Research Article 4589 Protein 4.1R binds to CLASP2 and regulates dynamics, organization and attachment of microtubules to the cell cortex Ana Ruiz-Saenz1, Jeffrey van Haren2, C. Laura Sayas1, Laura Rangel1, Jeroen Demmers3, Jaime Milla´n1, Miguel A. Alonso1, Niels Galjart2,*,` and Isabel Correas1,*,` 1Centro de Biologı´a Molecular Severo Ochoa and Departamento de Biologı´a Molecular, Consejo Superior de Investigaciones Cientı´ficas and Universidad Auto´noma de Madrid (CSIC and UAM), Nicola´s Cabrera 1, 28049 Madrid, Spain 2Department of Cell Biology, Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands 3Proteomics Center, Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands *These authors contributed equally to this work `Authors for correspondence ([email protected]; [email protected]) Accepted 17 July 2013 Journal of Cell Science 126, 4589–4601 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.120840 Summary The microtubule (MT) cytoskeleton is essential for many cellular processes, including cell polarity and migration. Cortical platforms, formed by a subset of MT plus-end-tracking proteins, such as CLASP2, and non-MT binding proteins such as LL5b, attach distal ends of MTs to the cell cortex. However, the mechanisms involved in organizing these platforms have not yet been described in detail. Here we show that 4.1R, a FERM-domain-containing protein, interacts and colocalizes with cortical CLASP2 and is required for the correct number and dynamics of CLASP2 cortical platforms. Protein 4.1R also controls binding of CLASP2 to MTs at the cell edge by locally altering GSK3 activity. Furthermore, in 4.1R-knockdown cells MT plus-ends were maintained for longer in the vicinity of cell edges, but instead of being tethered to the cell cortex, MTs continued to grow, bending at cell margins and losing their radial distribution. Our results suggest a previously unidentified role for the scaffolding protein 4.1R in locally controlling CLASP2 behavior, CLASP2 cortical platform turnover and GSK3 activity, enabling correct MT organization and dynamics essential for cell polarity. Key words: Microtubule dynamics, Protein 4.1R, CLASP2, Cortical platforms, Microtubule organization Journal of Cell Science Introduction binds membrane proteins and lipids (Bennett and Baines, 2001). The microtubule (MT) cytoskeleton is essential for many cellular Protein 4.1R was originally identified as an 80-kDa functions including cell polarity and migration. MTs are polarized multifunctional protein of the membrane skeleton of human red filaments that switch between phases of polymerization and blood cells. In these cells, protein 4.1R stabilizes the spectrin– depolymerization (Howard and Hyman, 2003), a behavior known actin network and mediates the attachment of the underlying as dynamic instability (Mitchison and Kirschner, 1984). A diverse cytoskeleton to the overlying lipid bilayer through interactions group of proteins called MT plus-end-tracking proteins (+TIPs) bind with integral membrane proteins and lipids (Conboy, 1993). It to growing MT plus-ends and regulate MT dynamics was subsequently discovered, however, that nucleated cells and interactions of MTs with other structures (Galjart, 2010). express multiple isoforms of protein 4.1R (Anderson et al., Some +TIPs, including cytoplasmic linker-associated protein-2 1988) and that the complex expression pattern of 4.1R in these (CLASP2), are also part of ‘cortical’ or ‘peripheral’ platforms, cells is mainly due to the extensive alternative splicing of the which are located near the plasma membrane and, in motile cells, 4.1R-encoding pre-mRNA (Conboy, 1999; Tang et al., 1990). are strongly enriched at the leading edge. A major function of these Subsequent studies showed that, in nucleated cells, protein 4.1R cortical platforms, which also contain non-MT-binding proteins isoforms localize at multiple subcellular sites including the such as LL5b and ELKS (also known as ERC1), is to attach MTs to nucleus (de Ca´rcer et al., 1995), the centrosome (Krauss et al., the cell cortex (Lansbergen et al., 2006). CLASP2 acts as a bridging 1997; Pe´rez-Ferreiro et al., 2004), MTs (Huang et al., 2004; protein between MT distal ends and cortical platforms, ensuring MT Pe´rez-Ferreiro et al., 2001) and the endoplasmic reticulum capture and attachment, and thereby regulating asymmetric MT (Luque et al., 1999). A role for 4.1R in organizing nuclear organization, cell polarity and migration (Akhmanova et al., 2001; architecture was established from studies showing interactions Lansbergen et al., 2006; Mimori-Kiyosue et al., 2005). A major aim between 4.1R and components of the splicing machinery in this field of research is to determine the molecular mechanisms (Lallena et al., 1998). Further analyses determined that 4.1R involved in organizing cortical platforms. is essential for proper nuclear assembly (Krauss et al., 2002) Protein 4.1R is the founding member of the large band 4.1 and centrosome–nucleus association (Meyer et al., 2011). superfamily, all of whose members contain a highly conserved In addition, a role for 4.1R as a linker between the actin region known as the ‘FERM domain’ (Chishti et al., 1998) that cytoskeleton and components of tight junctions (Mattagajasingh 4590 Journal of Cell Science 126 (20) et al., 2000) and adherens junctions (Yang et al., 2009) has been Results reported. Recently, we showed that 4.1R selectively Protein 4.1R is involved in MT organization accumulates at the leading edge of migrating cells and that its Migrating cells are characterized by the repositioning of the depletion affects cell motility (Ruiz-Sa´enz et al., 2011). All centrosome and of a large population of MTs toward the leading these findings indicate that 4.1R is not only an adaptor protein edge (Gundersen and Bulinski, 1988; Schmoranzer et al., 2009). linking transmembrane proteins and the actin cytoskeleton but We have recently shown that protein 4.1R selectively also a multifunctional protein acting at different subcellular accumulates at the leading edge and is necessary for cell sites. migration (Ruiz-Sa´enz et al., 2011). It is therefore plausible that In this study, we show that protein 4.1R associates with 4.1R has a role in MT organization at the cell edge. To explore CLASP2 independently of MTs, and that protein 4.1R is required this possibility, we adopted a loss-of-function strategy in human for the correct localization and dynamics of CLASP2 in cortical epithelial ECV304 cells using small interfering RNAs (siRNAs) platforms as well as for the organization, dynamics and specific to 4.1R (Fig. 1A), either used individually (siRNA1, attachment of MTs to the cell cortex. Our data indicate that siRNA2) or as a pool (siRNA_pool, or si4.1R). The same siRNAs protein 4.1R controls CLASP2 binding to MT plus-ends by were previously employed to characterize the role of 4.1R in locally affecting GSK3 activity. As a result, upon reduction of cell migration (Ruiz-Sa´enz et al., 2011). Immunofluorescence 4.1R expression, cells lose polarity, the association of CLASP2 analysis of control migratory cells showed that MTs formed a with the MT lattice increases, and growing MTs that approach the dense radial array oriented roughly perpendicular to the cell edge cell periphery are not captured properly, but continue to grow, (Fig. 1B). Unlike control cells, 4.1R-knockdown (KD) cells bending and curling at cell margins and losing their radial exhibited an abnormal MT organization with many MTs oriented distribution. Our results suggest a key role of the scaffolding more parallel to the membrane, often deviating less than 25˚from protein 4.1R in establishing MT network asymmetry at the the cell edge (Fig. 1C,D). leading edge, thereby ensuring the correct MT organization and We used CLIP-170, the prototype +TIP (Perez et al., 1999), to dynamics essential for cell polarity. mark growing MT plus-ends in control and 4.1R-KD cells Fig. 1. 4.1R knockdown alters MT architecture. (A) Lysates of ECV304 cells transfected with either Journal of Cell Science control siRNA or siRNAs targeting 4.1R were immunoblotted for 4.1R. GAPDH was used as a control of protein loading. (B,C) Control (B) or 4.1R-KD cells (C) were stained for 4.1R and a- tubulin. The boxed region of the cell edge is shown enlarged in the upper right panels. MT orientation was determined by measuring the angle between the MT plus-end and the cell edge (lower right panels). (D) The histogram represents the average angle in control and 4.1R-KD cells transfected with the indicated siRNAs. Values are means + s.d. (347 MTs were analyzed). (E,F) Control (E) and 4.1R- KD cells (F) were triple-stained for CLIP-170, a- tyrosinated-tubulin and 4.1R. Staining of the cell edge (boxed region) is shown in more detail in the bottom panels. Arrows indicate CLIP-170 binding to MT plus-ends, regardless of MT orientation. (G) Centrosomes in the forward-facing 90˚ sector (green zone) were scored as reoriented. (H) Cell monolayers were wounded and after 8 hours, fixed and triple-stained for c-tubulin, a-tyrosinated- tubulin and 4.1R. Arrows indicate the centrosome, one of which (boxed) is enlarged in the inset. (I) The histogram represents the percentage of wound-edge cells with reoriented centrosomes (196 cells were analyzed). Data in D and I were from three independent experiments and are presented as the means + s.d. **P,0.01; ***P,0.001. Scale bars: 20 mm. 4.1R and MTs at the cell edge 4591 (Fig. 1E,F). CLIP-170 was associated with MT plus-ends in the found in the cell extract, only the ,80-kDa 4.1R species was margin of the 4.1R-KD cells, indicating that many of the detected in the GFP–CLASP2 immunoprecipitates (Fig.