Protein EB1 Research Articles

EB3 helps daughter cells spread their wings

![Figure][1] In the absence of EB3, one daughter cell (arrow) often fails to spread out at the end of mitosis. Ferreira et al. reveal how Aurora B and the microtubule plus-end binding protein EB3 coordinate cytokinesis and daughter cell adhesion at the end of mitosis. EB3 and its relative

Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels

In cells, a complex network of proteins regulates the dynamic growth of microtubules that is essential for division and migration. In vitro approaches with purified components have so far been unable to reconstitute fast microtubule growth observed in vivo . Here we show that two well-studied plus-end-binding proteins-end-tracking protein EB1 and microtubule polymerase XMAP215-act together to strongly promote microtubule growth to cellular rates. Unexpectedly, the combined effects of XMAP215 and EB1 are highly synergistic, with acceleration of growth well beyond the product of the individual effects of either protein. The synergistic growth promotion does not rely on any of the canonical EB1 interactions, suggesting an allosteric interaction through the microtubule end. This hypothesis is supported by the finding that taxol and XMAP215, which have non-overlapping binding sites on tubulin, also act synergistically on growth. The increase in growth rates is accompanied by a strong enhancement of microtubule catastrophe by EB1, thereby rendering the fast and dynamic microtubule behaviour typically observed in cells.

Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End-Binding Protein EB1

Microtubules and their associated proteins play important roles in vesicle and organelle transport, cell motility and cell division. Perturbation of these processes by mutation typically gives rise to severe pathological conditions. In our efforts to obtain atomic information on microtubule-associated protein/microtubule interactions with the goal to understand mechanisms that might potentially assist in the development of treatments for these diseases, we have determined the three-dimensional structure of CAP-Gly (cytoskeleton-associated protein, glycine-rich) domain of mammalian dynactin by magic angle spinning NMR spectroscopy. We observe two conformations in the β2 strand encompassing residues T43-V44-A45, residues that are adjacent to the disease-associated mutation, G59S. Upon binding of CAP-Gly to microtubule plus-end tracking protein EB1, the CAP-Gly shifts to a single conformer. We find extensive chemical shift perturbations in several stretches of residues of CAP-Gly upon binding to EB1, from which we define accurately the CAP-Gly/EB1 binding interface. We also observe that the loop regions may exhibit unique flexibility, especially in the GKNDG motif, which participates in the microtubule binding. This study in conjunction with our previous reports suggests that conformational plasticity is an intrinsic property of CAP-Gly likely due to its unusually high loop content and may be required for its biological functions.

BRCA1 regulates microtubule dynamics and taxane-induced apoptotic cell signaling

The taxanes are effective microtubule-stabilizing chemotherapy drugs used in the treatment of various solid tumors. However, the emergence of drug resistance hampers their clinical efficacy. The molecular basis of clinical taxane resistance remains poorly understood. Breast cancer 1, early onset gene, BRCA1, is a tumor-suppressor gene, whose expression has been correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer. However, the molecular mechanism underlying the relationship between BRCA1 (B1) expression and taxane activity remains unclear. To this end, we created a stable B1 knockdown A549 cell line (B1-KD) to investigate B1's role in microtubule biology and response to taxane treatment. We show that B1-KD rendered A549 cells resistant to paclitaxel (PTX), phenocopying clinical studies showing that low B1 expression correlated with taxane resistance. As previously reported, we show that loss of B1 enhanced centrosomal γ-tubulin localization and microtubule nucleation. Interestingly, we found that the B1-KD cells exhibited increased microtubule dynamics as compared with parental A549 cells, as assessed by live-cell confocal microscopy using enhanced green fluorescent protein-tagged α-tubulin or EB1 protein. In addition, we showed that loss of B1 impairs the ability of PTX to induce microtubule polymerization using immunofluorescence microscopy and a cell-based tubulin polymerization assay. Furthermore, B1-KD cells exhibited significantly lower intracellular binding of a fluorescently labeled PTX to microtubules. Recent studies have shown that PTX-stabilized microtubules serves as a scaffold for pro-caspase-8 binding and induction of apoptosis downstream of induced-proximity activation of caspase-8. Here we show that loss of B1 reduces the association of pro-caspase-8 with microtubules and subsequently leads to impaired PTX-induced activation of apoptosis. Taken together, our data show that B1 regulates indirectly endogenous microtubule dynamics and stability while its loss leads to microtubules that are more dynamic and less susceptible to PTX-induced stabilization conferring taxane resistance.

Retraction: The association between the microtubule plus end protein EB1 and cortactin controls podosomes and bone resorption

Retraction: The following abstract from the Journal of Bone and Mineral Research supplement, “The Association Between the Microtubule Plus End Protein EB1 and Cortactin Controls Podosomes and Bone Resorption” by Martin Biosse-Duplan, Sebastien Stephens, Frank PL Lai, Margit Oelkers, Daisuke Kitamura, Klemens Rottner, William Horne, and Roland Baron, published online on 17 August 2011 in ASBMR 2011 Annual Meeting Abstracts (ASBMR.org), has been retracted by agreement between the authors, the journal Editor in Chief, Juliet E Compston, the American Society for Bone and Mineral Research, and Wiley Periodicals, Inc. As a result of a PI-initiated inquiry within Harvard, the U.S. Office of Research Integrity (ORI) has determined that Biosse-Duplan falsified histomorphometric and microCT results.

XMAP215 and EB1 act in Synergy to Promote Microtubule Growth

In cells, a complex network of microtubule-associated-proteins regulates the dynamic growth and shrinkage of microtubules that is essential for division and migration. In vitro approaches with purified components have helped to elucidate the mechanisms and the effects of individual microtubule plus-end-localizing proteins (+TIPs) on microtubule dynamics. Because microtubule dynamics observed in vitro with individual +TIPs does not account for the dynamics observed in vivo, it is important to study the combined effects of +TIPs. Here we show that two well-studied +TIPs - microtubule plus-end-tracking protein EB1, and the microtubule polymerase XMAP215 - act together to strongly promote microtubule growth to rates never before observed with purified proteins. Unexpectedly, we find that the combined effects of XMAP215 and EB1 are highly synergistic, with acceleration of growth well beyond the product of the individual effects of either protein. The synergy remains after EB1's C-terminal 20 amino acids have been removed, showing that it does not rely on any of the canonical EB1 interactions. The increase in growth rates is accompanied by a strong enhancement of microtubule catastrophe, thereby rendering the fast and dynamic microtubule behavior typically observed in cells.

The Microtubule-Associated Protein EB1 Affects both Kinesin Translocation and Microtubule Stiffness In Vitro

The plus end interacting protein (+TIP) EB1 localizes to the microtubule plus end by recognizing the nucleotide state of the microtubule lattice, and acts as a coordinator of the localization of many other +TIP proteins. Using GMPCPP-stabilized microtubules that recapitulate the end binding behavior of EB1 along their entire length, we demonstrate how EB1 changes key microtubule properties: stiffness and kinesin translocation. We find that EB1 increases microtubule stiffness by ∼50%, measured by fluorescent imaging of diffusively fluctuating microtubules. We also find that EB1 significantly slows the velocity at which kinesin proteins move, measured by imaging of quantum dot coupled kinesin with a custom built total internal reflection fluorescence (TIRF) microscope. These data cannot be explained by simple models of steric hindrance, leading us to propose a new mechanism for the regulation of microtubule function by EB1 in which plus end microtubule mechanics and kinesin motility are directly modulated through structural changes of the microtubule lattice. Surprisingly, the addition of taxol diminishes both effects, raising important questions about its use as an experimental reagent and anti-cancer chemotherapeutic.

Chapter 23 - Using MTBindingSim as a Tool for Experimental Planning and Interpretation

MTBindingSim is a program that enables users to simulate experiments in which proteins or other ligands (e.g., drugs) bind to microtubules or other polymers under various binding models. The purpose of MTBindingSim is to help researchers and students gain an intuitive understanding of binding behavior and design experiments to distinguish between different binding mechanisms. MTBindingSim is open-source, freely available software and can be found at bindingtutor.org/mtbindingsim. This chapter first describes the capabilities of MTBindingSim, including the experimental designs and protein-binding models that it simulates, and then discusses two examples in which MTBindingSim is utilized in an experimental context. In the first, MTBindingSim is used to investigate potential explanations for unusual behavior observed in the binding of the neuronal protein Tau to microtubules, demonstrating that some potential explanations are incompatible with the experimental data. In the second example, MTBindingSim is used to design experiments to examine the question of whether the plus-end tracking protein EB1 binds preferentially to the microtubule seam.

Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.

Cdc42 regulates neutrophil migration via crosstalk between WASp, CD11b, and microtubules

AbstractChemotaxis promotes neutrophil participation in cellular defense by enabling neutrophil migration to infected tissue and is controlled by persistent cell polarization. One long-standing question of neutrophil polarity has been how the pseudopod and the uropod are coordinated. In our previous report, we suggested that Rho GTPase Cdc42 controls neutrophil polarity through CD11b signaling at the uropod, albeit through an unknown mechanism. Here, we show that Cdc42 controls polarity, unexpectedly, via its effector WASp. Cdc42 controls WASp activation and its distant localization to the uropod. At the uropod, WASp regulates the reorganization of CD11b integrin into detergent resistant membrane domains; in turn, CD11b recruits the microtubule end binding protein EB1 to capture and stabilize microtubules at the uropod. This organization is necessary to maintain neutrophil polarity during migration and is critical for neutrophil emigration into inflamed lungs. These results suggest unrecognized mechanism of neutrophil polarity in which WASp mediates long-distance control of the uropod by Cdc42 to maintain a proper balance between the pseudopod and the uropod. Our study reveals a new function for WASp in the control of neutrophil polarity via crosstalk between CD11b and microtubules.

Amer2 Protein Interacts with EB1 Protein and Adenomatous Polyposis Coli (APC) and Controls Microtubule Stability and Cell Migration

EB1 is key factor in the organization of the microtubule cytoskeleton by binding to the plus-ends of microtubules and serving as a platform for a number of interacting proteins (termed +TIPs) that control microtubule dynamics. Together with its direct binding partner adenomatous polyposis coli (APC), EB1 can stabilize microtubules. Here, we show that Amer2 (APC membrane recruitment 2), a previously identified membrane-associated APC-binding protein, is a direct interaction partner of EB1 and acts as regulator of microtubule stability together with EB1. Amer2 binds to EB1 via specific (S/T)xIP motifs and recruits it to the plasma membrane. Coexpression of Amer2 and EB1 generates stabilized microtubules at the plasma membrane, whereas knockdown of Amer2 leads to destabilization of microtubules. Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated down-regulation of Xenopus Amer2 blocks convergent extension cell movements, suggesting that the Amer2-EB1-APC complex regulates cell migration by altering microtubule stability.

Microtubules: Sizing Up the GTP Cap

The 'GTP cap' of the microtubule has long been postulated to exist, but a recent experiment gives us the first quantitative measurements of the cap size in thecell.

Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics.

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO(-) present in TBCB, which is similar to the EEY/F-COO(-) element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.

Estimating the Microtubule GTP Cap Size In Vivo

Microtubules (MTs) polymerize via net addition of GTP-tubulin subunits to the MT plus end, which subsequently hydrolyze to GDP-tubulin in the MT lattice. Relatively stable GTP-tubulin subunits create a "GTP cap" at the growing MT plus end that suppresses catastrophe. To understand MT assembly regulation, we need to understand GTP hydrolysis reaction kinetics and the GTP cap size. In vitro, the GTP cap has been estimated to be as small as one layer (13 subunits) or as large as 100-200 subunits. GTP cap size estimates in vivo have not yet been reported. Using EB1-EGFP as a marker for GTP-tubulin in epithelial cells, we find on average (1) 270 EB1 dimers bound to growing MT plus ends, and (2) a GTP cap size of ∼750 tubulin subunits. Thus, in vivo, the GTP cap is far larger than previous estimates in vitro, and ∼60-fold larger than a single layer cap. We also find that the tail of a large GTP cap promotes MT rescue and suppresses shortening. We speculate that a large GTP cap provides a locally concentrated scaffold for tip-tracking proteins and confers persistence to assembly in the face of physical barriers such as the cell cortex.

Proteomic-based identification of the APC-binding protein EB1 as a candidate of novel tissue biomarker and therapeutic target for colorectal cancer

Novel candidates of biomarker and therapeutic target in colorectal cancer (CRC) were investigated using a proteomic approach. The proteome of normal colorectal epithelial tissues was compared with that of the tumor ones in 59 CRC patients using two-dimensional difference gel electrophoresis. Of 3458 protein spots, 110 exhibited statistically significant (p<0.01) differences in intensity (more than 2.5-folds) between the normal and tumor tissue groups. Of 67 unique gene products that were identified for 105 of the 110 protein spots, we focused on the higher expression of the adenoma polyposis coli-binding protein EB1 (EB1). EB1 was originally discovered as a binding protein of APC, which is a tumor suppressor gene product, and the expression of EB1 has been associated with poor prognosis in several malignancies but not in CRC. Immunohistochemical analysis of the 132 CRC cases revealed that EB1 was overexpressed in tumor cells in correlation with poor prognosis. Suppression of EB1 by RNAi inhibited CRC cell proliferation and invasion. In this study, the overexpression of EB1 in CRC tissues correlating with prognosis, and its functional contribution to the malignant phenotypes of CRC cells are described. The present findings indicate that EB1 is a potential biomarker and therapeutic target in CRC.

Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends

Invasion of normal brain tissue by tumor cells is a major contributing factor to the recurrence of glioblastoma and its resistance to therapy. Here, we have assessed the efficacy of the microtubule (MT) targeting agent Epothilone B (patupilone) on glioblastoma cell migration, a prerequisite for invasive tumor cell behavior. At non-cytotoxic concentrations, patupilone inhibited glioblastoma cell movement, as shown by transwell cell migration, random motility and spheroid assays. This anti-migratory effect was associated with a reduced accumulation of EB1 and other MT plus end tracking proteins at MT ends and with the induction of MT catastrophes, while the MT growth rate and other MT dynamic instability parameters remained unaltered. An increase in MT catastrophes led to the reduction of the number of MTs reaching the leading edge. Analysis of the effect of patupilone on MT dynamics in a reconstituted in vitro system demonstrated that the induction of MT catastrophes and an alteration of EB1 accumulation at MT plus end are intrinsic properties of patupilone activity. We have thus demonstrated that patupilone antagonizes glioblastoma cell migration by a novel mechanism, which is distinct from suppression of MT dynamic instability. Taken together, our results suggest that EB proteins may represent a new potential target for anti-cancer therapy in highly invasive tumors.

Abstract 2791: Disruption of EB1 comets from microtubule “plus” ends and induction of microtubule catastrophes are intrinsic mechanisms of patupilone-induced inhibition of glioblastoma cell migration

Abstract Glioblastoma are the most frequent primary tumors of central nervous system. They are very aggressive, highly angiogenic and associated with very bad prognostic. One of the main biological features of a glioblastoma is the local invasion of its constituent neoplastic cells into the surrounding brain tissue. This invasive behavior represents a major obstacle to an effective treatment of intrinsic brain tumors. Therefore, it seems very important to target glioblastoma cell migration. Although many of the genetic alterations that deregulate the cell processes of growth and death, and are involved in tumor initiation have been elucidated in recent years, less progress has been done to understand the complex processes of migration. As a consequence, no specific therapeutic strategy against tumor cell migration is currently used to manage glioblastoma invasion. The Epothilones are a new class of microtubule (MT) stabilizing agents, which exert their cytotoxic activity by suppressing microtubule dynamics and that have strong anti-cancer activity both in vitro and in vivo. This drug family is active against cancer cells resistant to paclitaxel or to cells developing resistance to taxanes. Among the Epothilones, Patupilone (Epothilone B, EPO 906) is able to cross the blood-brain barrier since its activity is fully P-gp independent. Moreover, patupilone specifically targets αIII-tubulin, which is over expressed in glioblastomas. Here, we investigated the anti-migratory potential of patupilone through its effects on MT dynamics in glioblastoma cells. At non-cytotoxic concentrations, Patupilone inhibited glioblastoma cell migration, as shown by transwell cell migration, random motility and spheroids assay. This was associated with EB1 and related MT “plus” end tracking proteins comet shortening; and with MT catastrophes induction, without modification of any other MT dynamic instability parameters, including the microtubule growth rate. Such effect on MT catastrophes led to the disruption of MT stabilization at the leading edge. Evaluation of the effect of patupilone on EB comets and MT dynamics in a reconstituted in vitro system revealed that MT stabilizing drugs intrinsically induce MT catastrophes and disrupt EB comets at MT “plus” end. We have demonstrated that patupilone antagonizes glioblastoma cell migration by a new mechanism i.e. disruption of EB1 comets and induction of MT catastrophes distinct from its cytotoxic main mechanism i.e suppression of microtubule dynamic instability. Altogether, our results suggest that EB proteins may represent new potential target for anti-cancer therapy in highly invasive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2791. doi:1538-7445.AM2012-2791

Lateralized gap junctions in pulmonary hypertension: Lost but not alone

t f f t c c i o s g i p c w t l a It has long been recognized that some states of cardiac disease are associated with a dislocation of gap junctions from the intercalated disc (ID) to the lateral sides, known as gap junction lateralization or disarray (for recent review, see Fontes et al). Lateralization was first directly linked to arrhythmia in the border zone of healing infarcts, where Peters et al showed that areas prone to inducible arrhythmia also exhibited gap junction lateralization. It is, however, still uncertain what causes the dislocation of gap junctions, how they get there, and whether lateralization is arrhythmogenic per se. In this issue of HeartRhythm, Chkourko et al show that pulmonary hypertension is associated with the lateralization of not only the connexins making up the gap junction channels but also a number of structural proteins that are normally located at the ID and proteins involved in the transport of connexin 43 (Cx43, the most abundant cardiac gap junctional protein) to the plasma membrane. Cx43 can be transported along microtubules, and recently it was shown that the microtubule-associated protein EB1 is essential for correct trafficking of Cx43 to the ID. The study also showed that EB1 disappears from the ID during ischemia, abrogating the trafficking of Cx43 to the disc. The study by Chkourko et al shows that EB1 is most ikely responsible for redirecting Cx43 to the lateral memranes during remodeling in pulmonary hypertension. The inking of EB1 to the membrane is reported to involve everal components ending with N-cadherin as the memrane anchor. Does this imply that N-cadherin is the first omponent to arrive at the lateral membrane? Maybe in the ase of pulmonary hypertension, but in some models of gap unction lateralization, N-cadherin stays at the ID (eg, see olussi et al). Still, N-cadherin remains an interesting andidate, raising the question why it translocates. Recent vidence implies that cadherins are involved in mechanoensing at cell-to-cell interfaces and maybe cadherin-de-

Remodeling of mechanical junctions and of microtubule-associated proteins accompany cardiac connexin43 lateralization

Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization"). To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling. Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp. Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells. Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells

Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.