EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.

Geethu E Thomas,Steny Simon,Hindol Gupta,M R Renjith,Binshad Badarudeen,Sabyasachi Sutradhar,K K Gireesh,J Mitra,K Bandopadhyay,Raja Paul,Puja Singh,Tapas K Manna,Manidipa Banerjee

doi:10.1038/ncomms11665

Abstract

Kinetochore couples chromosome movement to dynamic microtubules, a process that is fundamental to mitosis in all eukaryotes but poorly understood. In vertebrates, spindle-kinetochore-associated (Ska1–3) protein complex plays an important role in this process. However, the proteins that stabilize Ska-mediated kinetochore-microtubule attachment remain unknown. Here we show that microtubule plus-end tracking protein EB1 facilitates Ska localization on microtubules in vertebrate cells. EB1 depletion results in a significant reduction of Ska1 recruitment onto microtubules and defects in mitotic chromosome alignment, which is also reflected in computational modelling. Biochemical experiments reveal that EB1 interacts with Ska1, facilitates Ska1-microtubule attachment and together stabilizes microtubules. Structural studies reveal that EB1 either with Ska1 or Ska complex forms extended structures on microtubule lattice. Results indicate that EB1 promotes Ska association with K-fibres and facilitates kinetochore-microtubule attachment. They also implicate that in vertebrates, chromosome coupling to dynamic microtubules could be mediated through EB1-Ska extended structures.

Highlights

Kinetochore couples chromosome movement to dynamic microtubules, a process that is fundamental to mitosis in all eukaryotes but poorly understood
A plethora of KT- and spindle-associated factors involved in KT–MT attachment have been identified in recent years[3,4,5,6,7], the mechanisms by which the KT remains attached to the spindles despite the rapid dynamics of polymerization and depolymerization of MTs and how the KT couples these fast changing structures to chromosome movements remain unclear
Ska complex promotes the formation of curved protofilaments[16], a process that is likely to facilitate further depolymerization of the MT lattice and should destabilize KT–MT attachments

Summary

Introduction

Kinetochore couples chromosome movement to dynamic microtubules, a process that is fundamental to mitosis in all eukaryotes but poorly understood. We report that EB1 functions in chromosome alignment by recruiting Ska[1] to the spindle–KT interface and stabilizing Ska[1] interactions with the MTs. Biochemical analyses indicate that EB1 stimulates Ska[1] recruitment onto MTs by forming complex with Ska[1] and by imparting stabilization onto MTs. High-resolution atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses further reveal the distinct structural identities of EB1–Ska[1] and EB1–Ska complexes on the MTs. The results demonstrate EB1 as a critical regulator of Ska-mediated MT–KT attachment and provide new insights into the molecular details of MT interactions with spindle-KT proteins in vertebrates

Methods

Results

Conclusion