Activation of human CENP-E determined by cell Förster resonance energy transfer and optical tweezers.

Session

Poster Session Two

Authors

Dr Conor Treacy (1)

Affiliations

1. University of Edinburgh

Keywords

CENP-E, Kinetochore, Microtubules, FRET, Single-Molecule dynamics and Optical Tweezers

Abstract text

Correct and equal distribution of the genetic material in mitosis is essential to ensure genomic stability. Chromosome segregation takes place once all chromosomes have aligned and are bioriented, with attachments to opposite poles. Centromere-associated protein E (CENP-E) is a very large 316 kDa, processive, kinesin-like motor protein that walks along microtubules. During prometaphase, CENP-E is recruited to and attaches to chromosomes at specialised locations known as kinetochores. Once bound to the unattached outer kinetochore, CENP-E can capture microtubules and processively walk towards their plus ends, translocating chromosomes from the spindle poles towards the spindle equator in the process of chromosomal congression. Human CENP-E is thought to exchange between an active state, where it mediates chromosome congression, to an inactive autoinhibited state which allows its dynein-dependent removal from kinetochores. 

This study seeks to understand how CENP-E activity is controlled using a range of in vivo and in vitro techniques. A potential model is that a head-to-tail interaction stabilises CENP-E in a closed conformation, similar to Kinesin-1, allowing its removal and transport away from kinetochores. I am using both in vitro and in vivo approaches to analyse how CENP-E switches from activation to inhibition of state in vivo using microscopy.  

As mechanical force loading-reflecting load bearing of chromosomes- across CENP-E is one of its hypothesised activation mechanisms, we have also developed an optical tweezers-based methodology to determine the pulling force of the N-terminal motor domain of human CENP-E as CENP-E processively walks along the fluorescently labelled microtubules in vitro. This has furthered our understanding of the activation of CENP-E and the role that force-loading plays in successful chromosomal congression.

References

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