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Connecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data on the multiscale dynamics and structure of such systems. We approach this problem by studying the impact of depletion attraction on bundles of microtubules and kinesin-14 molecular motors. For all depletant concentrations, kinesin-14 bundles generate comparable extensile dynamics. However, this invariable mesoscopic behavior masks the transition in the microscopic motion of microtubules. Specifically, with increasing attraction, we observe a transition from bi-directional sliding with extension to pure extension with no sliding. Small-angle X-ray scattering shows that the transition in microtubule dynamics is concurrent with a structural rearrangement of microtubules from an open hexagonal to a compressed rectangular lattice. These results demonstrate that bundles of microtubules and molecular motors can display the same mesoscopic extensile behaviors despite having different internal structures and microscopic dynamics. They provide essential information for developing multiscale models of active matter.

Standard of care for triple-negative breast cancer (TNBC) involves the use of microtubule poisons such as paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug-resistant tumors. Identifying agents against targets that limit aneuploidy may be a valuable approach for therapeutic development. One potential target is the microtubule depolymerizing kinesin, MCAK, which limits aneuploidy by regulating microtubule dynamics during mitosis. Using publicly available datasets, we found that MCAK is upregulated in triple-negative breast cancer and is associated with poorer prognoses. Knockdown of MCAK in tumor-derived cell lines caused a two- to five-fold reduction in the IC 50 for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 50 k library and discovered three putative MCAK inhibitors. These compounds reproduced the aneuploidy-inducing phenotype of MCAK loss, reduced clonogenic survival of TNBC cells regardless of taxane-resistance, and the most potent of the three, C4, sensitized TNBC cells to paclitaxel. Collectively, our work shows promise that MCAK may serve as both a biomarker of prognosis and as a therapeutic target.

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/β as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/β interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/β increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/β have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/β spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal-containing spindle assembly factors.

Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate segregation of chromosomes to daughter cells. Errors in these processes can lead to aneuploidy, which is a hallmark of cancer. Understanding the mechanisms that drive spindle assembly will provide fundamental insights into how accurate chromosome segregation is achieved. One challenge in elucidating the complexities of spindle assembly is to visualize protein interactions in space and time. The Xenopus egg extract system has been a valuable tool to probe protein function during spindle assembly in vitro. Tagging proteins with fluorescent proteins and utilizing fluorescence-based approaches, such as Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), have provided visual clues about the mechanics of spindle assembly and its regulators. However, elucidating how spindle assembly factors are spatially regulated is still challenging. Combining the egg extract system and visual FRET approaches provides a powerful tool to probe the processes involved in spindle assembly. Here we describe how a FLIM-FRET biosensor can be used to study protein-protein interactions in spindles assembled in Xenopus egg extracts. This approach should be readily adaptable to a wide variety of proteins to allow for new insights into the regulation of spindle assembly., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.

High RanGTP around chromatin is important for governing spindle assembly during meiosis and mitosis by releasing the inhibitory effects of importin α/β. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin α/β form an effector gradient that is highest at the poles and diminishes toward the chromatin, which is opposite the RanGTP gradient. Importin α and β preferentially inhibit XCTK2 antiparallel microtubule cross-linking and sliding by decreasing the microtubule affinity of the XCTK2 tail domain. This change in microtubule affinity enables RanGTP to target endogenous XCTK2 to the spindle. We propose that these combined actions of the Ran pathway are critical to promote Kinesin-14 parallel microtubule cross-linking to help focus spindle poles for efficient bipolar spindle assembly. Furthermore, our work illustrates that RanGTP regulation in the spindle is not simply a switch, but rather generates effector gradients where importins α and β gradually tune the activities of spindle assembly factors., (© 2019 Ems-McClung et al.)

Proper cell division and the equal segregation of genetic material are essential for life. Cell division is mediated by the mitotic spindle, which is composed of microtubules (MTs) and MT-associated proteins that help align and segregate the chromosomes. The localization and characterization of many spindle proteins have been greatly aided by using GFP-tagged proteins in vivo, but these tools typically do not allow for understanding how their activity is regulated biochemically. With the recent explosion of the pallet of GFP-derived fluorescent proteins, fluorescence-based biosensors are becoming useful tools for the quantitative analysis of protein activity and protein-protein interactions. Here, we describe solution-based Förster resonance energy transfer (FRET) and fluorescence assays that can be used to quantify protein-protein interactions and to characterize protein conformations of MT-associated proteins involved in mitosis.

Cytoskeletal networks are foundational examples of active matter and central to self-organized structures in the cell. In vivo, these networks are active and densely crosslinked. Relating their large-scale dynamics to the properties of their constituents remains an unsolved problem. Here, we study an in vitro active gel made from aligned microtubules and XCTK2 kinesin motors. Using photobleaching, we demonstrate that the gel's aligned microtubules, driven by motors, continually slide past each other at a speed independent of the local microtubule polarity and motor concentration. This phenomenon is also observed, and remains unexplained, in spindles. We derive a general framework for coarse graining microtubule gels crosslinked by molecular motors from microscopic considerations. Using microtubule-microtubule coupling through a force-velocity relationship for kinesin, this theory naturally explains the experimental results: motors generate an active strain rate in regions of changing polarity, which allows microtubules of opposite polarities to slide past each other without stressing the material., Competing Interests: Competing interests The authors declare no competing interests.

Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly. It remains unclear, however, whether BuGZ could regulate additional proteins to promote spindle assembly. In this study, we report that BuGZ promotes Aurora A (AurA) activation in vitro. Depletion of BuGZ in cells reduces the amount of phosphorylated AurA on spindle MTs. BuGZ also enhances MCAK phosphorylation. The two zinc fingers in BuGZ directly bind to the kinase domain of AurA, which allows AurA to incorporate into the coacervates formed by BuGZ in vitro. Importantly, mutant BuGZ that disrupts the coacervation activity in vitro fails to promote AurA phosphorylation in Xenopus laevis egg extracts. These results suggest that BuGZ coacervation promotes AurA activation in mitosis., (© 2018 Huang et al.)

To ensure proper spindle assembly, microtubule (MT) dynamics needs to be spatially regulated within the cell. The kinesin-13 MCAK is a potent MT depolymerase with a complex subcellular localization, yet how MCAK spatial regulation contributes to spindle assembly is not understood. Here we show that the far C-terminus of MCAK plays a critical role in regulating MCAK conformation, subspindle localization, and spindle assembly in Xenopus egg extracts. Alteration of MCAK conformation by the point mutation E715A/E716A in the far C-terminus increased MCAK targeting to the poles and reduced MT lifetimes, which induced spindles with unfocused poles. These effects were phenocopied by the Aurora A phosphomimetic mutation, S719E. Furthermore, addition of the kinesin-14 XCTK2 to spindle assembly reactions rescued the unfocused-pole phenotype. Collectively our work shows how the regional targeting of MCAK regulates MT dynamics, highlighting the idea that multiple phosphorylation pathways of MCAK cooperate to spatially control MT dynamics to maintain spindle architecture., (© 2016 Zong et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Ran is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1] and subsequently found to be important for spindle formation [2-5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation and organization and regulate MT dynamics [7-9]. Although many SAFs are non-motile MT-associated proteins, such as NuMA, TPX2, and HURP [7, 10-12], Ran also controls motor proteins, including Kid and HSET/XCTK2 [13, 14]. The Kinesin-14 XCKT2 is important for spindle assembly and pole organization [15-20], and Ran-GTP is proposed to promote XCKT2 MT crosslinking activity by releasing importin α/β from a bipartite nuclear localization signal (NLS) located in the tail domain [14]. Here, we show that the Ran-GTP gradient spatially regulates XCTK2 within the spindle. A flattened Ran-GTP gradient blocked the ability of excess XCTK2 to stimulate bipolar spindle assembly and resulted in XCTK2-mediated bundling of free MTs. These effects required the XCTK2 tail, which promoted the motility of XCTK2 within the spindle independent of the Ran-GTP gradient. In addition, the turnover kinetics of XCTK2 were spatially controlled: they were faster near the poles relative to the chromatin, but not with a mutant XCTK2 that cannot bind to importin α/β. Our results support a model in which the Ran-GTP gradient spatially coordinates motor localization with motility to ensure efficient spindle formation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Proper spindle assembly and chromosome segregation rely on precise microtubule dynamics, which are governed in part by the kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood., Results: Here, we develop the first Förster resonance energy transfer (FRET)-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging, we find that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule., Conclusions: Unlike motile kinesins, which are open when doing work, the high-affinity binding state for microtubule-depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule-depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of kinesin-13s can be rapidly modulated to control cellular microtubule dynamics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9]. Kinesin-8 proteins are length-specific, plus-end-directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off rate from the MT end are important for Kif18A function. In addition, our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.

Successful cytokinesis is critical for maintaining genome stability and requires the assembly of a robust central spindle to specify the cleavage furrow position, to prevent separated chromosomes from coming back together, and to contribute to midbody abscission. A proper central spindle is assembled and maintained by a number of microtubule-associated and molecular motor proteins that sort microtubules into bundles with their plus ends overlapping at the center. The mechanisms by which different factors organize the central spindle microtubules remain unclear. We found that perturbation of the minus-end-directed Kinesin-14 HSET increased the frequency of polyploid cells, which resulted from a failure in cytokinesis. In addition, HSET knockdown resulted in severe midzone microtubule organization, most notably at microtubule minus ends, as well as mislocalization of several midbody-associated proteins. Biochemical analysis showed that both human HSET and Xenopus XCTK2 cofractionated with the gamma-tubulin ring complexes on sucrose gradients and that XCTK2 associated with gamma-tubulin and Xgrip109 by immunoprecipitation. Our data reveal the novel finding that a minus-end-directed motor contributes to the organization and stability of the central spindle, which is needed for proper cytokinesis., ((c) 2010 Elsevier Ltd. All rights reserved.)

Dynamic microtubules are essential for the process of mitosis. Thus, elucidating when, where, and how microtubule dynamics are regulated is key to understanding this process. One important class of proteins that directly regulates microtubule dynamics is the Kinesin-13 family. Kinesin-13 proteins induce depolymerization uniquely from both ends of the microtubule. This activity coincides with their cellular localization and with their ability to regulate microtubule dynamics to control spindle assembly and kinetochore-microtubule attachments. In this review, we highlight recent findings that dissect the important actions of Kinesin-13 family members and summarize important studies on the regulation of their activity by phosphorylation and by protein-protein interactions., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin alpha/beta, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (NLS) of human Kinesin-14 HSET caused an accumulation of HSET in the cytoplasm, which resulted in strong microtubule bundling. HSET overexpression in HeLa cells resulted in longer spindles, similar to what was seen with NLS mutants of XCTK2 in extracts, suggesting that Kinesin-14 proteins play similar roles in extracts and in somatic cells. Conversely, HSET knockdown by RNAi resulted in shorter spindles but did not affect pole formation. The change in spindle length was not dependent on K-fibers, as elimination of the K-fiber by Nuf2 RNAi resulted in an increase in spindle length that was partially rescued by co-RNAi of HSET. However, these changes in spindle length did require microtubule sliding, as overexpression of an HSET mutant that had its sliding activity uncoupled from its ATPase activity resulted in cells with spindle lengths shorter than cells overexpressing wild-type HSET. Our results are consistent with a model in which Ran regulates the association of Kinesin-14s with importin alpha/beta to prevent aberrant cross-linking and bundling of microtubules by sequestering Kinesin-14s in the nucleus during interphase. Kinesin-14s act during mitosis to cross-link and slide between parallel microtubules to regulate spindle length.

During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

Chromosome congression and segregation require the proper attachment of microtubules to the two sister kinetochores. Disruption of either Aurora B kinase or the Kinesin-13 mitotic centromere-associated kinesin (MCAK) increases chromosome misalignment and missegregation due to improper kinetochore-microtubule attachments. MCAK localization and activity are regulated by Aurora B, but how Aurora B phosphorylation of MCAK affects spindle assembly is unclear. Here, we show that the binding of MCAK to chromosome arms is also regulated by Aurora B and that Aurora B-dependent chromosome arm and centromere localization is regulated by distinct two-site phosphoregulatory mechanisms. MCAK association with chromosome arms is promoted by phosphorylation of T95 on MCAK, whereas phosphorylation of S196 on MCAK promotes dissociation from the arms. Although targeting of MCAK to centromeres requires phosphorylation of S110 on MCAK, dephosphorylation of T95 on MCAK increases the binding of MCAK to centromeres. Our study reveals a new role for Aurora B, which is to prevent excess MCAK binding to chromatin to facilitate chromatin-nucleated spindle assembly. Our study also shows that the interplay between multiple phosphorylation sites of MCAK may be critical to temporally and spatially control MCAK function.

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.

MCAK belongs to the Kinesin-13 family, whose members depolymerize microtubules rather than translocate along them. We defined the minimal functional unit of MCAK as the catalytic domain plus the class specific neck (MD-MCAK), which is consistent with previous reports. We used steady-state ATPase kinetics, microtubule depolymerization assays, and microtubule.MCAK cosedimentation assays to compare the activity of full-length MCAK, which is a dimer, with MD-MCAK, which is a monomer. Full-length MCAK exhibits higher ATPase activity, more efficient microtubule end binding, and reduced affinity for the tubulin heterodimer. Our studies suggest that MCAK dimerization is important for its catalytic cycle by promoting MCAK binding to microtubule ends, enhancing the ability of MCAK to recycle for multiple rounds of microtubule depolymerization, and preventing MCAK from being sequestered by tubulin heterodimers.

Kin Is, kinesins with an internal catalytic domain, de-polymerize microtubules from both ends, and the KIF2C crystal structure presented by ([this issue of Cell]) provides provocative evidence to support the theory that the highly conserved sequences are critical structural elements in these catastrophic kinesins.

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.

Kin I kinesins are members of the diverse kinesin superfamily of molecular motors. Whereas most kinesins use ATP to move along microtubules, Kin I kinesins depolymerize microtubules rather than walk along them. Functionally, this distinct subfamily of kinesins is important in regulating cellular microtubule dynamics and plays a crucial role in spindle assembly and chromosome segregation. The molecular mechanism of Kin I-induced microtubule destabilization is as yet unclear. It is generally believed that Kin Is induce a structural change on the microtubule that leads to microtubule destabilization. Recently, much progress has been made towards understanding how Kin Is may cause this structural change, and how ATPase activity is employed in the catalytic cycle.

Prolamins containing a highly conserved cysteine-rich C-terminal domain have been poorly expressed as soluble protein in model systems such as Escherichia coli. Possible reasons have included a combination of the reducing environment of the bacterial cytoplasm and protein secondary structure. Using a bacterial thioredoxin fusion expression system, full-length native gamma zein, native gamma zein C-terminus, and modified gamma zein C-terminus, containing 13 amino acid changes, were found to accumulate up to 58, 50, and 42% of the total cellular protein, respectively. The native gamma zein C-terminus fusion protein was six times more soluble (70%) than the full-length fusion protein (12%), four times more soluble than the N-terminus (19%), and eight times more soluble than the modified C-terminus (9%). The modified C-terminal domain contained amino acid changes that improved the lysine, isoleucine, and tryptophan content, while removing two evolutionarily conserved cysteines and one nonconserved cysteine. Expression of the native C-terminal domain without thioredoxin resulted in decreased solubility (13%) and decreased expression (8%). In contrast, coexpression with thioredoxin resulted in a sevenfold increase in solubility (86%). These results suggest that insolubility of full-length gamma zein results from structural interactions of the N-terminus and that solubility of the C-terminal domain is dependent on proper disulfide bond formation. The ability to express the C-terminal domain of gamma zein as soluble protein should allow future identification of important structural elements in gamma zein and similar proteins.