Molecular Delineation Of Ndc80 Complex Plasticity And Assembly In Mitosis

Loss or gain of whole chromosome,the form of chromosome instability commonly associated with cancers is thought to arise from aberrant chromosome segregation during cell division.Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules.Our recent study shows that Nek2A locates at the kinetochore in mitosis and possibly functions as a novel integrator of the spindle checkpoint signaling.Hecl is highlighted as a key component of Ndc80 complex as well as overexpressional hallmark of colorectal cancer.However,it remains unknown how Nek2A plays a role in kinetochore assembly and what triggers Hecl's overexpression in cancer.Here,we reported that Nek2A-mediated phosphorylation of Hecl modulates the interaction of Hecl with CENP-H,an inner kinetochore component,in vitro and in vivo.We demonstrated that Spc24-Spc25 subcomplex attaches directly to CENP-H's 1-105 aa,while Hec1 binds to CENP-H's 106-148 aa via its C-terminal coiled coil and leucine zip rich region. Nek2A phosphorylates Hecl at Ser¹⁶⁵ during mitosis while such phosphorylation regulates Hec1-CENP-H interaction in vitro and in vivo.Interestingly,phosphory -lation of Ser¹⁶⁵ is not essential for the assembly of Hecl to kinetochore.However, there was a significant increase in syntelic attachments together with lagging chromosomes in Hec1^S165A-overexpressing cells,as well as chromosome bridge phenotype in anaphase.These findings revealed a key role for the Nek2A-mediated phosphor-regulation of Hec1 in microtubule affinity,as we validated by in vitro microtubule cosedimentaion assay.Taken together,we suggested a novel model based on Nek2A-mediated orientational change of Hec1's globular domain,to trigger its interaction with CENP-H and enhance the affinity to microtubules simultaneously,by which Nek2A integrates KT-MT attachments into spindle checkpoint signaling.By SNAP-labeling,a novel approach for real time chasing,we made our latest progression on Ndc80 in vivo.We illustrated two pools of Spc24-Spc25 in nucleoli and cytoplasm portioned for primary targeting and saturating onto kinetochore prior to Hec1-Nuf2,respectively,and elucidated the scheduling assembly of Ndc80 as an optimized integration of kinetochore consolidation with microtubule association.