Microtubule Dynamic Instability Research Articles

Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Osteosarcoma is the most frequently occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common. Eribulin is a synthetic microtubule inhibitor that has demonstrated activity in preclinical osteosarcoma models. The effects of eribulin were evaluated in two human osteosarcoma cell lines as well as in eribulin-sensitive and -resistant osteosarcoma xenograft tumors of the Pediatric Preclinical Testing Program (PPTP) by characterizing cell viability, microtubule destabilization, mitotic arrest and mechanism of cell death. Eribulin demonstrated cytotoxic activity in vitro, through promotion of microtubule dynamic instability, arrest of cells in the G2/M phase, mitotic catastrophe and cell death. The microtubule-destabilizing protein stathmin-1 (STMN1) was coimmunoprecipitated with the cyclin-dependent kinase inhibitor p27 indicating that these cytoplasmic complexes can protect cells from the microtubule destabilizing effect of eribulin. Increased tumoral expression of P-glycoprotein (P-gp) and TUBB3 were also associated with lower drug sensitivity. In summary, eribulin successfully blocked cells in G2/M phase but interfered with mitochondria activity to inhibit proteins involved in apoptosis. Understanding the complex and inter-related mechanisms involved in the overall drug response to eribulin may help in the design of therapeutic strategies that enhance drug activity and improve benefits of eribulin in pediatric patients with osteosarcoma.

Five factors can reconstitute all three phases of microtubule polymerization dynamics.

Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behavior involving all three phases by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit). When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.

Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture

Microtubule dynamic instability allows search and capture of kinetochores during spindle formation, an important process for accurate chromosome segregation during cell division. Recent work has found that microtubule rotational diffusion about minus-end attachment points contributes to kinetochore capture in fission yeast, but the relative contributions of dynamic instability and rotational diffusion are not well understood. We have developed a biophysical model of kinetochore capture in small fission-yeast nuclei using hybrid Brownian dynamics/kinetic Monte Carlo simulation techniques. With this model, we have studied the importance of dynamic instability and microtubule rotational diffusion for kinetochore capture, both to the lateral surface of a microtubule and at or near its end. Over a range of biologically relevant parameters, microtubule rotational diffusion decreased capture time, but made a relatively small contribution compared to dynamic instability. At most, rotational diffusion reduced capture time by 25%. Our results suggest that while microtubule rotational diffusion can speed up kinetochore capture, it is unlikely to be the dominant physical mechanism for typical conditions in fission yeast. In addition, we found that when microtubules undergo dynamic instability, lateral captures predominate even in the absence of rotational diffusion. Counterintuitively, adding rotational diffusion to a dynamic microtubule increases the probability of end-on capture.

A Case for Microtubule Vulnerability in Amyotrophic Lateral Sclerosis: Altered Dynamics During Disease

Amyotrophic lateral sclerosis (ALS) is an aggressive multifactorial disease converging on a common pathology: the degeneration of motor neurons (MNs), their axons and neuromuscular synapses. This vulnerability and dysfunction of MNs highlights the dependency of these large cells on their intracellular machinery. Neuronal microtubules (MTs) are intracellular structures that facilitate a myriad of vital neuronal functions, including activity dependent axonal transport. In ALS, it is becoming increasingly apparent that MTs are likely to be a critical component of this disease. Not only are disruptions in this intracellular machinery present in the vast majority of seemingly sporadic cases, recent research has revealed that mutation to a microtubule protein, the tubulin isoform TUBA4A, is sufficient to cause a familial, albeit rare, form of disease. In both sporadic and familial disease, studies have provided evidence that microtubule mediated deficits in axonal transport are the tipping point for MN survivability. Axonal transport deficits would lead to abnormal mitochondrial recycling, decreased vesicle and mRNA transport and limited signaling of key survival factors from the neurons peripheral synapses, causing the characteristic peripheral “die back”. This disruption to microtubule dependant transport in ALS has been shown to result from alterations in the phenomenon of microtubule dynamic instability: the rapid growth and shrinkage of microtubule polymers. This is accomplished primarily due to aberrant alterations to microtubule associated proteins (MAPs) that regulate microtubule stability. Indeed, the current literature would argue that microtubule stability, particularly alterations in their dynamics, may be the initial driving force behind many familial and sporadic insults in ALS. Pharmacological stabilization of the microtubule network offers an attractive therapeutic strategy in ALS; indeed it has shown promise in many neurological disorders, ALS included. However, the pathophysiological involvement of MTs and their functions is still poorly understood in ALS. Future investigations will hopefully uncover further therapeutic targets that may aid in combating this awful disease.

Self-repair promotes microtubule rescue

SummaryThe dynamic instability of microtubules is characterised by slow growth phases stochastically interrupted by rapid depolymerisations called catastrophes. Rescue events can arrest the depolymerisation and restore microtubule elongation. However the origin of these rescue events remain unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints.

The structured core of human β tubulin confers isotype-specific polymerization properties.

Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a "tubulin code" in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human β isotype IIB and characterize polymerization dynamics. Microtubules with βIIB have catastrophe frequencies approximately threefold lower than those with isotype βIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric β tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved β tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin's polymerization properties across a wide range.

The Actin-Binding Protein Cofilin and Its Interaction With Cortactin Are Required for Podosome Patterning in Osteoclasts and Bone Resorption In Vivo and In Vitro.

The adhesion of osteoclasts (OCs) to bone and bone resorption require the assembly of specific F-actin adhesion structures, the podosomes, and their dense packing into a sealing zone. The OC-specific formation of the sealing zone requires the interaction of microtubule (MT) + ends with podosomes. Here, we deleted cofilin, a cortactin (CTTN)- and actin-binding protein highly expressed in OCs, to determine if it acts downstream of the MT-CTTN axis to regulate actin polymerization in podosomes. Conditional deletion of cofilin in OCs in mice, driven by the cathepsin K promoter (Ctsk-Cre), impaired bone resorption in vivo, increasing bone density. In vitro, OCs were not able to organize podosomes into peripheral belts. The MT network was disorganized, MT stability was decreased, and cell migration impaired. Active cofilin stabilizes MTs and allows podosome belt formation, whereas MT disruption deactivates cofilin via phosphorylation. Cofilin interacts with CTTN in podosomes and phosphorylation of either protein disrupts this interaction, which is critical for belt stabilization and for the maintenance of MT dynamic instability. Accordingly, active cofilin was required to rescue the OC cytoskeletal phenotype in vitro. These findings suggest that the patterning of podosomes into a sealing zone involves the dynamic interaction between cofilin, CTTN, and the MTs + ends. This interaction is critical for the functional organization of OCs and for bone resorption. © 2016 American Society for Bone and Mineral Research.

Abstract P5-03-08: Eribulin impairs the transport of TGF-β type I receptor leading to inhibition of downstream non-canonical TGF-β signaling necessary for cancer metastasis and survival

Abstract Microtubule targeting agents (MTAs) continue to be some of the most valuable drugs used in the treatment of breast cancers. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt microtubule-dependent transport of key signaling components, including proteins and microRNAs, in interphase cells likely contributes to their anticancer actions. TGF-β receptors are known to undergo constant cycling from the plasma membrane to intracellular portions of the cell, a process which is microtubule dependent. This microtubule-dependent trafficking has been shown to regulate the nuclear translocation of the TGF-β type I receptor, TGFβR1. Nuclear translocation of TGFβR1 activates the expression of genes including Snail and MMP2, which facilitate the invasiveness, motility and metastasis of cancer cells. We tested the hypothesis that a 2 h treatment of breast cancer cells with eribulin or 4 other clinically relevant MTAs, would differentially disrupt interphase microtubules and alter the internalization and trafficking of TGFβR1 to the nucleus; thereby impacting downstream signaling events. Cells were serum starved for 12 hours and then treated for 2 h with concentrations of MTAs that caused comparable disruption of the interphase microtubule network; 100 nM was used for the destabilizers, eribulin and vinorelbine and 1 μM was used for the stabilizers, paclitaxel, docetaxel and ixabepilone. Following the 2 h treatment, cells were stimulated with 10 ng/mL TGF-β1 for 30 min. The results show that there are distinct differences between the effects of microtubule stabilizers and destabilizers on TGFβR1 trafficking. Eribulin and vinorelbine decreased the nuclear localization of TGFβR1 in a panel of breast cancer cell lines with initial studies suggesting that eribulin impairs this trafficking to a greater extent. In contrast, the microtubule stabilizers, particularly ixabepilone, increased TGFβR1 localization in the nucleus. Additionally, TGFβR1 was extensively localized along stabilizer-induced microtubule bundles. Overall, our work suggests that eribulin is the most effective MTA at inhibiting TGFβR1 nuclear accumulation and subsequent phosphorylation of Smads 2 and 3. The downstream signaling effects of these MTAs on TGFβR1 induced transcription of Snail and MMP2 are also being investigated. Eribulin induced inhibition of non-canonical TGF-β signaling is consistent with previous studies that show that a 7 day treatment with eribulin reversed TGF-β-mediated EMT in breast cancer cells.1,2 Our data suggest that inhibition of the nuclear transport of TGFβR1 could be a potential mechanism for the eribulin-mediated EMT reversal. These studies begin to shed light into the diverse mechanisms of MTAs and lay the groundwork to identify patient populations that might respond optimally to different MTAs. 1. Yoshida T, et al., Brit J Cancer 110(6): 1497-505, 2014. 2. Dezso Z, et al., PloS One, 9(8): e106131, 2014. This work is funded by Eisai Inc. Citation Format: Rohena CC, Kaul R, Risinger AL, Mooberry SL. Eribulin impairs the transport of TGF-β type I receptor leading to inhibition of downstream non-canonical TGF-β signaling necessary for cancer metastasis and survival. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-08.

Mitotic Activation of a Novel Histone Deacetylase 3-Linker Histone H1.3 Protein Complex by Protein Kinase CK2

Histone deacetylase 3 (HDAC3) and linker histone H1 are involved in both chromatin compaction and the regulation of mitotic progression. However, the mechanisms by which HDAC3 and H1 regulate mitosis and the factors controlling HDAC3 and H1 activity during mitosis are unclear. Furthermore, as of now, no association between class I, II, or IV (non-sirtuin) HDACs and linker histones has been reported. Here we describe a novel HDAC3-H1.3 complex containing silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor 1 (N-CoR) that accumulated in synchronized HeLa cells in late G2 phase and mitosis. Nonetheless, the deacetylation activity by HDAC3 in the complex was evident only in mitotic complexes. HDAC3 associated with H1.3 was highly phosphorylated on Ser-424 only during mitosis. Isolation of inactive HDAC3-H1.3 complexes from late G2 phase cells, and phosphorylation of HDAC3 in the complexes at serine 424 by protein kinase CK2 (also known as casein kinase 2) activated the HDAC3 in vitro. In vivo, CK2α and CK2α' double knockdown cells demonstrated a significant decrease in HDAC3 Ser-424 phosphorylation during mitosis. HDAC3 and H1.3 co-localized in between the chromosomes, with polar microtubules and spindle poles during metaphase through telophase, and partially co-localized with chromatin during prophase and interphase. H1 has been reported previously to associate with microtubules and, therefore, could potentially function in targeting HDAC3 to the microtubules. We suggest that phosphorylation of HDAC3 in the complex by CK2 during mitosis activates the complex for a dual role: compaction of the mitotic chromatin and regulation of polar microtubules dynamic instability.

Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals

BackgroundTankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front.ResultsHere we report a new function for tankyrases, namely, regulation of directional cell locomotion. Using a panel of lung cancer cell lines as a model system, we found that abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors (one introduced and characterized here for the first time) or by RNA interference-based genetic silencing weakened cell migration, invasion, and directional movement induced by the motogenic cytokine hepatocyte growth factor. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted (1) microtubule dynamic instability; (2) adenomatous polyposis coli plasma membrane targeting; and (3) centrosome reorientation.ConclusionsCollectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the subcellular distribution of related polarity signals. These results encourage the further exploration of tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0226-9) contains supplementary material, which is available to authorized users.

Measuring the Effects of Microtubule-Associated Proteins on Microtubule Dynamics In Vitro.

Microtubule dynamic instability, the process by which individual microtubules switch between phases of growth and shrinkage, is essential for establishing the architecture of cellular microtubule structures, such as the mitotic spindle. This switching process is regulated by a complex network of microtubule-associated proteins (MAPs), which modulate different aspects of microtubule dynamic behavior. To elucidate the effects of MAPs and their molecular mechanisms of action, in vitro reconstitution approaches with purified components are used. Here, I present methods for measuring individual and combined effects of MAPs on microtubule dynamics, using purified protein components and total-internal-reflection fluorescence (TIRF) microscopy. Particular focus is given to the experimental design, proper parameterization, and data analysis.

Regulation of Focal Adhesion Dynamics and Cell Motility by the EB2 and Hax1 Protein Complex

Cell migration is a fundamental cellular process requiring integrated activities of the cytoskeleton, membrane, and cell/extracellular matrix adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2, and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe, whereas, in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about the cellular function of EB2. By quantitative proteomics, we identified mammalian HCLS1-associated protein X-1 (HAX1) as an EB2-specific interacting protein. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover require interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration.

Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

An electron microscopy journey in the study of microtubule structure and dynamics.

Structural characterization of microtubules has been the realm of three-dimensional electron microscopy and thus has evolved hand in hand with the progress of this technique, from the initial 3D reconstructions of stained tubulin assemblies, and the first atomic model of tubulin by electron crystallography of 2D sheets of protofilaments, to the ever more detailed cryoelectron microscopy structures of frozen-hydrated microtubules. Most recently, hybrid helical and single particle image processing techniques, and the latest detector technology, have lead to atomic models built directly into the density maps of microtubules in different functional states, shading new light into the critical process of microtubule dynamic instability.

Effects of eribulin on microtubule binding and dynamic instability are strengthened in the absence of the βIII tubulin isotype.

Eribulin mesylate (Halaven) is a microtubule-targeted anticancer drug used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline. It binds at the plus ends of microtubules and has been shown to suppress plus end growth selectively. Because the class III β tubulin isotype is associated with resistance to microtubule targeting drugs, we sought to determine how βIII tubulin might mechanistically influence the effects of eribulin on microtubules. We found that while [(3)H]eribulin bound to bovine brain soluble tubulin depleted of βIII tubulin in a manner similar to that of unfractionated tubulin, it bound to plus ends of microtubules that were depleted of βIII-depleted tubulin with a maximal stoichiometry (20 ± 3 molecules per microtubule) higher than that of unfractionated microtubules (9 ± 2 molecules per microtubule). In addition, eribulin suppressed the dynamic instability behavior of βIII-depleted microtubules more strongly than and in a manner different from that of microtubules containing βIII tubulin. Specifically, with βIII tubulin present in the microtubules, 100 nM eribulin suppressed the growth rate by 32% and marginally reduced the catastrophe frequency (by 17%) but did not modulate the rescue frequency. However, in the absence of βIII tubulin, eribulin not only reduced the growth rate but also strongly reduced the shortening rate (by 43%) and the catastrophe and the rescue frequencies (by 49 and 32%, respectively). Thus, when present in microtubules, βIII tubulin substantially weakens the effects of eribulin.

A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics.

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.

TOG Proteins Are Spatially Regulated by Rac-GSK3β to Control Interphase Microtubule Dynamics.

Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and we understand their basic contributions to microtubule dynamics, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. In this study, we have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. We show that microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on our findings, we propose that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics.

Abstract 4148: Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals

Abstract Tankyrases are poly(ADP)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and spindle pole assembly (1,2). Here we report a new function for tankyrases, namely, regulation of directional cell locomotion. Oriented migration is established by polarized reshaping of the microtubule network, which in turn is attained by phosphorylation-dependent inhibition of GSK3β at the leading edge, the ensuing association of APC with the plus end of microtubules at the cortex of cell membrane protrusions, and microtubule-based centrosome reorientation towards the migrating front (3,4). Using a panel of lung cancer cell lines as a model system, we found that pharmacologic abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors weakened cell migration, invasion and directional movement induced by the motogenic cytokine HGF. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted: i) microtubule dynamic instability; ii) HGF-triggered GSK3β phosphorylation; iii) APC plasma membrane targeting; iv) centrosome reorientation. Collectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the activity and localization of related polarity signals. In perspective, these results pave the way for challenging tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells. 1. Riffell JL, Lord CJ, Ashworth A. Tankyrase-targeted therapeutics: expanding opportunities in the PARP family. Nat Rev Drug Discov 2012;11(12):923-36. 2. Haikarainen T, Krauss S, Lehtio L. Tankyrases: structure, function and therapeutic implications in cancer. Curr Pharm Des 2014;20(41):6472-88. 3. Etienne-Manneville S. Microtubules in cell migration. Annu Rev Cell Dev Biol 2013;29:471-99. 4. Etienne-Manneville S. Polarity proteins in migration and invasion. Oncogene 2008;27(55):6970-80. Citation Format: Barbara Lupo, Jorge Vialard, Andrea Bertotti, Letizia Lanzetti, Livio Trusolino. Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4148. doi:10.1158/1538-7445.AM2015-4148

Abstract 1666: Increased sensitivity of βIII tubulin-containing microtubules to cabazitaxel compared with docetaxel: A possible role of βIII tubulin in specifying good efficacy of cabazitaxel for docetaxel-resistant tumors

Abstract Tumor progression and resistance to taxane-based therapies have been shown to correlate with overexpression of the class III β-tubulin isotype (βIII tubulin). Elevated expression of βIII tubulin is often associated with tumor aggressiveness in patients with metastatic docetaxel-resistant prostate cancer. Cabazitaxel is a novel taxane recently approved for the treatment of metastatic prostate cancer that failed docetaxel therapy. To understand the mechanistic distinctions between cabazitaxel and docetaxel, we analyzed the effects of both drugs on bovine brain microtubules consisting of unfractionated tubulin, which contains 25% βIII tubulin, and with microtubules depleted of βIII tubulin. BetaIII tubulin was depleted using immunoaffinity chromatography purification technique. Both cabazitaxel and docetaxel enhanced polymerization of unfractionated tubulin to similar extents. The stoichiometry of 14C-cabazitaxel and 14C-docetaxel binding to microtubules composed of unfractionated tubulin were similar at very low concentrations (≤ 10 μM). The Kds for binding were 8.4 μM and 7.5 μM for 14C-cabazitaxel and 14C-docetaxel, respectively. However, at higher concentrations (10 - 40 μM) significantly more 14C-cabazitaxel bound to the microtubules than 14C-docetaxel, with an apparent higher affinity. We also analyzed the effects of cabazitaxel and docetaxel on in vitro dynamic instability of microtubules assembled from unfractionated and βIII-depleted tubulin. We found that 100 nM cabazitaxel or docetaxel stabilized the dynamics of microtubules assembled from unfractionated tubulin similarly. However, cabazitaxel suppressed the dynamics of microtubules containing βIII tubulin more strongly than docetaxel. For example, 100 nM cabazitaxel suppressed the shortening rate of unfractionated microtubules by 49% and the shortening rate of βIII-depleted microtubules by only 31%. The overall suppression of dynamicity of microtubules assembled from unfractionated βIII-rich tubulin was 49% vs. 33% for βIII-depleted microtubules. In contrast with cabazitaxel, 100 nM docetaxel suppressed the shortening rate of microtubules assembled from unfractionated and βIII-depleted tubulin by 48% and 52%, respectively. The overall suppression of dynamicity of microtubules assembled from unfractionated βIII-rich tubulin was 56% vs. 50% for βIII-depleted microtubules. Experiments to examine the binding of cabazitaxel and docetaxel to microtubules assembled from βIII-depleted tubulin are ongoing. Our results indicate that the more potent activity of cabazitaxel for microtubules containing βIII tubulin may contribute to its superior efficacy in prostate tumors overexpressing βIII tubulin as compared with that of docetaxel. Supported by a research award from Sanofi Oncology. Citation Format: Leslie Wilson, Olga Azarenko, Gregoriy Smiyun, Herbert Miller, Mary Ann Jordan. Increased sensitivity of βIII tubulin-containing microtubules to cabazitaxel compared with docetaxel: A possible role of βIII tubulin in specifying good efficacy of cabazitaxel for docetaxel-resistant tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1666. doi:10.1158/1538-7445.AM2015-1666

Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins

Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics.