The direct binding of Orbit to tubulin in microtubule overlay assays was assessed using phosphocellulose purified MAP-free tubulin. Recombinant Orbit protein containing the putative tubulin binding domain and the two GTP binding motifs were transferred to PVDF membranes which were preincubated with GDP, GTP, or its nonhydrolyzable analog GTP-gamma-S. Recombinant Asp protein was used as a positive control for microtubule binding and BSA as a negative control. The filters were then incubated with polymerized microtubules and binding detected using antitubulin antibodies. This segment of Asp protein binds microtubules irrespective of the preincubation treatment. In contrast, Orbit binds microtubules only when first incubated with GTP, but not with GDP or GTP-gamma-S (Inoue, 2000).
To confirm the results obtained by the microtubule overlay assays, whether Orbit would bind microtubules in a GTP-dependent way when in solution was assessed. Microtubules were polymerized with taxol in the absence of GTP and then incubated with soluble Orbit protein in the presence of GTP, GTP, or GTP-gamma-S. Binding to microtubules was detected by Western blots after sedimentation of the tubulin polymers by centrifugation. In the presence of GTP, Orbit was found exclusively in the microtubule pellet, whereas the protein was in the supernatant when either GDP or GTP-gamma-S were used. This was independent of the microtubule concentration. It is concluded that to bind microtubules, Orbit must bind GTP (Inoue, 2000).
To determine whether the Orbit protein is a component of the mitotic spindle, immunostaining of syncytial blastoderm embryos was performed using the affinity-purified antibody and the staining pattern was compared with the distribution of tubulin. As syncytial embryos enter mitosis at prophase, Orbit protein accumulates distinctly at the periphery of nuclei in the polar regions, showing extensive colocalization with tubulin as the spindle forms. Throughout metaphase to anaphase, Orbit colocalizes with microtubules throughout the entire region of the mitotic spindle and its asters. The microtubule association remains with the midbody, and some residual midbody staining appears to remain in interphase (Inoue, 2000).
At present the essential mitotic function of Orbit remains highly speculative, but this study has revealed a fascinating property of this novel protein; that it binds microtubules in a GTP-dependent manner. It is surprising that Orbit will bind to microtubules in the presence of GTP, but not in the presence of the nonhydrolyzable GTP-gamma-S form since it is generally assumed that these nucleotides have similar structures. However, the vinca-alkaloid self-association of tubulin and microtubule assembly is sensitive to the precise modification of guanine nucleotide analogues and the salt concentration suggestive of an allosteric interaction. The GTP binding site of Orbit has similarities to that of tubulin and so may also be very sensitive to modifications of the nucleotide. Another possibility is that Orbit is itself a GTPase and that its association with microtubules requires GTP hydrolysis. It is well established that tubulin and its prokaryotic counterpart, the FstZ protein, are GTPases. The hydrolysis of GTP complexed to ß-tubulin dimers at the plus ends of microtubules leads to their increased curvature and destabilization of the tubulin lattice. Microtubule destabilizing agents, such as colchicine and nocodazole, are known to promote GTPase activity, whereas taxol binding to the inner surface of the microtubule counteracts the effects of GTP hydrolysis. It is of considerable interest to know whether Orbit has GTPase activity either intrinsically or in association with tubulin. Irrespective of whether Orbit can hydrolyze GTP, the finding of a new GTP binding MAP raises possible new complexities for the role of this nucleotide in regulating microtubule behavior (Inoue, 2000).
It would be of interest to determine whether there is any interaction between Orbit and the Awd protein (Abnormal wing discs) a microtubule-associated NDP kinase that converts GDP to GTP. awd mutants display hypercondensed chromosomes typical of those seen in colchicine-treated cells, suggesting activity of this enzyme is required for microtubule polymerization. Given the present findings, however, it is also likely that the Awd protein can influence other aspects of microtubule behavior. The availability of hypomorphic orbit1 mutants now raises the future possibility of using genetic screens to search for mutations that either enhance or suppress the orbit phenotype. Such mutations could identify genes encoding proteins that interact with or regulate Orbit protein function in the mitotic spindle (Inoue, 2000).
Sequence analysis of the Mast protein has revealed that it shares conservation with both bovine and mouse MAP4. The homology is restricted to a domain rich in proline and basic residues thought to be involved in microtubule (MT) binding. This domain falls outside the conserved regions CR-1, CR-2 and CR-3. Although it has not determined experimentally whether this region of the protein is responsible for MT binding, the results indicate that Mast co-sediments with polymerized MTs in a salt-stable complex. The immunolocalization and transfection studies provide further evidence that Mast is a MAP since the protein shows clear co-localization with both interphase and spindle MTs. In agreement with this, Stu1p was also demonstrated to interact with MTs (Pasqualone, 1994). However, Mast also localizes to other compartments of the mitotic apparatus even in the presence of MT poisons. The presence of two putative p34cdc2 phosphorylation sites suggests that Mast might undergo specific post-translational modifications during the G2/M transition, allowing it to reach specific mitotic structures. A number of MAPs have been shown to be phosphorylated upon entry into mitosis, allowing for modifications in MT dynamics to take place (Lemos, 2000).
Mast shares limited homology with members of the dis1-TOG family; however, like Mast, all these proteins localize to centrosomes and/or to spindle MTs during mitosis. In some aspects, Mast shows patterns of localization closer to those of ZYG-9, p93dis1 and XMAP215. These three proteins co-localize with interphase MTs and, during mitosis, ZYG-9 and p93dis1 localize to the centrosome in the absence of MTs. Nevertheless, during late stages of mitosis, Mast shows strong localization to the spindle midzone, like Msps and ch-TOG, two other members of the dis1-TOG family. Mast and ZYG-9, however, do show some unique features since both proteins remain localized to the centromeres of mitotic chromosomes when cells are incubated in the presence of MT-depolymerizing agents. The localization of Mast to MTs, centrosomes, centromeres and the spindle midzone suggests very strongly that this protein might play a role in the regulation of MT dynamics, as has been shown in vitro and in cell-free extracts for some members of the dis1-TOG family (Lemos, 2000 and references therein).
The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. Using the Drosophila S2 cell line, proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) were either depleted or overexpressed in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, a reduction in spindle size was found after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, a conclusion was reached that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force (Goshima, 2005).
Metaphase spindle length was examined in cells depleted of various proteins by RNAi. One general caveat of RNAi approach is that the targeted proteins are not completely depleted and that the observed phenotypes (especially 'no phenotype') could be still 'hypomorphic' as a result of the residual proteins. Although this is also the case of the current study, significant protein-level reduction was confirmed for most of the genes, and specific spindle/chromosome phenotypes were observed. First, it was found that reduction of Rad21, a protein essential for sister-chromatid cohesion, led to longer spindles, as documented in yeast. After Rad21 RNAi, anti-Cid staining (an inner-kinetochore marker) revealed no paired sister-kinetochore dots, and thin chromosomal masses were scattered, strongly suggesting the precocious separation of sister chromatids. Statistically significant effects also were observed for regulators of MT dynamics. In agreement with previous qualitative descriptions, RNAi of the MT stabilizers EB1, Msps, and Mast caused shortening of metaphase spindle, whereas knockdowns of MT depolymerases (Klp10A and Klp67A) caused expansion. Notably, the outer-kinetochore-enriched regulators of MT plus ends (Klp67A, Mast) affected the length more severely than the global or centrosome-localized MT regulators EB1, Msps, and Klp10A. Klp67A and EB1 RNAi sometimes causes centrosome detachment from the kinetochore microtubules (kMTs), and this detachment could skew the measured centrosome-to-centrosome distance from the actual spindle length. However, the degree of centrosome separation from the kMT for EB1 RNAi is small, only accounting for a 0.2 microm increase to the measurement of spindle length. In the case of Klp67A, the centrosome is detached, but is not always localized along the axis. Indeed, by comparing measurements of centrosome-to-centrosome distance to the distances of the minus ends of the kMT in 32 bipolar spindles, it was found that the centrosome-to-centrosome distance in Klp67A RNAi cells underestimates spindle length by 0.3 µm compared with control cells. Nevertheless, these effects are small and do not affect the conclusions that EB1 and Klp67A RNAi treatments shorten and lengthen spindle length, respectively (Goshima, 2005).
If spindle length is controlled by a balance of MT polymerization and depolymerization, it was reasoned that a phenotype generated by depletion of the MT-depolymerizing protein Klp67A might be rescued by depletion of MT-polymerizing proteins (e.g., Msps or Mast). In accordance with this idea, the simultaneous knockdown of Klp67A and Msps or Mast produced an average length that was intermediate between single Klp67A and single Msps or Mast knockdowns. The important role played by MT-polymerization dynamics in determining spindle length was also discovered recently in Xenopus egg extracts where it was also argued that an unidentified non-MT tensile element, possibly a 'spindle matrix', may constrain spindle length. Although the existence of such an element cannot be excluded, the current results have not uncovered evidence for its existence. For example, shortening of MT length by EB1 or Msps RNAi produced short metaphase spindles without significantly perturbing its shape (Goshima, 2005).
In summary, quantitative analyses indicate that MT dynamics are the major determinants of metaphase spindle length, with MT sliding playing a relatively minor role in determining length. Sister-chromatid cohesion is also critical to restrain the length. The model also provides a possible explanation for the surprising insensitivity of spindle length to sliding forces. This model uses a simple set of force-balance equations combined with a 'coupling assumption' in which increasing motor-driven forces leads to faster rates of depolymerization of MTs. The sliding-depolymerization-coupling model also might explain the difference between the current results showing no change in spindle length with varying Kinesin-5 levels, and those in yeast, where varying the concentration of this motor affects spindle length. Unlike metazoan cells, budding yeast displays no detectable MT depolymerization at spindle poles and thus may lack a means of buffering spindle length in response to pushing effects of microtubule motors. Undoubtedly, aspects of the model will need to be revised, and additional parameters will need to be incorporated (e.g., astral microtubule forces are not considered here). Nevertheless, the model in its present state has already served to predict a 'bistabilty' in spindle morphology in which a threshold amount of Kinesin-5-generated force is needed to prevent spindle collapse. With a combination of RNAi and protein overexpression, this prediction was experimentally verified. The model may make other predictions that could be explored in the future. For example, the model predicts that the degree of central-spindle MT overlap varies in response to Kinesin-5 levels, which could be examined in the future by 3D reconstructions of the spindle by electron or deconvolusion microscopy. As another example, a polymer ratchet mechanism was incorporated to explain sliding-depolymerization coupling. Such a coupling could be achieved by a gradient of the Klp10A MT depolymerase activity at the spindle poles, which would allow stronger sliding forces generated by Klp61F to push MT minus ends farther into the interior region of the pole, where the depolymerase activity might be correspondingly higher. Thus, the current model, although not providing a final answer to how mitotic spindle length is controlled and buffered in response to changing forces, provides a useful framework for additional experimentation and more detailed modeling of this important problem in the future (Goshima, 2005).
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