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Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation.

Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density., Molecular Biology of the Cell, 32 (9), ISSN:1939-4586, ISSN:1059-1524

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. Prior work has identified key molecular players in this process, including the kinesin-18 Kif18b and the kinesin-13 Kif2C/MCAK, which both promote microtubule depolymerization. MCAK acts as a potent microtubule depolymerase diffusing short distances on microtubules, while Kif18b is a mitotic processive motor with weak depolymerase activity. However the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 act in an integrated manner to potently promote microtubule depolymerization. We find Kif18b acts as a microtubule plus end delivery factor for its cargo MCAK, and that Kif18b also promotes EB accumulation to plus ends independently of lattice nucleotide state. Together, our work defines the mechanistic basis for a cooperative Kif18b-EB-MCAK network with emergent properties, that acts to efficiently shorten microtubules in mitosis.

Kinetochores that are not attached to microtubules prevent chromosome missegregation via the spindle assembly checkpoint. We show that they also promote their own capturing. Similar to what governs the localization of spindle assembly checkpoint proteins, the phosphorylation of Spc105 by Mps1 allows unattached kinetochores to sequester Stu1 in cooperation with Slk19. The withdrawal of Stu1, a CLASP essential for spindle integrity, from microtubules and attached kinetochores disrupts the organization of the spindle and thus allows the enhanced formation of dynamic random microtubules that span the nucleus and are ideal to capture unattached kinetochores. The enhanced formation of nuclear random microtubules does not occur if Stu1 sequestering to unattached kinetochores fails and the spindle remains uncompromised. Consequently, these cells exhibit a severely decreased capturing efficiency. After the capturing event, Stu1 is relocated to the capturing microtubule and prevents precocious microtubule depolymerization as long as kinetochores are laterally or incompletely end-on attached., Kinetochores (KT) that are not attached to microtubules prevent chromosome missegregation via the spindle assembly checkpoint. Here the authors show that Mps1 localizes Stu1 at unattached KTs together with Slk19, causing a reorganization of the nuclear MT network that favors the capturing of unattached KT.

Microtubules are highly dynamic filaments assembled from αβ-tubulin heterodimers and play important roles in many cellular processes, including cell division and migration. Microtubule dynamics is tightly regulated by microtubule-associated proteins (MAPs) that function by binding to microtubules or free tubulin dimers. Here, we report that FOR20 (FOP-related protein of 20 kDa), a conserved protein critical for ciliogenesis and cell cycle progression, is a previously uncharacterized MAP that facilitates microtubule depolymerization and promotes cell migration. FOR20 not only directly binds to microtubules but also regulates microtubule dynamics in vitro by decreasing the microtubule growth rate and increasing the depolymerization rate and catastrophe frequency. In the in vitro microtubule dynamics assays, FOR20 appears to preferentially interact with free tubulin dimers over microtubules. Depletion of FOR20 inhibits microtubule depolymerization and promotes microtubule regrowth after the nocodazole treatment in HeLa cells. In addition, FOR20 knockdown significantly inhibits both individual and collective migration of mammalian cells. Taken together, these data suggest that FOR20 functions as a MAP to promote microtubule depolymerization and cell migration.

Rabies virus (RABV) is the cause of rabies, and is associated with severe neurological symptoms, high mortality rate, and a serious threat to human health. Although cellular tubulin has recently been identified to be incorporated into RABV particles, the effects of RABV infection on the microtubule cytoskeleton remain poorly understood. In this study, we show that RABV infection induces microtubule depolymerization as observed by confocal microscopy, which is closely associated with the formation of the filamentous network of the RABV M protein. Depolymerization of microtubules significantly increases viral RNA synthesis, while the polymerization of microtubules notably inhibits viral RNA synthesis and prevents the viral M protein from inducing the formation of the filamentous network. Furthermore, the histone deacetylase 6 (HDAC6) expression level progressively increases during RABV infection, and the inhibition of HDAC6 deacetylase activity significantly decreases viral RNA synthesis. In addition, the expression of viral M protein alone was found to significantly upregulate HDAC6 expression, leading to a substantial reduction in its substrate, acetylated α-tubulin, eventually resulting in microtubule depolymerization. These results demonstrate that HDAC6 plays a positive role in viral transcription and replication by inducing microtubule depolymerization during RABV infection.

Summary To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.

// Long-Xian Lv 1, 2, * , Zhen-Xing Zhou 1, * , Zhan Zhou 1 , Li-Jiang Zhang 1 , Ren Yan 2 , Zhao Zhao 1 , Li-Ya Yang 2 , Xiao-Yuan Bian 2 , Hui-Yong Jiang 2 , Yu-Dong Li 1 , Yi-Sheng Sun 1 , Qin-Qin Xu 1 , Gui-Li Hu 3 , Wen-Jun Guan 1 , Yong-Quan Li 1 1 Institute of Pharmaceutical Biotechnology and College of Pharmaceutical Sciences, Zhejiang University, 310058 Hangzhou, China 2 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China 3 Department of Basic Medicine, College of Medicine, Zhejiang University, 310058 Hangzhou, China * These authors contributed equally to this work Correspondence to: Yong-Quan Li, email: lyq@zju.edu.cn Wen-Jun Guan, email: guanwj@zju.edu.cn Keywords: hispidin, autophagy, necrotic cell death, lysosomal membrane permeabilization, microtubule depolymerization Received: February 03, 2016 Accepted: February 20, 2017 Published: March 06, 2017 ABSTRACT Hispidin and its derivatives are widely distributed in edible mushrooms. Hispidin is more cytotoxic to A549, SCL-1, Bel7402 and Capan-1 cancer cells than to MRC5 normal cells; by contrast, hispidin protects H9c2 cardiomyoblast cells from hydrogen peroxide-induced or doxorubicin-induced apoptosis. Consequently, further research on how hispidin affects normal and cancer cells may help treat cancer and reduce chemotherapy-induced side effects. This study showed that hispidin caused caspase-independent death in SGC-7901 cancer cells but not in GES-1 normal cells. Hispidin-induced increases in LC3-II occurred in SGC-7901 cells in a time independent manner. Cell death can be partially inhibited by treatment with ATG5 siRNA but not by autophagy or necroptosis inhibitors. Ultrastructural evidence indicated that hispidin-induced necrotic cell death involved autophagy. Hispidin-induced lysosomal membrane permeabilization (LMP) related to complex cell death occurred more drastically in SGC-7901 cells than in GES-1 cells. Ca 2+ rather than cathepsins from LMP contributed more to cell death. Hispidin induced microtubule depolymerization, which can cause LMP, more drastically in SGC-7901 cells than in GES-1 cells. At 4.1 μM, hispidin promoted cell-free tubulin polymerization but at concentrations higher than 41 μM, hispidin inhibited polymerization. Hispidin did not bind to tubulin. Alterations in microtubule regulatory proteins, such as stathmin phosphorylation at Ser 16 , contributed to hispidin-induced SGC-7901 cell death. In conclusion, hispidin at concentrations higher than 41 μM may inhibit tubulin polymerization by modulating microtubule regulatory proteins, such as stathmin, causing LMP and complex SGC-7901 cell death. This mechanism suggests a promising novel treatment for human cancer.

Chromosome segregation requires the formation of K-fibres, microtubule bundles that attach sister kinetochores to spindle poles. Most K-fibre microtubules originate around the chromosomes through a non-centrosomal RanGTP-dependent pathway and become oriented with the plus ends attached to the kinetochore and the minus ends focused at the spindle poles. The capture and stabilization of microtubule plus ends at the kinetochore has been extensively studied but very little is known on how their minus-end dynamics are controlled. Here we show that MCRS1 is a RanGTP-regulated factor essential for non-centrosomal microtubule assembly. MCRS1 localizes to the minus ends of chromosomal microtubules and K-fibres, where it protects them from depolymerization. Our data reveal the existence of a mechanism that stabilizes the minus ends of chromosomal microtubules and K-fibres, and is essential for the assembly of a functional bipolar spindle.

The precise regulation of microtubule dynamics is essential during cell division. The kinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergent non-motor regions flanking the ATPase domain are critical in regulating its targeting and activity. However, the molecular basis for the function of the non-motor regions within the context of full-length MCAK is unknown. Here, we determine the structure of MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals that the MCAK C-terminus binds to two motor domains in solution and is displaced allosterically upon microtubule binding, which allows its robust accumulation at microtubule ends. These results demonstrate that MCAK undergoes long-range conformational changes involving its C-terminus during the soluble to microtubule-bound transition and that the C-terminus-motor interaction represents a structural intermediate in the MCAK catalytic cycle. Together, our work reveals intrinsic molecular mechanisms underlying the regulation of kinesin-13 activity. DOI: http://dx.doi.org/10.7554/eLife.06421.001, eLife digest Within a cell, there is a scaffold-like structure called the cytoskeleton that provides shape and structural support, and acts as a transport network for the movement of molecules around the cell. This scaffold contains highly dynamic polymers called microtubules that are made from a protein called tubulin. The constant growth and shrinking of the ends of the microtubules is essential to rebuild and adapt the cytoskeleton according to the needs of the cell. A protein called MCAK belongs to a family of motor proteins that can move along microtubules. It generally binds to the ends of the microtubules to shorten them. Previous studies have found that a single MCAK protein binds to another MCAK protein to form a larger molecule known as a dimer. Part of the MCAK protein forms a so-called motor domain, which enables this protein to bind to the microtubules. One end of the protein, known as the C-terminus, controls the activity of this motor domain. However, it is not clear how this works. Talapatra et al. have now revealed the three-dimensional structure of MCAK's motor domain with the C-terminus using a technique called X-ray crystallography. The experiments show that the C-terminus binds to the motor domain, which promotes the formation of the dimers. A short stretch of amino acids—the building blocks of proteins—in the C-terminus interacts with two motor molecules. This ‘motif’ is also found in other similar proteins from a variety of animals. However, once MCAK binds to a microtubule, the microtubule triggers the release of the C-terminus from the motor domain. This allows MCAK to bind more strongly to the microtubule. The experiments also show that the binding of the C-terminus to the motor domain alters the ability of MCAK to associate with microtubules, which encourages the protein to reach the ends of the polymers. Future work is required to see whether other motor proteins work in a similar way. DOI: http://dx.doi.org/10.7554/eLife.06421.002

Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles and provide similar structural information, our method is fast and simple enough to allow for a full quantitative reconstruction of the microtubule organization of several X. laevis spindles—which have volumes tens of thousands of times larger than spindles whose structures have been previously solved by electron microscopy—in a single experimental session, as well as to explore how the architecture of these structures changes in response to biochemical perturbations.

All kinesins share a conserved core motor domain implying a common mechanism for generating force from ATP hydrolysis. How is it then that kinesins exhibit such divergent activities: motility, microtubule cross-linking and microtubule depolymerization? Although conventional motile kinesins have served as the paradigm for understanding kinesin function, the unconventional kinesins exploit variations on the motile theme to perform unexpected tasks. This review summarizes the biological functions and examines the possible molecular mechanisms of Kin C and Kin I unconventional kinesins. We also discuss the possible differences between the microtubule destabilization models proposed for Kar3 and Kin I kinesins.

Whereas most kinesins motor along microtubules, KinI kinesins are microtubule depolymerizing machines. Surprisingly, we found that a KinI fragment consisting of only the motor core is capable of ATP-dependent depolymerization. The motor binds along microtubules in all nucleotide states, but in the presence of AMPPNP, microtubule depolymerization also occurs. Structural characterization of the products of AMPPNP-induced destabilization revealed a snapshot of the disassembly machine in action as it precisely deformed a tubulin dimer. While conventional kinesins use the energy of ATP binding to execute a "powerstroke," KinIs use it to bend the underlying protofilament. Thus, the relatively small class-specific differences within the KinI motor core modulate a fundamentally conserved mode of interaction with microtubules to produce a unique depolymerizing activity.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

More than 50 years ago, microtubule depolymerization was proposed as the force responsible for chromosome movement. New studies measure the force produced by depolymerization and show that protein ring complexes can couple depolymerization to movement. These results have implications for anaphase chromosome motility and mitotic evolution.

Increased microtubule density, through viscous loading of active myofilaments, causes contractile dysfunction of hypertrophied and failing pressure-overloaded myocardium, which is normalized by microtubule depolymerization. We have found this to be based on augmented tubulin synthesis and microtubule stability. We show here that increased tubulin synthesis is accounted for by marked transcriptional up-regulation of the beta1- and beta2-tubulin isoforms, that hypertrophic regulation of these genes recapitulates their developmental regulation, and that the greater proportion of beta1-tubulin protein may have a causative role in the microtubule stabilization found in cardiac hypertrophy.

In this paper I have presented results of experiments in which spindle microtubules were depolymerized by hydrostatic pressure, in order to examine the Inoué dynamic equilibrium concept of spindle assembly and the possible role of microtubule depolymerization-polymerization in the movement of chromosomes. Using a newly developed optical hydrostatic pressure chamber, I investigated with polarization microscopy the quantitative effects of pressure on the polymerization of spindle microtubules and, with phase contrast microscopy, the relationship of pressure-induced spindle microtubule depolymerization to chromosome movement in living cells. From results of earlier experiments, principally those of Inoué et al. with low temperature and colchicine as microtubule-depolymerizing agents, and from results of my own research, I have concluded that: (1) spindle fiber microtubules are sensitive to depolymerization by pressure (3000-7000 psi), spindle microtubules do exist in a labile equilibrium with a pool of subunits, and the Inoué simple equilibrium model does predict changes in spindle microtubule assembly at metaphase induced by pressure; (2) the stability of microtubules depends on the number of "attached ends;" (3) the longest interpolar microtubules and the longest chromosomal fiber microtubules regulate the spindle interpolar length and the chromosome-to-pole positions; (4) chromosome velocity is independent of the number of spindle microtubules, as well as of the drag force of the chromosomes; (5) the chromosomal fiber microtubules transmit the forces between the poles and between the chromosomes and the poles; and (6) polymerization of microtubules does produce pushing forces and, if controlled microtubule depolymerization does not actually produce pulling forces, at least it governs the velocity of chromosome-to-pole movement.

Microtubule disassembly has been studied using a rapid dilution technique (Karr, T.L., and Purich, D.L. (1979) J. Biol. Chem. 254, 10885-10888). Disassembly curves, generated by computer from the solution of series first order differential equations (see following paper), were fit to experimental data with excellent agreement when the diluted microtubules contained no microtubule-associated proteins. The rate constant for dimer release from microtubules was found to be 154 s-1. Assuming a critical tubulin concentration of 8 to 9 x 10(-6) M, the apparent bimolecular rate constant (2 x 10(7) M-1 s-1) for assembly is near the diffusion limit. It was also possible to use the rapid dilution technique for quantitatively correlating the disassembly rate to the number concentration of microtubule ends. These findings suggest that the dynamics of tubulin interactions with microtubules may be characterized in terms of an endwise depolymerization model. A re-evaluation of cold induced depolymerization kinetics (see miniprint supplement) is also fully consistent with our analysis of disassembly dynamics.

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis.In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.

Membranes are common components of the mitotic apparatus of dividing cells (Harris 1975; Hepler 1977). Although they have been recognized for over twenty years in ultrastructural studies of the mitotic apparatus, they have received much less attention than the spindle microtubules and have occupied a relatively minor role in our effort to elucidate the mechanism and control of mitosis.

We examined severed axons of cat sympathetic nerves and severed neurites of cultured chick sensory neurons for evidence of extensive microtubule depolymerization. Cat sympathetic fibres fixed at various times after severing were cross-sectioned for electron microscopy from both cut ends. The number density of microtubules was determined at various times after severing for matched proximal and distal regions equidistant from cut ends. These data show that the number density of microtubules was nearly identical in proximal and distal fragments at 10, 20 and 60 min and at distances between 10 and 50 micron from the cut ends. In chick sensory neurites the microtubule array was examined in longitudinal sections. In order to define objectively a normal microtubule array, the distance between 100 microtubule pairs was measured in seven control neurites, giving a mean distance (+/- S.D.) of 33 nm +/- 19 nm. A normal array of microtubules was defined as having microtubules within 52 nm of their nearest lateral neighbour. Among 11 neurites at times from 1 to 15 min after transection, the mean distance from the cut tip to the first microtubule was 1.3 micron in proximal fragments and 0.5 micron in distal fragments. The mean distance to the normal microtubule array was 2.8 micron in proximal fragments and 2.1 micron in distal fragments. There was no trend or pattern with respect to the times after severing that the neurite was fixed and the amount of microtubule depolymerization. Our results show no evidence for stabilization of axonal/neurite microtubules by capping structures at their ends. We conclude that microtubule instability is unlikely to play a role in the response of axons to axotomy.

The ability of kinetochores (KTs) to maintain stable attachments to dynamic microtubule structures (‘straight’ during microtubule polymerization and ‘curved’ during microtubule depolymerization) is an essential requirement for accurate chromosome segregation. Here we show that the kinetochore-associated Ska complex interacts with tubulin monomers via the carboxy-terminal winged-helix domain of Ska1, providing the structural basis for the ability to bind both straight and curved microtubule structures. This contrasts with the Ndc80 complex, which binds straight microtubules by recognizing the dimeric interface of tubulin. The Ska1 microtubule-binding domain interacts with tubulins using multiple contact sites that allow the Ska complex to bind microtubules in multiple modes. Disrupting either the flexibility or the tubulin contact sites of the Ska1 microtubule-binding domain perturbs normal mitotic progression, explaining the critical role of the Ska complex in maintaining a firm grip on dynamic microtubules.