Signalling during cell division
Jaqui Wood Cancer Centre
University of Dundee
James Arrott Drive
Ninewells Hospital and Medical School
+(44) 01382 383963
Adrian graduated with first class honours from the University of Leeds in 1997 before moving to London to study for a PhD at Kings College in Professor Michael Marber’s laboratory at the Department of Cardiology. Following his PhD, Adrian moved to the Protein Phosphorylation laboratory at CRUK’s London Research Institute (headed by Professor Peter Parker), where he first became fascinated by the process of cell division. He subsequently moved to UMC Utrecht, in The Netherlands, to study the role of protein kinases during mitosis in the lab of Professor Geert Kops. Adrian relocated back to the UK in March 2013 to set up his own lab in Dundee studying the spatial regulation of mitotic signalling networks.
The external website for the Saurin Lab can be viewed at https://www.saurinlab.com/
WHAT WE STUDY
Cell division, or mitosis, is a fantastically dynamic process that can be broken down into a series of distinct phases. In brief, the chromosomes condense and the nuclear envelope breaks down (prophase), the sister chromatids attach to microtubules from opposite spindle poles (prometaphase), key proteins which preserve the mitotic state are degraded (metaphase), the sister chromatids are pulled apart (anaphase), the nuclear envelopes reform (telophase) and the cell is finally segregated into two new daughter cells (cytokinesis). What controls such dramatic rearrangements in the cell and how is this so well coordinated in time and space? These are two fundamental questions that we aim to address in the lab by investigating how the underlying signalling network is spatially regulated.
We are particularly interested in understanding how kinase-phosphatase coupling controls signal responses. The widely-held belief that kinases simply activate signals and phosphatases silence them is somewhat misleading because these enzymes frequently work together to shape the right responses. We showed recently how two major mitotic kinases (Mps1 and Aurora B) couple to two phosphatases (PP1 and PP2A-B56) in a way that allows the mitotic checkpoint signal to switch on and off rapidly (Nijenhuis et al. Nature Cell Biology, 2014). This is an example of a simple binary switch-like response that nevertheless still relies on tight kinase-phosphatase coupling to be optimal. Different signals will require much more complex responses (e.g. graded, pulsatile, dynamic) with specific properties (e.g. ultrasensitivity, robustness) that need to be coordinated with other signals in time and/or space. This range of complexity would simply not be possible without effective integration of kinase and phosphatase activities. Kinase-phosphatase coupling is therefore a fundamental aspect of cell biology and yet, rather surprisingly, it is an area of signalling that we know almost nothing about.
THE METHODS WE USE
We use new cutting-edge techniques to visualise endogenous signalling proteins (genome-editing with AAV and CRISPR/Cas9), quantify their activity in different areas of the cell (localised FRET reporters) and modify their localisation using small molecule compounds (the rapalog dimerization system). We work in mammalian cell lines using specialised live-cell microscopy techniques coupled with an array of biochemical and cell biological approaches to characterise mitotic signalling networks. An example of these techniques can be seen in the movies below.
WHY THIS IS IMPORTANT FOR CANCER RESEARCH
The majority of cancer cells (more than 80%) divide with errors during mitosis. This allows the tumours to evolve because they can constantly shuffle their genetic make-up (by gaining, losing or damaging chromosomes) to find combinations capable of adapting to challenging growth conditions. This Darwinian-style evolution is particularly dangerous because it can enable a subset of tumour cells to survive chemo- or radio-therapy. We are searching for defects in the signalling network that could cause these mitotic errors because this may allow us to either halt the evolutionary process or to use it to selectively target tumour cells (by making the errors worse so the cancer cells can’t survive).
SOME MOVIES TO EXPLAIN ALL OF THIS
Cell division is highly dynamic.
The above movie depicts microtubules and DNA which are dramatically reorganised during cell division.
The underlying signalling network is also dynamic
The localisation of the Cyclin B/Cdk1 – the master regulator of mitosis.
Cyclin B1 is known as the master regulator of mitosis because along with its catalytic subunit, Cdk1, it phosphorylates thousands of proteins to coordinated most aspects of cell division. In the movies above endogenous Cyclin B1 is tagged with EYFP, which shows how Cyclin B/Cdk1 triggers mitotic entry by translocating onto spindle poles and then into the nucleus. Cyclin B can then be visualised on the mitotic spindle, chromosomes and kinetochores (the region of chromosomes that attached to microtubules). As chromosomes become attached to microtubules, Cyclin B is then removed from kinetochores (which causes the dots to progressively disappear). Finally, when chromosome alignment is complete, Cyclin B is degraded and the cell can exit mitosis.
The localised activity of Aurora B kinase.
FRET reporters that change confirmation depending upon phosphorylation can be used to monitor Aurora B kinase activity live. These reporters can be placed in specific areas of the cell to measure localised kinase activity. Aurora B activity on chromatin is depicted above (pseudocoloured to show activity; blue - inactive, red - active). Activity rises on chromatin as cells enter mitosis and then drops as soon as chromosomes are separated (at which time Aurora B moves to the central spindle to control cytokinesis).
Modifying protein localisation using small molecule drugs.
The movie above shows the FKBP-FRB dimerization system that we use in the lab to control protein localisation. Rapamycin (added after 2 mins) causes FKBP-FRB interaction within seconds. This can be used to relocalise proteins to different areas of the cell (centromere translocation is depicted above). This is a powerful approach that we are using to alter the localisation of endogenous kinases and phosphatases.
What goes wrong in cancer cells
The movie above depicts a typical error seen in cancer cells. The chromosomes fail to align fully, which allows the tumour cells to gain (upper cell) or lose (lower cell) chromosomes. This constant shuffling of the chromosomes allow tumours to become genetically diverse. If all individual cells within a tumour are slightly different then they can adapt well to changing conditions – if chemotherapy drugs are applied for example, then a few cells may survive and grow to make the tumour resistant. If we understand what goes wrong with the signalling network to cause these errors then perhaps we can stop them happening (i.e. prevent evolution) or make them slightly worse (and tip tumour cells over the edge).
We are using a combination of techniques to visualise and modify the localisation and activity of different mitotic signalling proteins. This allows us to tackle and number of important questions:
1) How are kinases and phosphatases spatially regulated to control mitosis?
2) How do kinases and phosphatases cross-talk in a way that ensures mitosis is co-ordinated, reliable and robust?
3) Are these properties lost in cancer cells that divide with persistent errors?
4) Can this information be used to treat tumours be preventing or exacerbating the errors during mitosis?
Vallardi G, Cordeiro MH and Saurin AT. A kinase-phosphatase network that regulates kinetochore-microtubule attachments and the SAC. Progress in Molecular and Subcellular Biology. In Press
Saurin AT and Kops GJPL. Studying kinetochore kinases. Methods in Molecular Biology. 2016; 1413:333-47.
Mchedlishvili N, Joank K and Saurin AT. Meeting report – Getting Into and Out of Mitosis. J Cell Sci. 2015; 128(22): 4035-8
Vallardi G and Saurin AT. Mitotic kinases and phosphatases cooperate to shape the right response. Cell Cycle. 2015. 14(6): 795-6
Nijenhuis W, Vallardi G, Teixeira A, Kops GJ, Saurin AT. Negative feedback at kinetochores underlies a responsive spindle checkpoint signal. Nature Cell Biology. 2014. Dec;16(12):1257-64.
Mullers E, Cascales HS, Jaiswal H, Saurin AT, Lindqvist A. Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase. Cell Cycle. 2014 13:17, 2733-2743
Kuijt T, Omerzu M, Saurin AT, and Kops GJPL. Conditional targeting of Mad1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase. Chromosoma. 123(5):471-80.
Shaltiel IA, Aprelia M, Saurin AT, Chowdhury D, Kops GJPL, Voest EE, Medema RH. Distinct phosphatases antagonize the p53 response in different phases of the cell cycle. PNAS. 2014. 20;111(20):7313-8.
Akopyan K, Silva Cascales H, Hukasova E, Saurin AT, Müllers E, Jaiswal H, Hollman DA, Kops GJ, Medema RH, Lindqvist A. Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the s/g2 transition. Molecular Cell. 2014 53(5); 843-853.
*Van der Waal MS, *Saurin AT, Vromans MJ, Vleugel M, Wurzenberger C, Gerlich D Kops GJPL, Lens SMA. Mps1 promotes rapid centromere accumulation of Aurora B. EMBO reports. 2012 Sep;13(9):847-54. *equal contribution
Cameron AJ, Linch MD, Saurin AT, Escribano C, Parker PJ. mTORC2 targets AGC kinases through Sin1-dependent recruitment. Biochem J. 2011 Oct 15;439(2):287-97.
Saurin AT, van der Waal MS, Medema RH, Lens SMA, Kops GJPL. Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis. Nature Communications. 2011 May;.2:316 doi: 10.1038/ncomms1319
Kops GJ, Saurin AT, Meraldi P. Finding the middle ground: how kinetochores power chromosome congression. Cellular and Molecular Life Sciences. 2010 Jul;67(13):2145-61.
Kostelecky B, Saurin AT, Purkiss A, Parker PJ, McDonald NQ. Recognition of an intra-chain tandem 14-3-3 binding site within PKCepsilon. EMBO reports. 2009 Sep;10(9):983-9.
Cameron AJ, Escribano C, Saurin AT, Kostelecky B, Parker PJ. PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity. Nature Structural and Molecular Biology. 2009 Jun;16(6):624-30.
Saurin AT, Durgan J, Cameron AJ, Faisal A, Marber MS, Parker PJ. The regulated assembly of a PKCepsilon complex controls the completion of cytokinesis. Nature Cell Biology. 2008 Aug;10:891-901.
Saurin AT, Brownlow N, Parker PJ. Protein kinase C epsilon in cell division: control of abscission. Cell Cycle. 2009 Feb 15;8(4):549-55.
Saurin, AT, Neubert, H, Brennan, JP, Eaton P. Widespread sulfenic acid formation in tissue in response to hydrogen peroxide. PNAS. 2004. Dec:101(52):17982-87.
Saurin, AT., Pennington, DJ., Raat NJH., Owen MJ., Marber MS. Targeted disruption of the protein kinase C-epsilon gene abolishes the infarct size reduction that follows ischemic preconditioning of isolated buffer-perfused mouse hearts. Cardiovascular Research. 2002. Aug;55(3):672-680.
Saurin, AT., Martin, JL., Heads, RJ., Foley, C., Mockridge, JW., Wright, MJ., Wang, Y., Marber MS. The role of differential activation of p38-Mitogen-activated Protein Kinase in preconditioned ventricular myocytes. FASEB Journal. 2000 Nov;14(14):2237-46.
I am currently full time research with minimal teaching commitments
Magda Camacho Reis, Spatial feedback controls during mitosis, (started 2013).
Giulia Vallardi, Spatial control of kinetochore phosphatases during mitosis (started 2014)
Richard Smith. Understanding phosphatase specificity at kinetochores (started 2016)
Recent invited lectures:
Cancer Research UK Fellow Meeting, York (2016). Understanding how kinases and phosphatases work together to safeguard the genome.
Phosphatases and signalling in health and disease, Bath (2016). Perkin Elmer sponsored lecture: How kinases and phosphatase work together to shape a signal response.
Dynamic kinetochore Workshop, Copenhagen (2015). How mitotic kinases and phosphatases cooperate to shape the right response.
IFOM-IEO, Milan (2015): Mitotic kinases and phosphatases work together to shape the right response.
Department of Genetics, Cambridge (2015): Mitotic kinases and phosphatases work together to shape the right response.
Dynamic kinetochore workshop, Porto (2013): The kinetochore as a regulator of Cyclin B/Cdk1 signalling during mitosis.
MRC Cancer Unit, Cambridge (2012): Localisation-based feedback controls during mitosis and the maintenance of chromosomal stability
Wellcome Trust Centre for Cell Biology, Edinburgh (2011): Feedback controls in mitosis: when kinase, phosphatase and ubiquitin ligase meet.
Department of Biochemistry, Oxford (2011): Feedback controls in mitosis: when kinase, phosphatase and ubiquitin ligase meet.
CRUK London Research Institute, London (2011): Kinase phosphatase and ubiquitin ligase meet to control mitosis.
“A computer game about cancer progression”
Along with a team of software developers at Abertay University we created a strategic game (called Cell Cycle) that allows the player to learn the fundamental concepts of cancer progression in a fun and addictive way. I embarked on this project because I was excited by the prospect of using a novel form of media to target teenagers with important messages: namely that cancer is a progressive disease that can be prevented by making important lifestyle choices. Although most people are aware of these lifestyle choices, many don’t actually know why smoking or UV exposure is so bad for you. That is why revealing this within a game, by allowing players to experience it for themselves, could provide an empowering message to exactly the right age group.
The game depicts dividing cells that occupy a hexagonal grid system, and the aim is to control these dividing cells by using various powers: the players can decide when and where the cells divide, create blood vessels to supply energy to expand the playing grid, move stem cells around the grid to populate new areas, temporarily or permanently arrest cells (quiescence/senescence) or kill them (apoptosis). The cells will occasionally randomly mutate to gain hallmarks of cancer (continued growth, evading apoptosis and replicative immortality) and the aim is to control the spread of these mutant cells. The game becomes more challenging as hallmarks are gained (you can’t kill or arrest them etc.), and levels of difficulty can be chosen based on “risk factors” (e.g. smokers experience more frequent mutations). The players therefore learn exactly why risk factors are bad for you, which is important if they are to feel empowered to make the right lifestyle choices. The game also depicts how mutations are a natural part of cell division/growth that occur well before cancer is ever diagnosed, which explains why making lifestyle changes at this stage is so important.
Public engagement events were used to make a direct connection between project members, student game designers, and the wider public. At all events, attendees were invited to play the game prototypes and engage with games technologies that were used by the student teams to demonstrate serious game design. The primary public engagement events were:
1. 1st International Joint Conference of DiGRA and FDG (August 2016)
2. Dare Indie Fest (August 2016)
3. Perth Museum and Art Gallery Tech Day (September 2016)
4. Digital and Gaming Weekend, Dundee Science Centre (November 2016)
Click on the link below for a trailer of the game