[Cell Cycle 7:15, 2377-2383; 1 August 2008]; ©2008 Landes Bioscience Report Signaling function of α-catenin in microtubule regulation Michael Shtutman,1,* Alexander Chausovsky,2 Masha Prager-Khoutorsky,2 Natalia Schiefermeier,2,† Shlomit Boguslavsky,2 Zvi Kam,2 Elaine Fuchs,3 Benjamin Geiger,2 Gary G. Borisy4 and Alexander D. Bershadsky2 1Cancer Center, Ordway Research Institute; Albany, New York USA; 2Department of Molecular and Cellular Biology; The Weizmann Institute of Science; Rehovot, Israel; 3Howard Hughes Medical Institute; Laboratory of Mammalian Cell Biology and Development; The Rockefeller University; New York, New York USA; 4Marine Biological Laboratory; Woods Hole; Massachusetts, USA †Current address: Innsbruck Medical University; Biocenter, Innsbruck, Austria Abbreviations: APC, adenomatous polyposis coli protein; AJ, adherens junction; CHO, chinese hamster ovary cells; DMEM, Dulbecco’s Modified Eagle’s Medium; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; IL2R, interleukin-2 receptor; MAPK, mitogen-activated protein kinase; mDia1, mouse diaphanous related formin 1; MT, microtubule; PBS, phosphate buffered saline; SD, stan- dard deviation; SEM, standard error of mean Key words: alpha-catenin, microtubules, beta-catenin, p120ctn, adherens junction, centrosome, cadherins, cytoplasts Centrosomes control microtubule dynamics in many cell stability depends on the minus end being anchored in the centrosome, types, and their removal from the cytoplasm leads to a shift from more precisely in the pericentriolar material surrounding the mother dynamic instability to treadmilling behavior and to a dramatic centriole.1,2 In contrast, epithelial and neuronal cells maintain decrease of microtubule mass (Rodionov et al., 1999; PNAS large populations of MTs that have no apparent connection to the 96:115). In cadherin-expressing cells, these effects can be reversed: centrosome.2-4 While several mechanisms of the stabilization of non- non-centrosomal cytoplasts that form cadherin-mediated adherens centrosomal MTs have been proposed,5 the pathways activating these junctions display dense arrays of microtubules (Chausovsky et al., or other mechanisms of this kind specifically in epithelial or neural 2000; Nature Cell Biol 2:797). In adherens junctions, cadherin’s cells are not known. Recent observations suggest that stabilization of cytoplasmic domain binds p120 catenin and β-catenin, which in noncentrosomal microtubules is promoted via their interaction with turn binds α-catenin. To elucidate the roles of the cadherin-asso- basal cortex of polarized epithelial cells.6 ciated proteins in regulating microtubule dynamics, we prepared A characteristic feature of epithelial cells is that they form GFP-tagged, plasma membrane targeted or untargeted p120 cadherin-based contacts (adherens junctions or AJs) with neighboring catenin, α-catenin and β-catenin and tested their ability to rescue cells. Cadherins and associated AJ proteins are serving not only as the loss of microtubule mass caused by centrosomal removal in adhesion molecules but produce a variety of signals determining the the poorly adhesive cell line CHO-K1. Only membrane targeting major features of epithelial cell phenotype.7-9 Cytoplasmic domains of α-catenin led to a significant increase in microtubule length of AJ cadherins bind armadillo family proteins, β-catenin and plako- and density in centrosome-free cytoplasts. Expression of non- globin at the C-terminus,10 and p120-catenin at the juxtamembranal membrane-targeted α-catenin produced only a slight effect, while part.11,12 β-catenin and plakoglobin are in turn associated with both membrane-targeted and non-targeted p120 and β-catenin α-catenin, a protein known to interact with numerous cytoskeletal were ineffective in this assay. Together, these findings suggest partners, such as α-actinin, vinculin, actin and formin-1.13 Though that α-catenin is able to regulate microtubule dynamics in a α-catenin was thought for many years to form a direct link between centrosome-independent manner. actin cytoskeleton and cadherin-based junctional complexes, more Introduction recent studies do not confirm this idea, stressing that α-catenin interacts with β-catenin only in monomeric and with actin only in The dependence of microtubules (MTs) on the presence of the dimeric form.14-16 At the same time α-catenin can potentially affect centrosome is related to the ‘lifestyle’ of the cell. In cells that maintain the actin cytoskeleton both by regulating Arp2/3 complex-dependent a centrosomally focused©2008 array of MTs,LANDES such as most BIOSCIENCE. fibroblasts, MT actin polymerization,DO NOT14 DISTRIBUTE.and via interaction with an actin-regulatory protein, formin-1.17 In addition, α-catenin is involved in several *Correspondence to: Michael Shtutman; Cancer Center; Ordway Research Institute; other signaling pathways.18 150 New Scotland Ave; Albany, New York USA; Tel.: 518.641.6484; Fax: In our previous study19 we investigated whether the non- 518.641.6305; Email: [email protected]/ Alexander Bershadsky; The Weizmann Institute of Science; Rehovot 76100 Israel; Email: alexander.bershad- centrosomal pattern of MT organization characteristic of the epithelial [email protected] ‘lifestyle’ could be induced by driving formation of cadherin cell-cell contacts in a non-epithelial cell. We used well-known Chinese Submitted: 05/21/08; Accepted: 05/30/08 Hamster Ovary cell line (CHO) and its cloned subline CHO-K1 as Previously published online as a Cell Cycle E-publication: a model system because these fibroblast-like cells display the radial, http://www.landesbioscience.com/journals/cc/article/6362 www.landesbioscience.com Cell Cycle 2377 Alpha-catenin in microtubule regulation Figure 1. Quantification of the MT length and density in CHO-K1 cells and cytoplasts. (A, C and E) α-tubulin antibody staining of MTs in intact cell (A), centrosome-containing (C), and centrosome-lacking (E) cytoplasts. (B, D and F) Processed images of MT networks produced from A, C and E images, respectively, using the “FiberScore” program described by Lichtenstein et al. (2003). Graphs on the right represent (top) average total MT length per cell or per cytoplast with or without centrosome; (bottom) average MT densities (total MT length per cell or cytoplast area) in cells and in cytoplasts. Error bars correspond to standard errors of mean (SEM). centrosomal pattern of MTs characteristic of the ‘fibroblast lifestyle’ prepared from control CHO-K1 cells, the MT array is random and contain no or low levels of E- and N-cadherin. We found that and sparse (Fig. 1E and F). We used total length and density of the expression of classic cadherins and formation of cell-cell contacts MTs in centrosome-free cytoplasts as quantitative parameters for the induced a change in the pattern of MT organization to the epithelial- assessment of the effects of experimental manipulations on the non- type ‘lifestyle’. Based on this result, we proposed that cadherins centrosomal MTs. initiate a signaling pathway that alters MT organization, promoting In our previous study, we estimated the density of MTs in a cell formation and/or stabilization of non-centrosomal MTs.19 Here, we or cytoplast by total tubulin immunofluorescence after triton X-100 begin testing this hypothesis©2008 by attempting LANDES to identify BIOSCIENCE. the immediate extraction. DO This NOT procedure DISTRIBUTE. gave a value for MT density in terms of downstream steps in the signaling pathway. Our results point to an arbitrary fluorescence units per unit area.19,20 It provided reliable important role for α-catenin. relative measurements for examination of variables within a single Results experiment, but did not provide data that could be directly compared between experiments. In this study, we improved our measurements Assessment of microtubule total length in cytoplasts containing of tubulin polymer by means of custom software21 that recognized and lacking centrosomes. In cytoplasts containing a centrosome, and segmented fibrillar elements in the immunofluorescence image the MT array is radial and the density is high, similar to that of satisfying the criteria of MT staining (Fig. 1A–F). The derived intact cells (Fig. 1A–D). In cytoplasts lacking the centrosome, if images (Fig. 1B, D and F) allowed measurement of total MT 2378 Cell Cycle 2008; Vol. 7 Issue 15 Alpha-catenin in microtubule regulation Targeting of cadherin-catenin complex compo- nents to plasma membrane. Previously, we suggested that cadherins regulation of MT dynamics was medi- ated by the cadherin cytoplasmic tail.19 Here, we intend to investigate, which cytoplasmic partner(s) of cadherin are responsible for the cadherin-depen- dent survival of free MTs in the centrosome-lacking cytoplasts. Since formation of cadherin-mediated cell-cell junctions recruits to the membrane cadherin partners, β-catenin and p120 catenin, as well as the β-catenin partner α-catenin, we decided to deliver these proteins one by one to the membrane in a cadherin-independent manner and to check whether such targeting would affect microtubule length and density in the centrosome-free cytoplasts. A require- ment of this experimental approach was to prepare constructs of the cadherin-catenin complex compo- nents containing a membrane targeting domain. For this purpose we chose a membrane targeting domain consisting of the extracellular and transmembrane portions of the α subunit of the interleukin 2 receptor (IL2R) described previously22 and