Publications by authors named "Ali Badache"

25 Publications

  • Page 1 of 1

Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

J Cell Sci 2022 Jan 10;135(1). Epub 2022 Jan 10.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM, Institut Paoli-Calmettes, Aix Marseille Univ, CNRS, 13009 Marseille, France.

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.258850DOI Listing
January 2022

iASPP contributes to cell cortex rigidity, mitotic cell rounding, and spindle positioning.

J Cell Biol 2021 12 27;220(12). Epub 2021 Oct 27.

Centre de Recherche en Cancérologie de Marseille, Institut National de la Santé et de la Recherche Médicale, Institut Paoli-Calmettes, Aix-Marseille Université, Centre National de la Recherche Scientifique, Marseille, France.

iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.
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http://dx.doi.org/10.1083/jcb.202012002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8562848PMC
December 2021

Structural and dynamic characterization of the C-terminal tail of ErbB2: Disordered but not random.

Biophys J 2021 05 17;120(10):1869-1882. Epub 2021 Mar 17.

Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Saclay, Gif-sur-Yvette, France. Electronic address:

ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers and associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last 20 years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, the ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics, and detailed functional mechanism. Yet, it is where signal transduction is triggered via phosphorylation of tyrosine residues and carried out via interaction with adaptor proteins. Here, we report the first description, to our knowledge, of the ErbB2 disordered tail at atomic resolution using NMR, complemented by small-angle x-ray scattering. We show that although no part of CtErbB2 has any fully populated secondary or tertiary structure, it contains several transient α-helices and numerous transient polyproline II helices, populated up to 20 and 40%, respectively, and low but significant compaction. The presence of some structural elements suggests, along the lines of the results obtained for EGFR (ErbB1), that they may have a functional role in ErbB2's autoregulation processes. In addition, the transient formation of polyproline II helices is compliant with previously suggested interactions with SH3 domains. All in all, our in-depth structural study opens perspectives in the mechanistic understanding of ErbB2.
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http://dx.doi.org/10.1016/j.bpj.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204338PMC
May 2021

EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK.

Cells 2021 02 13;10(2). Epub 2021 Feb 13.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM, Institut Paoli-Calmettes, Aix-Marseille University, CNRS, 13009 Marseille, France.

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.
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http://dx.doi.org/10.3390/cells10020388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918453PMC
February 2021

A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells.

Sci Rep 2020 04 22;10(1):6787. Epub 2020 Apr 22.

Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, INSERM, Institut Paoli-Calmettes, CNRS, Marseille, France.

Metastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5β). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5β interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape.
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http://dx.doi.org/10.1038/s41598-020-63926-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176661PMC
April 2020

The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

J Cell Biol 2019 10 30;218(10):3415-3435. Epub 2019 Aug 30.

Department of Biology, Brandeis University, Waltham, MA

Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin's roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state "primed" for microtubule-induced turnover.
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http://dx.doi.org/10.1083/jcb.201904165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781439PMC
October 2019

Memo1-Mediated Tiling of Radial Glial Cells Facilitates Cerebral Cortical Development.

Neuron 2019 09 2;103(5):836-852.e5. Epub 2019 Jul 2.

UNC Neuroscience Center and the Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. Electronic address:

Polarized, non-overlapping, regularly spaced, tiled organization of radial glial cells (RGCs) serves as a framework to generate and organize cortical neuronal columns, layers, and circuitry. Here, we show that mediator of cell motility 1 (Memo1) is a critical determinant of radial glial tiling during neocortical development. Memo1 deletion or knockdown leads to hyperbranching of RGC basal processes and disrupted RGC tiling, resulting in aberrant radial unit assembly and neuronal layering. Memo1 regulates microtubule (MT) stability necessary for RGC tiling. Memo1 deficiency leads to disrupted MT minus-end CAMSAP2 distribution, initiation of aberrant MT branching, and altered polarized trafficking of key basal domain proteins such as GPR56, and thus aberrant RGC tiling. These findings identify Memo1 as a mediator of RGC scaffold tiling, necessary to generate and organize neurons into functional ensembles in the developing cerebral cortex.
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http://dx.doi.org/10.1016/j.neuron.2019.05.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728225PMC
September 2019

EB1-binding-myomegalin protein complex promotes centrosomal microtubules functions.

Proc Natl Acad Sci U S A 2017 12 21;114(50):E10687-E10696. Epub 2017 Nov 21.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM, Institut Paoli-Calmettes, CNRS, Aix-Marseille Université,13009 Marseille, France;

Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (γ-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression.
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http://dx.doi.org/10.1073/pnas.1705682114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740655PMC
December 2017

Syntenin mediates SRC function in exosomal cell-to-cell communication.

Proc Natl Acad Sci U S A 2017 11 6;114(47):12495-12500. Epub 2017 Nov 6.

Department of Human Genetics, K. U. Leuven, B-3000 Leuven, Belgium;

The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin-syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell-cell communication, supported by syntenin exosomes, which is likely to contribute to tumor-host interactions.
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http://dx.doi.org/10.1073/pnas.1713433114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703317PMC
November 2017

H, C and N assignments of the C-terminal intrinsically disordered cytosolic fragment of the receptor tyrosine kinase ErbB2.

Biomol NMR Assign 2018 04 13;12(1):23-26. Epub 2017 Sep 13.

Institut de Chimie des Substances Naturelles, Structural Chemistry and Biology team, CNRS, Université Paris-Saclay, 1 av. de la terrasse, 91190, Gif-sur-Yvette, France.

ErbB2 (or HER2) is a receptor tyrosine kinase that is involved in signaling pathways controlling cell division, motility and apoptosis. Though important in development and cell growth homeostasis, this protein, when overexpressed, participates in triggering aggressive HER2+ breast cancers. It is composed of an extracellular part and a transmembrane domain, both important for activation by dimerization, and a cytosolic tyrosine kinase, which activates its intrinsically disordered C-terminal end (CtErbB2). Little is known about this C-terminal part of 268 residues, despite its crucial role in interacting with adaptor proteins involved in signaling. Understanding its structural and dynamic characteristics could eventually lead to the design of new interaction inhibitors, and treatments complementary to those already targeting other parts of ErbB2. Here we report backbone and side-chain assignment of CtErbB2, which, together with structural predictions, confirms its intrinsically disordered nature.
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http://dx.doi.org/10.1007/s12104-017-9773-4DOI Listing
April 2018

Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover.

J Cell Biol 2017 09 29;216(9):2859-2875. Epub 2017 Jun 29.

Department of Biology, Brandeis University, Waltham, MA

Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT-actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration.
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http://dx.doi.org/10.1083/jcb.201702007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584174PMC
September 2017

Eribulin targets a ch-TOG-dependent directed migration of cancer cells.

Oncotarget 2015 Dec;6(39):41667-78

Centre de Recherche en Cancérologie de Marseille, Inserm, Marseille, France.

Non-cytotoxic concentrations of microtubule targeting agents (MTAs) interfere with the dynamics of interphase microtubules and affect cell migration, which could impair tumor angiogenesis and metastasis. The underlying mechanisms however are still ill-defined. We previously established that directed cell migration is dependent on stabilization of microtubules at the cell leading edge, which is controlled by microtubule +end interacting proteins (+TIPs). In the present study, we found that eribulin, a recently approved MTA interacting with a new class of binding site on β-tubulin, decreased microtubule growth speed, impaired their cortical stabilization and prevented directed migration of cancer cells. These effects were reminiscent of those observed when +TIP expression or cortical localization was altered. Actually, eribulin induced a dose-dependent depletion of EB1, CLIP-170 and the tubulin polymerase ch-TOG from microtubule +ends. Interestingly, eribulin doses that disturbed ch-TOG localization without significant effect on EB1 and CLIP-170 comets, had an impact on microtubule dynamics and directed migration. Moreover, knockdown of ch-TOG led to a similar inhibition of microtubule growth speed, microtubule capture and chemotaxis. Our data suggest that eribulin binding to the tip of microtubules and subsequent loss of ch-TOG is a priming event leading to alterations in microtubule dynamics and cancer cell migration.
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http://dx.doi.org/10.18632/oncotarget.6147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747180PMC
December 2015

Memo is a copper-dependent redox protein with an essential role in migration and metastasis.

Sci Signal 2014 Jun 10;7(329):ra56. Epub 2014 Jun 10.

Friedrich Miescher Institute for Biomedical Research, Basel 4058, Switzerland. University of Basel, Basel 4002, Switzerland.

Memo is an evolutionarily conserved protein with a critical role in cell motility. We found that Memo was required for migration and invasion of breast cancer cells in vitro and spontaneous lung metastasis from breast cancer cell xenografts in vivo. Biochemical assays revealed that Memo is a copper-dependent redox enzyme that promoted a more oxidized intracellular milieu and stimulated the production of reactive oxygen species (ROS) in cellular structures involved in migration. Memo was also required for the sustained production of the ROS O2- by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 1 (NOX1) in breast cancer cells. Memo abundance was increased in >40% of the primary breast tumors tested, was correlated with clinical parameters of aggressive disease, and was an independent prognostic factor of early distant metastasis.
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http://dx.doi.org/10.1126/scisignal.2004870DOI Listing
June 2014

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase.

FEBS Lett 2014 Jun 8;588(12):2031-6. Epub 2014 May 8.

LISM, CNRS UMR 7255, Aix-Marseille Université, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France. Electronic address:

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis.
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http://dx.doi.org/10.1016/j.febslet.2014.04.029DOI Listing
June 2014

Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration.

Mol Biol Cell 2014 Mar 8;25(5):658-68. Epub 2014 Jan 8.

Centre de Recherche en Cancérologie de Marseille, INSERM U1068, 13009 Marseille, France Institut Paoli-Calmettes, 13009 Marseille, France Aix-Marseille Université, 13009 Marseille, France Centre National de la Recherche Scientifique UMR7258, 13009 Marseille, France Department of Biology, Brandeis University, Waltham, MA 02454.

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.
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http://dx.doi.org/10.1091/mbc.E13-08-0482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937091PMC
March 2014

ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways.

PLoS One 2013 29;8(1):e55211. Epub 2013 Jan 29.

Centre de Recherche en Cancérologie de Marseille, Inserm U1068, Marseille, France.

Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055211PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558493PMC
July 2013

MEMO associated with an ErbB2 receptor phosphopeptide reveals a new phosphotyrosine motif.

FEBS Lett 2011 Sep 10;585(17):2688-92. Epub 2011 Aug 10.

UPR3243-CNRS, Marseille, France.

Tyrosine phosphorylations are essential in signal transduction. Recently, a new type of phosphotyrosine binding protein, MEMO (Mediator of ErbB2-driven cell motility), has been reported to bind specifically to an ErbB2-derived phosphorylated peptide encompassing Tyr-1227 (PYD). Structural and functional analyses of variants of this peptide revealed the minimum sequence required for MEMO recognition. Using a docking approach we have generated a structural model for MEMO/PYD complex and compare this new phosphotyrosine motif to SH2 and PTB phosphotyrosine motives.
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http://dx.doi.org/10.1016/j.febslet.2011.07.048DOI Listing
September 2011

ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells.

Proc Natl Acad Sci U S A 2010 Oct 11;107(43):18517-22. Epub 2010 Oct 11.

Centre de Recherche en Cancérologie de Marseille, Institut National de la Santé et de la Recherche Médicale U891, 13009 Marseille, France.

Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.
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http://dx.doi.org/10.1073/pnas.1000975107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2972954PMC
October 2010

A simplified, 96-well-adapted, ATP luminescence-based motility assay.

Biotechniques 2009 Oct;47(4):871-5

Inserm, U891, TrGET, Centre de Recherche en Cancérologie de Marseille, Marseille, France, Institut Paoli-Calmettes, Marseille, France.

Directional motility assays make use of Boyden chambers or Transwell culture inserts with porous membranes that separate cells seeded in the upper chamber from a chemoattractant supplied in a lower chamber. These assays are often time-consuming and are associated with several limitations due to manual counting and inconsistent results; low signal-to-noise ratio and fluorescence interference; and high cost and the need for specific equipment. Here, we describe a simple, direct, and easy ATP luminescence-based motility assay (ALMA), which can be used for 96-well plate quantification.
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http://dx.doi.org/10.2144/000113250DOI Listing
October 2009

Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells.

J Cell Biol 2008 Nov 27;183(3):401-8. Epub 2008 Oct 27.

French National Institute for Health and Medical Research Unit 891, Centre de Recherche en Cancérologie de Marseille, 13009 Marseille, France.

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as alpha-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.
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http://dx.doi.org/10.1083/jcb.200805107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575782PMC
November 2008

Memo is homologous to nonheme iron dioxygenases and binds an ErbB2-derived phosphopeptide in its vestigial active site.

J Biol Chem 2008 Feb 28;283(5):2734-40. Epub 2007 Nov 28.

Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA.

Memo (mediator of ErbB2-driven cell motility) is a 297-amino-acid protein recently shown to co-precipitate with the C terminus of ErbB2 and be required for ErbB2-driven cell motility. Memo is not homologous to any known signaling proteins, and how it mediates ErbB2 signals is not known. To provide a molecular basis for understanding Memo function, we have determined and report here the 2.1A crystal structure of human Memo and show it be homologous to class III nonheme iron-dependent dioxygenases, a structural class that now includes a zinc-binding protein of unknown function. No metal binding or enzymatic activity can be detected for Memo, but Memo does bind directly to a specific ErbB2-derived phosphopeptide encompassing Tyr-1227 using its vestigial enzymatic active site. Memo thus represents a new class of phosphotyrosine-binding protein.
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http://dx.doi.org/10.1074/jbc.M703523200DOI Listing
February 2008

The ErbB2 signaling network as a target for breast cancer therapy.

J Mammary Gland Biol Neoplasia 2006 Jan;11(1):13-25

UMR599 Inserm, Centre de Recherche en Cancérologie de Marseille, 27 bd Leï Roure, 13009 Marseille, France.

Overexpression of the ErbB2/Her2 receptor tyrosine kinase in breast cancers is associated with the most aggressive tumors. Experimental studies have revealed that ErbB2 shows many features of a therapeutic target: ErbB2 is able to confer many of the characteristics of a cancerous cell, including uncontrolled proliferation, resistance to apoptosis and increased motility; ErbB2 overexpression is specific to tumor cells; as a cell surface-associated protein, it is easily accessible to drugs and as a kinase it is amenable to targeted inhibition by small molecules. Recent clinical results demonstrate the efficacy of ErbB2-targeting therapy and promise an expanding use of ErbB2-targeting drugs for breast cancer treatment. However, as only a fraction of patients responds successfully to therapy and risks of recurrence are still high, further investigation is required for an improved understanding of the complex network of signaling pathways underlying ErbB2-driven cancer progression.
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http://dx.doi.org/10.1007/s10911-006-9009-1DOI Listing
January 2006

TEL/ETV6 is a signal transducer and activator of transcription 3 (Stat3)-induced repressor of Stat3 activity.

J Biol Chem 2004 Sep 30;279(37):38787-96. Epub 2004 Jun 30.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel CH-4058, Switzerland.

The signal transducer and activator of transcription 3 (Stat3) transcription factor is required for the antiproliferative effects induced by cytokines, such as the interleukin-6 type. In order to investigate the role of Stat3 in inhibition of cell proliferation, we have used an inducible Stat3 construct in A375 melanoma cells. We found that activation of Stat3 to moderate levels was sufficient to repress A375 proliferation, by slowing cell transit through the cell cycle. Enhanced and prolonged Stat3 activity led to cell cycle arrest and apoptosis. Genes whose expression was altered by Stat3 activation were identified by oligonucleotide microarray analysis. We found that TEL (ETV6), a novel Stat3 target identified in this study, is a negative regulator of Stat3 activity. Small interfering RNA-mediated inhibition of TEL expression resulted in increased Stat3-dependent transcriptional activity and stronger Stat3 antiproliferative activity. Confirming these results, overexpression of TEL repressed Stat3 transcriptional activity. Intriguingly, Stat3 repression did not require TEL DNA binding and appeared to proceed via recruitment of TEL to Stat3. Inhibition of Stat3 activity by TEL represents a novel mechanism regulating the Stat3 signaling pathway.
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http://dx.doi.org/10.1074/jbc.M312581200DOI Listing
September 2004

Memo mediates ErbB2-driven cell motility.

Nat Cell Biol 2004 Jun 23;6(6):515-22. Epub 2004 May 23.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.

Clinical studies have revealed that cancer patients whose tumours have increased ErbB2 expression tend to have more aggressive, metastatic disease, which is associated with parameters predicting a poor outcome. The molecular basis underlying ErbB2-dependent cell motility and metastases formation, however, still remains poorly understood. In this study, we show that activation of a set of signalling molecules, including MAPK, phosphatidylinositol-3-OH kinase (PI(3)K) and Src, is required for Neu/ErbB2-dependent lamellipodia formation and for motility of breast carcinoma cells. Stimulation of these molecules, however, failed to induce efficient cell migration in the absence of Neu/ErbB2 phosphorylation at Tyr 1201 or Tyr 1227. We describe a novel molecule, Memo (mediator of ErbB2-driven cell motility), that interacts with a phospho-Tyr 1227-containing peptide, most probably through the Shc adaptor protein. After Neu/ErbB2 activation, Memo-defective cells form actin fibres and grow lamellipodia, but fail to extend microtubules towards the cell cortex. Our data suggest that Memo controls cell migration by relaying extracellular chemotactic signals to the microtubule cytoskeleton.
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http://dx.doi.org/10.1038/ncb1134DOI Listing
June 2004

A new therapeutic antibody masks ErbB2 to its partners.

Cancer Cell 2004 Apr;5(4):299-301

Friedrich Miescher Institute of the Novartis Research Foundation, P.O. Box 2543, CH-4002 Basel, Switzerland.

In cancer cells, the ErbB2 receptor tyrosine kinase can be activated in two ways: by overexpression or by ligand-mediated stimulation of another ErbB receptor. The ErbB2-targeting antibody trastuzumab (Herceptin) is used for treatment of metastatic breast cancer patients whose tumors overexpress ErbB2. A new structural study in this issue of Cancer Cell reveals how targeting ErbB2 with another antibody, pertuzumab (Omnitarg), prevents ligand-induced dimerization of ErbB2 with the other ErbB receptors. Pertuzumab's novel mode of action might offer additional therapeutic opportunities for treatment of tumors expressing ligand-activated ErbB2.
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http://dx.doi.org/10.1016/s1535-6108(04)00088-1DOI Listing
April 2004
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