Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin. Both experimentally and theoretically we study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. We find that to be an effective suppressor of microtubule dynamics a drug must primarily suppress the loss of GDP tubulin from the microtubule tip.Comment: 17 pages, 11 figures, to appear in Phys. Bio

A Conserved Cytoskeletal Signaling Cascade Mediates Neurotoxicity of FTDP-17 Tau Mutations In Vivo

The microtubule binding proteintau is strongly implicated in multiple neurodegenerative disorders, includingfrontotemporal dementia and parkinsonism linkedto chromosome 17 (FTDP-17), which is caused by mutations intau.In vitro, FTDP-17 mutant versions oftau can reduce microtubule binding and increase the aggregation of tau, but the mechanism by which these mutations promote disease in vivo is not clear. Here we take a combined biochemical and in vivo modeling approach to define functional properties of tau driving neurotoxicity in vivo. We express wild-type human tau and five FTDP-17 mutant forms of tau inDrosophila using a site-directed insertion strategy to ensure equivalent levels of expression. We then analyze multiple markers of neurodegeneration and neurotoxicity in transgenic animals, including analysis of both males and females. We find that FTDP-17 mutations act to enhance phosphorylation of tau and thus promote neurotoxicity in an in vivo setting. Further, we demonstrate that phosphorylation-dependent excess stabilization of the actin cytoskeleton is a key phosphorylation-dependent mediator of the toxicity of wild-type tau and of all the FTDP-17 mutants tested. Finally, we show that important downstream pathways, including autophagy and the unfolded protein response, are coregulated with neurotoxicity and actin cytoskeletal stabilization in brains of flies expressing wild-type human and various FTDP-17 tau mutants, supporting a conserved mechanism of neurotoxicity of wild-type tau and FTDP-17 mutant tau in disease pathogenesis.This work wassupported by National Institutes of Health-National Institute of Neurological Disorders and Stroke Grant R01-NS-08339

Photodegradation of carbendazim sensitized by aromatic ketones

Carbendazim (1) is a benzimidazole extensively used as post-harvest fungicide on fruits and vegetables. The aim of the present work is to study the photodegradation of I sensitized by aromatic ketones, with special attention to mechanistic aspects and to the possible detoxification associated with photochemical treatment. Laser flash photolysis (LPF) lambda(exc) = 355 nm of xanthone (XA) and anthraquinone (AQ) was performed in MeCN solutions, in the presence of 1. A new transient absorbing at lambda(max) 500 and 320 nm was obtained and assigned to the semioxidized radical cation 1(+center dot). An exergonic thermodynamics for electron transfer quenching was confirmed by means of the Rehm-Weller equation. The same species was observed by direct LFP of 1 at 266 nm in polar solvents. Conversely, when a deoxygenated solution of 1 was submitted to LFP in cyclohexane the transient spectrum presented a band with maximum at 380 nm; it was assigned to the triplet excited state ((3)1*) on the basis of energy transfer to oxygen and beta-carotene. The photodegradation of 1 was achieved using XA and AQ as electron acceptors in a solar-simulator, in aerated aqueous medium; the reaction was faster with XA. Formation of a new photoproduct was initially observed; its structure was assigned as carbendazim N-C5 dimer (2). A balance of the total organic carbon (TOC) after prolonged irradiation indicated that mineralization does not occur to a significant extent, pointing to oxidative fragmentation of 1 and 2 to give a variety of low molecular weight products. To check whether the observed photodegradation of 1 results in a decreased toxicity, biological assays were performed using an established model based on the inhibition of mobility of Daphnia magna. The results demonstrate that photodegradation leads to a diminished toxicity, indicating that the photoproducts are less toxic than the parent compound. (C) 2013 Elsevier B.V. All rights reserved.Financial support from the MICINN (Grant: CTQ2010-19909) and the Generalitat Valenciana (Prometeo Program) is gratefully acknowledged.Jornet Olivé, MD.; Castillo López, MÁ.; Sabater Marco, C.; Tormos Faus, RE.; Miranda Alonso, MÁ. (2013). Photodegradation of carbendazim sensitized by aromatic ketones. Journal of Photochemistry and Photobiology A: Chemistry. 256:36-41. https://doi.org/10.1016/j.jphotochem.2013.02.004S364125

TD-60 links RalA GTPase function to the CPC in mitosis

TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore–microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies

Carbendazim Inhibits Cancer Cell Proliferation by Suppressing Microtubule Dynamics

Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G2/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC50, 10 μM) and half-maximally arrests mitosis at a similar concentration (8 μM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 μM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (Kd, 42.8 ± 4.0 μM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest

A Molecular and Structural Mechanism for G Protein-mediated Microtubule Destabilization*

The heterotrimeric, G protein-coupled receptor-associated G protein, Gαs, binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activated form of Gαs binds tubulin with a KD of 100 nm, stimulates tubulin GTPase, and promotes microtubule dynamic instability. Moreover, the data reveal that the α3–β5 region of Gαs is a functionally important motif in the Gαs-mediated microtubule destabilization. Indeed, peptides corresponding to that region of Gαs mimic Gαs protein in activating tubulin GTPase and increase microtubule dynamic instability. We have identified specific mutations in peptides or proteins that interfere with this process. The data allow for a model of the Gαs/tubulin interface in which Gαs binds to the microtubule plus-end and activates the intrinsic tubulin GTPase. This model illuminates both the role of tubulin as an “effector” (e.g. adenylyl cyclase) for Gαs and the role of Gαs as a GTPase activator for tubulin. Given the ability of Gαs to translocate intracellularly in response to agonist activation, Gαs may play a role in hormone- or neurotransmitter-induced regulation of cellular morphology