Ubiquitin-mediated events in cell fate decisions

Lead Research Organisation: University of Cambridge
Department Name: Pharmacology

Abstract

The most critical decision that a cell takes when it divides is whether to go ahead and divide again as soon as possible (making more cells), or not to divide for now. Errors in making this decision of cell fate cause cancer and other diseases of cell proliferation.
Our general objective is to understand the molecular regulation of pathways that control the cell division cycle in human cells. These pathways are essential, during and after cell division, to generate daughter cells with correct chromosome numbers, correct cell architecture and correct control of future division decisions. We have developed techniques that allow us to probe critical events that involve modification of cellular components through a process known as ubiquitination. Our pilot study has shown that many ubiquitin-mediated pathways control events immediately following cell division and are likely to influence or determine the decision to divide again.
We propose research that studies ubiquitination of cellular components in cells committed to the two alternative cell fates. This can provide us with a better understanding of cancer. Current cancer treatments that target dividing cells do not distinguish between dividing cancer cells and dividing healthy cells that retain control over their cell fate. Therefore the ability to distinguish dividing cells with different fates would lead to better targeting of future therapies.

Technical Summary

Recent work in the field has demonstrated that cell fate choices are not restricted to G1 phase, since cells exit mitosis already committed to enter a subsequent round of cell division. Our own recent work has identified cellular components that are specifically ubiquitinated during the wave of proteolysis that accompanies exit from mitosis. Many of these components are predicted to be involved in rebuilding the interphase cell, and we hypothesize that ubiquitination of these components contributes to organization of the interphase cell in a way that depends on cell fate. We propose here to identify components that are specifically targeted in cells of differing fate, and to understand 1) how they are targeted 2) how their ubiquitination contributes to interphase 3) how their ubiquitination marks and influences cell fate choices.

Planned Impact

Our research will have beneficiaries within academia and within the commercial sector (biotechnology, pharmaceutical) sectors and will benefit the general public on various timescales.
Academia will benefit through our acquisition and dissemination of new knowledge, through training of young scientists and future scientists in a world-class environment and through the new tools and methodology that we will generate, that will be shared with fellow academics upon request.
The commercial sector will benefit through new knowledge of cellular mechanisms important to disease and to treatment of disease. Moreover, the critical methodologies that we develop and promote in this research (purification of ubiquitin conjugates via a biotin tag, measurement of protein turnover via tandem Fluorescent Protein Timers, tFTs) are highly exploitable for drug discovery in ubiquitin-mediated pathways.
The general public will benefit on different timescales: In the short term, they will benefit from our dissemination and outreach activities aimed to engage a wider audience with our research. In the medium term from the training we provide that will contribute to the global future of science. In the long term, from the discovery and exploitation of new therapeutic pathways in cancer or benign proliferative diseases such as arthritis, psoriasis.
 
Description BBSRC targeted DTP studentship
Amount £85,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Characterizing mitotic phenotype of a novel E3 
Organisation University of Bath
Department Department of Pharmacy and Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution Dr Licchesi's PhD student Natalie Vaughan spent two weeks working in our lab in August 2016, to carry out timelapse fluorescence imaging assays of mitotic cells. We have contributed to the analysis of data and design of ongoing studies to understand mitotic phenotypes. I visited Dr Licchesi's lab in December 2016 to discuss planning of future experiments.
Collaborator Contribution The work of Dr Licchesi's lab is elucidating the activity of a novel ubiquitin ligase in mitosis that will contribute to our understanding of ubiquitin-mediated events in mitosis, and help us in identifying new targets of ubiquitin pathways that might be involved in subsequent cell fate decisions.
Impact This project was presented at an EU COST Proteostasis Network Workshop held in Lisbon in November 2016. Natalie Vaughan gave a 'speed presentation' of her project that included the data from our collaboration. Her accompanying poster won the best poster competition.
Start Year 2016
 
Description Role of deubiquitinases in regulating mitotic targets 
Organisation Free University of Brussels
Country Belgium 
Sector Academic/University 
PI Contribution Providing imaging facilities for cell-based assays of a DUB targeting Aurora kinases
Collaborator Contribution Providing molecular reagents for cell-based assays of a DUB targeting Aurora kinases
Impact Very exciting preliminary data
Start Year 2016
 
Description Testing K11-ubiquitin linkages on Aurora kinases 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology (LMCB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Purifying ubiquitinated Aurora kinases from human cells for testing in DUBs restriction assay for ubiquitin linkage types
Collaborator Contribution DUBs restriction assays on ubiquitinated material conjugated on Aurora kinases from mitotic cells
Impact Publication Min et al. (2015). doi: 10.1091/mbc.E15-02-0102
Start Year 2014
 
Description Ubiquitination of nuclear lamins 
Organisation Technical University of Dresden
Country Germany 
Sector Academic/University 
PI Contribution Identifying lamins as substrates of mitotic ubiquitination, characterizing pathways involved. Providing cell-based tools for this work.
Collaborator Contribution Characterizing APC/C-mediated ubiquitination of Lamin A
Impact manuscript in preparation
Start Year 2017
 
Description Science Festival display 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Cambridge Science Festival 'Highlighting Biology' event designed by my lab and hosted along with colleagues from the Department of Genetics in the Plant and Life Sciences Marquee. Purpose to explain and illustrate the use of GFP in cell biology, with opportunity for hands-on activity. Our display featured in the October 2016 edition of The Biologist magazine of Royal Society of Biology, which had awarded funds for this event. The Plant and Life Sciences Marquee exhibits received a Cambridge BID (Business Improvement District) Award in 2016 for exceptional customer experience.
Year(s) Of Engagement Activity 2016,2017
URL http://www.phar.cam.ac.uk/news/pharmsciencefestival
 
Description Senior Members' Research Forum (SMRS) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Informal seminar to academics from all research fields at Newnham College
Year(s) Of Engagement Activity 2014
 
Description Work experience for Yr 11 and 12 students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Two students, one from London and the other from Kent, spent one week in the lab shadowing lab members and carrying out simple experiments on the microscope.
Both of the students are keen to study science at University. The Year 12 student went on to secure a University place to study Biochemical Engineering, the other will make her UCAS applications later this year.
Year(s) Of Engagement Activity 2016