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«Essential and non-redundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration Pascale Daoua,b,c,d, Salma ...»

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Essential and non-redundant roles for Diaphanous formins in cortical microtubule

capture and directed cell migration

Pascale Daoua,b,c,d, Salma Hasana,b,c,d, Dennis Breitsprechere, Emilie Baudeleta,b,c,d, Luc

Camoina,b,c,d, Stéphane Audeberta,b,c,d, Bruce L. Goodee, and Ali Badachea,b,c,d


Centre de Recherche en Cancérologie de Marseille, Inserm U1068; bInstitut Paoli-Calmettes;


Aix-Marseille Université; dCNRS UMR7258, 13009 Marseille, France; and eDepartment of

Biology, Brandeis University, Waltham, MA 02454.

Address correspondence to: Ali Badache, Centre de Recherche en Cancérologie de Marseille, 27 bd Lei Roure, 13009, Marseille, France. Phone: 33 1 486 97 73 21; Fax: 33 1 486 97 74 99 Running title: mDia formins in microtubule capture


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 have further investigated the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2dependent breast carcinoma cell migration. We found that depletion of each of these three formins strongly disrupted chemotaxis without significantly impacting actin-based structures.

Further, all three formins were required for the formation of cortical microtubules, in a nonredundant fashion, and formin proteins defective in actin polymerization remained active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identified differential binding partners of the FH2 domains of mDia1, mDia2, and mDia3, which may explain their non-redundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacted with Rab6-Interacting Protein 2 (Rab6IP2). Further, mDia1 was required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupted cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.

Keywords: formins, microtubules, cell motility, actin, proteomics Abbreviations used: APC, adenomatous Polyposis Coli; FH1/2, formin homology domain 1/2;

Rab6IP2, Rab6-Interacting Protein 2


Cell motility depends on the coordinated reorganization of the actin and microtubule cytoskeletons (Waterman-Storer and Salmon, 1999; Wittmann and Waterman-Storer, 2001).

In response to a pro-migratory stimulus, cells assemble actin based protrusions, lamellipodia and filipodia, at the leading edge. Following formation of adhesion sites, thick acto-myosin cables called stress fibers connect the adhesion sites and facilitate contraction of the cell body and, upon detachment of the cell rear, cell movement in the forward direction (Le Clainche and Carlier, 2008). In migrating cells, microtubules are nucleated from microtubule organizing centers and their dynamic plus ends explore the cytoplasmic area through alternating phases of growth and shrinkage, until stabilized near the cell cortex at the leading edge. Microtubules contribute to directional motility in several ways, including the establishment of cell polarity, and the regulation of adhesion sites turnover (Kaverina and Straube, 2011) The molecular mechanisms underlying actin-microtubule crosstalk remain largely unknown.

Supplemental Material can be found at:

http://www.molbiolcell.org/content/suppl/2014/01/06/mbc.E13-08-0482v1.DC1.html Formins are a family of ubiquitous and conserved actin filament assembly factors that nucleate linear, unbranched filaments, and stay processively attached to the growing barbed ends of filaments, protecting them from capping proteins (Breitsprecher and Goode, 2013).

Accordingly, formins have diverse roles in forming primarily unbranched actin structures, such as those found in filopodia, cytokinetic rings, cables, and stress fibers (Pellegrin and Mellor, 2005; Schirenbeck et al., 2005; Hotulainen and Lappalainen, 2006). Formins are large, multi-domain proteins defined primarily by the presence of an actin filament endbinding formin homology 2 (FH2) domain. Mammalian cells have 15 different formin genes, which fall into seven phylogenetic subfamilies (Higgs and Peterson, 2005). Most of our current knowledge of mammalian formins stems from studies on Diaphanous-related formins or DRFs which includes 8 out of the 15 formins: three ‘mammalian Diaphanous’ formins, (mDia1/DRF1, mDia2/DRF3, and mDia3/DRF2), two ‘disheveled-associated activators of morphogenesis’ (DAAM1 and DAAM2) and three ‘Formin-related proteins identified in leucocytes’ (FRL1 FRL2, and FRL3) (Higgs, 2005). The FH2 domain of formins directly nucleates actin nucleation and remains bound to the growing barbed end during elongation of the filament, a phenomenon referred to as processive capping (Pruyne et al., 2002; Sagot et al., 2002; Zigmond et al., 2003; Kovar and Pollard, 2004; Moseley et al., 2004). DRFs also contain, a proline-rich FH1 domain, adjacent to the FH2, which greatly accelerates actin assembly by allowing formins to efficiently recruit profilin-bound actin monomers (Kovar et al., 2006). Finally, DRFs are also characterized by the presence of a C-terminal Diaphanousautoregulatory domain (DAD), which interacts with an N-terminal Diaphanous inhibitory domain (DID) (Alberts, 2001) and contributes to actin nucleation {Gould, 2011 #1484.

Multiple lines of evidence indicate that autoinhibitory DID-DAD interactions in DRFs are released by binding of RhoGTPases to an N-terminal GTPase-binding domain (GBD), and therefore N-terminally truncated DRFs are constitutively active in vitro and in vivo (Watanabe et al., 1999; Sagot et al., 2002).

A growing body of evidence shows that formins, and DRFs in particular, are critical for directly regulating microtubule dynamics in migrating cells, suggesting that formins are potential mediators of actin-microtubule interplay during cell motility. In Hela cells mDia1 induces the co-alignment of stress fiber and microtubule arrays (Ishizaki et al., 2001). In NIH3T3 cells, active mDia2 induces the formation of oriented stable (detyrosinated) microtubules, through its interaction with microtubule plus-end tracking proteins EB1 and APC (adenomatous polyposis coli) (Palazzo et al., 2001; Wen et al., 2004). Our own work has previously shown that, in migrating breast carcinoma cells, mDia1 facilitates capture of microtubules at the cell cortex downstream of the ErbB2 receptor tyrosine kinase signaling.

ErbB2 activation leads to the recruitment of mDia1 via the Memo adaptor and RhoA (Zaoui et al., 2008). In turn, this complex controls the cortical localization of a microtubule capture complex that includes APC and the spectraplakin ACF7 (Zaoui et al., 2010). mDia1 was also shown to control microtubule dynamics downstream of integrin-linked kinase and IQGAP1 in keratinocytes (Wickstrom et al., 2010), and downstream of Gα12/13 and the RhoGEF LARG in fibroblasts (Goulimari et al., 2008), but downstream effectors were not described.

In the present study, we have investigated the contribution of mDia1, mDia2 and mDia3, to breast carcinoma cell motility and microtubule capture, mapped the functional domains and identified critical amino-acid residues and characterized a novel mDia1 ligand involved in cortical capture of microtubules.


mDia1, mDia2 and mDia3 contribute to ErbB2-dependent chemotaxis Heregulin β1 (HRG), a ligand for the ErbB3 and ErbB4 receptor tyrosine kinases, induces breast carcinoma cell motility, via transactivation of ErbB2 (Spencer et al., 2000; Marone et al., 2004). When applied to cells in a gradient, HRG triggers a chemotactic response, which requires a functional Memo/ACF7 pathway (Benseddik et al., 2013). mDia1 is a critical component of the Memo/ACF7 pathway (Zaoui et al., 2008; Zaoui et al., 2010), but the specific functional roles of mDia2 and mDia3 in this pathway are unknown.

We designed siRNAs that efficiently and specifically inhibit the expression of each mDia, as verified by qRT-PCR and Western blotting (Figure 1A and Figure S1). Then, we evaluated their roles in HRG-dependent chemotaxis by tracking SKBr3 breast carcinoma cells as they migrated in response to a HRG gradient in Dunn chambers. In contrast to control cells, cells depleted of mDia1, mDia2 or mDia3 failed to detect the source of the pro-migratory ligand and therefore migrated randomly (Figure 1B), demonstrating that all three proteins are required for directed cell migration, non-redundantly.

Roles of mDia proteins in the formation of actin cellular structures Formins are known regulators of actin assembly with critical roles in the formation of filopodia, lamellipodia, and stress fibers (Schirenbeck et al., 2005; Hotulainen and Lappalainen, 2006; Yang et al., 2007). We evaluated whether depletion of mDia proteins affected the organization of the actin cytoskeleton of migrating SKBr3 cells. Depletion of mDia1, mDia2 or mDia3 individually (data not shown) or together (Figure S2) had no effect on HRG-induced (i) cell protrusion, (ii) lamellipodial actin network, (iii) filopodia-like structures (microspikes), or (iv) stress fiber formation. Thus, silencing of the three mDia formins does not alter the general organization of the actin cytoskeleton in SKBr3 cells.

Role of the mDia formins in ErbB2-induced microtubule capture We have recently observed that defective microtubule capture in cells impairs the chemotactic response (Benseddik et al., 2013). Thus, we investigated the role of mDia formins in microtubule capture.

Activation of ErbB2 led to the formation of cell protrusions that contain microtubules extending towards and stabilized at the cell cortex. Silencing of mDia1 expression led to the expected defect in cortical microtubules, as previously reported (Zaoui et al., 2008).

Inhibition of mDia2 and mDia3 led to a similar absence of cortical microtubules (Figure 2A and 2B). Expression of a N-terminally truncated, constitutively active, form of mDia1 (ΔNmDia1), not targeted by the siRNA, restored cortical microtubules. ΔNmDia2 and ΔNmDia3 also restored cortical microtubules in mDia2-depleted and mDia3-depleted cells respectively, confirming that the observed effect is specifically caused by decreased mDia expression (Figure 2A and 2B). To ensure that restoration of function was not simply due to unregulated mDia activity (caused by the ∆N truncation), we verified in similar experiments that full-length wild type mDia proteins can restore microtubule capture, using a set of siRNAs targeting the 3’UTR (Figure S3).

Importantly, when the three mDia formins were concomitantly depleted (Figure 1A), reexpression of active mDia1, mDia2 or mDia3, individually, was not sufficient to restore cortical microtubules (Fig. S4). Moreover, expression of active mDia1 only restored cortical microtubules in mDia1-, but not in mDia2- or mDia3-depleted cells. In the same manner, expression of active mDia2 and mDia3 compensated for loss of mDia2 and mDia3 respectively, but not the loss of other mDia proteins (Figure 2A and 2B). Thus, mDia1, mDia2 and mDia3 are each required for cortical microtubules, in a non-redundant manner.

Contribution of mDia formins to the Memo/ACF7 pathway We have previously shown that ErbB2-induced cortical microtubules were dependent on the Memo/ACF7 signaling pathway and that inactivation of the Memo pathway at any level can be compensated for by expression of a shortened, internally deleted ACF7 construct that is constitutively targeted to the cell membrane (called ACF7-CCKVL) (Zaoui et al., 2010).

Accordingly, we observed that expression of ACF7-CCKVL restored microtubule capture in cells lacking mDia1. In contrast, ACF7-CCKVL failed to restore microtubule capture in the absence of mDia1 or mDia3 (Figure 3A and S5A), suggesting that mDia2 and mDia3 function via different effectors.

The FH2 domain of mDia formins is sufficient for microtubule capture Next, we asked what domains in mDia proteins are required for microtubule capture. We already showed that ΔNmDia1, which contains FH1 and FH2 domains, is functional (Figure 2A). Gradual truncations of the FH1 domain revealed that the FH1 domain of mDia1 is not required to mediate microtubule capture (Figure S6A and B). Similarly, we found that the FH2 domains of mDia2 and mDia3 were sufficient to restore cortical microtubules in mDia2 and mDia3 depleted cells, respectively (Figure 3B and S5B). Interestingly, expression of the FH2 domain of an mDia did not compensate the loss of the other mDia formins (Figure 3B), demonstrating the specificity of the microtubule-regulatory functions contained in each separate mDia FH2 domain.

The FH2 domain dimerizes; each functional half of an FH2 dimer contains two distinct actinbinding surfaces, marked by conserved Ile and Lys residues, respectively, that are important for actin nucleation and elongation.(Xu et al., 2004) In murine mDia1, the first actin-binding site is centered on Ile845 and the second on Lys994 (Shimada et al., 2004; Xu et al., 2004;

Otomo et al., 2005). We introduced the I845A and K994A mutations in mDia1. These mutations abolished the activity of recombinant mDia1 FH2 domains in bulk pyrene-actin assembly assay (Figure 3C), but the same mutations did not affect microtubule function in vivo, when endogenous mDia1 was silenced and cells were rescued with mutant versus wildtype constructs (Figures 3D and S6C). These results show that functions of the FH2 domain in microtubule capture are independent of its functions in actin assembly. Moreover, a mutation of the conserved Trp767 that greatly weakens FH2 dimerization and abolish actin assembly activity (Moseley et al., 2004) did not alter mDia1 function in cortical microtubule capture (Figure 3D), indicating that the mDia1 FH2 domain can perform its microtubule function, but not its actin function, as a monomer.

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