ErbB2 receptor controls 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) (MTs) contribute to key processes during cell motility, outgrowing MTs. We have recently described a signaling pathway including the regulation of 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- , such as α- (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 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-–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 -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 , 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). (GSK3βS9A) prevented ErbB2-induced formation of peripheral In cells expressing active GSK3β, EGFP-CLASP2 was still asso- MTs (Fig. 2 E and G). It is noteworthy that whereas APC or ciated with the tip of defective MTs but was no longer seen on CLASP2 expression restored MTs in Memo-depleted cells (Fig. 1 E membrane and ruffles (Fig. S2A and Movie S5), confirming that and F), they did not in GSK3S9A-expressing cells (Fig. 2 F and G the association of EGFP-CLASP2 with the plasma membrane is and Fig. S2A), suggesting that GSK3-mediated phosphorylation regulated by phosphorylation. of APC and CLASPs prevented their function on MT stability. We then investigated whether GSK3β localization was also Interestingly, we observed that LiCl treatment of Memo-depleted regulated. We found that upon ErbB2 activation, phosphorylated cells before they extended protrusions restored the ability of the GSK3 showed cytoplasmic staining, as well as plasma membrane cells to form peripheral MTs (Fig. 2B and Movie S1), whereas LiCl labeling (Fig. 3C). GSK3 showed similar labeling (Fig. S3). When treatment of cells after they have extended protrusions did not allow expression of Memo was decreased, phosphorylated GSK3 the recovery of defective MTs (Movie S2). This result suggested that membrane labeling was strongly diminished (Fig. 3F and Fig. S2B), inhibition of GSK3 did not simply promote the activity of some whereas GSK3 staining was mostly unchanged (Fig. S3). In con- progrowth MT-associated proteins. Instead, this observation is in trast, inhibition of APC or CLASP2 expression did not modify accordance with a model of early MT capture at the cell cortex phosphorylated GSK3 staining (Fig. S3), as expected for down- followed by coincident growth of cell protrusions and MTs. stream targets of GSK3. Thus, Memo signaling controls local- Altogether these results show that down-regulation of GSK3 ization of phosphorylated GSK3, APC, and CLASP2 to the activity by Memo-mDia1 regulates MT capture at the cell cortex plasma membrane. via APC and CLASPs. APC Is Required for ACF7 Localization. Our data show that APC and Regulation of APC and CLASP Localization. Our data show that GSK3 CLASP2 are mediators of ErbB2-dependent stabilization of MTs activity regulates peripheral MT outgrowth via APC and CLASP2. at the cell cortex. This could be mediated by direct interaction of Because previous studies have shown that phosphorylation of APC these proteins with EB1 (11, 16). However, we found that the EB1- and CLASPs regulates their ability to associate with MTs, we an- binding domain of APC was not required for this function (Fig. alyzed APC and CLASP2 localization as a function of Memo S1). Furthermore, recent work suggests that the spectraplakin signaling and GSK3 activity. ACF7/MACF1 is involved in MT dynamics in endodermal and fi We observed localization of endogenous CLASP2 and APC by epidermal cells, guiding MTs along actin stress bers to focal immunofluorescence. In HRG-treated cells, CLASP2 localized to adhesions, possibly via direct interaction with EB1 (7, 17). We both the cell plasma membrane and MTs (Fig. 3A). Besides the investigated whether EB1 and ACF7 were involved in the targeting cytoplasmic staining, APC was mostly associated with the plasma of MTs to the cell cortex, downstream of Memo signaling. membrane and not with MTs (Fig. 3B). Depletion of Memo (Fig. 3 Inhibition of EB1 or ACF7 expression prevented formation of A–C A D and E and Fig. S2B) or expression of active GSK3β (Fig. 3 G and ErbB2-induced peripheral MTs (Fig. 4 and Fig. S4 ), simi- H larly to what was seen upon inhibition of Memo, APC, or CLASP2.

) led to a loss of membrane-associated staining of both APC CELL BIOLOGY However, in contrast to what was seen upon APC or CLASP2 and CLASP2. E F The typical localization of +TIPs is better observed in live cells. overexpression (Fig. 1 and ), expression of EB1 or ACF7 did not restore MT outgrowth in Memo-depleted cells (Fig. 4 D–F and EGFP-CLASP2 formed comets at the tips of outgrowing MTs, as A expected, but was also associated with specific areas of the plasma Fig. S4 ). We then investigated ACF7 and EB1 subcellular lo- membrane and ruffles (Fig. S2A and Movie S3). When GSK3 ac- calization (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 lo- calization 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. 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, la- Fig. 3. Memo signaling controls APC, CLASP2, and pGSK3 membrane lo- fl A calization. Cells transfected with control (Ctrl) or Memo siRNA or GSK3βS9A beling the plasma membrane and ruf es (Fig. 5 and Movie S8). cDNA construct were treated with HRG for 30 min before analysis by im- Cells expressing higher levels of EGFP-ACF7 also show MT plus- munofluorescence with APC, CLASP2, or phosphorylated GSK3β (pGSK3) end tracking (Movie S9). In contrast, EGFP-ACF7-NC decorated antibodies. Arrows indicate plasma membrane. (Scale bars, 5 μm.) both cortical actin in lamellipodia and the entire length of MTs

Zaoui et al. PNAS | October 26, 2010 | vol. 107 | no. 43 | 18519 Downloaded by guest on September 28, 2021 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 pe- ripheral 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 res- cues 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, confirm- ing that the function of Memo signaling is to allow the formation of an ACF7-comprising membrane-bound protein complex that captures and stabilizes MTs. 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 phosphory- lation 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). Fig. 4. ACF7 is required for MT outgrowth and associates with the plasma The involvement of GSK3 in the control of MT dynamics has membrane in a Memo- and APC-dependent manner. (A–F) Cells expressing been suggested (19–22) . Interestingly, in each of these examples, EGFP-tubulin (A–E) or EB1-EGFP (F) were transfected with the indicated APC was postulated to be one of the main effectors of GSK3. siRNAs or cDNA constructs 48 h before addition of HRG and analysis by time- Although phosphorylation by GSK3 of both APC and CLASPs lapse fluorescence microscopy. Still images are shown. Quantification of was shown to control their association with MTs (14, 15, 23), we these data is shown in Fig. S4A.(G–L) Cells were transfected with the in- fl propose that it can also control their association with the plasma dicated siRNAs or cDNA constructs before analysis by immuno uorescence membrane. In recent years, APC has been involved in several with ACF7 (G–K) or APC (L) antibodies. Arrows indicate plasma membrane. (Scale bars, 5 μm.) 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, (Fig. 5B and Movie S10). Thus, ACF7-NC localization is strikingly which includes the basic region [a domain of interaction for MTs different from localization of the full-length protein. Moreover, (12) and actin (13)] and the last two 20-aa repeats. Actually, EGFP-ACF7-NC strongly disturbed MT dynamics, induced MT APC’s ability to stabilize MTs correlated with its ability to asso- bundling, and prevented MT outgrowth (Fig. 5B and Movie S10). ciate with the plasma membrane (Fig. S1 A and B). Some reports As a consequence, and in contrast to expression of ectopic full- suggested that localization of APC at cortical clusters was de- length ACF7 (Fig. 5D and Fig. S4B), expression of ACF7-NC did pendent on transport along MTs (25). Yet we find that APC is still not allow recovery of MTs in cells depleted of endogenous ACF7 targeted to the membrane when peripheral MTs are disrupted [e. (Fig. 5E and Fig. S4B). Thus, the MT and actin-binding domains of g., when EB1 (Fig. S5A) or ACF7 (Fig. 4L) are knocked down]. ACF7 do not reconstitute a functional protein for MT capture. Interestingly, in preliminary experiments, we observed that It was recently demonstrated that ACF7 central region was β- is also recruited to the front of migrating cells and that endowed with ATPase activity, which was involved in cytoskeletal β-catenin expression is required for recruitment of APC, sug- and focal adhesion dynamics (17). The absence of the ATPase gesting that APC membrane localization might be dependent on domain could explain why ACF7-NC cannot functionally replace the presence of a β-catenin–containing complex at the plasma endogenous ACF7 for MT outgrowth. Alternatively, ACF7-NC membrane. It would be interesting to explore the relationship defective function might be due to its disturbed localization between this membrane-bound complex and the β-catenin deg- compared with wild-type ACF7. To explore the latter possibility, radation complex (26), especially because APC and ACF7 were we engineered a membrane-targeted ACF7-NC by fusing it to the proposed to be part of a larger Wnt-regulated complex (27). C-terminal domain of RhoB that confers a palmitoylation signal We show that APC, CLASPs, ACF7, and EB1 are all required for (ACF7-NC-CCKVL). ACF7-NC-CCKVL localized to the plasma MT capture downstream of ErbB2. However, overexpression of membrane and ruffles (Fig. 5C and Movie S11). Expression of these proteins in Memo-depleted cells yield different outcomes ACF7-NC-CCKVL in ACF7 knockdown cells restored the capa- (APC and CLASP2 reverse Memo-dependent MT defect, whereas

18520 | www.pnas.org/cgi/doi/10.1073/pnas.1000975107 Zaoui et al. Downloaded by guest on September 28, 2021 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 CELL BIOLOGY 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.

ACF and EB1 do not), which can be interpreted in regard to their trast, we show that a mutant ACF7, comprising only the very respective modes of regulation. Indeed, Memo depletion results in N-terminal and C-terminal domains and thus devoid of ATPase increased GSK3 activity. Consequently APC and CLASPs will be activity, is functional for MT capture when targeted to the mem- phosphorylated and, since their localization depends on their brane. This suggests that either ATPase activity is dispensable for phosphorylation state, displaced from the membrane because their ACF7 function or that ATPase activity is required for targeting localization is controlled by phosphorylation, Overexpressing APC ACF7 to the plasma membrane. However, the fact that ACF7WB, and CLASPs overcomes GSK3, leaving a pool of unphosphorylated a mutant ACF7 with reduced ATPase activity [due to mutations in functional APC and CLASPs at the membrane. Ectopically the Walker B domain (17)], localizes to the plasma membrane expressed APC and CLASP2 can in turn be inactivated by over- and captures/stabilizes peripheral MTs (Fig. S6E) demonstrates expressing active GSK3 (Fig. 2 F and G). In contrast, overexpressing that ACF7 ATPase activity is dispensable for MT capture down- ACF7 is not sufficient to restore function, because ACF7 localiza- stream of Memo signaling. Thus ACF7 seems to use two different tion to the membrane is not determined by GSK3-dependent modes of action associated with distinct functions: displacement phosphorylation, but depends on the presence of membrane-bound along actin might require motor-like activity, whereas MT capture APC. Finally, overexpression of EB1 cannot restore a functional might not. However, because little is known about the function of MT-capture complex, because EB1 is not involved in the formation ACF7 multiple domains, further investigations are required to of this complex but represents its counterpart on MTs. understand the molecular mechanisms underlying ACF7 re- An intriguing observation was that APC and CLASPs seemed spective contributions to MT guidance and MT capture. to control MTs by different means. First, APC and CLASP as- sociate with the membrane independently (Fig. S5B). Moreover, Materials and Methods ACF7 localization to the membrane was dependent on APC but Analysis of MT Dynamics and Immunofluorescence. SKBr3 breast carcinoma not on CLASPs (Fig. 4 J and K). Finally, overexpression of CLASP cells, grown in DMEM and 10% FCS, were transfected by nucleofection in Memo-depleted cells rescued peripheral MT without recruiting (Lonza). siRNAs and cDNA constructs used and Western blotting analysis are SI Materials and Methods APC or ACF7 to the membrane (Fig. S5C). These data suggest described in . the existence of two distinct mechanisms controlling MT capture: 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× one dependent on ACF7 recruitment to the leading edge via APC, (plan apochromat NA 1.4) objective of a fluorescence microscope (Zeiss Axio- the other dependent on CLASPs via a mechanism that remains vert 200). Images were acquired at 4-s intervals using a digital camera (Cool- to be described. snap HQ; Roper Scientific). Plus-ends of individual MTs were tracked with time ACF7 ATPase domain was shown to be required for MT using Metamorph software (Universal Imaging Corporation). Number of cat- tracking along actin fibers toward focal adhesions (17). In con- astrophes (transitions from growth or pause to shortening) and rescues

Zaoui et al. PNAS | October 26, 2010 | vol. 107 | no. 43 | 18521 Downloaded by guest on September 28, 2021 (transitions from shortening to pause or growth) was calculated as previously with Hoechst dye (Sigma). Images were recorded with an ApoTome described (28) from 30 MTs in three independent experiments and analyzed ImagerZ1 Zeiss microscope, 63× plan apo objective (1.4 NA) coupled to an with GraphPad Prism. Means and SEM are shown. AxioCam MRm camera, driven by AxioVision LE software. For immunofluorescence, cells grown on collagen I-coated coverslips were fixed in cold methanol or 4% paraformaldehyde and permeabilized in 0.2% ACKNOWLEDGMENTS. We thank E. Fuchs (Rockefeller University, New York), Triton-X-100 before addition of antibodies directed toward APC C terminus N. Galjart (Erasmus Medical Center, Rotterdam), B. Vogelstein (Johns Hopkins (Santa Cruz Biotechnology) or N terminus (Chemicon), CLASP2 (kindly pro- Kimmel Cancer Center, Baltimore), and J. Woodgett (Mount Sinai Hospital, vided by N. Galjart, Erasmus Medical Center, Rotterdam), ACF7 (C20 from 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 Santa Cruz Biotechnology and ACF7 polyclonal antibody from E. Fuchs, β β Médicale (INSERM) Avenir Program, Association pour la Recherche sur le Can- Rockefeller University, New York), GSK3 , or phospho-GSK3 (Cell Signaling cer, and Institut National du Cancer (INCa) (to A.B.), predoctoral fellowships Technology). Alexa secondary antibodies were from Invitrogen-Molecular from INSERM/Région Provence-Alpes-Côte d’Azur (to K.Z.), Ministère de la Probes. When required, signal was amplified using a biotinylated secondary Recherche et de l’Enseignement Supérieur (to P.D.), and programme de antibody and streptavidin-FITC (BD Biosciences). DNA was counterstained bourses franco-algérien BFA (to K.B.).

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