Molecular Delineation Of Microtubule And Kinetochore Interface

During cell division, chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore, a macromolecular complex located at the centromere of chromosomes. Although previous studies show that mitotic kinesin CENP-E forms a link between attachment of spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying kinetochore-microtubule interactions in mammalian cells remains elusive.Here we identify a novel human kinetochore protein CENP-V that functions synergistically with CENP-E in governing kinetochore-microtubule interactions. CENP-V accumulates in the G2 phase of the cell cycle and associates with kinetochores in early mitosis. CENP-V departs from the kinetochore upon metaphase alignment and is quantitatively degraded at the end of mitosis. Immuno-electron microscopic analysis indicates that CENP-V is a constituent of the kinetochore corona fibers of mammalian centromeres. CENP-V binds to the C-terminal tail of CENP-E in vitro and is essential for a stable kinetochore-microtubule connection in vivo. CENP-V binding to microtubules occurs in vitro, and is synergistically enhanced by CENP-E. Depletion of CENP-V prevents formation of a mechanically stable kinetochore-microtubule interface and causes chromosome mis-segregation with a prolonged delay in metaphase alignment. CENP-V interacts with PLK1 and becomes phosphorylated at the kinetochore. Importantly, PLK1-mediated phosphorylation of CENP-V maintained a stable kinetochore-microtubule attachment essential for faithful chromosome congression. Thus, our studies suggest a novel regulatory mechanism in which CENP-V links kinetochore-spindle attachment to mitotic checkpoint.Mitotic kinase NEK2A interacts with MAD1 and participates in mitotic spindle checkpoint. However, it has remained elusive whether and how NEK2A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates Hec1 and such phosphorylation is essential for faithful chromosome congression in mitosis. Using phospho-specific antibody, our studies show that NEK2A phosphorylates Hec1 at Ser165 during mitosis. Although such phosphorylation is not required for assembly of Hec1 to the kinetochore, expression of non-phosphorylatable mutant Hec1S165 perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. Importantly, the phosphorylation does not modulate Ndc80 complex assembly but increases the affinity of Ndc80 complex for microtubule in vitro. Based on these findings, we reason that NEK2A-mediated phosphorylation of Hec1 governs a stable microtubule-kinetochore attachment essential for chromosome congression in mitosis.