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).
Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and their basic contributions to microtubule dynamics are understood, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. This study have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. Microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on these findings, it is proposed that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics (Trogden, 2015).
Microtubules interact with the small GTPase Rac in a complex pattern of cross-talk at the leading edge of motile cells. Growing microtubules induce cortical Rac activation by locally activating a guanine exchange factor (GEF) to induce protrusion and directional migration. In what is thought to be a positive feedback loop, active Rac promotes persistent microtubule growth in the lamellae and lamellipodia by locally regulating the activity of microtubule-associated proteins (MAPs). At least three MAPs have been implicated in regulation of microtubule dynamics downstream of Rac. The first is CLIP-170, a microtubule plus end interacting protein (+TIP) that interacts with the Rac effector IQGAP1 to capture microtubule plus ends at the plasma membrane. The second is Stathmin/OP18, a microtubule destabilizing factor that is locally inhibited at the leading edge due to phosphorylation by the Rac effector kinase Pak. The third is CLASP, another +TIP that suppresses dynamics, leading to increased stabilization of microtubules at the leading edge of polarized fibroblasts. CLASP binds directly to microtubules through a central lattice-binding domain and localizes to growing plus ends through an interaction with EB1. In the cell cortex, CLASP is phosphorylated by GSK3β, which blocks its ability to bind to the microtubule lattice, thus targeting it to growing plus ends. At the leading edge, GSK3β is locally inhibited by Rac and dephosphorylated CLASP binds along the microtubule lattice. Although local inhibition of Stathmin and activation of CLASP seem to be necessary for persistent microtubule growth at the leading edge, neither factor is sufficient, suggesting that other regulatory mechanisms remain to be discovered (Trogden, 2015 and references therein).
The present study identified the XMAP215 homolog, Msps, as a downstream effector of the Rac pathway and describes a novel regulatory mechanism for Msps through a protein-protein interaction with the microtubule stabilizer Orbit and the scaffolding protein Sentin. In S2 cells, the Drosophila CLASP homolog Orbit localizes to microtubule plus ends, but binds to the microtubule lattice upon expression of active Rac1 or depletion of GSK3β. These observations are similar to the dynamics of CLASP in mammalian cells and suggest that this mode of regulation is conserved. Activation of Rac or depletion of GSK3β promotes Msps binding to the microtubule lattice and this localization requires Orbit. The data suggests that, like CLASP, Orbit is directly phosphorylated by GSK3β which prevents it from interacting with and recruiting Msps to the microtubule lattice. The Orbit-Msps interaction further requires another +TIP, Sentin. As the localization of Sentin at the microtubule plus ends is not regulated by Rac-GSK3β, it is likely serving a scaffolding function to promote interactions between Msps and Orbit. This study mapped the protein-protein interaction sites on Msps and Orbit to their C-termini and found that mutations that block their interaction severely perturb microtubule dynamics. Both a non-phosophorylatable Orbit mutant and a mutant that prevented the Msps-Orbit interaction lead to more persistent growth, with the non-phosphorylatable Orbit mutant also causing an increase in microtubule pause. This may indicate that this interaction is important for persistent microtubule growth downstream of Rac-GSK3β (Trogden, 2015).
A growing body of evidence indicates that +TIPs exhibit cross-talk with one another to regulate microtubule dynamics in response to upstream regulatory cues. The current results indicate that Msps and Orbit function together during interphase to regulate dynamic instability in response to Rac and GSK3β activity. It is well established that members of the XMAP215 family promote microtubule dynamics by catalyzing microtubule polymerization and depolymerization. These activities are conserved in Drosophila Msps; microtubules in S2 cells lacking Msps are less dynamic, spending most of their lifetime in a pause state. In contrast, Orbit acts to suppress microtubule dynamics and promotes their stability. Thus, Msps and Orbit would be seem to regulate microtubule dynamics antagonistically, a functional relationship supported by recent genetic studies. However, the current results indicate that the two proteins share a more complex interaction (Trogden, 2015).
Msps exhibits two distinct localization patterns on microtubules in S2 cells- at the plus ends of microtubules and along the distal microtubule lattice in the periphery. The data support the model that these different modes of microtubule association represent functionally distinct pools of Msps. First, the Msps-Orbit interaction sites were identified, they were mutated to ablate the interaction, and these mutants were used to rescue cells depleted of either endogenous Msps or Orbit. Expression of either Msps-GFP 3A or 3K3A was able to rescue microtubule dynamics as compared to Msps-depleted cells. However, microtubules in these cells exhibited abnormally high frequencies of rescue and low frequencies of catastrophe, spending more time in growth and less in shrinkage compared to control cells. Cells expressing GFP-Orbit with the GSK3β phospho-acceptor sites mutated to alanine (5S->A) suppressed Orbit RNAi-induced increases in dynamic instability, but microtubules in these cells also exhibited higher frequencies of rescue, lower frequencies of catastrophe, and more time in the pause state compared to control cells. These results indicate that Msps must interact with Orbit in order to properly regulate microtubule dynamics in the cell periphery. Second, when the growth rates of microtubules were examined by tracking EB1-GFP, it was noted that EB1 comets in the cell periphery exhibit slower velocities than those in the cell cortex. These differences likely reflect interactions between growing microtubules and lamellipodial actin undergoing retrograde centripetal flow in the cell periphery. Recent work has also shown that EB1 comets are structurally different in the cortex versus the periphery, so the differences may be explained by changes in the microtubule as well. However, when EB1 comets on microtubules were compared with Msps at the plus end to those that had Msps localized along the distal lattice, it was discovered that the latter exhibited a significantly slower rate of growth. Collectively, the results indicate that the Msps-Orbit interaction 'tunes' microtubule dynamics in response to Rac activation in the cell periphery. It is suggested that Msps could be shunted onto the lattice to act as a localized 'sink' that attenuates its activity as a microtubule polymerase. This inactive pool may serve as a mechanism to partially suppress Msps activity so that microtubules grow at specific rates upon reaching the edge of the cell. The mechanism of how Msps regionally governs microtubule dynamics presents an intriguing problem; future studies employing biochemical reconstitution of microtubule dynamics with recombinant +TIPs and their regulators will likely be required to address these models (Trogden, 2015).
One puzzling aspect of this study is that, despite the biochemical and functional evidence for the Msps-Orbit interaction, colocalization of Msps and Orbit on the microtubule lattice in the cell periphery was not detected under unperturbed conditions. It is speculated that this protein-protein interaction is transient, occurring at the plus end, but is required for some conformational change in Msps that unmasks its microtubule lattice-binding activity. Two lattice-binding sites have been detected in the inter-TOG linker regions that seem to be inactive while the protein is localized to plus ends. This results were also confirmed using in vitro reconstitution assays. It is possible, however, that Orbit does localize to the lattice in the cell periphery, but at levels so low it was not possible to detect in living cells using the available probes. A third possibility is that Orbit is able to alter the structure of the microtubule lattice proximal to the plus end in order to promote lattice binding of Msps. This alteration could represent a change in the local nucleotide state of the polymer as a recent study indicated that mammalian CLASPs are able to promote GTP hydrolysis by polymerized tubulin. Alternatively, it has been shown that EB1 family members promote structural transitions within the microtubule lattice that favor GTP hydrolysis and compaction of the lattice itself. Perhaps similar localized changes in microtubule structure signal to Msps to transition from tip-association to lattice binding. Further work will be required to understand how these proteins interact to regulate their respective functions and it is expected that in vitro reconstitution assays will prove valuable to advance understanding of this protein-protein interaction (Trogden, 2015).
It is interesting to note that the localization patterns for Msps and Orbit observed in Drosophila cells seem to exhibit the converse relationships to those described for XMAP215/CH-TOG and CLASP in mammalian cells. Msps also differs from XMAP215 and ch-TOG through its lack of ability to either bind directly to EB1 or independently recognize growing microtubule ends. It is possible that the interaction with Orbit developed to increase Msps' ability to target the microtubule plus end. This interaction may also be present in mammalian cells, where it may serve to modulate growth rates of microtubules. Although Msps and XMAP215/CH-TOG exhibit high degrees of identity overall, it will be interesting to compare their relative activities in living cells using Msps to replace CH-TOG, and vice-versa, using heterologous systems (Trogden, 2015).
The data point to an outstanding question about how this localized regulation of dynamic instability impact behavior at the level of the cell. Dynamic microtubules exhibit a complex, bidirectional cross-talk with the Rho family of small G proteins. It is suggested that Msps and other XMAP215 family members are critical components of these pathways. In migrating cells, for example, Rac activity promotes processive microtubule growth while microtubule dynamics also promote Rac activation. It is predicted that Msps/XMAP215 family members are likely to participate in this positive feedback look and are, therefore, likely to play crucial roles in cell motility. Microtubules are also essential for directed membrane traffic to the leading edge. Msps-induced microtubule growth may also contribute to this polarized delivery of cargo to the front of motile cells. In order to address these fascinating questions, the Msps-Orbit interaction will have to be addressed in the context of migratory cell lines or, better still, within the developing embryo (Trogden, 2015).
In the current model in the cortex of the cell, Rac activity is low and therefore GSK3β is active, leading to phosphorylation of Orbit on 5 serine residues. Both Orbit and Msps are at the plus end, but cannot interact with each other. Msps is localized to the plus end through its interaction with Sentin. Orbit can bind either Sentin or EB1 to target the plus end. In the periphery of an S2 cell (or the leading edge of a migrating cell), Rac is active, which leads to the local inactivation of GSK3β and dephosphorylation of Orbit. Orbit is still on the plus end, but is now able to interact with Msps, allowing Msps to bind to the lattice. How this interaction allows Msps to bind the lattice with Orbit remaining on the plus end remains to be determined. It is hypothesized that when Msps is at the plus end it is in a closed conformation where the C-terminus covers the Linker4-TOG5 region that can bind the microtubule lattice. When Msps and Orbit bind to one another, this causes Msps to adopt an open conformation, exposing the lattice binding region which allows Msps to diffuse along to lattice (Trogden, 2015).
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