Portrait of Dr Campbell Gourlay

Dr Campbell Gourlay

Reader in Cell Biology
Director of Graduate Studies

About

Dr. Campbell Gourlay is a Reader in the School of Biosciences and the current Director of Graduate Studies. He began his career at The John Innes Centre in 1996 where he completed a PhD in plant development, studying the genetic control of leaf development. Following this he began to work with budding yeast as a model eukaryote in the lab of Prof. Kathryn Ayscough, where he investigated the role of actin in the process of endocytosis. During this time he discovered a link between actin, the regulation of mitochondrial function and the control of ageing and apoptosis. This led to his involvement in the emerging field of yeast apoptosis, which has popularised the novel concept that unicellular organisms possess the ability to undergo programmed cell death as an altruistic act for the betterment of a population. He established his own research group with the aid of Wellcome Trust Value in People Award and a Medical Research Council Career Development Fellowship in 2006. He is a founding member of the Kent Fungal Group (KFG) which represents one of the largest collections of yeast research groups in the UK (http://www.kentfungalgroup.com/)

ORCID ID: 0000-0002-2373-6788

Research interests

Recent and current funding from the BBSRC, EPSRC, Wellcome Trust, FWF (Austrian Science Foundation), Kent Cancer Trust, Scottish MND Society and the Luxembourg National Research Fund (FNR) supports a range of projects. The group uses yeast, human cell culture and whole animal models within a range of research activities. Current research projects include: 

  1. The regulation of mitochondrial health and production of reactive oxygen species 
  2. The role of translational accuracy in healthy ageing and apoptosis 
  3. Roles for the actin cytoskeleton in regulating stress response mechanisms 
  4. Yeast as a model for motor neuron disease 
  5. Using yeast to understand the development of multi-drug resistance 
  6. The role of mitochondrial function in the pathogenicity of Candida albicans
  7. The detection and management of biofilms on airway management devices 

Dr. Gourlay has established solid foundations within the East Kent Hospital University Foundation Trust (EKHUFT) and works within a multi-disciplinary team to tackle fungal colonisation of medical devices, including voice prostheses and tracheostomy tubing. These studies have led to the implementation of fully ratified NHS treatment guidelines for the management of fungal growth on voice prosthesis and have been adopted throughout the UK as well as being implemented overseas. 

Teaching

Undergraduate

  • BI324 – Genetics and Evolution (Stage 1) 
  • BI501 - Gene Expression (Stage 2) 
  • BI503 – Cell Biology (Stage 2) 
  • BI601 – Cell Biology (Stage 3) 
  • BI639 – Frontiers in Cancer Research (Stage 3) 
  • BI600 – Final Year Project (Stage 3) 

Postgraduate

  • BI854 - Fungi as Human Pathogens (MSc in Infectious Disease)

Supervision

MSc-R projects available for 2020/21

The roles of RAS in controlling cell fate – a yeast model of oncogenic potential RAS proteins are small GTPases that couple cell signals to fate. Mutations in Ras that cause a loss of its regulation are found in around 30% of all human cancers. The role of RAS as an oncogene can be attributed to it being a master regulator of proliferation and viability, however the processes by which RAS controls cell fate are not fully understood. In this MSc project you will investigate the consequences of altering RAS activity in a yeast model system to help understand its oncogenic potential. The project will involve the use of a number of techniques such as advanced live cell imaging techniques, gene editing technology, flow cytometry and cell culture.

Additional research costs: £1500 

Investigating the role of mitochondria in regulating cell health and ageing 
Yeast can be used as an effective cell based system to study the basis of ageing. This project will investigate how mitochondria are used as sensors of metabolic fitness in cells and how this fascinating organelle controls cell fate during the ageing process. The project will build on our recent and published data that place mitochondria at the heart of processes such as autophagy, lipid metabolism and reactive oxygen species production, all of which are essential for cell health and survival. 

Additional research costs: £1500  

Publications

Showing 50 of 53 total publications in the Kent Academic Repository. View all publications.

Article

  • Makarovaite, V., Hillier, A., Holder, S., Gourlay, C. and Batchelor, J. (2020). Passive UHF RFID Voice Prosthesis Mounted Sensor for Microbial Growth Detection. IEEE Journal of Radio Frequency Identification [Online]:1-6. Available at: https://doi.org/10.1109/JRFID.2020.3011900.
    Capacitive loading due to human tissue can lead to low efficiency for implantable Passive Radio Frequency Identification (RFID) antennas. The presented passive UHF antenna sensor provides read distances above 0.5 meters (within a body phantom) by utilizing a convoluted half-wave dipole design. It is able to detect simulated early to mature Candida albicans biofilm growth when mounted upon a voice prosthesis (up to a 30 μm biofilm thickness). Depending on the propagation frequency of interest, as early 4-hour growth (5 to 10 μm biofilm thickness) equivalent could be detected and before any device failure could occur due to the colonization. This was accomplished by utilising thin layers of polyurethane to decouple the saliva from the presented UHF sensor (biofilm growth is known to increase layer hydrophobicity). This presented sensor has better functionality within the US UHF frequency band as it detects changes above 5 μm. If there is a need for implantation within additional tissues with variable dielectric properties, a shunt capacitance of 2.6 pF could allow the system functionality within the permittivity range of 21 to 58. Allowing for immediate medical intervention before medical prosthesis failure.
  • Makarovaite, V., Hillier, A., Holder, S., Gourlay, C. and Batchelor, J. (2020). Passive UHF RFID Voice Prosthesis Mounted Sensor for Microbial Growth Detection. IEEE Journal of Radio Frequency Identification [Online]. Available at: http://dx.doi.org/10.1109/JRFID.2020.3011900.
    Capacitive loading due to human tissue can lead to low efficiency for implantable Passive Radio Frequency Identification (RFID) antennas. The presented passive UHF antenna sensor provides read distances above 0.5 meters (within a body phantom) by utilizing a convoluted half-wave dipole design. It is able to detect simulated early to mature Candida albicans biofilm growth when mounted upon a voice prosthesis (up to a 30 μm biofilm thickness). Depending on the propagation frequency of interest, as early 4-hour growth (5 to 10 μm biofilm thickness) equivalent could be detected and before any device failure could occur due to the colonization. This was accomplished by utilising thin layers of polyurethane to decouple the saliva from the presented UHF sensor (biofilm growth is known to increase layer hydrophobicity). This presented sensor has better functionality within the US UHF frequency band as it detects changes above 5 μm. If there is a need for implantation within additional tissues with variable dielectric properties, a shunt capacitance of 2.6 pF could allow the system functionality within the permittivity range of 21 to 58. Allowing for immediate medical intervention before medical prosthesis failure
  • Pentland, D., Stevens, S., Williams, L., Baker, M., McCall, C., Makarovaite, V., Balfour, A., Mühlschlegel, F. and Gourlay, C. (2020). Precision Antifungal Treatment Significantly Extends Voice Prosthesis Lifespan in Patients Following Total Laryngectomy. Frontiers in Microbiology [Online]. Available at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.00975/full?utm_source=F-NTF&utm_medium=EMLX&utm_campaign=PRD_FEOPS_20170000_ARTICLE.
    Indwelling silicone valves called voice prostheses (VPs) are the gold standard for speech rehabilitation in patients with laryngeal cancer following total laryngectomy. Reported VP lifespans amongst these patients are highly variable but when devices fail patients experience loss of voice and an increase risk of chest infection. Early failure of VP is a current clinical concern that is associated with regular hospital visits, reduced quality of life and associated medical cost. Poly-microbial biofilms comprised of both bacterial and fungal microorganisms readily colonize VPs and are linked to loss of device performance and its early failure in addition to providing a reservoir for potential infection. Our detailed analysis of poly-microbial biofilm composition on 159 early failing VPs from 48 total laryngectomy patients confirmed Candida albicans as the predominant fungal species and Staphylococcus aureus as the most common bacterial colonizer within our patient cohort. Using a combination of microbiological analysis, patient data and a high-throughput antifungal test assay mimicking in vivo conditions we established an evidence based precision antifungal treatment approach to VP management. Our approach has allowed us to implement a personalized VP management pathway, which increases device in situ lifespan by an average of 270%. Our study represents a significant step forward in both our understanding of the cause of VP failure and a new effective treatment pathway that offers tangible benefit to patients.
  • Duvenage, L., Munro, C. and Gourlay, C. (2019). High Resolution Respirometry in Candida albicans. Bio-protocol [Online] 9. Available at: https://doi.org/10.21769/BioProtoc.3361.
    Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterisation of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the “mode” of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance.
  • Hillier, A., Makarovaite, V., Gourlay, C., Holder, S. and Batchelor, J. (2019). A Passive UHF RFID Dielectric Sensor for Aqueous Electrolytes. IEEE Sensors Journal [Online] 19:5389-5395. Available at: http://dx.doi.org/10.1109/JSEN.2019.2909353.
    The one step modification of a commercial RFID sensing tag is demonstrated using polydimethylsiloxane based thin film chemistry to construct reusable passive RFID sensors for changes in the dielectric properties of electrolyte solutions as a function of concentration. The effects of PDMS film thickness were characterized as a function of RFID sensor code value. The output sensor code of the RFMicron RFM2100-AER wireless flexible moisture sensor (taken between 800-860 MHz) was compared to readings taken when the tag was dry and when the tag had a water deposition on the sensor area. The effect of the direct application of liquid water to the tag was to alter the capacitance presented to the integrated chip which auto-tunes to correct for the reactance. By varying the thickness of the PDMS film between the interdigitated sensor and deposited liquid, the sensitivity of the tag to a high dielectric medium could be controlled. Aqueous salt solutions were tested on a 500 m thickness film. It was found that the sensing platform could be used as a means of measuring the concentration of various salt solutions within the range 0-2M, and in turn could be used as a passive UHF RFID dielectric measuring tool. The measurement capability of the platform was subsequently demonstrated using a reduced frequency range (845-865 MHz).
  • Duvenage, L., Munro, C. and Gourlay, C. (2019). The potential of respiration inhibition as a new approach to combat human fungal pathogens. Current Genetics [Online]. Available at: https://doi.org/10.1007/s00294-019-01001-w.
    The respiratory chain has been proposed as an attractive target for the development of new therapies to tackle human fungal pathogens. This arises from the presence of fungal-specific electron transport chain components and links between respiration and the control of virulence traits in several pathogenic species. However, as the physiological roles of mitochondria remain largely undetermined with respect to pathogenesis, its value as a potential new drug target remains to be determined. The use of respiration inhibitors as fungicides is well developed but has been hampered by the emergence of rapid resistance to current inhibitors. In addition, recent data suggest that adaptation of the human fungal pathogen, Candida albicans, to respiration inhibitors can enhance virulence traits such as yeast-to-hypha transition and cell wall organisation. We conclude that although respiration holds promise as a target for the development of new therapies to treat human fungal infections, we require a more detailed understanding of the role that mitochondria play in stress adaption and virulence.
  • Makarovaite, V., Hillier, A., Holder, S., Gourlay, C. and Batchelor, J. (2019). Passive Wireless UHF RFID Antenna Label for Sensing Dielectric Properties of Aqueous and Organic Liquids. IEEE Sensors Journal [Online]. Available at: http://dx.doi.org/10.1109/JSEN.2019.2896481.
    The in situ wireless sensing of dielectric properties for organic aqueous solutions with a wide range of relative permittivities is presented. The use of a UHF passive label antenna design attached to either clear borosilicate glass bottle or petri plate is proposed and which allows for the unobtrusive, safe monitoring of the liquid solutions. The meandered dipole antenna (with a parasitic loop matching component) frequency is highly reliant on the chosen container as well as on the liquid present within, and adjusts with shifting dielectric properties. Tested solutions of high relative permittivity (such as water) along with low permittivity, lossy liquids (such as xylene) presented distinctive frequency characteristics with read distances of up to 7 meters for each type of container tested. The sensor was also able to detect ‘unknown’ solutions and determine the dielectric properties by utilizing standard curve analysis with an accuracy of ± 0.834 relative permittivity and ± 0.050 S·m-1 conductivity (compared to a standard dielectric measurement system available on the market). With the accuracy known, tuning the design to fit any necessary frequency is possible as a means to detect specific changes in any one liquid system. This sensor is a possible candidate for discreet real-time monitoring of liquid storage containers and an alternative for low-cost bulk liquid dielectric property identification which could be implemented in areas requiring, constant, or remote monitoring as needed.
  • Duvenage, L., Walker, L., Bojarczuk, A., Johnston, S., Maccallum, D., Munro, C. and Gourlay, C. (2019). Inhibition of classical and alternative modes of respiration in Candida albicans leads to cell wall remodelling and increased macrophage recognition. mBio [Online]. Available at: http://dx.doi.org/10.1128/mBio.02535-18.
    The human fungal pathogen Candida albicans requires respiratory function for normal growth, morphogenesis and virulence. Mitochondria therefore represent an enticing target for the development of new antifungal strategies. This possibility is bolstered by the presence of characteristics specific to fungi. However, respiration in C. albicans, as in many fungal organisms, is facilitated by redundant electron transport mechanisms, making direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we make use of chemicals with low toxicity to efficiently inhibit respiration in C. albicans. We find that use of the Nitric Oxide donor, Sodium Nitroprusside (SNP), and the alternative oxidase inhibitor, SHAM, prevents respiration, leads to a loss of viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo. We propose that SNP+SHAM treatment leads to transcriptional changes that drive cell wall re-arrangement but which also prime cells to activate transition to hyphal growth. In line with this we find that pre-treatment of C. albicans with SNP+SHAM leads to an increase in virulence. Our data reveals strong links between respiration, cell wall remodelling and activation of virulence factors. Our findings demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals, which are not damaging to the mammalian host, but that we need to develop a deeper understanding of the roles of mitochondria in cellular signalling if they are to be developed successfully as a target for new antifungals.
  • Tsaousis, A., Hamblin, K., Elliot, C., Gourlay, C., Moore, A. and van der Geizen, M. (2018). The human gut colonizer Blastocystis respires using Complex II and alternative oxidase to buffer transient oxygen fluctuations in the gut. Frontiers in Cellular and Infection Microbiology [Online]. Available at: http://dx.doi.org/10.3389/fcimb.2018.00371/full.
    Blastocystis is the most common eukaryotic microbe in the human gut. It is linked to irritable bowel syndrome (IBS), but its role in disease has been contested considering its widespread nature. This organism is well adapted to its anoxic niche and lacks typical eukaryotic features such as a cytochrome-driven mitochondrial electron transport. Although generally considered a strict or obligate anaerobe, its genome encodes an alternative oxidase. Alternative oxidases are energetically wasteful enzymes as they are non-protonmotive and energy is liberated in heat, but they are considered to be involved in oxidative stress protective mechanisms. Our results demonstrate that the Blastocystis cells themselves respire oxygen via this alternative oxidase thereby casting doubt on its strict anaerobic nature. Inhibition experiments using alternative oxidase and Complex II specific inhibitors clearly demonstrate their role in cellular respiration. We postulate that the alternative oxidase in Blastocystis is used to buffer transient oxygen fluctuations in the gut and that it likely is a common colonizer of the human gut and not causally involved in IBS. Additionally the alternative oxidase could act as a protective mechanism in a dysbiotic gut and thereby explain the absence of Blastocystis in established IBS environments.
  • Rockenfeller, P. and Gourlay, C. (2018). Lipotoxicty in yeast: a focus on plasma membrane signalling and membrane contact sites. FEMS Yeast Research [Online] 18. Available at: https://doi.org/10.1093/femsyr/foy034.
    Lipotoxicity is a pathophysiological process triggered by lipid overload. In metazoans, lipotoxicity is characterised by the ectopic deposition of lipids on organs other than adipose tissue. This leads to organ dysfunction, cell death, and is intimately linked to lipid-associated diseases such as cardiac dysfunction, atherosclerosis, stroke, hepatosteatosis, cancer and the metabolic syndrome. The molecules involved in eliciting lipotoxicity include FAs and their acyl-CoA derivatives, triacylglycerol (TG), diacylglycerol (DG), ceramides, acyl-carnitines and phospholipids. However, the cellular transport of toxic lipids through membrane contact sites (MCS) and vesicular mechanisms as well as lipid metabolism that progress lipotoxicity to the onset of disease are not entirely understood. Yeast has proven a useful model organism to study the molecular mechanisms of lipotoxicity. Recently, the Rim101 pathway, which senses alkaline pH and the lipid status at the plasmamembrane, has been connected to lipotoxicity. In this review article, we summarise recent research advances on the Rim101 pathway and MCS in the context of lipotoxicity in yeast and present a perspective for future research directions.
  • Guaragnella, N., Stirpe, M., Burhans, W., Côrte-Real, M., Gourlay, C., Ludovico, P., Madeo, F., Petranovic, D., Winderickx, J., Mazzoni, C. and Giannattasio, S. (2018). New perspectives from South-Y-East, not all about death. A report of the 12lnternational Meeting on Yeast Apoptosis in Bari, Italy, May 14th-18th, 2017. Microbial cell [Online] 5:112-115. Available at: http://dx.doi.org/10.15698/mic2018.02.616.
  • Carmona-Gutierrez, D., Bauer, M., Zimmermann, A., Aguilera, A., Austriaco, N., Ayscough, K., Balzan, R., Bar-Nun, S., Barrientos, A., Belenky, P., Blondel, M., Braun, R., Breitenbach, M., Burhans, W., Büttner, S., Cavalieri, D., Chang, M., Cooper, K., Côrte-Real, M., Costa, V., Cullin, C., Dawes, I., Dengjel, J., Dickman, M., Eisenberg, T., Fahrenkrog, B., Fasel, N., Fröhlich, K., Gargouri, A., Giannattasio, S., Goffrini, P., Gourlay, C., Grant, C., Greenwood, M., Guaragnella, N., Heger, T., Heinisch, J., Herker, E., Herrmann, J., Hofer, S., Jiménez-Ruiz, A., Jungwirth, H., Kainz, K., Kontoyiannis, D., Ludovico, P., Manon, S., Martegani, E., Mazzoni, C., Megeney, L., Meisinger, C., Nielsen, J., Nyström, T., Osiewacz, H., Outeiro, T., Park, H., Pendl, T., Petranovic, D., Picot, S., Pol?ic, P., Powers, T., Ramsdale, M., Rinnerthaler, M., Rockenfeller, P., Ruckenstuhl, C., Schaffrath, R., Segovia, M., Severin, F., Sharon, A., Sigrist, S., Sommer-Ruck, C., Sousa, M., Thevelein, J., Thevissen, K., Titorenko, V., Toledano, M., Tuite, M., Vögtle, F., Westermann, B., Winderickx, J., Wissing, S., Wölfl, S., Zhang, Z., Zhao, R., Zhou, B., Galluzzi, L., Kroemer, G. and Madeo, F. (2018). Guidelines and recommendations on yeast cell death nomenclature. Microbial Cell [Online] 5:4-31. Available at: http://dx.doi.org/10.15698/mic2018.01.607.
    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.
  • Pentland, D., Piper-Brown, E., Mühlschlegel, F. and Gourlay, C. (2017). Ras signalling in pathogenic yeasts. Microbial Cell [Online] 5:63-73. Available at: http://dx.doi.org/10.15698/mic2018.02.612.
    The small GTPase Ras acts as a master regulator of growth, stress response and cell death in eukaryotic cells. The control of Ras activity is fundamental, as highlighted by the oncogenic properties of constitutive forms of Ras proteins. Ras also plays a crucial role in the pathogenicity of fungal pathogens where it has been found to regulate a number of adaptions required for virulence. The importance of Ras in fungal disease raises the possibility that it may provide a useful target for the development of new treatments at a time when resistance to available antifungals is increasing. New findings suggest that important regulatory sequences found within fungal Ras proteins that are not conserved may prove useful in the development of new antifungals. Here we review the roles of Ras protein function and signalling in the major human yeast pathogensandand discuss the potential for targeting Ras as a novel approach to anti-fungal therapy.
  • Rockenfeller, P., Smolnig, M., Diessl, J., Bashir, M., Schmiedhofer, V., Knittelfelder, O., Ring, J., Franz, J., Foessl, I., Khan, M., Rost, R., Graier, W., Kroemer, G., Zimmermann, A., Carmona- Gutierrez, D., Eisenberg, T., Büttner, S., Sigrist, S., Kühnlein, R., Kohlwein, S., Gourlay, C. and Madeo, F. (2017). Diacylglycerol triggers Rim101 pathway dependent necrosis in yeast: a model for lipotoxicity. Cell Death and Differentiation [Online]. Available at: http://dx.doi.org/10.1038/s41418-017-0014-2.
    The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21 dependent sensing complex and activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity i.e. high fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.
  • Saintas, E., Abrahams, L., Ahmed, G., Ajakaiye, A., AlHumaidi, A., Ashmore-Harris, C., Clark, I., Dura, U., Fixmer, C., Ike-Morris, C., Mato Prado, M., Mccullough, D., Mishra, S., Schoeler, K., Timur, H., Williamson, M., Alatsatianos, M., Bahsoun, B., Blackburn, E., Hogwood, C., Lithgow, P., Rowe, M., Yiangou, L., Rothweiler, F., Cinatl jr, J., Zehner, R., Baines, A., Garrett, M., Gourlay, C., Griffin, D., Gullick, W., Hargreaves, E., Howard, M., Lloyd, D., Rossman, J., Smales, C., Tsaousis, A., von der Haar, T., Wass, M. and Michaelis, M. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE [Online] 12:e0172140. Available at: http://dx.doi.org/10.1371/journal.pone.0172140.
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • von der Haar, T., Leadsham, J., Sauvadet, A., Tarrant, D., Adam, I., Saromi, K., Laun, P., Rinnerthaler, M., Breitenbach-Koller, H., Breitenbach, M., Tuite, M. and Gourlay, C. (2017). The control of translational accuracy is a determinant of healthy ageing in yeast. Open Biology [Online] 7:160291. Available at: http://dx.doi.org/10.1098/rsob.160291.
    Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.
  • Tarrant, D., Stirpe, M., Rowe, M., Howard, M., von der Haar, T. and Gourlay, C. (2016). Inappropriate expression of the translation elongation factor 1A disrupts genome stability and metabolism. Journal of Cell Science [Online] 129:4455-4465. Available at: http://doi.org/10.1242/jcs.192831.
    The translation elongation factor eEF1A is one of the most abundant proteins found within cells and its role within protein synthesis is well documented. Levels of eEF1A are tightly controlled, with inappropriate expression linked to oncogenesis. However the mechanisms by which increased eEF1A expression alter cell behaviour are unknown. Our analyses in yeast suggest that elevation of eEF1A levels lead to stabilisation of the spindle pole body and changes in nuclear organisation. Elevation of eEF1A2 also leads to altered nuclear morphology in cultured human cells suggesting a conserved role in maintaining genome stability. Gene expression and metabolomic analyses reveal that the level of eEF1A is crucial for the maintenance of metabolism and amino acid levels in yeast, most likely via its role in the control of vacuole function. Increased eEF1A2 levels trigger lysosome biogenesis in cultured human cells, also suggesting a conserved role within metabolic control mechanisms. Together our data suggest that the control of eEF1A levels is important for the maintenance of a number of cell functions out-with translation, whose de-regulation may contribute to its oncogenic properties.
  • Bastow, E., Peswani, A., Tarrant, D., Pentland, D., Chen, X., Morgan, A., Staniforth, G., Tullet, J., Rowe, M., Howard, M., Tuite, M. and Gourlay, C. (2016). New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis. Journal of cell science [Online] 129:4118-4129. Available at: http://dx.doi.org/10.1242/jcs.190298.
    A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a Caenorhabditis elegans model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.
  • Jastrzebska, Z., Kaminska, J., Chelstowska, A., Domanska, A., Rzepnikowska, W., Sitkiewicz, E., Cholbinski, P., Gourlay, C., Plochocka, D. and Zoladek, T. (2015). Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization. European Journal of Cell Biology [Online] 94:576-588. Available at: http://doi.org/10.1016/j.ejcb.2015.10.005.
    Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1? tpk3? mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28 °C and did not grow at 37 °C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.
  • Talpaert, M., Balfour, A., Stevens, S., Baker, M., Mühlschlegel, F. and Gourlay, C. (2015). Candida Biofilm Formation on Voice Prostheses. Journal of medical microbiology [Online] 64:199-208. Available at: http://dx.doi.org/10.1099/jmm.0.078717-0.
    Laryngopharyngeal malignancy is treated with radiotherapy and/or surgery. When total laryngectomy is required major laryngeal functions (phonation, airway control, swallowing, and coughing) are affected. The insertion of a silicone rubber voice prosthesis in a surgically created tracheoesophageal puncture is the most effective method for voice rehabilitation. Silicone has the propensity to become rapidly colonised by microorganisms (mainly Candida albicans) forming a biofilm which leads to the failure of the devices. Valve failure, as well as compromising speech, may result in aspiration pneumonia, and repeated valve replacement may lead to either tract stenosis or insufficiency. Prevention and control of biofilm formation is therefore crucial for the lifespan of the prosthesis and promotion of tracheosophageal tissue and lung health. To date the mechanisms of biofilm formation on voice prostheses are not fully understood. Further studies are therefore required to identify factors influencing Candida biofilm formation. This review describes the factors known to influence biofilm formation on voice prosthesis and current strategies employed to prolong their life by interfering with microbial colonisation.
  • Ayer, A., Gourlay, C. and Dawes, I. (2014). Cellular redox homeostasis, reactive oxygen species and replicative ageing inSaccharomyces cerevisiae. FEMS Yeast Research [Online] 14:60-72. Available at: http://doi.org/10.1111/1567-1364.12114.
    Ageing cells undergo changes in redox homeostasis and acquire high levels of reactive oxygen species (ROS). Because accumulation of ROS involves a change in redox state of cells, functions that are involved in setting redox and maintaining redox homeostasis are very relevant to an understanding of the possible roles of redox homeostasis and ROS in ageing. This review discusses these aspects of ROS in relation to replicative ageing in the model organism Saccharomyces cerevisiae, with reference to ROS generated in cells; cellular responses to oxidative stress; and how cells maintain redox homeostasis in different cellular compartments. It also considers when ROS generation begins as cells age, which ROS species are relevant to ageing and which cellular compartments and processes may contribute ROS to the ageing process. The discussion also covers the heterogeneity of cells with respect to ROS accumulation at particular cell ages, and the possibility of testing the oxidative theory of ageing in yeast cells.
  • Smethurst, D., Dawes, I. and Gourlay, C. (2014). Actin - a biosensor that determines cell fate in yeasts. FEMS Yeast Research [Online] 14:89-95. Available at: http://doi.org/10.1111/1567-1364.12119.
    The decision to proliferate, to activate stress response mechanisms or to initiate cell death lies at the heart of the maintenance of a healthy cell population. Within multicellular and colony-forming single-celled organisms, such as yeasts, the functionality of cellular compartments that connect signalling to cell fate must be maintained to maximise adaptability and survival. The actin cytoskeleton is involved in processes such as the regulation of membrane microcompartments, receptor internalisation and the control of master regulatory GTPases, which govern cell decision-making. This affords the actin cytoskeleton a central position within cell response networks. In this sense, a functional actin cytoskeleton is essential to efficiently connect information input to response at the level of the cell. Recent research from fungal, plant and mammalian cells systems has highlighted that actin can trigger apoptotic death in cells that become incompetent to respond to environmental cues. It may also be the case that this property has been appropriated by microorganisms competing for niche environments within a human host. Here, we discuss the research that has been carried out in yeast that links actin to signalling processes and cell fate that supports its role as a biosensor.
  • Leadsham, J., Sanders, G., Giannaki, S., Bastow, E., Hutton, R., Naemi, W., Breitenbach, M. and Gourlay, C. (2013). Loss of Cytochrome c Oxidase Promotes RAS-Dependent ROS Production from the ER Resident NADPH Oxidase, Yno1p, in Yeast. Cell Metabolism [Online] 18:279-286. Available at: http://dx.doi.org/10.1016/j.cmet.2013.07.005.
    Many disease states, including the aging process, are associated with the accumulation of mitochondria harboring respiratory dysfunction. Mitochondrial dysfunction is often accompanied by increased ROS levels that can contribute to cellular dysfunction and disease etiology. Here we use the model eukaryote S. cerevisiae to investigate whether reduced cytochrome c oxidase (COX) activity, commonly reported in aging organisms and associated with neurodegenerative disorders, leads to ROS production from mitochondria. We provide evidence that although reduced COX complex activity correlates with ROS accumulation, mitochondria are not the major production center. Instead we show that COX-deficient mitochondria activate Ras upon their outer membrane that establishes a pro-ROS accumulation environment by suppressing antioxidant defenses and the ERAD-mediated turnover of the ER-localized NADPH oxidase Yno1p. Our data suggest that dysfunctional mitochondria can serve as a signaling platform to promote the loss of redox homeostasis, ROS accumulation, and accelerate aging in yeast.
  • Guaragnella, N., Palermo, V., Burhans, W., Gourlay, C., Ludovico, P., Madeo, F., Giannattasio, S. and Mazzoni, C. (2013). Yeast between life and death: a summary of the Ninth International Meeting on Yeast Apoptosis in Rome, Italy, 17–20 September 2012. Cell Death and Differentiation [Online] 20:1281-1283. Available at: https://doi.org/10.1038/cdd.2013.71.
    Remembering the ancient latin saying omnes viae Romam
    ducunt, the yeast cell death community came to the eternal
    city to attend the 9th International Meeting on Yeast Apoptosis
    (IMYA), from 17–20 September 2012. More than one hundred
    investigators from around the world presented and discussed
    their researches on programmed cell death (PCD) and its role
    in stress responses, aging and development employing yeast
    as model organism. On the first day, the meeting took place at
    the historical Angelicum Congress Center, sharing its opening
    session with the last session of the 20th Euroconference on
    Apoptosis (ECDO).
  • Kotiadis, V., Leadsham, J., Bastow, E., Gheeraert, A., Whybrew, J., Bardsley, M., Lappalainen, P. and Gourlay, C. (2012). Identification of new surfaces of cofilin that link mitochondrial function to the control of multi-drug resistance. Journal of Cell Science [Online] 125:2288-2299. Available at: http://dx.doi.org/10.1242/jcs.099390.
    ADF/cofilin family proteins are essential regulators of actin cytoskeletal dynamics. Recent evidence also implicates cofilin in the regulation of mitochondrial function. Here, we identify new functional surfaces of cofilin that are linked with mitochondrial function and stress responses in the budding yeast Saccharomyces cerevisiae. Our data link surfaces of cofilin that are involved in separable activities of actin filament disassembly or stabilisation, to the regulation of mitochondrial morphology and the activation status of Ras, respectively. Importantly, charge alterations to conserved surfaces of cofilin that do not interfere with its actin regulatory activity lead to a dramatic increase in respiratory function that triggers a retrograde signal to upregulate a battery of ABC transporters and concurrent metabolic changes that support multi-drug resistance. We hypothesise that cofilin functions within a bio-sensing system that connects the cytoskeleton and mitochondrial function to environmental challenge.
  • Rinnerthaler, M., Buttner, S., Laun, P., Heeren, G., Felder, T., Klinger, H., Weinberger, M., Stolze, K., Grousl, T., Hasek, J., Benada, O., Frydlova, I., Klocker, A., Simon-Nobbe, B., Jansko, B., Breitenbach-Koller, H., Eisenberg, T., Gourlay, C., Madeo, F., Burhans, W. and Breitenbach, M. (2012). Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast. Proceedings of the National Academy of Sciences [Online] 109:8658-8663. Available at: http://dx.doi.org/10.1073/pnas.1201629109.
    The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.
  • Di, Y., Holmes, E., Butt, A., Dawson, K., Mironov, A., Kotiadis, V., Gourlay, C., Jones, N. and Wilkinson, C. (2011). H2O2 stress-specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4. The EMBO Journal [Online] 31:563-575. Available at: http://dx.doi.org/10.1038/emboj.2011.438.
    In fission yeast, the stress?activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H2O2 but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N?terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H2O2, resulting in the full?length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress?specific nature of the regulation of Sty1 by Ptc4.
  • Jossé, L., Li, X., Coker, R., Gourlay, C. and Evans, I. (2011). Transcriptomic and phenotypic analysis of the effects of T-2 toxin on Saccharomyces cerevisiae: evidence of mitochondrial involvement. FEMS Yeast Research [Online] 11:133-150. Available at: http://dx.doi.org/10.1111/j.1567-1364.2010.00699.x.
    At 5 ?g mL?1, T-2 toxin significantly upregulated the transcription of 281 genes and downregulated 86. Strongly upregulated genes included those involved in redox activity, mitochondrial functions, the response to oxidative stress, and cytoplasmic rRNA transcription and processing. Highly repressed genes have roles in mitochondrial biogenesis, and the expression and stability of cytoplasmic rRNAs. T-2 toxin inhibition of growth was greater in a medium requiring respiration, and was antagonized by antioxidants. T-2 toxin treatment induced reactive oxygen species, caused nucleolytic damage to DNA, probably mitochondrial, and externalization of phosphatidylserine. Deletion mutations causing respiratory deficiency substantially increased toxin tolerance, and deletion of some TOR (target of rapamycin) pathway genes altered T-2 toxin sensitivity. Deletion of FMS1, which plays an indirect role in cytoplasmic protein synthesis, markedly increased toxin tolerance. Overall, the findings suggest that T-2 toxin targets mitochondria, generating oxy-radicals and repressing mitochondrial biogenesis genes, thus inducing oxidative stress and redox enzyme genes, and triggering changes associated with apoptosis. The large transcriptional changes in genes needed for rRNA transcription and expression and the effects of deletion of FMS1 are also consistent with T-2 toxin damage to the cytoplasmic translational mechanism, although it is unclear how this relates to the mitochondrial effects.
  • Leadsham, J. and Gourlay, C. (2010). cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation. BMC Cell Biology [Online] 11:1471-2121. Available at: http://dx.doi.org/10.1186/1471-2121-11-92.
    Background
    Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death.

    Results
    We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1.

    Conclusions
    We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast.
  • Sudarsha, S., Sourbier, C., Kong, H., Block, K., Valera Romero, V., Yang, Y., Galindo, C., Mollapour, M., Scroggins, B., Goode, N., Lee, M., Gourlay, C., Trepel, J., Linehan, W. and Neckers, L. (2009). Fumarate hydratase deficiency in renal cancer induces glycolytic addiction and hypoxia-inducible transcription factor 1 alpha stabilization by glucose-dependent generation of reactive oxygen species. Molecular and Cellular Biology [Online] 29:4080-4090. Available at: http://dx.doi.org/10.1128/MCB.00483-09.
    Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited cancer syndrome linked to biallelic inactivation of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH). Individuals with HLRCC are at risk to develop cutaneous and uterine leiomyomas and an aggressive form of kidney cancer. Pseudohypoxic drive-the aberrant activation of cellular hypoxia response pathways despite normal oxygen tension-is considered to be a likely mechanism underlying the etiology of this tumor. Pseudohypoxia requires the oxygen-independent stabilization of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1alpha). Under normoxic conditions, proline hydroxylation of HIF-1alpha permits VHL recognition and subsequent targeting for proteasomal degradation. Here, we demonstrate that inactivating mutations of FH in an HLRCC-derived cell line result in glucose-mediated generation of cellular reactive oxygen species (ROS) and ROS-dependent HIF-1alpha stabilization. Additionally, we demonstrate that stable knockdown of FH in immortalized renal epithelial cells results in ROS-dependent HIF-1alpha stabilization. These data reveal that the obligate glycolytic switch present in HLRCC is critical to HIF stabilization via ROS generation.
  • Leadsham, J., Miller, K., Ayscough, K., Colombo, S., Martegani, E., Sudbery, P. and Gourlay, C. (2009). Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast. Journal of Cell Science [Online] 122:706-715. Available at: http://dx.doi.org/10.1242/jcs.042424.
    Elucidating the mechanisms by which eukaryotic cells coordinate environmental signals with intracellular ;fate' decisions, such as apoptosis, remains one of the important challenges facing cell biologists. It has recently emerged that the dynamic nature of the actin cytoskeleton is an important factor in the linkage of sensation of extracellular stimuli to signalling mechanisms that regulate programmed cell death. In yeast, actin has been shown to play a role in the regulation of apoptosis as cells prepare themselves for quiescence in the face of nutritional exhaustion, by facilitating the shutdown of Ras-cAMP-PKA pathway activity. Here, we demonstrate that the loss of Whi2p function, a protein known to influence cell cycle exit under conditions of nutritional stress, leads to cell death in yeast that displays the hallmarks of actin-mediated apoptosis. We show that actin-mediated apoptosis occurs as a result of inappropriate Ras-cAMP-PKA activity in Deltawhi2 cells. Cells lacking Whi2p function exhibit an aberrant accumulation of activated Ras2 at the mitochondria in response to nutritional depletion. This study provides evidence that the shutdown of cAMP-PKA signalling activity in wild-type cells involves Whi2p-dependent targeting of Ras2p to the vacuole for proteolysis. We also demonstrate for the first time that Whi2p-dependent regulation of cAMP-PKA signalling plays a physiological role in the differentiation of yeast colonies by facilitating elaboration of distinct zones of cell death.
  • Franklin-Tong, V. and Gourlay, C. (2008). A role for actin in regulating apoptosis/programmed cell death: evidence spanning yeast, plants and animals. Biochemical Journal [Online] 413:389-404. Available at: http://dx.doi.org/10.1042/BJ20080320.
    Achieving an understanding of how apoptosis/PCD (programmed cell death) is integrated within cellular responses to environmental and intracellular signals is a daunting task. From the sensation of a stimulus to the point of no return, a programme of cell death must engage specific pro-death components, whose effects can in turn be enhanced or repressed by downstream regulatory factors. In recent years, considerable progress has been made in our understanding of how components involved in these processes function. We now know that some of the factors involved in PCD networks have ancient origins that pre-date multicellularity and, indeed, eukaryotes themselves. A subject attracting much attention is the role that the actin cytoskeleton, itself a cellular component with ancient origins, plays in cell death regulation. Actin, a key cellular component, has an established role as a cellular sensor, with reorganization and alterations in actin dynamics being a well known consequence of signalling. A range of studies have revealed that actin also plays a key role in apoptosis/PCD regulation. Evidence implicating actin as a regulator of eukaryotic cell death has emerged from studies from the Animal, Plant and Fungal Kingdoms. Here we review recent data that provide evidence for an active, functional role for actin in determining whether PCD is triggered and executed, and discuss these findings within the context of regulation of actin dynamics.
  • Leadsham, J. and Gourlay, C. (2008). Cytoskeletal induced apoptosis in yeast. Biochimica et biophysica acta Molceular Cell Research [Online] 1783:1406-1412. Available at: http://dx.doi.org/10.1016/j.bbamcr.2008.01.019.
    The influence of the cytoskeleton reaches into almost every aspect of eukaryotic cell function. It is a little surprise therefore that links between the regulation of the cytoskeleton and apoptosis have been found in a variety of eukaryotic systems. Studies from yeast have made a significant contribution to this new field of research and have highlighted the importance of interactions between the cytoskeleton and mitochondria in determining cell fate. In yeast both the actin and microtubular cytoskeletons have been shown to influence mitochondrial function and the commitment to apoptosis. In this review we discuss the recent advances and speculate that apoptotic mechanisms that feed off the ability of the cytoskeleton to respond to environmental signals may represent a useful mechanism to remove weak or damaged individuals from a population.
  • Gourlay, C., Du, W. and Ayscough, K. (2006). Apoptosis in yeast--mechanisms and benefits to a unicellular organism. Molecular Microbiology [Online] 62:1515-1521. Available at: http://dx.doi.org/10.1111/j.1365-2958.2006.05486.x.
    Initial observations that the budding yeast Saccharomyces cerevisiae can be induced to undergo a form of cell death exhibiting typical markers of apoptosis has led to the emergence of a thriving new field of research. Since this discovery, a number of conserved pro- and antiapoptotic proteins have been identified in yeast. Indeed, early experiments have successfully validated yeasts as a powerful genetic tool with which to investigate mechanisms of apoptosis. However, we still have little understanding as to why programmes of cell suicide exist in unicellular organisms and how they may be benefit such organisms. Recent research has begun to elucidate pathways that regulate yeast apoptosis in response to environmental stimuli. These reports strengthen the idea that physiologically relevant mechanisms of programmed cell death are present, and that these function as important regulators of yeast cell populations.
  • Gourlay, C. and Ayscough, K. (2006). Actin-Induced Hyperactivation of the Ras Signaling Pathway Leads to Apoptosis in Saccharomyces cerevisiae. Molecular and Cellular Biology [Online] 26:6487-6501. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16914733.
    Recent research has revealed a conserved role for the actin cytoskeleton in the regulation of aging and apoptosis among eukaryotes. Here we show that the stabilization of the actin cytoskeleton caused by deletion of Sla1p or End3p leads to hyperactivation of the Ras signaling pathway. The consequent rise in cyclic AMP (cAMP) levels leads to the loss of mitochondrial membrane potential, accumulation of reactive oxygen species (ROS), and cell death. We have established a mechanistic link between Ras signaling and actin by demonstrating that ROS production in actin-stabilized cells is dependent on the G-actin binding region of the cyclase-associated protein Srv2p/CAP. Furthermore, the artificial elevation of cAMP directly mimics the apoptotic phenotypes displayed by actin-stabilized cells. The effect of cAMP elevation in inducing actin-mediated apoptosis functions primarily through the Tpk3p subunit of protein kinase A. This pathway represents the first defined link between environmental sensing, actin remodeling, and apoptosis in Saccharomyces cerevisiae.
  • Gourlay, C. and Ayscough, K. (2005). The actin cytoskeleton in ageing and apoptosis. FEMS Yeast Research [Online] 5:1193-1198. Available at: http://dx.doi.org/10.1016/j.femsyr.2005.08.001.
    Regulated cell death, or apoptosis, has evolved to fulfil a myriad of functions amongst multicellular organisms. It is now apparent that programmed cell death occurs in unicellular organisms such as yeast. In yeast, as in higher eukaryotes, the actin cytoskeleton is an essential component of a number of cellular activities, and many of the regulatory proteins involved are highly conserved. Recent evidence from diverse eukaryotic systems suggests that the actin cytoskeleton has a role in regulating apoptosis via interactions with the mitochondria. This interaction also appears to have a significant impact on the management of oxidative stress and so cellular ageing. In this mini-review we summarise some of the work, which suggests that actin is a key regulator of apoptosis and ageing in eukaryotic cells.
  • Gourlay, C. and Ayscough, K. (2005). A role for actin in aging and apoptosis. Biochemical Society Transactions [Online] 33:1260-1264. Available at: http://dx.doi.org/10.1042/BST0331260.
    The actin cytoskeleton is central to many cell processes including membrane trafficking and generation of cell polarity. We have identified a role for actin in cell death and in promoting longevity of the budding yeast, Saccharomyces cerevisiae. Aging in yeast appears to occur via an apoptotic-like pathway with changes including DNA fragmentation, loss of mitochondrial membrane permeability, increase in levels of ROS (reactive oxygen species) and exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This pathway can be induced by alterations in actin dynamics, such that reduced dynamics correlates with increased levels of ROS and decreased viability. Conversely, increased actin dynamics correlates with low ROS levels and increased survival. Our current studies have focused on identifying pathways which couple changes in actin dynamics to cell death.
  • Gourlay, C. and Ayscough, K. (2005). The actin cytoskeleton: a key regulator of apoptosis and ageing?. Nature Reviews Molecular Cell Biology [Online] 6:583-589. Available at: http://dx.doi.org/10.1038/nrm1682.
    Evidence from many organisms has shown that the accumulation of reactive oxygen species (ROS) has a detrimental effect on cell well-being. High levels of ROS have been linked to programmed cell death pathways and to ageing. Recent reports have implicated changes to the dynamics of the actin cytoskeleton in the release of ROS from mitochondria and subsequent cell death.
  • Gourlay, C. and Ayscough, K. (2005). Identification of an upstream regulatory pathway controlling actin-mediated apoptosis in yeast. Journal of Cell Science [Online] 118:2119-2132. Available at: http://dx.doi.org/10.1242/jcs.02337.
    The build up of reactive oxygen species (ROS) is known to contribute to a reduction in the lifespan of a cell and to their degeneration in diseases such as Alzheimer's and tissue ischaemia. It is therefore important to elucidate pathways that regulate cellular oxidative stress. We have previously shown that actin dynamics can affect the oxidative-stress burden on a yeast cell and thereby its potential lifespan. To elucidate further the connection between actin dynamics and oxidative stress, we sought to identify regulators of this process. The actin regulatory proteins Sla1p and End3p are important in maintaining a rapid turnover of F-actin in cortical patches. We show that cells expressing a mutated form of Sla1p or lacking End3p display markers of apoptosis such as depolarized mitochondrial membranes and elevated levels of reactive oxygen species. Overexpression of the ubiquitin ligase RSP5 can alleviate the oxidative-stress phenotype observed in cells lacking End3p by targeting Sla1p to the cortex and restoring actin remodelling capability. We also demonstrate that overexpression of PDE2, a negative regulator of the Ras/cAMP pathway rescues actin dynamics, reduces oxidative stress sensitivity and restores viability in deltaend3 cells. Our data suggest, for the first time, that a physiological link exists between actin regulation and cAMP signalling that regulates apoptosis in yeast.
  • Gourlay, C., Carpp, L., Timpson, P., Winder, S. and Ayscough, K. (2004). A role for the actin cytoskeleton in cell death and aging in yeast. Journal of Cell Biology [Online] 164:803-809. Available at: http://dx.doi.org/10.1083/jcb.200310148.
    Several determinants of aging, including metabolic capacity and genetic stability, are recognized in both yeast and humans. However, many aspects of the pathways leading to cell death remain to be elucidated. Here we report a role for the actin cytoskeleton both in cell death and in promoting longevity. We have analyzed yeast strains expressing mutants with either increased or decreased actin dynamics. We show that decreased actin dynamics causes depolarization of the mitochondrial membrane and an increase in reactive oxygen species (ROS) production, resulting in cell death. Important, however, is the demonstration that increasing actin dynamics, either by a specific actin allele or by deletion of a gene encoding the actin-bundling protein Scp1p, can increase lifespan by over 65%. Increased longevity appears to be due to these cells producing lower than wild-type levels of ROS. Homology between Scp1p and mammalian SM22/transgelin, which itself has been isolated in senescence screens, suggests a conserved mechanism linking aging to actin stability.
  • Gourlay, C., Dewar, H., Warren, D., Costa, R., Satish, N. and Ayscough, K. (2003). An interaction between Sla1p and Sla2p plays a role in regulating actin dynamics and endocytosis in budding yeast. Journal of Cell Science [Online] 116:2551-2564. Available at: http://dx.doi.org/10.1242/jcs.00454.
    The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly recognised in mammalian cells. However, the mechanism for actin recruitment and the role it plays in endocytosis is unclear. Here we show the importance of two yeast proteins in this process. We demonstrate that Sla1p and Sla2p interact in vitro and in vivo and that this interaction is mediated by the central domain of Sla2p, which includes its coiled-coil region, and by a domain of Sla1p between residues 118 and 361. Overexpression of the interacting fragment of Sla1p causes reduced fluid-phase endocytosis and, interestingly, defects in subsequent trafficking to vacuoles. We show that Sla2p is required for the polarised localisation of Sla1p in cells but not for its cortical localisation or for its overlapping localisation with actin. Generation of an Deltasla1Deltasla2 double mutant demonstrates that Sla2p is likely to act upstream of Sla1p in endocytosis, whereas sensitivity to latrunculin-A suggests that the proteins have opposite effects on actin dynamics. We propose that Sla2p recruits Sla1p to endocytic sites. Sla1p and its associated protein Pan1p then regulate actin assembly through interactions with Arp2/3 and Arp2/3-activating proteins Abp1p and Las17/Bee1p.
  • Dewar, H., Warren, D., Gardiner, F., Gourlay, C., Satish, N., Richardson, M., Andrews, P. and Ayscough, K. (2002). Novel proteins linking the actin cytoskeleton to the endocytic machinery in Saccharomyces cerevisiae. Molecular Biology of the Cell [Online] 13:3646-3661. Available at: http://dx.doi.org/10.1091/mbc.E02-05-0262.
    The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

Book section

  • Makarovaite, V., Gourlay, C., Hillier, A., Batchelor, J. and Holder, S. (2019). Adjustable Passive RFID Skin Mounted Sticker. In: 2019 IEEE 16th International Conference on Wearable and Implantable Body Sensor Networks. New York, USA: IEEE, pp. 1-4. Available at: https://doi.org/10.1109/BSN.2019.8771069.
    Passive Radio Frequency Identification (RFID) can be limited by such aspects as size that make overcoming the capacitive loading introduced by human tissue difficult when intending to produce an efficient antenna with a high read range. It is still possible to design a small and thin RFID tag with a read range above half a meter. Here we present such a design with a diameter of less than 3 cm, By utilizing a breathable polyurethane polymer with two thicknesses, it is possible to use a single tag design for human tissue (dielectric constants ranging from a 22 to 40) with only a slight loss in antenna read range. It is more cost effective to just adjust the polyurethane thickness for a single design than to tune the antenna design itself. Volunteers one and two had read ranges above one meter while volunteer three (with permittivity of 40) caused the largest shift in antenna resonance from 865 MHz (EU band) and required an increase in polyurethane thickness from 1.1 mm to 1.5 mm. However, both designs still produced read ranges above 0.5 meters which could be further tuned with future adjustments to increase read range. This led to a passive RFID design that can be worn by most people with a simple thickness adjustment if needed.

Conference or workshop item

  • Makarovaite, V., Hillier, A., Holder, S., Gourlay, C. and Batchelor, J. (2019). Voice Prosthesis Implantable UHF RFID Self-Sensing Tag for Microbial Growth Detection. In: 2019 IEEE International Conference on RFID Technology and Applications (RFID-TA). New York, NY, USA: IEEE, pp. 314-317. Available at: https://doi.org/10.1109/RFID-TA.2019.8891963.
    Passive Radio Frequency Identification (RFID) implantable antennas experience low efficiency due to the capacitive loading of human tissue that the implantable design has to overcome. Here we present a passive UHF implantable device able to produce a read range of 65 cm (EU frequency band) and 80 cm (US frequency band) from within the body. With a slight adjustment, this self-sensing tag can be utilized to sense microbial growth within the body once mounted upon a voice prosthesis. Simulated Candida albicans biofilm growth can be detected within the body with this sensor from early and up to a mature biofilm thickness of 30 µm (0 to 48-hour growth in vitro equivalent); allowing for detection before any infection symptoms within a patient become apparent (as early as 4-hour biofilm colonization (5 µm thickness)) and before the voice prosthesis experiences failure. This passive UHF device would permit cost-effective immediate medical intervention before any serious consequence could occur to a voice prosthesis patient.

Thesis

  • Lee, O. (2018). Investigating the Regulation of Multidrug Resistance by Cofilin in Saccharomyces Cerevisiae.
    The actin cytoskeleton is a dynamic structure which is capable of rapid assembly and disassembly, and has roles in the structural stability and migration of the cell. Cofilin 1 (Cof1) is an intracellular protein which depolymerises F-actin whilst inhibiting the polymerisation of G-actin, and as such dissembling the actin cytoskeleton, in a pH dependant way. Cofilin 1 has also been linked to the function and regulation of mitochondria in Saccharomyces cerevisiae. Mutations in the COF1 gene have been connected to drug resistance to numerous antifungals in Saccharomyces cerevisiae. One such mutant is the cof1-6 strain whose mutations are present outside of the charge - dependant PtdIns(4,5)P2 binding site of Cofilin 1, and has been linked to the up-regulation of several ABC transporters that have roles in drug resistance. We investigate the role of genes involved in lipid biosynthesis and the role of the mitochondrial outer membrane protein Porin 1 in cof1-6 linked drug resistance. We show that the Cof1-6 mediated drug resistance is PKC-regulated MAPK pathway independent. We further observe an increase in the expression of the sphingoid long chain base efflux pump Rsb1 in the cof1-6 strain. This up-regulation of Rsb1 is pH-independent but Porin dependent. Previous literature has demonstrated a link between peroxisomal and mitochondrial function. Our work suggests that peroxisome function does not regulate drug resistance whereas mitochondrial function is a key mediator in the resistance phenotype of the cof1-6 strain. Furthermore, we demonstrate that cof1-6 linked drug resistance is very complex with the ABC transporter Pdr5 involved in resistance to antifungals which operate through different modes of action. We hope that this work will further understanding into drug resistance against common antifungal drugs.
  • Piper-Brown, E. (2015). The Effects of Phosphorylation on the Regulation of Ras Localisation in Eukaryotic Cells.
    Ras proteins are small GTPases that function as regulatory switches linking external environmental stimuli with intracellular effectors to control cell growth and proliferation. Mutations that lead to the constitutive activation of Ras proteins are associated with the development of several human cancers. The localisation of Ras is crucial for its function and this is controlled by post-translational modifications. The genes encoding Ras proteins are highly conserved and yeast serves as a useful model to study the control of localisation and activation. Serine225 has been implicated as a potential phosphorylation site which affects Ras2p localization in S. cerevisiae. The substitution of serine225 with alanine or glutamate suggests that serine225 plays a key role in Ras2p localisation. Although there is significant sequence divergence outside of the GTPase domain and C terminal CAAX box. Sequence alignment between yeast Ras2 and human N-Ras suggests that serine225 and the surrounding sequence are conserved. This study aims to determine whether phosphorylation of human N-Ras regulates activity and localisation when expressed in yeast cells. We have successfully expressed N-Ras in yeast cells and find that it is targeted to the plasma membrane, suggesting that is correctly processed by yeast cells. Modification of the residue equivalent to yeast Ras2 serine225 in N-Ras leads to senescence in the yeast system suggesting that this conserved serine residue is important for function. As the N-Ras human oncogene is linked to many cancers, especially malignant melanomas, the data collected in this study could potentially provide a greater understanding into the activity and regulation of Ras2p and N-Ras, which could be used to aid in the formulation of new strategies for cancer treatment.
  • Peswani, A. (2015). Dissecting the Molecular Mechanisms of Motor Neurone Disease in Yeast and Worms.
    Superoxide dismutase 1 (SOD1) is an enzyme responsible for intracellular breakdown of toxic reactive oxygen species in prokaryotes and eukaryotes. Mutations in SOD1 are linked to the motor neurone disease Amyotrophic lateral sclerosis (ALS), with around 20% of familial (fALS) cases and, it is proposed, a significant proportion of sporadic cases caused by SOD1 mutations, however the mechanism of neuronal death remains unknown. Misfolding and aggregation of SOD1 leading to a ‘toxic gain of function’ is the most widely accepted mechanism, but further work is needed. To gain new insights into the underlying mechanism of cell toxicity, I am using two model systems: the yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans. One hypothesis being tested was proposed by Ghadge et al. (2006) who reported that truncated forms of SOD1 (both wild type and fALS–linked mutants) form toxic aggregates in spinal cords of chick embryos, hypothesising that proteolytic digestion of unstable mutant SOD1 creates these toxic fragments. My project aims to elucidate how fALS–linked SOD1 mutants and SOD1 truncations exert toxicity on S. cerevisiae and C. elegans. In S. cerevisiae, SOD1 truncations do not specifically localise to, or disrupt the functioning of, mitochondria nor reduce viability of yeast although cell growth is slowed. Conversely, a motor-defective phenotype is exhibited in transgenic fALS–linked SOD1 mutant (G85R) C. elegans accompanied by vulval defects, which lead to reduced lifespan. My findings suggest that amino acid supplementation of G85R C. elegans mutants improves their motor function and vulval defects, implicating a role of amino acid signalling in mutant SOD1-mediated toxicity.
  • Tarrant, D. (2015). Investigating the Effects of Increased Levels of the Translation Elongation Factor eEF1A Within Eukaryotic Cells.
    The highly conserved eukaryotic elongation factor 1A (eEF1A) plays a canonical role in translation elongation, where it is responsible for delivering the aminoacylated tRNA to the A-site of the 80S ribosome. Further to this essential role it is reported to be involved in a plethora of moonlighting functions that are not fully characterised or understood. One of the human isoforms, eEF1A2, is known to induce cancer when expressed in non-native tissues, although the mechanism by which it promotes tumour growth is not yet known.
    In this study we have characterised eEF1A overexpression in yeast and provided evidence to suggest that elevated levels of eEF1A result spindle defects which lead to chromosomal abnormalities that have the potential to induce uncontrolled cell growth in human cells. Moreover, we have confirmed conservation of this chromosomal abnormality in human cell lines suggesting that the mechanism that eEF1A utilises to induce these effects are highly conserved. We have also observed that in yeast, eEF1A overexpression results in increased metabolic activity, a hallmark of cancer cells.
    We hypothesise that eEF1A interacts with the dynactin complex, a regulator of spindle dynamics, resulting in aberrant spindle formation. This in turn leads to chromosomal abnormalities that appear toxic to the cell. Cells appear to overcome the toxicity induced by eEF1A by suppressing plasmid copy number to the lowest levels possible. This however, brings its own problems and appears to result in synthetic effects together with eEF1A overexpression.
  • Smethurst, D. (2014). The Dynamic Status of Actin in the Regulation of Environmental Sensing and Homeostatic Control in Saccharomyces Cerevisiae.
    Actin is a highly conserved protein in eukaryotes which forms dynamic cytoskeletal structures. Rapid remodeling of actin filaments is important for the regulation of a broad range of critical cellular processes. The cytoskeleton is acutely responsive to stresses and there are multiple interactions between actin and signaling pathways, positioning it centrally to a cells ability to adapt and respond to their environment. Here I provide further evidence that a dynamic actin cytoskeleton regulates processes including endocytosis, mitochondrial respiration, and signal transduction. Results presented here show that actin is embedded in the signaling networks which control the responses to environmental change. Change to the dynamic status of actin modulates the activity of the transcription factor Ste12p which regulates both the mating and filamentous/invasive growth responses. The activity of these pathways are linked to cortical patch organisation, and our data suggests that there is crosstalk between multiple pathways. I propose that the links between actin dynamics and environmental sensing pathways leave it well positioned for a role as a biosensor. Actin dynamics is altered by changes in internal or external conditions, leading to adapted of cellular responses which may provide a protective function for a population.

Forthcoming

  • Pentland, D. (2019). Investigating the Colonisation of Voice Prostheses by Candida Albicans Following Total Laryngectomy.
    A total laryngectomy is a surgical procedure for the treatment of advanced laryngeal cancer. The vocal cords are removed during the procedure, leaving patients with an inability to form speech. The gold standard of speech rehabilitation involves the use of small silicone valves called voice prostheses (VPs) which are inserted into the throat. C. albicans is a major coloniser of VPs, often being found in polymicrobial biofilms, causing loss of device function as well as creating a reservoir for infections. As a result, VPs have to be regularly changed at great patient stress and clinical cost. In this study, we present precision antifungal-focussed treatment guidelines which significantly increase VP in situ lifespan from a mean of 71.9 to 192.0 days, a 2.7-fold increase, in a 20 patient cohort. In addition, we find that the high CO2 environment of the throat (due to the CO2 content of exhaled breath) increases the biofilm-forming capacity of C. albicans. All stages of biofilm growth (attachment, maturation and dispersal) are enhanced in 5% CO2 and this is accompanied by increased antifungal tolerance; providing a possible explanation for the success of C. albicans in the VP niche. Finally, we explore potential treatment options for high CO2 biofilms, identifying the glycolytic inhibitor 2-deoxyglucose as a promising candidate. Overall, our study characterises a clinically relevant CO2-mediated increase in C. albicans biofilm formation which could influence future biofilm studies and therapeutic discovery.
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