Accurate chromosome segregation is critical to ensure genomic integrity during cell division. This process is facilitated by the kinetochore, a multiprotein structure that is assembled on centromeric regions of chromosomes. The kinetochore establishes a mechanical link between the chromosomes and spindle microtubules and modulates cell cycle progression by regulating spindle assembly checkpoint (SAC). Defects in this process result in an aneuploidy, leading to miscarriages, infertility and various genetic disorder such as Down’s syndrome. Although the numerous kinetochore proteins have been identified and studied, the mechanisms that engaged in kinetochore assembly and chromosome segregation are poorly understood. Here we investigated the function of kinetochore protein Zwint-1 on homologous chromosome segregation during oocyte meiotic maturation. We found that Zwint-1 was localized at the kinetochore during meiotic maturation. Knockdown of Zwint-1 caused premature polar body extrusion, indicating acceleration of meiosis I. Interestingly, Zwint-1 knockdown impaired the recruitment of Mad2 at the kinetochores. However, BubR1 localization at the kinetochores was not affected by Zwint-1 knockdown, suggesting that Zwint-1 selectively regulates the recruitment of SAC components into the kinetochores. We also found that Zwint-1 knockdown abrogated chromosome alignment and segregation, thereby resulting in a high incidence of aneuploidy. These chromosomal defects were mostly due to the abnormal kinetochore-microtubule (kMT) attachments. Intriguingly, chromosome misalignment mediated by SAC inactivation was repaired, when anaphase onset was delayed by treating oocytes with proteasome inhibitor MG132. However, surprisingly, chromosomal defects following Zwint-1 knockdown were not restored by delaying anaphase onset. This result suggests that chromosomal defects induced by Zwint-1 knockdown are less likely associated with the failure of SAC activation. In addition, we observed that Aurora B/C kinase activity was not affected by Zwint-1 knockdown. Nevertheless, the meiotic defects induced by Zwint-1 knockdown were similar to those observed in Aurora B/C inhibition, suggesting that Zwint-1 is a downstream effector of Aurora B/C kinase during meiosis. Consistent with this, in Zwint-1 knockdown oocytes chromosomal defects following Aurora B/C inhibition were not restored when Aurora B/C inhibitor was removed, whereas the defects were well rescued in control oocytes after removing Aurora B/C inhibitor. This result suggests that the role of Aurora B/C kinases that correct erroneous kMT attachment is primarily regulated by Zwint-1. Collectively, our results demonstrated for the first time that Zwint-1 is an essential downstream effector of Aurora B/C kinase that corrects erroneous kMT attachment and regulates SAC activity, which ensures accurate homologous chromosome segregation during oocyte meiosis.

난자에서 감수분열이 일어나는 동안 centromere와 microtuble의 부착이 일어난다. 이때 잘못된 부착이 일어나면, 염색체 비분리로 인해, 배아의 염색체 이수성, 착상실패 및 자연유산을 야기시킨다. 그러나, 이러한 중요성에도 불구하고, 난자의 감수분열에서의 동원체와 미세소관의 부착에 대한 연구가 미미한 실정이다. 이에 본 연구에서는 난자의 감수분열동안 염색체와 미세소관 부착의 연결고리 역할을 하는 단백질 Zwint-1에 관하여 분석하였다. Zwint-1은 바깥 표면의 동원체 단백질로써 RZZ복합체(Rod/Zwilch/Zw10), KMN(KNL-1/Mis12/ Ndc80)복합체와 상호작용을 한다고 알려져 있다. 본 연구에서는 미세주입법을 통한 RNAi 방법을 사용하여 난자에서 Zwint-1의 발현을 Knock down 시켰다. 그 후 RT-PCR법을 이용하여 난자내의 Zwint-1의 발현감소를 확인하였으며, 면역형광법을 이용하여 Zwint-1의 위치와 단백질 발현 등을 관찰하였다. 또한 Zwint-1과 상호작용하는 단백질인 Zw10의 위치를 확인하였으며, Spindle assembly checkpoint(SAC)의 활성을 조사하였다. 이를 통해, 난자의 감수분열 동안, Zwint-1이 kinetochore와 microtubule의 부착을 매개함으로써, SAC의 활성을 조절하는 것을 확인하였다. 따라서, 본 연구결과는 Zwint-1이 체세포 분열뿐 아니라, 난자의 감수분열에서도 kinetochore와 microtubule의 부착을 조절하는 중요한 매개인자임을 보여준다.