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␤-Catenin plays important roles in cell adhesion and gene transcription, and has been shown recently to be essential for the establishment of a bipolar mitotic spindle. Here we show that ␤-catenin is a component of interphase centrosomes and that stabilization of ␤-catenin, mimicking mutations found in cancers, induces centrosome splitting. Centrosomes are held together by a dynamic linker regulated by Nek2 kinase and its substrates C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin. We show that ␤-catenin binds to and is phosphorylated by Nek2, and is in a complex with Rootletin. In interphase, ␤-catenin colocalizes with Rootletin between C-Nap1 puncta at the proximal end of centrioles, and this localization is dependent on C-Nap1 and Rootletin. In mitosis, when Nek2 activity increases, ␤-catenin localizes to centrosomes at spindle poles independent of Rootletin. Increased Nek2 activity disrupts the interaction of Rootletin with centrosomes and results in binding of ␤-catenin to Rootletin-independent sites on centrosomes, an event that is required for centrosome separation. These results identify ␤-catenin as a component of the intercentrosomal linker and define a new function for ␤-catenin as a key regulator of mitotic centrosome separation.[Keywords: ␤-Catenin; centrosome; Nek2; mitosis; C-Nap1; Rootletin] Supplemental material is available at http://www.genesdev.org. it has been shown that ␤-catenin localizes to centrosomes in mitosis and has a role in establishing a bipolar spindle (Kaplan et al. 2004); however, the regulation and function for ␤-catenin at centrosomes in normal cells and how this function is perturbed in cancers are not understood.Centrosomes undergo a highly regulated duplication cycle in interphase cells so that at the onset of mitosis a cell has two centrosomes that can separate to establish a bipolar spindle (Hinchcliffe and Sluder 2001;Tsou and Stearns 2006). In general, mechanisms of centrosome cohesion and separation are not well understood. It is thought that in interphase the two centrosomes, each with a pair of centrioles, are held together by a dynamic physical linker composed of C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin (Bahe et al. 2005;Yang et al. 2006). In mitosis, activation of the NIMArelated centrosomal kinase Nek2A results in the phosphorylation of C-Nap1 and Rootletin, which is thought to cause their dissociation from centrosomes and enable centrosome separation (Fry et al. 1998a;Mayor et al. 2002

β-Catenin is essential for the function of cadherins, a family of Ca2+-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH2-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH2-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH2-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH2-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH2 terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.

Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by ϵ-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, our data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole.

Abstract. A centrifugal force-based adhesion assay has been used to quantitatively examine the kinetics of formation of cell-cell contacts mediated specifically by expression of E-cadherin under the control of a glucocorticoid-inducible promoter in mouse fibroblasts. Analysis of cells expressing maximal or minimal levels of E-cadherin showed that the strength of E-cadherinmediated adhesion developed in a single exponential step over a short time (half-maximal adhesion, 13-17 min). At 37°C, adhesion strength increased rapidly in the first 20 min without an apparent lag phase. After 90 min, adhesion strength reached a plateau. Differences in final strengths of adhesion were commensurate with the level of E-cadherin expression. Strengthening of adhesion was temperature dependent. At 19°C, strengthening of adhesion was delayed and subsequently developed with a slower rate compared to adhesion at 37°C. At 4°C, adhesion was completely inhibited. Strengthening of adhesion was absolutely dependent on a functional actin cytoskeleton since adhesion did not develop when cells were treated with cytochalasin D. Together, our current and previous (McNeill et al., 1993. J. Cell Biol. 120:1217-1226) studies indicate that the rate of initial strengthening of E-cadherin-mediated adhesion is neither dependent on the amount of E-cadherin expressed nor on long-range protein diffusion in the membrane to the adhesion site. However, initial strengthening of adhesion is dependent on temperature-sensitive cellular activities that may locally couple clusters of E-cadherin to the actin cytoskeleton.