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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.

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.

Proteins from the kinesin-8 family promote microtubule (MT) depolymerization, a process thought to be important for the control of microtubule length in living cells. In addition to this MT shortening activity, kinesin 8s are motors that show plus-end directed motility on MTs. Here we describe a simple model that incorporates directional motion and destabilization of the MT plus end by kinesin 8. Our model quantitatively reproduces the key features of length-vs-time traces for stabilized MTs in the presence of purified kinesin 8, including length-dependent depolymerization. Comparison of model predictions with experiments suggests that kinesin 8 depolymerizes processively, i.e., one motor can remove multiple tubulin dimers from a stabilized MT. Fluctuations in MT length as a function of time are related to depolymerization processivity. We have also determined the parameter regime in which the rate of MT depolymerization is length dependent: length-dependent depolymerization occurs only when MTs are sufficiently short; this crossover is sensitive to the bulk motor concentration., 34 pages, 11 figures

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

A reinvestigation of Auletta sp. yielded the novel compound milnamide C (3) plus the known compounds milnamide A (1), milnamide B (hemiasterlin) (2), jasplakinolide (5), and geodiamolides A (6), D (7), E (8), and G (9). The isolation work was guided by cytoskeletal bioactivity data. Compounds 2 and 3 were shown to cause microtubule depolymerization, and 6-9 were shown to cause microfilament disruption. This biological activity and the structural elucidation of 3, including X-ray analysis, are reported here. [structure: see text]

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.

The synthesis of a limited number of novel isothiazolyl-substituted 1,3,4-oxadiazoles (V) is described.

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as dynamic instability. Microtubule-associated proteins (MAPs) regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2 on in vitro MT dynamics, we correlated the distribution of MAP2 on individual MTs with the dynamic phase changes of the same MTs. MAP2 was modified selectively on its projection region by X-rhodamine iodoacetamide without altering the MT-binding activity. When the labeled MAP2 was added to MTs, the fluorescence was distributed along almost the entire length of individual MTs. However, the inhomogeneity of the distribution gradually became obvious due to the fluorescence bleaching, and the MTs appeared to consist of rapidly bleached portions (RBPs) and slowly bleached portions (SBPs), which were distributed randomly along the MT. By measuring the duration of fluorescence bleaching, the density of MAP2 in SBP was estimated to be approximately 2.5 times higher than the RBP. The average tubulin:MAP2 ratio in SBP was calculated to be 16. When the MT dynamics were observed by dark-field microscopy after determining the MAP2 distribution, rescues were always found to occur only at the SBPs. MTs also displayed intermittent shortening by repeated depolymerization phases separated by pause phases. In these cases, depolymerization phases stopped only at the SBPs. Not every SBP stopped depolymerization, but depolymerization always stopped at an SBP. Taken together, we suggest that there is a minimum density of MAP2 that is necessary to stop depolymerization.

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.

A new class of borneol esters that might be considered as biological analogs of paclitaxel regarding their action on microtubules has been found. By structure-activity optimizations, compounds stabilizing microtubules much better than paclitaxel while showing a remarkably reduced cytotoxic activity were obtained. This dissoziation will open completely new therapeutic areas.

The synthesis, biological evaluation and molecular modeling of a series of pyrrole compounds related to 3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid that evaluates and optimizes C-4 substituents are reported. The key factor for microtubule depolymerization activity appears to be the presence of an appropriately positioned acceptor for Cys241β in the otherwise hydrophobic subpocket A.

The marked effects of ethylene on pea stem growth have been investigated. Low temperature and colchicine, both known microtubule depolymerization agents, reverse the effects of ethylene in straight growth tests. Low temperature (6 C) also profoundly reduces the effects of gas in terms of swelling, hook curvature, and horizontal nutation. Deuterium oxide, an agent capable of rigidifying microtubular structure, mimics the effects of ethylene. Electron microscopy shows that microtubule orientation is strikingly altered by ethylene. These findings indicate that some of the ethylene responses may be due to a stabilizing effect on microtubules in plant cells.

The rate of depolymerization of microtubules upon lowering the temperature was found to depend on the amount of time elapsed since the beginning of the assembly process. In the first minutes following self-assembly at 37 degrees C, microtubules are more cold sensitive and depolymerize faster than later at the steady state. In the meanwhile, no change occurred in the average length nor in the shape of the distribution of microtubules. On the other hand, the evolution with time of the apparent dissociation rate constant of tubulin from microtubules was in good correlation with the GTP content of microtubules following assembly, showing that GTP hydrolysis modifies the tubulin-tubulin interactions. Microtubule-bound GTP was not exchangeable for GDP, but steady-state GTP hydrolysis was inhibited by GDP. This result indicates that GDP and GTP exhibit different affinities for tubulin in the body and at the ends of microtubules. It is proposed that GTP-tubulin dissociates faster from microtubules than GDP-tubulin. In other words GTP hydrolysis contributes to the stabilization of microtubules.

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.

Roots of Pisum sativum L. were treated with 0.1% caffeine for 2 hr and 1 hr later some roots were also treated with 0.01% colchicine for 1 hr. Binucleate cells, induced by caffeine, were seen in division 16 hr after the end of treatment; their mitotic index (MI) was 1%. By 20 hr their MI was 8.2%. In roots treated with colchicine after caffeine MI of binucleate cells was 36.3% at 16 hr and was >33% at 18 and 20 hr. Colchicine releases cells from a block imposed by caffeine early in interphase. Parallels are drawn between the antagonism of taxol, a microtubule stabilizing drug, and colchicine and that of caffeine and colchicine, the release induced by colchicine is postulated to be related to microtubule depolymerization.

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.