David supports Goliath: Investigating the role of Zwint-1 in kinetochore assembly mediated by KNL1

Abstract

Proper assembly of the kinetochore is essential for correct microtubule attachment and stable chromosome segregation during mitosis. KNL1 is a large scaffolding protein central to formation of the kinetochore which initiates spindle assembly checkpoint signalling to halter anaphase onset until microtubule attachment has been established. Given its important role in ensuring stable chromosome segregation, functioning of KNL1’s recruiters and close interactors Zwint-1 and Mis12 should also be acknowledged. Here, the role of Zwint-1 in the kinetochore is addressed, as previous research hasn’t been able to robustly identify the function of Zwint-1. A Zwint-1 knockout RPE1 cell line is presented, which exhibits increased chromosome segregation defects and a decreased time in mitosis. Immunofluorescence assessment of the kinetochore structure reveals disturbed localization of many kinetochore proteins, namely Bub1 and BubR1, as well as fibrous corona proteins CENP-E and Mad1. Furthermore, interkinetochore distance was increased in the Zwint-1 KO line, and formation of fibrous corona structures is impaired. Expressing Zwint-1 rescue constructs in the knockout line restores both localization of the outer kinetochore proteins and chromosome segregation integrity. These findings illustrate the relevance of Zwint-1 in the kinetochore, and warrant further research in the precise mechanics behind its interaction with KNL1 to stabilize the kinetochore structure.