Soluble Tubulin Research Articles

Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line

BackgroundArsenic trioxide (As2O3) is a well-known and effective treatment that can result in clinical remission for patients diagnosed with acute promyelocytic leukemia (APL). The biologic efficacy of As2O3 in APL and solid tumor cells has been explained through its actions on anti-proliferation, anti-angiogenesis, and apoptotic signaling pathways. We theorize that As2O3 activates a pathway that disrupts microtubule dynamics forming abnormal, nonfunctioning mitotic spindles, thus preventing cellular division. In this study, we investigated how As2O3 induces apoptosis by causing microtubule dysfunction.MethodsCultured NB4 cells were treated with As2O3, paclitaxel, and vincristine. Flow cytometric analysis was then performed. An MTT assay was used to determine drug-mediated cytotoxicity. For tubulin polymerization assay, each polymerized or soluble tubulin was measured. Microtubule assembly-disassembly was measured using a tubulin polymerization kit. Cellular microtubules were also observed with fluorescence microscopy.ResultsAs2O3 treatment disrupted tubulin assembly resulting in dysfunctional microtubules that cause death in APL cells. As2O3 markedly enhanced the amount of depolymerized microtubules. The number of microtubule posttranslational modifications on an individual tubulin decreased with As2O3 concentration. Immunocytochemistry revealed changes in the cellular microtubule network and formation of polymerized microtubules in As2O3-treated cells.ConclusionThe microtubules alterations found with As2O3 treatment suggest that As2O3 increases the depolymerized forms of tubulin in cells and that this is potentially due to arsenite's negative effects on spindle dynamics.

Hypoxia decreased chemosensitivity of breast cancer cell line MCF-7 to paclitaxel through cyclin B1

Hypoxia, frequently found in the center of solid tumors, may lead to enhance the production of key factor in cell survival, invasion, angiogenesis and loss of apoptosis. The low oxygen tension in hypoxic tumors is also known to interfere with the efficacy of chemotherapy, but the underlying mechanisms are not very clear. Paclitaxel (PTX) is an active agent used in breast cancer chemotherapy, which disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. In the present study, we try to determine whether hypoxia can decrease the chemosensitivity of human breast carcinoma cells to PTX and elucidate the underlying mechanism. We found that hypoxia could decrease PTX-induced cell death and G2/M arrest. Furthermore, our results showed that hypoxia inhibit PTX-induced soluble tubulin polymerized. In addition, we also found hypoxia could suppress PTX-induced cell cycle protein—cyclin B1 expression in MCF-7 cells. To further investigate whether the inhibitory effect of hypoxia on PTX-induced cell death is mediated by decreasing levels of cyclin B1, cyclin B1-transfected MCF-7 cells were used under hypoxic condition. The data showed that the hypoxia-based decreasing chemosensitivity of breast cancer cells to PTX was reversed by cyclin B1. We also found that overexpression of cyclin B1 could significantly increase the sensitivity of MCF-7 cells to PTX by stimulating soluble polymerized tubulin. Overall, hypoxia decreases cyclin B1, which could in turn reverse hypoxia-induced decreasing chemosensitivity to PTX in breast cancer cell line MCF-7.

Panobinostat (LBH589)-Mediated Acetylation of Tubulin Plays a Role in Megakaryocyte Maturation and Platelet Release

Abstract 704Panobinostat (LBH589) is a histone deacetylase inhibitor (HDACi) which has been shown to have therapeutic effects against several types of human cancers. One of the major dose-limiting toxicities associated with the use of LBH589 has been thrombocytopenia. In this study, we investigated the mechanisms underlying thrombocytopenia induced by LBH589. Thrombocytopoiesis is the result of a highly regulated process in which bone marrow (BM) megakaryocyte (MK) progenitors proliferate and acquire lineage-specific markers in the early stages, followed by polyploidization, maturation and platelet formation in the later stages. We evaluated the effects of LBH589 on thrombocytopoiesis, by utilizing an in vitro system in which MK were generated from human BM-derived CD34+ hematopoietic progenitor cells. At low nanomolar concentrations, LBH589 did not interfere with either cellular proliferation or the ability of CD34+ cells to generate MK: both control and LBH589-treated CD34+ cells expanded 2.5 to 5 fold and generated a comparable number of CD61+ MK progenitors. This was also confirmed by MK colonies assay and suggested that LBH589 treatment does not affect the proliferation and commitment of CD34+ cells towards MK lineage. We next evaluated the effects of LBH589 on MK maturation and platelet production. The percentage of mature CD61+/CD42b+ MK generated in the presence of LBH589 was 3-fold lower compared to that generated in the absence of LBH589. Interestingly, the fraction of CD61+/CD42b+ MK generated in cultures in which LBH589 was withdrawn during the maturation step was only 1.4 lower than that found in control cultures, indicating that the negative effects of LBH589 on MK maturation are reversible, confirming similar observations made during clinical trials of LBH589. Culture-derived platelets were analyzed phenotypically and quantitated after dual labeling with anti-CD41 antibodies and thiazole orange (TO). The percentage of CD41+/TO+ platelets derived from MK generated in the presence of LBH589 was significantly reduced (11%) as compared with MK generated in the absence of LBH589 (18.5%). These findings were consistent with the presence of significant numbers of proplatelet-bearing MKs being observed in control cultures but not in LBH 589-treated cultures. Collectively, these data indicate that LBH589 impairs the ability of MK to mature and release platelets.To better understand the mechanisms responsible for these later stage effects on thrombocytopoiesis, we investigated the potential molecular targets of LBH589 in MK lineage. MK maturation is highly dependent on microtubule (MT) cytoskeleton which represents the structural scaffold for proplatelet extension and ensures the transport of cytoplasmic organelles into nascent platelets. Acetylation of tubulin, a non-histone protein which is critical for proper MT function, is a hallmark of polymerized MT and is mediated by HDAC6, a target of LBH589 inhibition. Based on this, we hypothesized that LBH589 treatment might induce changes in tubulin acetylation that in turn result in alterations in MT cytoskeleton. To test this hypothesis, we assessed tubulin acetylation and demonstrated that treatment with LBH589 resulted in an increase in the levels of acetylated tubulin. To determine if LBH589-induced changes in tubulin acetylation resulted in alterations in MT dynamics, we performed Western blot analysis of polymerized vs. soluble (depolymerized) tubulin in the presence or absence of LBH589. The ratio of soluble:polymerized tubulin in control MK was up to 2-fold greater than that found in MK treated with LBH589. In other words, LBH589 treatment induced an increase in the levels of polymerized MT which correlated with an increase in tubulin acetylation. These changes in MT dynamics at molecular levels were reflected by morphological alterations of cytoskeleton organization as visualized by microscopic analysis.Based on these findings we suggest that LBH589-induced changes in tubulin acetylation results in aberrant cytoskeletal organization that in turn leads to defective MK maturation and platelet release. In summary, we demonstrate that the negative effects of LBH589 on thrombocytopoiesis are mediated by targeting acetylation of tubulin. Furthermore, these studies suggest that tubulin can serve as a drug target for HDACi such as LBH589 to treat patients with extreme thrombocytosis. Disclosures:No relevant conflicts of interest to declare.

Eribulin mesylate for the treatment of late-stage breast cancer

Introduction: Chemotherapy agents, particularly anthracycline and taxane, have demonstrated their significance in metastatic breast cancer. However, improving overall survival in late-stage breast cancer remains a challenge. Eribulin mesylate, a new chemotherapy agent, has a proven significance in this setting. Eribulin mesylate is a synthetic analog of a macrolide isolated from a marine sponge. It inhibits microtubule polymerization, inducing mitosis arrest and apoptosis, and aggregates soluble tubulin in nonproductive form. In Phase II studies, this drug has shown a partial and stable response. The Phase III EMBRACE study showed that eribulin mesylate improved overall survival, compared with the physician's choice of treatment, in women who had received two to five prior chemotherapy regimens, including anthracycline and taxane for advanced breast cancer (median overall survival: 13.1 versus 10.6 months HR 0.81, p = 0.041). This compound is well tolerated. The most common adverse event is neutropenia.Areas covered: This paper provides an introduction to the drug, eribulin mesylate, along with an overview of the current drug market for late-stage breast cancer; it also reviews its pharmacodynamics, pharmacokinetics and clinical efficacy.Expert opinion: Currently, eribulin mesylate is only the third single-agent chemotherapy that has improved overall survival (after anthracycline and taxane) in advanced breast cancer. These results, particularly in heavily pretreated breast cancer, suggest that this drug could become a new standard in the treatment of metastatic breast cancer, and should, therefore, be further developed in its earlier stages.

Factors that influence primary cilium length.

Almost all mammalian cells carry one primary cilium that functions as a biosensor for chemical and mechanical stimuli. Genetic damages that compromise cilia formation or function cause a spectrum of disorders referred to as ciliapathies. Recent studies have demonstrated that some pharmacological agents and extracellular environmental changes can alter primary cilium length. Renal injury is a well-known example of an environmental insult that triggers cilia length modification. Lithium treatment causes primary cilia to extend in several cell types including neuronal cells;this phenomenon is likely independent of glycogen synthase kinase-3β inhibition. In renal epithelial cell lines, deflection of the primary cilia by fluid shear shortens them by reducing the intracellular cyclic AMP level, leading to a subsequent decrease in mechanosensitivity to fluid shear. Primary cilium length is also influenced by the dynamics of actin filaments and microtubules through the levels of soluble tubulin in the cytosol available for primary cilia extension. Thus, mammalian cells can adapt to the extracellular environment by modulating the primary cilium length, and this feedback system utilizing primary cilia might exist throughout the mammalian body. Further investigation is required concerning the precise molecular mechanisms underlying the control of primary cilium length in response to environmental factors.

The Determinants That Govern Microtubule Assembly from the Atomic Structure of GTP-Tubulin

Tubulin alternates between a soluble curved structure and a microtubule straight conformation. GTP binding to αβ-tubulin is required for microtubule assembly, but whether this triggers conversion into a straighter structure is still debated. This is due, at least in part, to the lack of structural data for GTP-tubulin before assembly. Here, we report atomic-resolution crystal structures of soluble tubulin in the GDP and GTP nucleotide states in a complex with a stathmin-like domain. The structures differ locally in the neighborhood of the nucleotide. A loop movement in GTP-bound tubulin favors its recruitment to the ends of growing microtubules and facilitates its curved-to-straight transition, but this conversion has not proceeded yet. The data therefore argue for the conformational change toward the straight structure occurring as microtubule-specific contacts are established. They also suggest a model for the way the tubulin structure is modified in relation to microtubule assembly.

Abstract 3832: Defining molecular mechanisms controlling FES activation and microtubule binding as a potential strategy to limit mast cell recruitment to tumors via the SCF/KIT axis

Abstract FES protein-tyrosine kinase (PTK) is activated downstream of the KIT receptor in mast cells, and promotes mast cell migration towards Stem cell factor (SCF), the ligand for KIT. Recently, tumor-derived SCF has been implicated in promoting mast cell recruitment leading to increased tumor vascularity and poor prognosis. FES is also a potential therapeutic target in leukemias driven by oncogenic KIT receptors that are constitutively active. Thus, defining FES activation and signaling mechanisms could lead to development of FES inhibitors as potential therapeutics in mast cell-involved tumors or KIT-driven leukemias. FES was previously shown to phosphorylate Tubulin, and localize to microtubules (MTs) in an SH2 domain-dependent manner. We hypothesized that defects in MT reorganization in FES-deficient mast cells may account for the observed defects in migration towards SCF. To map potential MT binding site(s) in FES, we performed MT binding assays, and identified two modes of FES binding, one via the N-terminal F-BAR/FX domains, and also via the SH2 domain. Interestingly, mutations predicted to disrupt oligomerization of the F-BAR/FX domains enhanced MT binding in vitro, and MT localization in vivo. Using in vitro kinase assays, we show that FES preferentially phosphorylates soluble tubulin compared to MTs, suggesting that FES recruitment to MTs may depend on their phosphorylation by other PTKs (such as Src family PTKs). In mast cells treated with SCF, FES co-localizes with MTs, including some localization to the MT organizing center (MTOC). We are currently comparing MTOC orientation in polarized wild-type and FES-deficient mast cells, and examining MT subsets marked by γ-tubulin, acetylation, and detyrosination. In conclusion, we find that FES interacts directly with MTs in vitro, and co-localizes with MTs in vivo, and hypothesize that disruption of FES-MT interaction and/or activation will be relevant therapeutic targets to limit mast cell recruitment in solid tumors, and growth of KIT-driven leukemias. Funded by an operating grant from Canadian Institutes for Health Research (MOP82882) to AWBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2011-3832

Mean-field study of the role of lateral cracks in microtubule dynamics

A link between dimer-scale processes and microtubule (MT) dynamics at macroscale is studied by comparing simulations obtained using computational dimer-scale model with its mean-field approximation. The novelty of the mean-field model (MFM) is in its explicit representation of inter-protofilament cracks, as well as in the direct incorporation of the dimer-level kinetics. Due to inclusion of both longitudinal and lateral dimer interactions, the MFM is two dimensional, in contrast to previous theoretical models of MTs. It is the first analytical model that predicts and quantifies crucial features of MT dynamics such as (i) existence of a minimal soluble tubulin concentration needed for the polymerization (with concentration represented as a function of model parameters), (ii) existence of steady-state growth and shortening phases (given with their respective velocities), and (iii) existence of an unstable pause state near zero velocity. In addition, the size of the GTP cap of a growing MT is estimated. Theoretical predictions are shown to be in good agreement with the numerical simulations.

Soluble levels of cytosolic tubulin regulate ciliary length control

The primary cilium is an evolutionarily conserved dynamic organelle important for regulating numerous signaling pathways, and, as such, mutations disrupting ciliogenesis result in a variety of developmental abnormalities and postnatal disorders. The length of the cilium is regulated by the cell through largely unknown mechanisms. Normal cilia length is important, as either shortened or elongated cilia have been associated with disease and developmental defects. Here we explore the importance of cytoskeletal dynamics in regulating cilia length. Using pharmacological approaches in different cell types, we demonstrate that actin depolymerization or stabilization and protein kinase A activation result in a rapid elongation of the primary cilium. The effects of pharmacological agents on cilia length are associated with a subsequent increase in soluble tubulin levels and can be impaired by depletion of soluble tubulin with taxol. In addition, subtle nocodazole treatment was able to induce ciliogenesis under conditions in which cilia are not normally formed and also increases cilia length on cells that have already established cilia. Together these data indicate that cilia length can be regulated through changes in either the actin or microtubule network and implicate a possible role for soluble tubulin levels in cilia length control.

2-Anilinonicotinyl linked 1,3,4-oxadiazole derivatives: Synthesis, antitumour activity and inhibition of tubulin polymerization

A series of 2-anilinonicotinyl linked 1,3,4-oxadiazoles was synthesized and evaluated for their antitumour activity against various cancer cell lines, inhibition of tubulin polymerization and cell cycle effects. Some of these compounds showed good antiproliferative activity with GI50 values ranging from 4.57 to 97.09 μM in the human cancer cell lines and one of the compounds 5m showed potent antitumour efficacy in all the cell lines tested. This compound also inhibited tubulin polymerization under both in vitro and in vivo conditions. Analysis of tubulin by Western blot experiments demonstrated that 5m depolymerizes microtubules by causing disturbances in the ratio of soluble versus polymerized tubulin in cells, leading to the cell cycle arrest at G2/M phase of the cell cycle followed by activation of caspase-3 activity and apoptotic cell death.

A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

Microtubule-Disrupting Chemotherapeutics Result in Enhanced Proteasome-Mediated Degradation and Disappearance of Tubulin in Neural Cells

We sought to examine the effects of microtubule-targeting agents (MTA) on neural cells to better understand the problem of neurotoxicity, their principal side effect, and to possibly develop a model of clinical toxicity. Studies showed that microtubule-depolymerizing agents (MDA) not only disassembled microtubules in neural HCN2 cells but also led to rapid disappearance of tubulin, and that this was specific for MDAs. Tubulin levels decreased to 20% as early as 8 hours after adding vincristine, and to 1% to 30% (mean, 9.8 +/- 7.6%; median of 7%) after 100 nmol/L vincristine for 24 hours. This disappearance was reversible. An increase in both glu-terminated and acetylated tubulin, markers of stable tubulin, preceded reaccumulation of soluble tubulin, suggesting a priority for stabilizing tubulin first as microtubules before replenishing the soluble pool. Similar results were shown with other MDAs. Furthermore, microtubule reassembly did not arise from a central focus but instead appeared to involve dispersed nucleation, as evidenced by the appearance of small, stable microtubule stubs throughout the cytoplasm. In contrast, experiments with four nonneural "normal" cell lines and four cancer cell lines resulted in microtubule destabilization but only modest tubulin degradation. Evidence for proteasome-mediated degradation was obtained by demonstrating that adding a proteasome inhibitor before vincristine prevented tubulin disappearance. In summary, MDAs lead to rapid disappearance of tubulin in neural but not in other normal or cancer cells. These results underscore the fine control that occurs in neural cells and may further our understanding of neurotoxicity following MDAs.

History-Dependent Catastrophes Regulate Axonal Microtubule Behavior

In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

Abstract 4421: The ability of the microtubule-targeting agent T0070907 to induce tubulin degradation in colorectal cancer cell lines is affected by the microtubule-destabilizing but not the tubulin sequestering functions of stathmin

Abstract Background: The compound T0070907 is a microtubule-targeting agent (MTA) that, unlike standard MTAs, induces proteasomal degradation of tubulin. Strikingly, tubulin loss occurs preferentially in tumor cells. One potential candidate for a tumor-specific function that affects the ability of T0070907 to induce tubulin loss is the microtubule-associated protein stathmin. Stathmin normally destabilizes microtubules and also sequesters tubulin in complexes unavailable for degradation. We therefore examined whether T0070907-induced stathmin inactivation is required for tubulin loss. Methods: 50μM T0070907 was added to the colorectal cancer cell lines HT-29, HCT-15, and SW620, and microtubule polymerization, tubulin loss, and stathmin phosphorylation measured at multiple time points by Western blot. When applicable, microtubules were depolymerized with 0.1 μM vinblastine or nocodazole or polymerized with 0.1 μM paclitaxel before addition of T0070907. Microtubule polymerization was quantified by cell lysis in microtubule stabilizing buffer followed by centrifugation to separate the insoluble microtubules from the soluble tubulin heterodimers. Stathmin levels were reduced using chemically synthesized stathmin-specific siRNAs; responsiveness to T0070907 was assessed after 72h knockdown. Results: T0070907 induced tubulin loss starting 8h after compound addition. Depolymerizing the microtubules eliminated the lag time before tubulin loss, while polymerizing the microtubules with paclitaxel prevented loss. T0070907 alone or in the presence of 25μM of the proteasome inhibitor MG132 increased the amount of tubulin found in the soluble, degradable fraction. T0070907 also increased phosphorylation of the stathmin regulatory ser16 site that normally inactivates both microtubule destabilizing and sequestering functions. Stathmin knockdown slowed the kinetics of T0070907-induced tubulin loss, as was expected given that stathmin normally destabilizes microtubules. However, stathmin knockdown did not increase the rate of T0070907-induced tubulin loss when microtubules were fully depolymerized with vinblastine, as would be expected if inactivation of the sequestering function were critical. Conclusion: T0070907-induced degradation requires microtubule depolymerization of the microtubules. T0070907 increases depolymerization, and also inactivates stathmin. However, this inactivation does not contribute to the ability of T0070907 to degrade tubulin, as inactivation of the stathmin sequestering function does not seem to be critical to T0070907's ability to degrade tubulin, and inactivation of the microtubule destabilizing function antagonizes tubulin degradation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4421.

Abstract 4415: Role for the protein farnesyltransferase in the regulation of microtubule dynamics

Abstract The protein farnesyltransferase (FTase) is well characterized for its prenylation activity whereby a lipid farnesyl moiety is added to substrate proteins with a C-terminal CaaX motif. Farnesylation of the Ras oncogene was shown to be important for both its plasma membrane localization and its downstream tumorigenic signaling. Inhibitors of FTase (FTIs) were subsequently developed to inhibit Ras-dependent tumorigenesis, but soon it was found that Ras could be alternatively geranylgeranylated and remain active. However, it was also found that FTIs retain some inhibition of tumor growth activity even when Ras is alternatively geranylgeranylated, suggesting that FTIs exert their antitumor activity through other proteins than Ras. In preclinical studies, FTIs appeared to be very synergistic with microtubule-stabilizing agent such as the taxanes. In trying to understand the underlying mechanism for this synergy, we have demonstrated that FTIs increase tubulin acetylation by inhibiting the tubulin deacetylase HDAC6 and stabilizing microtubules, which in turn favors paclitaxel binding and eventually leads to mitotic arrest and cell death. We have found that FTase, microtubules and HDAC6 are part of the same protein complex and more interestingly that FTase directly bind microtubules. The interaction between FTase and microtubules was abolished in vivo upon treatment of cells with FTIs. The direct binding of FTase to microtubules requires the presence of the C-terminal end of tubulin, a known binding site for Microtubule-Associated Proteins (MAPs), and is reduced in presence of MAPs. Together with the observed reduction of microtubules dynamics in cells after treatment with FTIs, these results suggest a role for FTase in the regulation of microtubule dynamics. To test this hypothesis, we used an in vitro tubulin polymerization assay using purified bovine brain tubulin in presence or absence of recombinant purified FTase. Preliminary experiments showed that FTase greatly reduced both the rate and extent of tubulin polymerization when FTase was added directly to soluble tubulin dimers, but had no effect on preformed microtubules. Interestingly, tubulin is not a substrate for farnesylation as it does not contain a CaaX motif, and thus the effect of FTase on tubulin polymerization appears to reveal a new role for FTase in cells. Experiments are underway to determine whether the farnesyltransferase activity is required for the regulation of microtubules and to further elucidate the importance of this regulation in cells. A better understanding of this FTase-tubulin interaction and their role in vitro and in vivo might lead to the development of inhibitors that could specifically affect this interaction without altering farnesylated proteins. (Supported by NCI 5P01CA116676 and NCI 5F32CA134148-02) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4415.

Drosophila Stathmins Bind Tubulin Heterodimers with High and Variable Stoichiometries

In vertebrates, stathmins form a family of proteins possessing two tubulin binding repeats (TBRs), which each binds one soluble tubulin heterodimer. The stathmins thus sequester two tubulins in a phosphorylation-dependent manner, providing a link between signal transduction and microtubule dynamics. In Drosophila, we show here that a single stathmin gene (stai) encodes a family of D-stathmin proteins. Two of the D-stathmins are maternally deposited and then restricted to germ cells, and the other two are detected in the nervous system during embryo development. Like in vertebrates, the nervous system-enriched stathmins contain an N-terminal domain involved in subcellular targeting. All the D-stathmins possess a domain containing three or four predicted TBRs, and we demonstrate here, using complementary biochemical and biophysical methods, that all four predicted TBR domains actually bind tubulin. D-stathmins can indeed bind up to four tubulins, the resulting complex being directly visualized by electron microscopy. Phylogenetic analysis shows that the presence of regulated multiple tubulin sites is a conserved characteristic of stathmins in invertebrates and allows us to predict key residues in stathmin for the binding of tubulin. Altogether, our results reveal that the single Drosophila stathmin gene codes for a stathmin family similar to the multigene vertebrate one, but with particular tubulin binding properties.

Eribulin Binds at Microtubule Ends to a Single Site on Tubulin To Suppress Dynamic Instability

Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. Using [(3)H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K(d) of 46 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 +/- 1.3 molecules per microtubule (K(d) = 3.5 microM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Preincubation of microtubules with 2 or 4 microM vinblastine induced additional lower-affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability.

Inhibition of Tau Polymerization with a Cyanine Dye in Two Distinct Model Systems

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3'-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 microm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 microm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 microm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 microm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 microm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.