University of Cambridge
Genome-wide microscopy screening identifies links across processes including a conserved connection between DNA damage control and the microtubule cytoskeleton
Abstract
dc:description.abstractPrevious PhD students in the lab created a method for large-scale, high-content microscopy screening of a cell library consisting of over 3000 single mutant strains of the fission yeast, Schizosaccharomyces pombe. Each strain has one nonessential gene knocked-out, allowing investigation of the resulting phenotypes. I report the implementation and completion of this screen; developing methods to ensure reliable and accurate results through inclusion of many controls across multiple screening repeats. In total, over 4.5 million images from approximately 19 000 biologically independent cell populations were imaged and analysed. All strains screened contained GFP-labelled tubulin (GFP-Atb2) allowing visualisation of the microtubule polymer network and its organisation in cells, a feature that is conserved across eukaryotes and simplified in S. pombe, making it easy to study. Examination of cell outlines and microtubule patterns was used to study three cell processes: the shape of cells, the organisational pattern of interphase microtubules and the cell cycle stage of cells, as judged by microtubule pattern. Comparison with extensive data from wild-type cells led to the identification of 262 factors that influence one or more of these cell processes. I go on to biologically validate some of the outcomes from the screen, leading to a publication in Developmental Cell reporting the screen, its findings and the online genomic resource SYSGRO. I then focus on a group of mutants that suggest a connection between the DNA damage response (DDR) and microtubule organisation. From here I show that the DDR induces elongation of microtubule bundles in response to the DDR kinases, ATM and ATR. I begin to reveal factors that may mediate this response and finally, I provide evidence to suggest that the same mechanism is conserved in cultured human cells (Hc3716-hTERT), which may go some way to explaining clinical results showing a beneficial effect of microtubule destabilisation in conjunction with cancer therapies.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2015
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Lawson, Jonathan Luke Done
Subjects
dc:subject × 2Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.16334
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/253007