Molecular Delineation Of HsMis12 Complex Architecture And Interacting Network

The cell cycle is the ordered sets of process through which cells divide,producing two daughter cells possessing the same set of genetic information.This process is fundamental for cell growth and differentiation.The most important events of the cell cycle are those concerned with the duplicating and partitioning of the hereditary materials.More specifically,the cell replicates its genomic DNA during S phase and separates the replicated genomes into two daughter cells during mitosis.And the chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore,a multi-protein complex assembled onto centromeric DNA of the chromosome.HsMis12 complex is a conserved four-protein complex consisting of HsMis12, HsMis13,HsMis14 and HsPMF1,which are essential for kinetochore assembly and chromosome segregation,but their loading manner to the kinetochore remains elusive. Our previous far western assay of Aurora-B interacting protein implied that the HsMis13 protein is a potential binding partner of Aurora-B kinase.Indeed,HsMis13 co-localized with Aurora-B kinase at the centromere from prophase to metaphase in HeLa cells.Aurora B interacts with HsMis13 in vitro and in vivo.HsMis13 is a cognate substrate of Aurora B and the phosphorylation sites were mapped to Ser100 and Ser109.Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore.In addition,non-phosphorylatable but not wild type and phospho-mimicking HsMis13,failed to localize to the kinetochore,demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore.In fact,localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase,which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly.Importantly,phosphomimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore and tension developed across the sister kinetochores in Aurora B-inhibited cells.Thus,we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.Functional studies have suggested that the HsMis12 complex may act as the core component of kinetochore,but its molecular architecture is less known.To delineate the interacting network within the HsMis12 complex,we adopt the co-expression system to purify and reconstitute this complex in vitro.Our co-purification results show that HsMis13 and HsMis14 can form a tight hetero-dimer,whereas the interaction between HsMis12 and HsPMF1 is quite weak.In spite of the low binding affinity between the Mis13/Mis14 dimer and the Mis12/PMF1 dimer,the four proteins form a heterotetramer with a stoichiometry of 1:1:1:1 when these four proteins expressed synchronously,implying that the four subunits can act synergically to form an intact Mis12 complex in vivo.Our yeast two hybrid experiments echo this hypothesis.Furthermore,both the spc24-spc25 hetero-dimer of HsNdc80 complex and the C terminal of KNL-1 protein can bind with the HsMis12 complex in vitro, suggesting that HsMis12 complex has the potency to form a huge KMN complex just as its homologue in C.elegans.Except for the KMN complex,the HsMis12 complex link the outer kinetochore protein Zwint-1 through the direct binding with HsMis12 and HsMis13 subunits.In addition,HsMis13 subunits interact with HsCENP-H protein,a subunit of CENP-H/I complex,but not the HsCENP-50.These results suggest that the HsMis12 and HsMis13 subunits may act as a bridge to link the HsMis12 complex with other kinetochore proteins.Finally,we propose that the phosphorylation of HsMis13 by Aurora B kinase lead to the accumulation of HsMis13 on kinetochore at the onset of mitosis,assembling an intact HsMis12 complex.Subsequently,the HsMis12 complex bind with the NDC80 complex and KNL-1 protein to form a stable kinetochore-microtubule interacting surface.In addition,the HsMis12 complex recruit the motor protein CENP-E to kinetochore indirectly,facilitating the movement and segregation of chromosome during mitosis.