Studying the kinetochore corona with super-resolution and fast lattice-light sheet imaging

Session

Poster Session Two

Authors

Lydia Daly (2, 1), Dr Sarah McClelland (1), Dr Susan Cox (2)

Affiliations

1. Bart's Cancer Institute, Queen Mary, University of London
2. King’s College London, Randall Centre for Cell and Molecular Biophysics

Keywords

STORM

SMLM

lattice-light sheet

super-resolution

deep-learning

kinetochore

corona

mitosis

Abstract text

During mitosis replicated chromosomes are aligned and separated with high fidelity into daughter cells. Incorrect chromosome segregation leads to aneuploidy, which is common in many cancers. The process is controlled by the sister kinetochores at the centromere of each chromosome pair. The fibrous corona is the outermost layer of the kinetochore; it controls the capture and attachment of spindle microtubule fibres and is the location of some of the spindle assembly checkpoint machinery. Structural information about the corona is currently limited to either diffraction-limited light microscopy or cryo-electron microscopy of recombinant proteins. This project aims to bridge this resolution gap by gathering spatial and temporal data about several corona proteins.

The kinetochore corona is a fibrous structure that forms during prometaphase following nuclear envelope breakdown (NEB). It expands to many times the size of the inner kinetochore then, following chromosome alignment in metaphase, contracts and disappears rapidly. The motor proteins dynein and CENP-E are responsible for controlling chromosome capture, conversion to stable end-on-attachment and congression to the metaphase plate. The Mad1/Mad2 complex is located in the corona and is a core component of the checkpoint which prevents entry into anaphase before correct chromosome alignment. Here we use super-resolution microscopy techniques to gather fixed and live-cell information about these proteins over the expansion, microtubule capture, and contraction phases of the corona.

In this project we are using two methods of super-resolution microscopy, lattice light-sheet live-cell imaging and STORM super-resolution immunofluorescence imaging, to investigate the changes that corona proteins undergo during prometaphase and metaphase of mitosis. The light-sheet microscopy allows live imaging of the locations of corona proteins and how they move as the corona expands and contracts, including transport off the kinetochore via the dynein motor. STORM microscopy provides an improvement in resolution by a factor of ten to standard confocal imaging and allows investigation at a length scale of tens of nanometres of the positions of the corona proteins relative to one another. By integrating the two types of data a nanoscale model of corona dynamics can be created.

High-resolution images of some corona proteins have suggested there are distinct domains of proteins throughout the corona. Simulated data enables construction of a workflow to analyse the relative positions of these domains and predict similarities or differences between different proteins. Live-cell imaging has successfully captured the location of corona proteins on kinetochores during mitosis. The transport of these proteins away from the chromosomes on microtubule fibres following metaphase alignment has been imaged. Automating the detection, intensity analysis and tracking of these protein foci allows us to build up an aggregate dataset of foci properties.

The kinetochore corona is a nanoscale structure, dynamic on a second timescale. This makes it challenging to image, requiring a multimodal approach to glean information at short length- and timescales. Such approaches to imaging and data synthesis are likely to find applications in many other areas of cell biology.