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requires expression of the plus-end binding protein EB1 and its re- ..... ability to interact with EB1 at MT ends (Fig. S7). The involvement of GSK3 in the control of ...
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 a Centre de Recherche en Cancérologie de Marseille, Institut National de la Santé et de la Recherche Médicale U891, 13009 Marseille, France; bInstitut PaoliCalmettes, 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)

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cell motility receptor tyrosine kinase kinase-3 adenomatous polyposis coli

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| Memo protein | glycogen synthase

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icrotubules (MTs) have several important functions during cell motility. They participate in cell detachment at the cell back, regulate the turnover of focal adhesions at the cell front, and contribute to the establishment and maintenance of cell orientation (1). MTs are polarized polymers, which in interphasic cells are assembled from the MT organizing center and elongate toward the cell periphery. MT plus-ends explore the cytoplasmic area in search of cortical targets, where they will be stabilized. MT assembly and stability are under the control of MT-associated proteins. Plus-end tracking proteins (+TIPs) are specialized MTassociated proteins that concentrate at the plus-end of MTs. They include several families of structurally unrelated proteins (2). Although some proteins, such as kinesin-13 family members (3), bind to depolymerizing MTs, most of them, including EB1, cytoplasmic linker protein-170, cytoplasmic linker-associated proteins (CLASPs), or the tumor suppressor APC (adenomatous polyposis coli), decorate only growing MTs. +TIPs interact with 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 proteins, such as RhoGTPases, via intermediate proteins, such as IQGAP1 or the mDia formins (4). +TIPs can also associate directly with cortical factors (5) or actin, as illustrated by the MT– actin crosslinking factor MACF/ACF7 (6, 7). Activation of the receptor tyrosine kinase ErbB2 stimulates breast carcinoma cell motility. Upon ErbB2 activation, breast carcinoma cells form extensive protrusions, which are populated by www.pnas.org/cgi/doi/10.1073/pnas.1000975107

outgrowing MTs. We have recently described a signaling pathway by which ErbB2 controls MT outgrowth and stabilization at the cell cortex, which involves the recruitment of Memo, mediator of ErbB2-driven motility (8), to the plasma membrane and the subsequent localization of the GTPase RhoA and its effector, the mDia1 formin, to the cell front (9). This pathway also controls the formation of nascent adhesions and the recruitment of actin-binding proteins, such as α-actinin (9). The mechanism by which the MemoRhoA-mDia1 signaling module regulates MT dynamics remained unsolved. In this study, we report that Memo signaling controls the localization of APC and CLASP2 to the plasma membrane, via the regulation of glycogen synthase kinase-3 (GSK3) activity. In turn, membrane-bound APC allows the localization of the spectraplakin ACF7/MACF1 to the plasma membrane, which is required and sufficient for MT capture and stabilization. Results GSK3 Regulates MT Outgrowth via APC and CLASP2. We have pre-

viously shown that Memo-RhoA-mDia1 signaling mediated ErbB2-regulated MT dynamics. We have further explored the underlying mechanism. We initially investigated the contribution of APC and CLASPs, plus-end binding proteins known to contribute to MT stability (10, 11), to MT outgrowth during ErbB2driven cell motility. Activation of ErbB2 by the addition of heregulin β (HRG) to EGFP-tubulin–expressing SKBr3 breast carcinoma cells resulted in the extension of cell protrusions, populated by MTs (Fig. 1A). siRNA-mediated inhibition of APC or CLASP1/2 expression did not affect formation of protrusions but resulted in defective MT outgrowth (Fig. 1 C, D, and G). MTs were assembled but remained at a distance from the cell periphery, at the basis of the lamella. This phenotype was reminiscent of Memo- or mDia1-depleted cells (Fig. 1B) (9). Interestingly, overexpression of APC or CLASP2 restored peripheral MTs in Memo-depleted cells, suggesting that APC and CLASP2 acted downstream of Memo (Fig. 1 E–G). The MT defect caused by APC siRNA could be prevented by reexpression of wild-type APC, confirming that the observed effect was actually a consequence of APC depletion (Fig. S1A). APC was previously described as a component of a trimolecular complex including mDia and EB1 (11). Interestingly, an Nterminal deletion mutant APC construct defective for the PDZ and EB1 binding sites (APC2644Δ) also complemented the depletion of APC for formation of peripheral MTs, suggesting that APC did not require direct interaction with EB1 for this function

Author contributions: A.B. designed research; K.Z., K.B., P.D., and D.S. performed research; K.Z. K.B, P.D., and A.B. analyzed data; and K.Z. and A.B. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

K.B. and P.D. contributed equally to this work.

2

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1000975107/-/DCSupplemental.

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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 linkerassociated 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.

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 independent 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 associated with decreased activity. Memo or mDia1 knockdown resulted in decreased ErbB2-induced phosphorylation of GSK3β on Ser9 (Fig. 2A). Thus, activation of ErbB2 inhibits GSK3 activity via Memo signaling. We then tested whether Memo-controlled GSK3 signaling was involved in MT outgrowth to the cell periphery. Inhibition of GSK3 activity, by LiCl pretreatment, restored the ability of Memo-depleted cells to grow MTs in response to HRG (Fig. 2 B and G). Similar results were obtained by overexpressing kinase dead GSK3β (GSK3βK85A; Fig. 2G). LiCl treatment of APC- or CLASP-depleted cells did not restore MT outgrowth as 18518 | www.pnas.org/cgi/doi/10.1073/pnas.1000975107

Fig. 2. Memo-dependent MT outgrowth is mediated by inhibition of GSK3 activity. (A) 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 with HRG for 30 min before Western blotting analysis with antibodies to phosphorylated GSK3β (pGSK3β), GSK3β, Memo, or mDia1. (B–F) EGFPtubulin–expressing SKBr3 cells, transfected with Memo, APC, or CLASP1/2 siRNAs and pretreated with 20 mM LiCl for 30 min (B–D) or with active GSK3 (GSK3βS9A or GSK3*) or APC cDNA constructs (E and F) were treated with HRG before analysis by time-lapse fluorescence microscopy. Still images are shown. (Scale bars, 5 μm.) (G) Percentage of cells showing peripheral MTs was determined and represented as in Fig. 1.

Zaoui et al.

Regulation of APC and CLASP Localization. Our data show that GSK3

activity regulates peripheral MT outgrowth via APC and CLASP2. Because previous studies have shown that phosphorylation of APC and CLASPs regulates their ability to associate with MTs, we analyzed APC and CLASP2 localization as a function of Memo signaling and GSK3 activity. We observed localization of endogenous CLASP2 and APC by immunofluorescence. In HRG-treated cells, CLASP2 localized to both the cell plasma membrane and MTs (Fig. 3A). Besides the cytoplasmic staining, APC was mostly associated with the plasma membrane and not with MTs (Fig. 3B). Depletion of Memo (Fig. 3 D and E and Fig. S2B) or expression of active GSK3β (Fig. 3 G and H) led to a loss of membrane-associated staining of both APC and CLASP2. The typical localization of +TIPs is better observed in live cells. EGFP-CLASP2 formed comets at the tips of outgrowing MTs, as expected, but was also associated with specific areas of the plasma membrane and ruffles (Fig. S2A and Movie S3). When GSK3 ac-

tivity was inhibited by LiCl treatment (Fig. S2A and Movie S4), EGFP-CLASP2 decorated the entire length of MTs, suggesting increased affinity for MTs, as described in previous studies (15). In cells expressing active GSK3β, EGFP-CLASP2 was still associated with the tip of defective MTs but was no longer seen on membrane and ruffles (Fig. S2A and Movie S5), confirming that the association of EGFP-CLASP2 with the plasma membrane is regulated by phosphorylation. We then investigated whether GSK3β localization was also regulated. We found that upon ErbB2 activation, phosphorylated GSK3 showed cytoplasmic staining, as well as plasma membrane labeling (Fig. 3C). GSK3 showed similar labeling (Fig. S3). When expression of Memo was decreased, phosphorylated GSK3 membrane labeling was strongly diminished (Fig. 3F and Fig. S2B), whereas GSK3 staining was mostly unchanged (Fig. S3). In contrast, inhibition of APC or CLASP2 expression did not modify phosphorylated GSK3 staining (Fig. S3), as expected for downstream targets of GSK3. Thus, Memo signaling controls localization of phosphorylated GSK3, APC, and CLASP2 to the plasma membrane. APC Is Required for ACF7 Localization. Our data show that APC and CLASP2 are mediators of ErbB2-dependent stabilization of MTs at the cell cortex. This could be mediated by direct interaction of these proteins with EB1 (11, 16). However, we found that the EB1binding domain of APC was not required for this function (Fig. S1). Furthermore, recent work suggests that the spectraplakin ACF7/MACF1 is involved in MT dynamics in endodermal and epidermal cells, guiding MTs along actin stress fibers to focal adhesions, possibly via direct interaction with EB1 (7, 17). We investigated whether EB1 and ACF7 were involved in the targeting of MTs to the cell cortex, downstream of Memo signaling. Inhibition of EB1 or ACF7 expression prevented formation of ErbB2-induced peripheral MTs (Fig. 4 A–C and Fig. S4A), similarly to what was seen upon inhibition of Memo, APC, or CLASP2. However, in contrast to what was seen upon APC or CLASP2 overexpression (Fig. 1 E and F), expression of EB1 or ACF7 did not restore MT outgrowth in Memo-depleted cells (Fig. 4 D–F and Fig. S4A). We then investigated ACF7 and EB1 subcellular localization (Fig. 4 and Fig. S5). Unexpectedly, ACF7 staining, as observed with two different antibodies, was not associated with actin-based structures or MTs but with the plasma membrane (Fig. 4G and Fig. S6). Interestingly, this localization was dependent on expression of APC (Fig. 4J). In addition, inhibition of Memo or expression of GSK3βS9A, which we have shown to affect APC redistribution to the plasma membrane, also prevented ACF7 localization to the membrane (Fig. 4 H and I and Fig. S2B). Reduced ACF7 expression did not affect the localization of phosphorylated GSK3 (Fig. S3) or APC (Fig. 4L), confirming that ACF7 is downstream of APC. In contrast to APC, CLASP expression was not required for ACF7 membrane labeling (Fig. 4K), suggesting that CLASPs function via a distinct mechanism. Of note, EB1 associated with MT plus-ends independently of Memo signaling (Movies S6 and S7). Furthermore, inhibition of EB1 expression did not disturb membrane-bound APC, CLASP2, or ACF7, showing that their redistribution to the membrane is not dependent on EB1 or peripheral MTs (Fig. S5A). ACF7 Targeting to the Plasma Membrane Is Sufficient for MT Capture.

Fig. 3. Memo signaling controls APC, CLASP2, and pGSK3 membrane localization. Cells transfected with control (Ctrl) or Memo siRNA or GSK3βS9A cDNA construct were treated with HRG for 30 min before analysis by immunofluorescence with APC, CLASP2, or phosphorylated GSK3β (pGSK3) antibodies. Arrows indicate plasma membrane. (Scale bars, 5 μm.)

Zaoui et al.

ACF7 harbors both MT and actin-binding domains (6, 18). To address the function of these regions, we have used a mutant ACF7 construct (ACF7-NC), consisting of the N-terminal actin-binding and the C-terminal MT/plus-end interacting domains (17). In live cells, EGFP-ACF7 localized similarly to endogenous ACF7, labeling the plasma membrane and ruffles (Fig. 5A and Movie S8). Cells expressing higher levels of EGFP-ACF7 also show MT plusend tracking (Movie S9). In contrast, EGFP-ACF7-NC decorated both cortical actin in lamellipodia and the entire length of MTs PNAS | October 26, 2010 | vol. 107 | no. 43 | 18519

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expected by the model that positioned APC and CLASP downstream of GSK3 (Fig. 2 C, D, and G). Conversely, expression of a constitutively active form of GSK3β (GSK3βS9A) prevented ErbB2-induced formation of peripheral MTs (Fig. 2 E and G). It is noteworthy that whereas APC or CLASP2 expression restored MTs in Memo-depleted cells (Fig. 1 E and F), they did not in GSK3S9A-expressing cells (Fig. 2 F and G and Fig. S2A), suggesting that GSK3-mediated phosphorylation of APC and CLASPs prevented their function on MT stability. Interestingly, we observed that LiCl treatment of Memo-depleted cells before they extended protrusions restored the ability of the cells to form peripheral MTs (Fig. 2B and Movie S1), whereas LiCl treatment of cells after they have extended protrusions did not allow the recovery of defective MTs (Movie S2). This result suggested that inhibition of GSK3 did not simply promote the activity of some progrowth MT-associated proteins. Instead, this observation is in accordance with a model of early MT capture at the cell cortex followed by coincident growth of cell protrusions and MTs. Altogether these results show that down-regulation of GSK3 activity by Memo-mDia1 regulates MT capture at the cell cortex via APC and CLASPs.

bility of the cells to form peripheral MTs (Fig. 5F and Fig. S4B), demonstrating that ACF7-NC is functional when targeted to the plasma membrane. ACF7 localization is dependent on Memo signaling and ACF7 localization at the plasma membrane is critical for its function in MT stabilization. To verify that Memo’s main function is to recruit ACF7 to the plasma membrane, we investigated whether forcing ACF7 to the membrane would substitute Memo signaling. We found that in contrast to ACF7-NC or wild-type ACF7, expression of ACF7-NC-CCKVL was sufficient to restore formation of peripheral MTs in Memo- (Fig. 5 G–I, Fig. S4B, and Movies S12– S14) and APC-knockdown cells (Fig. S4C). As we have previously shown (9), inhibiting Memo’s expression resulted in disturbed ErbB2-controlled MT dynamics, decreasing the frequency of rescues and increasing the frequency of catastrophes (Fig. 5 J and K). Expression of ACF7-NC failed to restore normal MT dynamics in Memo knockdown cells. In fact, ACF7-NC generally decreased MT dynamicity. Expression of membrane-targeted ACF7-NC (ACF7-NC-CCKVL) restored close to normal MT dynamics, preventing the decreased rescues and increased catastrophes due to the loss of Memo (Fig. 5 J and K). Thus, targeting ACF7 to the plasma membrane functionally mimics Memo signaling, confirming that the function of Memo signaling is to allow the formation of an ACF7-comprising membrane-bound protein complex that captures and stabilizes MTs.

Fig. 4. ACF7 is required for MT outgrowth and associates with the plasma membrane in a Memo- and APC-dependent manner. (A–F) Cells expressing EGFP-tubulin (A–E) or EB1-EGFP (F) were transfected with the indicated siRNAs or cDNA constructs 48 h before addition of HRG and analysis by timelapse fluorescence microscopy. Still images are shown. Quantification of these data is shown in Fig. S4A. (G–L) Cells were transfected with the indicated siRNAs or cDNA constructs before analysis by immunofluorescence with ACF7 (G–K) or APC (L) antibodies. Arrows indicate plasma membrane. (Scale bars, 5 μm.)

(Fig. 5B and Movie S10). Thus, ACF7-NC localization is strikingly different from localization of the full-length protein. Moreover, EGFP-ACF7-NC strongly disturbed MT dynamics, induced MT bundling, and prevented MT outgrowth (Fig. 5B and Movie S10). As a consequence, and in contrast to expression of ectopic fulllength ACF7 (Fig. 5D and Fig. S4B), expression of ACF7-NC did not allow recovery of MTs in cells depleted of endogenous ACF7 (Fig. 5E and Fig. S4B). Thus, the MT and actin-binding domains of ACF7 do not reconstitute a functional protein for MT capture. It was recently demonstrated that ACF7 central region was endowed with ATPase activity, which was involved in cytoskeletal and focal adhesion dynamics (17). The absence of the ATPase domain could explain why ACF7-NC cannot functionally replace endogenous ACF7 for MT outgrowth. Alternatively, ACF7-NC defective function might be due to its disturbed localization compared with wild-type ACF7. To explore the latter possibility, we engineered a membrane-targeted ACF7-NC by fusing it to the C-terminal domain of RhoB that confers a palmitoylation signal (ACF7-NC-CCKVL). ACF7-NC-CCKVL localized to the plasma membrane and ruffles (Fig. 5C and Movie S11). Expression of ACF7-NC-CCKVL in ACF7 knockdown cells restored the capa18520 | www.pnas.org/cgi/doi/10.1073/pnas.1000975107

Discussion We describe the signaling pathway whereby ErbB2 activation induces outgrowth and stabilization of peripheral MTs. We found that upon ErbB2 activation, the Memo-RhoA-mDia1 pathway elicits the phosphorylation and thus the inhibition of GSK3 at the plasma membrane. This prevents the phosphorylation of APC (and CLASP2), allowing its association with the plasma membrane and the recruitment of ACF7, which we show to be required and sufficient for MT capture, probably via its ability to interact with EB1 at MT ends (Fig. S7). The involvement of GSK3 in the control of MT dynamics has been suggested (19–22) . Interestingly, in each of these examples, APC was postulated to be one of the main effectors of GSK3. Although phosphorylation by GSK3 of both APC and CLASPs was shown to control their association with MTs (14, 15, 23), we propose that it can also control their association with the plasma membrane. In recent years, APC has been involved in several functions correlating with distinct subcellular locations, including the nucleus, MTs, tight junctions, and the plasma membrane (24). We found that APC function does not require the EB1 and Disk Large binding domains but involves amino acids 1941–2644, which includes the basic region [a domain of interaction for MTs (12) and actin (13)] and the last two 20-aa repeats. Actually, APC’s ability to stabilize MTs correlated with its ability to associate with the plasma membrane (Fig. S1 A and B). Some reports suggested that localization of APC at cortical clusters was dependent on transport along MTs (25). Yet we find that APC is still targeted to the membrane when peripheral MTs are disrupted [e. g., when EB1 (Fig. S5A) or ACF7 (Fig. 4L) are knocked down]. Interestingly, in preliminary experiments, we observed that β-catenin is also recruited to the front of migrating cells and that β-catenin expression is required for recruitment of APC, suggesting that APC membrane localization might be dependent on the presence of a β-catenin–containing complex at the plasma membrane. It would be interesting to explore the relationship between this membrane-bound complex and the β-catenin degradation complex (26), especially because APC and ACF7 were proposed to be part of a larger Wnt-regulated complex (27). We show that APC, CLASPs, ACF7, and EB1 are all required for MT capture downstream of ErbB2. However, overexpression of these proteins in Memo-depleted cells yield different outcomes (APC and CLASP2 reverse Memo-dependent MT defect, whereas Zaoui et al.

ACF and EB1 do not), which can be interpreted in regard to their respective modes of regulation. Indeed, Memo depletion results in increased GSK3 activity. Consequently APC and CLASPs will be phosphorylated and, since their localization depends on their phosphorylation state, displaced from the membrane because their localization is controlled by phosphorylation, Overexpressing APC and CLASPs overcomes GSK3, leaving a pool of unphosphorylated functional APC and CLASPs at the membrane. Ectopically expressed APC and CLASP2 can in turn be inactivated by overexpressing active GSK3 (Fig. 2 F and G). In contrast, overexpressing ACF7 is not sufficient to restore function, because ACF7 localization to the membrane is not determined by GSK3-dependent phosphorylation, but depends on the presence of membrane-bound APC. Finally, overexpression of EB1 cannot restore a functional MT-capture complex, because EB1 is not involved in the formation of this complex but represents its counterpart on MTs. An intriguing observation was that APC and CLASPs seemed to control MTs by different means. First, APC and CLASP associate with the membrane independently (Fig. S5B). Moreover, ACF7 localization to the membrane was dependent on APC but not on CLASPs (Fig. 4 J and K). Finally, overexpression of CLASP in Memo-depleted cells rescued peripheral MT without recruiting APC or ACF7 to the membrane (Fig. S5C). These data suggest the existence of two distinct mechanisms controlling MT capture: one dependent on ACF7 recruitment to the leading edge via APC, the other dependent on CLASPs via a mechanism that remains to be described. ACF7 ATPase domain was shown to be required for MT tracking along actin fibers toward focal adhesions (17). In conZaoui et al.

trast, we show that a mutant ACF7, comprising only the very N-terminal and C-terminal domains and thus devoid of ATPase activity, is functional for MT capture when targeted to the membrane. This suggests that either ATPase activity is dispensable for ACF7 function or that ATPase activity is required for targeting ACF7 to the plasma membrane. However, the fact that ACF7WB, a mutant ACF7 with reduced ATPase activity [due to mutations in the Walker B domain (17)], localizes to the plasma membrane and captures/stabilizes peripheral MTs (Fig. S6E) demonstrates that ACF7 ATPase activity is dispensable for MT capture downstream of Memo signaling. Thus ACF7 seems to use two different modes of action associated with distinct functions: displacement along actin might require motor-like activity, whereas MT capture might not. However, because little is known about the function of ACF7 multiple domains, further investigations are required to understand the molecular mechanisms underlying ACF7 respective contributions to MT guidance and MT capture. Materials and Methods Analysis of MT Dynamics and Immunofluorescence. SKBr3 breast carcinoma cells, grown in DMEM and 10% FCS, were transfected by nucleofection (Lonza). siRNAs and cDNA constructs used and Western blotting analysis are described in SI Materials and Methods. Cells grown on collagen-coated glass coverslips were maintained at 37 °C and observed upon addition of 5 nM HRG-β1 (R&D Systems) using the 100× (plan apochromat NA 1.4) objective of a fluorescence microscope (Zeiss Axiovert 200). Images were acquired at 4-s intervals using a digital camera (Coolsnap HQ; Roper Scientific). Plus-ends of individual MTs were tracked with time using Metamorph software (Universal Imaging Corporation). Number of catastrophes (transitions from growth or pause to shortening) and rescues

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Fig. 5. ACF7 membrane targeting is sufficient for MT capture at the cell cortex. (A–C) Wild-type and mutant EGFP-ACF7–expressing cells were treated with HRG and analyzed by time-lapse fluorescence microscopy. ACF7-NC, mutant ACF with only MT and actin-binding domains; ACF7-NC-CCKVL, membrane-targeted ACF7-NC. Still images from Movies S8–S10 are shown. (D–F) mCherry-tubulin–expressing cells were transfected with ACF7 siRNA and ACF7 cDNA constructs 48 h before addition of HRG and analysis by time-lapse fluorescence microscopy. (G–I) mCherry-tubulin–expressing cells were transfected with Memo siRNA and ACF7-derived constructs; still images from Movies S11–S13 are shown. Only membrane-targeted ACF7 can restore MT outgrowth. (Scale bars, 5 μm.) Quantification of these data is shown in Fig. S5. (J and K) MT dynamics was analyzed by tracking plus-ends of mCherry-tubulin–labeled MTs over time; frequency of rescues (J) and catastrophes (K) are shown. *P < 0.05; *** P < 0.005; ns, not significantly different relative to Ctrl siRNA by Student’s t test in three independent experiments.

(transitions from shortening to pause or growth) was calculated as previously described (28) from 30 MTs in three independent experiments and analyzed with GraphPad Prism. Means and SEM are shown. For immunofluorescence, cells grown on collagen I-coated coverslips were fixed in cold methanol or 4% paraformaldehyde and permeabilized in 0.2% Triton-X-100 before addition of antibodies directed toward APC C terminus (Santa Cruz Biotechnology) or N terminus (Chemicon), CLASP2 (kindly provided by N. Galjart, Erasmus Medical Center, Rotterdam), ACF7 (C20 from Santa Cruz Biotechnology and ACF7 polyclonal antibody from E. Fuchs, Rockefeller University, New York), GSK3β, or phospho-GSK3β (Cell Signaling Technology). Alexa secondary antibodies were from Invitrogen-Molecular Probes. When required, signal was amplified using a biotinylated secondary antibody and streptavidin-FITC (BD Biosciences). DNA was counterstained

with Hoechst dye (Sigma). Images were recorded with an ApoTome ImagerZ1 Zeiss microscope, 63× plan apo objective (1.4 NA) coupled to an AxioCam MRm camera, driven by AxioVision LE software.

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ACKNOWLEDGMENTS. We thank E. Fuchs (Rockefeller University, New York), N. Galjart (Erasmus Medical Center, Rotterdam), B. Vogelstein (Johns Hopkins Kimmel Cancer Center, Baltimore), and J. Woodgett (Mount Sinai Hospital, Toronto) for kindly providing cDNA constructs or antibodies. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM) Avenir Program, Association pour la Recherche sur le Cancer, and Institut National du Cancer (INCa) (to A.B.), predoctoral fellowships from INSERM/Région Provence-Alpes-Côte d’Azur (to K.Z.), Ministère de la Recherche et de l’Enseignement Supérieur (to P.D.), and programme de bourses franco-algérien BFA (to K.B.).

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