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Erbb2 Receptor Controls Microtubule Capture by Recruiting ACF7 to the Plasma Membrane of Migrating Cells

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Erbb2 Receptor Controls Microtubule Capture by Recruiting ACF7 to the Plasma Membrane of Migrating Cells ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells Kossay Zaouia,b,c, Khedidja Benseddika,b,c,1, Pascale Daoua,b,c,1, Danièle Salaüna,b,c, and Ali Badachea,b,c,2 aCentre de Recherche en Cancérologie de Marseille, Institut National de la Santé et de la Recherche Médicale U891, 13009 Marseille, France; bInstitut Paoli- Calmettes, 13009 Marseille, France; and cAix-Marseille Université, 13007 Marseille, France Edited by Elaine Fuchs, The Rockefeller University, New York, NY, and approved August 9, 2010 (received for review January 26, 2010) Microtubules (MTs) contribute to key processes during cell motility, outgrowing MTs. We have recently described a signaling pathway including the regulation of focal adhesion turnover and the estab- by which ErbB2 controls MT outgrowth and stabilization at the lishment and maintenance of cell orientation. It was previously dem- cell cortex, which involves the recruitment of Memo, mediator of onstrated that the ErbB2 receptor tyrosine kinase regulated MT ErbB2-driven motility (8), to the plasma membrane and the sub- outgrowth to the cell cortex via a complex including Memo, the sequent localization of the GTPase RhoA and its effector, the GTPase RhoA, and the formin mDia1. But the mechanism that linked mDia1 formin, to the cell front (9). This pathway also controls the this signaling module to MTs remained undefined. We report that formation ofnascentadhesions and the recruitment ofactin-binding ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) ac- proteins, such as α-actinin (9). The mechanism by which the Memo- tivity, mediated by Memo and mDia1, is required for MT capture and RhoA-mDia1 signaling module regulates MT dynamics remained stabilization. Memo-dependent inhibition of GSK3 allows the reloc- unsolved. In this study, we report that Memo signaling controls the alization of APC (adenomatous polyposis coli) and cytoplasmic linker- localization of APC and CLASP2 to the plasma membrane, via the associated protein 2 (CLASP2), known MT-associated proteins, to the regulation of glycogen synthase kinase-3 (GSK3) activity. In turn, plasma membrane and ruffles. Peripheral microtubule extension also membrane-bound APC allows the localization of the spectraplakin requires expression of the plus-end binding protein EB1 and its re- ACF7/MACF1 to the plasma membrane, which is required and cently described interactor, the spectraplakin ACF7. In fact, in migrat- sufficient for MT capture and stabilization. ing cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demon- Results strate that ACF7 targeting to the plasma membrane is both required GSK3 Regulates MT Outgrowth via APC and CLASP2. We have pre- and sufficient for MT capture downstream of ErbB2. This function of viously shown that Memo-RhoA-mDia1 signaling mediated ACF7 does not require its recently described ATPase activity. By de- ErbB2-regulated MT dynamics. We have further explored the fining the signaling pathway by which ErbB2 allows MT capture and underlying mechanism. We initially investigated the contribution stabilization at the cell leading edge, we provide insights into the of APC and CLASPs, plus-end binding proteins known to con- CELL BIOLOGY mechanism underlying cell motility and steering. tribute to MT stability (10, 11), to MT outgrowth during ErbB2- driven cell motility. cell motility | receptor tyrosine kinase | Memo protein | glycogen synthase Activation of ErbB2 by the addition of heregulin β (HRG) to kinase-3 | adenomatous polyposis coli EGFP-tubulin–expressing SKBr3 breast carcinoma cells resulted in the extension of cell protrusions, populated by MTs (Fig. 1A). icrotubules (MTs) have several important functions during siRNA-mediated inhibition of APC or CLASP1/2 expression did Mcell motility. They participate in cell detachment at the cell not affect formation of protrusions but resulted in defective MT back, regulate the turnover of focal adhesions at the cell front, outgrowth (Fig. 1 C, D,andG). MTs were assembled but remained and contribute to the establishment and maintenance of cell at a distance from the cell periphery, at the basis of the lamella. This orientation (1). MTs are polarized polymers, which in interphasic phenotype was reminiscent of Memo- or mDia1-depleted cells (Fig. cells are assembled from the MT organizing center and elongate 1B) (9). Interestingly, overexpression of APC or CLASP2 restored toward the cell periphery. MT plus-ends explore the cytoplasmic peripheral MTs in Memo-depleted cells, suggesting that APC and area in search of cortical targets, where they will be stabilized. CLASP2 acted downstream of Memo (Fig. 1 E–G). MT assembly and stability are under the control of MT-associated The MT defect caused by APC siRNA could be prevented by proteins. Plus-end tracking proteins (+TIPs) are specialized MT- reexpression of wild-type APC, confirming that the observed associated proteins that concentrate at the plus-end of MTs. They effect was actually a consequence of APC depletion (Fig. S1A). include several families of structurally unrelated proteins (2). Al- APC was previously described as a component of a trimolecular though some proteins, such as kinesin-13 family members (3), complex including mDia and EB1 (11). Interestingly, an N- bind to depolymerizing MTs, most of them, including EB1, cy- terminal deletion mutant APC construct defective for the PDZ toplasmic linker protein-170, cytoplasmic linker-associated pro- and EB1 binding sites (APC2644Δ) also complemented the de- teins (CLASPs), or the tumor suppressor APC (adenomatous pletion of APC for formation of peripheral MTs, suggesting that polyposis coli), decorate only growing MTs. +TIPs interact with APC did not require direct interaction with EB1 for this function MT ends and with each other, generating a network of proteins held together by selective and transient interactions (2). Plus-end associated complexes interact in turn with membrane-associated Author contributions: A.B. designed research; K.Z., K.B., P.D., and D.S. performed re- proteins, such as RhoGTPases, via intermediate proteins, such as search; K.Z. K.B, P.D., and A.B. analyzed data; and K.Z. and A.B. wrote the paper. IQGAP1 or the mDia formins (4). +TIPs can also associate di- The authors declare no conflict of interest. rectly with cortical factors (5) or actin, as illustrated by the MT– This article is a PNAS Direct Submission. actin crosslinking factor MACF/ACF7 (6, 7). 1K.B. and P.D. contributed equally to this work. Activation of the receptor tyrosine kinase ErbB2 stimulates 2To whom correspondence should be addressed. E-mail: [email protected]. breast carcinoma cell motility. Upon ErbB2 activation, breast car- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cinoma cells form extensive protrusions, which are populated by 1073/pnas.1000975107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000975107 PNAS | October 26, 2010 | vol. 107 | no. 43 | 18517–18522 Downloaded by guest on September 28, 2021 Fig. 1. APC and CLASP2 are required for peripheral MT outgrowth. (A–F) EGFP-tubulin–expressing SKBr3 cells were treated with 5 nM HRG before analysis by time-lapse fluorescence microscopy. Cells were cotransfected with control (Ctrl), Memo, APC, or CLASP1/2 siRNAs and APC or CLASP2 cDNA constructs, as indicated. Still images are shown. Inset: Enlargement of cell front to show EGFP-CLASP2 comets and membrane staining. (Scale bars, 5 μm.) (G) Percentage of cells showing peripheral MTs was determined in three in- dependent experiments. More than 150 cells were counted for each data point. Mean ± SEM is shown. *P < 0.005 relative to Ctrl, Student’s t test. (Fig. S1A). However, an APC mutant defective for the basic region (APC1941Δ), a putative MT and actin binding region (12, 13), was unable to substitute for endogenous APC (Fig. S1A). Several studies suggested that binding of APC and CLASPs to MTs was regulated by GSK3β-mediated phosphorylation (14, 15). We have thus evaluated whether GSK3 activity was involved in Memo/mDia1-dependent MT dynamics. HRG treatment resulted in increased phosphorylation of GSK3β on Ser9, which is associ- ated with decreased activity. Memo or mDia1 knockdown resulted in decreased ErbB2-induced phosphorylation of GSK3β on Ser9 Fig. 2. Memo-dependent MT outgrowth is mediated by inhibition of GSK3 A (Fig. 2A). Thus, activation of ErbB2 inhibits GSK3 activity via activity. ( ) SKBr3 cells, transfected with Memo or mDia1 siRNA for 48 h or pretreated with LY294002 (LY) for 60 min as a positive control, were treated Memo signaling. We then tested whether Memo-controlled GSK3 with HRG for 30 min before Western blotting analysis with antibodies to signaling was involved in MT outgrowth to the cell periphery. phosphorylated GSK3β (pGSK3β), GSK3β, Memo, or mDia1. (B–F) EGFP- Inhibition of GSK3 activity, by LiCl pretreatment, restored the tubulin–expressing SKBr3 cells, transfected with Memo, APC, or CLASP1/2 B–D ability of Memo-depleted cells to grow MTs in response to HRG siRNAs and pretreated with 20 mM LiCl for 30 min ( ) or with active GSK3 B G (GSK3βS9A or GSK3*) or APC cDNA constructs (E and F) were treated with (Fig. 2 and ). Similar results were obtained by overexpressing HRG before analysis by time-lapse fluorescence microscopy. Still images are kinase dead GSK3β (GSK3βK85A; Fig. 2G). LiCl treatment of shown. (Scale bars, 5 μm.) (G) Percentage of cells showing peripheral MTs APC- or CLASP-depleted cells did not restore MT outgrowth as was determined and represented as in Fig. 1. 18518 | www.pnas.org/cgi/doi/10.1073/pnas.1000975107 Zaoui et al. Downloaded by guest on September 28, 2021 expected by the model that positioned APC and CLASP down- tivity was inhibited by LiCl treatment (Fig. S2A and Movie S4), stream of GSK3 (Fig. 2 C, D,andG). EGFP-CLASP2 decorated the entire length of MTs, suggesting Conversely, expression of a constitutively active form of GSK3β increased affinity for MTs, as described in previous studies (15).
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