Human NUF2 Interacts with Centromere-associated Protein E and Is Essential for a Stable Spindle Microtubule-Kinetochore Attachment

Abstract

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here, we show that Homo sapiens (Hs) NUF2 is required for stable kinetochore localization of centromere-associated protein E (CENP-E) in HeLa cells. HsNUF2 specifies the kinetochore association of CENP-E by interacting with its C-terminal domain. The region of HsNUF2 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pulldown and yeast two-hybrid assays. Suppression of synthesis of HsNUF2 by small interfering RNA abrogated the localization of CENP-E to the kinetochore, demonstrating the requirement of HsNUF2 for CENP-E kinetochore localization. In addition, depletion of HsNUF2 caused aberrant chromosome segregation. These HsNUF2-suppressed cells displayed reduced tension at kinetochores of bi-orientated chromosomes. Double knockdown of CENP-E and HsNUF2 further abolished the tension at the kinetochores. Our results indicate that HsNUF2 and CENP-E are required for organization of stable microtubule-kinetochore attachment that is essential for faithful chromosome segregation in mitosis.

The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability

“…Hec1 was initially identified as an Rb-binding protein and subsequently demonstrated as an evolutionarily conserved kinetochore protein (Durfee et al, 1993). Our recent study revealed that Ndc80 complex provides a link between mitotic motor CENP-E and kinetochore (Liu et al, 2007).…”

Silencing of NUF2 Inhibits Tumor Growth and Induces Apoptosis in Human Hepatocellular Carcinomas

“…In particular, NUF2 was reported to stabilize microtubule attachment as part of a linker between the kinetochore and tubulin subunits of the spindle, and depletion of NUF2 could induce kinetochore attachment defects and spindle checkpoint activation, and finally deaths of mitotic cells (DeLuca et al, 2002). Further studies revealed that NUF2 binds to CENPE and is required for stabilizing CENPE in the kinetochore, and both proteins are essential for stable kinetochores and proper chromosome segregation during mitosis (Liu et al, 2007). As a tight subcomplex, NUF2-HEC1 was essential for organizing microtubule attachment sites, and recently more details concerning NUF2-HEC1 heterodimerization and their distinct contributions to kinetochore-microtubule attachment were studied (Ciferri et al, 2005;DeLuca et al, 2005).…”

Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

“…10,17,26,29 Parallel to mitosis, a surveillance pathway is needed to safeguard fidelity of chromosome separation in oocyte meiosis. The present study clearly demonstrated that Nuf2-RNAi caused misalignment of chromosomes and unattached microtubules, which may indicate failure of microtubule attachments to chromosomes.…”

“…13,25 Another RNAi study in HeLa cells demonstrated the necessity of HsNuf2 in stabilizing microtubule-kinetochore attachments, maintaining tension, and silencing the spindle checkpoint. 26 In addition, perturbation of Ndc80 and Spc25 in mouse oocytes disrupted chromosome alignment and proper spindle checkpoint signaling. 27,28 Therefore, we hypothesized that Nuf2 may be a critical player in the regulation of kinetochore-microtubule attachment and spindle assembly checkpoint function in female germ cell meiosis.…”