Understanding how cells trigger mitosis

Lead Research Organisation: University of Cambridge
Department Name: Gurdon Institute

Abstract

We hope to discover exactly how the cell makes the decision to divide into two. This is a crucial decision because when it goes wrong the two daughter cells that are formed can go on to cause cancer, or become resistant to current anti-cancer drugs. In the course of this research we hope to uncover new targets for anti-cancer drugs and to identify ways to make current anti-cancer drugs much more effective.

Technical Summary

We aim to understand what controls the entry to mitosis in human cells. Although many important regulators have been identified it is not known whether we have identified all the necessary factors, nor how they act on each other to make the final decision to enter mitosis. This question is important to the understanding and treatment of cancer because when cells enter mitosis with damaged DNA this can lead to chromosome breakage or loss and consequent aneuploidy, which can clearly contribute to the development of cancer. To provide a definitive answer to this problem we will combine single cell imaging with cell engineering and biochemistry to determine: how Cyclin A promotes mitosis; and how the decision to activate Cyclin B1-Cdk1 is finally made; and how the cell cycle machinery is coordinated in throughout the cell. By mass-spectrometry we have already identified the proteins specifically associated with Cyclin A and Cyclin B1 at each stage of the cell cycle, which has revealed a number of candidates for proteins that could be involved in the control of mitosis and will be pursued. Single cell imaging will provide the necessary temporal resolution to determine when and how cells enter mitosis and we will use FRET probes for CyclinA-Cdk and for Cyclin B1-Cdk1 activity to assay both the exact time when each kinase is turned on, and the kinetics with which it is activated in living cells. In addition to transgenic mouse embryos, recent advances in gene targeting in human cells now allow us to combine the resolution of single cell imaging with biochemical analysis in defined genetic backgrounds. Using gene targeting we will introduce epitope tags and point mutations into specific genes to generate cell lines containing analogue-sensitive kinases, conditionally unstable proteins, and epitope-tagged proteins that will enable us both to assay their level and localisation in living cells and to identify their interaction partners and post-translational modifications by mass spectroscopy. The analogue-sensitive kinases and conditionally unstable proteins will enable us specifically to inactivate a chosen protein at a defined point in the cell cycle, using an ATP-analogues for kinases, and either Cre-mediated excision or an inducible-degron for other proteins, such as phosphatases. With this multi-pronged approach we will be able to identify the molecular mechanisms that control entry to mitosis and how this is coordinated with the unreplicated DNA and damaged DNA checkpoint pathways.
 
Title Gene targeted cell lines 
Description We have used homologous recombination to introduce the coding region for a fluorescent protein into one allele of the genes for PCNA, Aurora A and Plk1. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact We now have cell lines in which we can monitor cell cycle phase by time-lapse video microscopy. We also have cell lines in which we can determine the localisation and behaviour of Aurora A and Plk1. 
 
Description 6th form school visit - March 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact 11 AS level students visited the Institute and Dr Bernhard Strauss talked about his work and that of the Institute.

Well-received by students.
Year(s) Of Engagement Activity 2013
 
Description 6th form school visit April 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact 12 students taking AS level Biology visited the Instiute and Dr Bernhard Strauss gave a seminar to them and introduced them to the work in the Institute.

Well-received by students.
Year(s) Of Engagement Activity 2013
 
Description 6th form visit - February 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Schools
Results and Impact 15 AS level students visited the Institute. Dr Bernhard Strauss talked about his work and the Institute, and the party then had discussions with current PhD students.

Good interaction with students.
Year(s) Of Engagement Activity 2013
 
Description Monograph 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach National
Primary Audience Policymakers/parliamentarians
Results and Impact Monograph published in Public Service Review: UK Science & Technology.

None as yet.
Year(s) Of Engagement Activity 2012
 
Description School student intern 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact A high school student from Parmiter's school in Watford shadowed my post-doctoral researcher, Dr Bernhard Strauss, for one week to gain experience of research.

At the end of her visit she told us that she had been inspired to work towards a career in clinical research at university.
Year(s) Of Engagement Activity 2013
 
Description School visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Schools
Results and Impact 20 Japanese high school students (Yokohama Science Frontier High School) attended the Institute and my post-doctoral researcher, Dr Bernhard Strauss gave a seminar on his work and showed the students the equipment we use.

Very good feedback from students.
Year(s) Of Engagement Activity 2013
 
Description School visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Schools
Results and Impact 1 student from the Netherlands and 1 student from Ecuador visited the Institute and were introduced to some of the research that we do.

Good feedback from students.
Year(s) Of Engagement Activity 2012
 
Description Science Uncovered at Natural History Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Poster Presentation
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Dr Bernhard Strauss, along with several other students and post-docs from the Gurdon Institute, presented their work at the Natural History Museum.

Very enthusiastic audience.
Year(s) Of Engagement Activity 2013
URL http://www.nhm.ac.uk/visit-us/whats-on/after-hours/science-uncovered/
 
Description Undergraduate visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact 8 students from Japan were introduced to our research.

Good feedback from students.
Year(s) Of Engagement Activity 2011