuSAP. To distinguish between these two possibilities, we repeated the experiment under conditions where de novo microtubule assembly is highly inefficient. As before, tubulin was polymerized at 50 μM and subsequently diluted to 5 μM in the presence or absence of 0.7 μM NuSAP. This time, however, the dilution buffer lacked GTP, and the reaction was stopped after 30 s. In these conditions, NuSAP did not induce microtubule production with soluble tubulin (B, bottom). B shows that when NuSAP was absent, microtubules had already disassembled 30 s after dilution to 5 μM. In contrast, in the presence of NuSAP, large fields of microtubules were seen. This suggests that NuSAP exerts its function by stabilizing prepolymerized microtubules and not by de novo polymerization.
To investigate NuSAP-mediated microtubule stabilization in a more quantitative way, we performed microtubule pelleting assays. The amount of tubulin in the pellet reflects the fraction of microtubules that are stabilized by NuSAP and thus remain in the polymerized, i.e., precipitable form. In agreement with the microscopy results in , A and B, most of the microtubules depolymerized in the absence of NuSAP, whereas with increasing concentrations of NuSAP, more and more microtubules were stabilized against depolymerization (C). NuSAP alone precipitated to some extent (C, top). However, when microtubules were present, it efficiently coprecipitated with the microtubules (C, bottom).
Remarkably, the microtubules that were stabilized by NuSAP did not remain individual, but instead were prominently cross-linked into large networks (, A and B). This NuSAP-dependent microtubule cross-linking activity was exceedingly fast, being detectable after 30 s (B). Importantly, other microtubule-interacting proteins such as TPX2 or EB1 did not produce such stabilized microtubule networks (our unpublished data), indicating that the capacity to both stabilize and cross-link microtubules is a specific property of NuSAP. Notably, not all tubulin structures in the networks had the compact and linear appearance of intact microtubules (, A and B, and 6). Instead, numerous fuzzy structures and aggregates were detectable. This suggests that NuSAP stabilizes and cross-links not only intact microtubules but also other polymerization intermediates. Indeed, as depicted in B, a significant proportion of NuSAP–microtubule bundles consisted of protofilament sheets.
The relative distribution of NuSAP and tubulin in the networks as detected by light microscopy is shown in . NuSAP formed small, compact aggregates when incubated in the absence of microtubules (, middle). In contrast, in the presence of microtubules, NuSAP no longer formed aggregates but instead localized along, and maybe also between, the cross-linked microtubules ().
Together, and show that NuSAP is able to efficiently protect microtubules against depolymerization and that NuSAP can efficiently cross-link microtubules and possibly also other polymerization intermediates.
Impα, Impβ, and Imp7 Interact Directly with Recombinant NuSAP
A potent effector such as NuSAP is likely to be tightly regulated within the cell. It is a key finding of recent cell biology that certain mitotic factors are kept inactive by importin binding and that they are activated by RanGTP-dependent importin release (Hetzer et al., 2002
; Zheng, 2004
). To determine whether potential regulators bind to NuSAP in a RanGTP-dependent manner, we immobilized NuSAP (zzNuSAP) and incubated it with meiotic Xenopus
extract in the presence or absence of 20 μM RanQ69L. Two main binding partners bound to NuSAP that were released by RanQ69L (A, lanes 2 and 3). Mass spectrometry identified them as Imp7 and Impβ. Subsequent Western blot analysis revealed Impα as one further interaction partner of NuSAP (our unpublished data; but see B). Using recombinant factors, we verified that Impβ, Imp7, and the Impα/β heterodimer can interact with NuSAP directly (B).
Figure 7. Recombinant NuSAP interacts directly with Impα, Impβ, and Imp7. (A) Immobilized zzNuSAP was incubated with CSF extract (start) in the presence or absence of 20 μM RanQ69L. The eluates from the zzNuSAP-matrix were analyzed by SDS-PAGE. (more ...)
The interaction of Impα with NuSAP was independent of the presence of RanQ69L. This is expected, because Impα lacks a RanGTP binding site and is not a direct target of RanGTP. Notably, the RanQ69L-mediated dissociation of Impβ and Imp7 from NuSAP was not complete and indeed rather inefficient when the importins were present simultaneously (B). This may indicate that complexes consisting of NuSAP and the two importins cannot be dissociated by RanGTP alone.
To examine whether the NuSAP–importin interactions are functional, we analyzed whether the identified importins could mediate import of Alexa 488-labeled NuSAP into nuclei of permeabilized HeLa cells in a standard nuclear import assay (C). In the absence of importins, NuSAP was largely excluded from the nuclei and remained cytoplasmic. This distribution did not significantly change when Impα was added. In contrast, in the presence of Impβ or Imp7 alone, NuSAP was efficiently removed from the cytoplasm and imported into the nuclei, where it accumulated at the nuclear rim and the nucleoli. Together, our data show that Impα, Impβ, and Imp7 can directly interact with NuSAP. The Impα/β heterodimer, as well as Impβ and Imp7 alone, can mediate nuclear import of NuSAP.
Impα, -β, and -7 Impair the Ability of NuSAP to Form Microtubule Networks
Next, we probed whether the importins affect the ability of NuSAP to stabilize and cross-link microtubules into networks. As in , tubulin was polymerized at 50 μM and subsequently diluted to trigger depolymerization. The dilution buffer contained 0.7 μM NuSAP alone or NuSAP and 5 μM of the individual importins. After 10-min incubation at RT, soluble and polymerized tubulin were separated by ultracentrifugation ().
Figure 8. Impα, Impβ, and Imp7 impair the ability of NuSAP to form microtubule networks. Tubulin (50 μM) was polymerized in 20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, and 1 mM GTP at 37°C for 10 min. Subsequently, the microtubules were (more ...)
The stabilizing and cross-linking activity of NuSAP, as determined by the amount of tubulin in the pellet, was decreased in the presence of either Impα, Impβ, or Imp7, indicating that each importin reduced the ability of NuSAP to form microtubule networks. Interestingly, Impβ not only suppressed the formation of the networks but also was able to rapidly dissolve preexisting NuSAP–microtubule networks (our unpublished data), suggesting that the microtubules within the NuSAP-induced networks are still potentially dynamic.
The inhibitory effect of the individual importins was not complete and did not increase when a threefold higher concentration of each importin was used (our unpublished data). Interestingly, the inhibition of microtubule stabilization was more efficient when Impβ and Imp7 were present simultaneously and nearly complete when all three receptors were provided (). This suggests that the importins operate in an additive manner to block NuSAP function.
Impα, -β, and -7 Appear to Inhibit Different Aspects of NuSAP Function
Do the importins affect NuSAP function by the same mechanism? To address this question, we returned to the observation that NuSAP can convert soluble tubulin (at 6 μM and higher) into two morphologically distinct structures, asters and linear fibers (). As mentioned, these two conformations possibly reflect two distinct activities of NuSAP. If so, this assay may be useful to probe the individual effects of the importins on NuSAP activity in more detail.
NuSAP (1 μM) was incubated with 15 μM soluble tubulin at 37°C for 15 min. Importins were present in the reactions at 5 μM and in combinations as indicated in . As shown before, NuSAP alone efficiently converted 15 μM tubulin into asters and thick microtubule fibers. When Impα was present, neither long microtubule fibers nor small aster-like structures were detectable. Instead, small patches of tubulin were seen (). In contrast, when Impβ was present, NuSAP almost exclusively produced long microtubule fibers, and only very few small asters emerged. Finally, on addition of Imp7, NuSAP apparently failed to generate long fibers and instead produced mainly small asters. These asters were on average smaller and consisted of fewer microtubules than the patches that emerged when Impα was present (). The importins alone did not promote the production of microtubule structures (Supplemental Figure 2).
Figure 9. Impα, Impβ, and Imp7 appear to inhibit different aspects of NuSAP function. Tubulin (15 μM) was incubated in BrB80 buffer with 1 mM GTP, and Ficoll and dextran as crowding reagents. The top panels show 15- and 30-min incubations (more ...)
Thus, Impα, Impβ, and Imp7 appear to affect different aspects of NuSAP function. In the presence of Impα or Imp7, NuSAP was able to efficiently produce tubulin asters and patches, but it failed to promote the production of long microtubule fibers. In contrast, in the presence of Impβ, NuSAP efficiently produced long microtubules fibers; however, it apparently had lost its ability to generate asters. These results suggest that asters and linear microtubule fibers reflect two distinct functions of NuSAP.
Finally, we tested whether the inhibitory effect of the importins could be reversed by 15 μM RanQ69L (). The addition of RanQ69L alone did not visibly affect the activity of NuSAP. The effect of Impα was not reversed by RanQ69L, indicating that the NuSAP–Impα interaction was not dissociated by RanQ69L. This is expected, because Impα does not directly interact with Ran. In contrast, the inhibitory effect of Impβ was efficiently reversed by RanQ69L, and NuSAP regained the ability to produce asters at an efficiency comparable with the control sample without importins. The inhibitory effect of Imp7 was only partially reversed by RanQ69L (, fourth panel, top and bottom). This may suggest that the interaction between NuSAP and Imp7 is too strong to be efficiently released by RanQ69L alone.
Figure 10. In the presence of microtubules, RanQ69L can efficiently reverse the effect of Impβ but not of Imp7. Tubulin (15 μM) was incubated in BrB80 buffer with 1 mM GTP, Ficoll and dextran as crowding reagents, and the indicated combinations of (more ...)