M

M. (RNAi)-mediated replacement with mutant blinkin reveals that the Bubs-binding domain is functionally important for chromosome alignment and segregation. We also provide evidence that hMis14 mediates hNdc80 binding to blinkin at the kinetochore. The C-terminal fragment of blinkin locates at kinetochores in a dominant-negative fashion by displacing endogenous blinkin from kinetochores. This negative dominance SR3335 is relieved by mutations of SR3335 the hMis14 Itga6 binding PPSS motif on the C terminus of blinkin or by fusion of the N sequence that binds to Bub1 and BubR1. Taken together, these results indicate that blinkin functions to connect Bub1 and BubR1 with the hMis12, Ndc80, and Zwint-1 complexes, and disruption of this connection may lead to tumorigenesis. The kinetochore is formed on centromeric DNA and is essential for microtubule attachment and mitotic checkpoint signaling during mitosis (3, 6, 23, 27, 34). Recent genetic SR3335 and mass spectrometric analyses have identified more than 80 kinetochore components in fungi, nematodes, insects, and mammalian cells and have revealed both conserved core components and species-specific kinetochore proteins (4, 14, 22, 25, 30). These proteins form several subcomplexes and assemble and disassemble in an ordered fashion to build functional kinetochores during mitosis. The Mis12 complex is a key conserved kinetochore subcomplex which consists of four proteins, Mis12, Mis13/Dsn1, Mis14/Nsl1, and Nnf1/PMF1 (4, 10, 11, 18, 25). The human Mis12 (hMis12) complex interacts with heterochromatin protein 1 (HP1) during interphase and dissociates from HP1 during mitosis to form SR3335 the inner centromere structure between sister kinetochores (16). During mitosis, the hMis12 complex associates with the microtubule-binding protein blinkin (alternatively called hSpc105, hKNL1, CASC5, and D40) and the Ndc80/Hec1 complex (2, 5, 33), as well as the mitotic checkpoint-related proteins Zwint-1 and Bub1 and BubR1 (Bub1-related protein) (17). Thus, the hMis12 complex plays a key role in inner centromere and kinetochore assembly as well as kinetochore-microtubule attachment and mitotic checkpoint signaling in human cells. Yeast two-hybrid (Y2H) mapping, localization dependency, and RNA interference (RNAi) rescue experiments have suggested that the hMis12 complex directly interacts with the C terminus of blinkin and that the N terminus of blinkin directly recruits Bub1 and BubR1 (here designated Bubs) to kinetochores (17). The molecular mechanisms of these interactions are elusive but are indispensable for understanding the mechanism of kinetochore assembly, checkpoint signaling, and regulation. In this study, we performed extensive Y2H analyses of blinkin and identified its SR3335 minimal binding sites for Bub1, BubR1, Zwint-1, and hMis14. Deletion constructs or substitution mutants of blinkin suggest a critical functional importance for these domains. Furthermore, we show that hMis14 links blinkin to the Ndc80 complex at kinetochores during mitosis. We discuss the conserved and species-specific function of the Mis12 complex. MATERIALS AND METHODS Strains and media. HeLa cells and strains that stably express the green fluorescent protein (GFP)-hMis14 wild type (WT) or GFP-hMis14 m2E mutant were grown at 37C in Dulbecco’s modified Eagle’s medium (DMEM; Kohjin Bio) supplemented with 10% fetal bovine serum (FBS; Biowest), 1% penicillin-streptomycin, and 1% antibiotic and antimycotic (Gibco). Stable T-Rex HeLa and Flip-in TRex 293 cells were constructed and cultured according to the manufacturer’s protocol (Invitrogen). To induce transgenes, cells were incubated with 1 g/ml tetracycline (tet; MP Biomedicals). Cells were treated with 100 ng/ml nocodazole (MP Biomedicals) or 150 ng/ml TN16 (Wako) for 15 to 18 h to obtain the mitotic arrest extract. Plasmids and siRNA transfection. The nucleotide sequences of all blinkin mutations were verified by DNA sequencing. Plasmid DNAs purified using an EndoFree Maxi kit (Qiagen) were transfected using an Effectene transfection kit (Qiagen) to obtain the stable cell lines. The conventional 21-nucleotide small interfering RNA (siRNA) for blinkin (siRNA 4) or stealth siRNA 3 (designed for the middle region of blinkin) (17) was transfected with Oligofectamine (Invitrogen) or RNAi Max (Invitrogen), respectively. The cell culture and transfection of siRNA were performed according to the manufacturer’s instructions. Yeast two-hybrid analysis. The two-hybrid analysis was performed according to the procedures described in the two-hybrid analysis kit (Matchmaker; Clontech).