Dynamics and interaction of cell-polarity landmark proteins and the Cdc42 GTPase module: a systems approach

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences


In the process of development from embryo to adult, cells of multicellular organisms undergo transitions from undifferentiated, embryonic-type cells into more specialised differentiated types, such as neurons, epithelial cells and muscle fibres. The term "cell polarity" refers to the ability of many types of cells to acquire an internal structure or architecture that distinguishes, for example, their front from their back, or their top from their bottom. Differentiation of cells into specialised cell types is typically accompanied by changes in cell polarity and cell shape, and these are important for the specific functions of different cell types.

Environmental stresses to the cell, such as extreme temperature, changes in oxygen, acidity or toxins, can lead to loss of cell polarity, and this loss is in fact a normal defensive reaction of the cell. However, if cell polarity does not recover back to normal after the stress subsides, the cell may be more likely to eventually undergo a malignant transformation into a rapidly-dividing non-differentiated form that could cause cancer. Therefore, understanding the detailed mechanisms that control the loss of cell polarity in response to stresses, and the subsequent recovery of cell polarity, is very important to succeed in our fight against cancer. However, human cells, and indeed all mammalian cells, are exceedingly complex and difficult to study experimentally. In this project we will use fission yeast, a model laboratory fungus that is much better understood than human cells, and significantly easier to work with, to study how cell polarity is established and re-established after the recovery from environmental stresses

In particular, we will focus on two systems that contribute to the regulation of cell polarity. One system involves a protein called Cdc42, which is found in all cells from yeasts to humans and acts as a "switch" on the cell membrane (surface). Regions of the cell membrane where Cdc42 is "switched on" have different properties to regions where Cdc42 is "switched off". These different regions generate signals to the cell interior to control internal cellular architecture, cell movement and growth. A second system involves filaments called microtubules, which are also found in all cells and reach from the cell interior to the cell membrane. Microtubules contribute to cell polarity by delivering various protein factors to the cell membrane, including a protein called Tea1. To date, the Cdc42 system and the Tea1/microtubules system and their associated proteins have been studied only in isolation. However, in our laboratory we have obtained evidence that the two systems are not completely independent, but instead "talk to each other". The goal of our research is to understand the interplay between the Cdc42 system and the Tea1/microtubules system during the establishment of cell polarity and in response to, and recovery from, stress. To achieve this goal we will use a combination of methods, including state-of-the art microscopy, genetics, mathematical modelling, and analysis of signal-dependent chemical modifications of proteins.

In spite of the obvious differences between human cells and yeast, the actual detailed mechanisms underlying their biological behaviours are remarkably similar. Therefore, an improved understanding of the regulation of these processes in yeast, a "model organism", will aid in the understanding of comparable processes in human cells. In addition, our work with yeast may also be useful to develop novel strategies to control growth of fungal pathogens that infect humans and economically important crops and animals.

Technical Summary

Establishment and maintenance of cell polarity are fundamental cellular phenomena directly related to health and disease. Loss of cell polarity is increasingly noted to be associated with neoplasia and cancerous transformation in epithelial organs. Maintenance of polarity is absolutely required for the function of motile immune cells, asymmetric division of stem cells, and growth and regeneration of neurons. Importantly, polarity is frequently lost in response to a variety of cellular stresses, and its recovery requires some form of cellular memory, often referred to as "landmarks". While an understanding of the mechanisms of polarity recovery is absolutely essential for bio-medical applications, it is hindered by the complexity of mammalian cells. Using fission yeast as a model organism, we propose to determine the mechanisms linking the fundamental Cdc42 cell-polarity module with the microtubule-mediated Tea1 polarity landmark. In addition, we will uncover the role of this interaction in the recovery of cell polarity after stress. We will address the issue of Tea1 landmark stability and its control by Tea1 phosphorylation.

Planned Impact

Who will benefit from this research? How will they benefit from this research?

1. This is a basic-science project that will use fission yeast as a model organism to investigate the interplay between two eukaryotic cell polarity regulation systems, at both mechanistic and systems levels, using a combination of quantitative imaging, molecular genetics, phosphoproteomics, and computational modelling. As such, the primary beneficiaries of project outcomes will be from the international scientific community. Importantly, because of the multidisciplinary nature of the project, beneficiaries will come from diverse groups: 1) Researchers using fungi as model organisms to understand principles of eukaryotic cell development, differentiation and cell cycle control will benefit from conceptual advances made in understanding how cell polarity is established de novo and re-established after recovery from stress. Because fission yeast offers a level of complexity intermediate between budding yeast and filamentous fungi, we our results will bridge research in budding yeast and in complex filamentous fungi, including pathogens. 2) Researchers working on related problems in mammalian cells will also benefit from an improved understanding of general principles controlling cell polarity after stress. 3) Researchers in imaging will benefit from new methods in image analysis developed in the project, e.g. methods for quantifying recruitment of proteins to the plasma membrane during de novo polarity establishment. 4) Researchers doing computational modelling will benefit from new tools generated, which may be applicable to other systems. 5) Researchers interested in post-translational modifications will benefit from our publicly available SILAC phosphoproteomics datasets as they relate to signalling by conserved protein kinases.

2. There will also be several types of beneficiaries outside the immediate academic community. For example, because fungi are economically important pathogens of both plants and animals, applied researchers working on developing novel chemical compounds to suppress growth and proliferation of fungal pathogens will benefit, as they will be able to leverage conceptual advances, datasets and, ultimately, possible novel fungal-specific drug targets identified in the project. In addition, due to the broader relevance of the project to understanding mechanisms related to cell polarity in cancer progression, the international communities of cancer biologists and medical oncologists will benefit, as they will be able to use paradigms emerging from our work as guiding principles in much more complex mammalian cells. Thus, in the long run, our work will benefit both agricultural and medical practitioners developing anti-fungal drugs and therapies.

3. There will also be wider benefits to society in the technology arena, because of the training and professional skills that staff (PDRAs) employed on the proposal will acquire in the course of carrying out multidisciplinary research. The project provides outstanding potential for staff to develop novel cross-disciplinary, quantitative and other skills required in the modern work environment. These will improve their employment potential in diverse sectors.

4. We also envision the potential for beneficiaries in the area of intellectual property. Although this is a basic-science project, specific areas that may lead to intellectual property include software tools for analysis and modelling, as well as methods of imaging of live cells, which could be useful in high-content screening.

5. Finally in the public sphere, results from the project will be of interest in the popular press, due to interest in both modern cell-imaging techniques and the importance of fungi in agricultural, ecological, and medical contexts. Communication of our results and methodologies, through engagement and the media, will raise awareness within the general public.
Description We identified a pathway of Cdc42-dependent cell polarity regulation in fission yeast that functions in parallel with regulation by the Cdc42 guanine-nucleotide exchange factor (GEF) Scd1. We showed that in this second pathway, the Cdc42 GEF Gef1 acts a global, cytosolic GEF that promotes isotropic rather than polarized growth. However, the tendency of Gef1 to promote isotropic growth is countered at cell sides by the Cdc42 GTPase-activating protein (GAP) Rga4. Localization of Rga4 to cell sides is regulated by cytoplasmic microtubules and the Tea1 landmark, via the protein kinase activity of the cell-polarity kinase Pom1. As a result of the Tea1-Pom1-Rga4 system, the "net" activity of Gef1 is channeled towards cell tips to promote polarized growth. These discoveries represent a significant advance in elucidating the previously poorly-understood relationship between the Cdc42 polarity module and the Tea1 cell-polarity landmark.

We performed theoretical analysis of the emergence of Cdc42 polarity as a symmetry-breaking transition of an unpolarized cell. We found that symmetry breaking requires convergence of at least two positive feedback loops, or the existence of one nonlinear positive feedback loop. Using mathematical analysis, we demonstrated existence of six discrete classes of potential Cdc42 polarization mechanisms, based on regulation of Cdc42 by GEFs, GAPs and membrane-cytoplasmic trafficking. Our analysis of available experimental evidence suggests that multiple symmetry-breaking mechanisms may function in parallel in distinct fungal species to provide robust Cdc42polarization. Importantly, this analysis suggests that, despite conservation of major polarity regulators, the establishment of cell polarity in fission yeast need not exploit precisely the same mechanisms that are used by budding yeast.

We discovered that a stress-signalling pathway involving the conserved p38 fission yeast MAP kinase Sty1 is a key regulator of the Cdc42 cell-polarity module. It was previously known that actin depolymerization (using the drug Latrunculin A) leads to depolarization of the Cdc42 module from cell tips. We showed that Cdc42 depolarization is due not to actin depolymerization per se but rather to activation of Sty1 as a result of cell stress. We developed a synthetic-biology approach to activate Sty1 independently of external stress and showed that this also led to Cdc42 depolarization. Further experiments suggested that Cdc42 depolarization involves uncoupling of the Cdc42 module from cell-polarity landmarks. This work linked, for the first time, two fundamental aspects of eukaryotic cell biology-stress stress signalling and cell polarity--and generated an entirely new paradigm in our understanding of mechanisms controlling the Cdc42 cell-polarity module.

We discovered new features of cell-polarity regulation by the conserved NDR fission yeast kinase Orb6. Previously it was reported that Orb6 regulates cell polarity through phosphorylation of Gef1. We generated an "analog-sensitive" orb6 mutant whose protein kinase activity can be inhibited in vivo by nucleotide-competitive analogs. Our analysis of Orb6-inhibited cells showed that many polarity-defective phenotypes after Orb6 inhibition do not involve Gef1, and that a major consequence of Orb6 inhibition is impaired exocytosis. To identify Orb6 substrates invovled in exocytosis, we carried out a quantitative phosphoproteomics analysis, comparing global protein phosphorylation in Orb6-inhibited vs. uninhibited cells. This generated a high-quality dataset of Orb6-dependent phosphorylation sites, many of which appear to be direct targets of Orb6. We demonstrated that the exocyst complex protein Sec3 is a key target of Orb6 for exocytosis.
Exploitation Route Our findings will be of greatest relevance to other researchers, primarily in academia. Our experimental and theoretical work indicates that there can be many routes to cell polarity regulation by Cdc42, and that different systems (organisms) may exploit different related mechanisms to different extents. Our work on stress-signalling regulation of cell polarity opens up new areas that may be of interest to healthcare, because to date, the effects of stress-kinase signalling are thought to be mediated largely through regulation of gene expression, whereas our work suggests that stress-regulation of cell polarity involves post-translational mechanisms. The identification of stress-kinase substrates involved in cell polarity regulation would be a key future area of interest. Our work on polarity regulation by NDR kinase Orb6 suggests that regulation of exocytosis may be a key target of NDR kinases, which again have largely been reported to function via regulation of gene expression. Our phosphoproteomics datasets will be useful to other researchers more broadly.
Sectors Healthcare

Description Regulation of fission yeast cell polarity by stress-signalling pathways
Amount £1,039,434 (GBP)
Funding ID 210659 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2023
Title SISA strain (fission yeast) 
Description To study in closer detail how fission yeast cells regulate cell polarity through stress-signaling, we developed a strain in which the stress-activated MAP kinase Sty1 (homolog of human p38) can be activated without any external stress. The motivation for developing this strain, which we termed SISA (for Stress-Independent Sty1 Activation), was that many external stresses are "broad spectrum" stresses (e.g. osmotic stress, heat stress) that may simultaneously activate multiple intracellular signaling pathways. As a result, after such a stress it can be difficult to distinguish how cell polarity is specifically affected by one pathway vs. another pathway. In the SISA strain, the Sty1 pathway can be specifically activated simply by removing a small-molecule inhibitor from the growth medium. The SISA strain contains four mutations, each of which is introduced into the only copy of the relevant endogenous gene (fission yeast are normally haploid). The first mutation is in the MAP kinase kinase Wis1, which phosphorylates and activates Sty1. The activation loop of Wis1 is mutated so that Wis1 becomes constitutively active, leading to constitutive phosphorylation of Sty1. Normally, this would also make Sty1 constitutively active; however, a second mutation, in the Sty1 ATP-binding pocket, makes Sty1 kinase activity sensitive to a membrane-permeable ATP-competitive analog. As a result, if cells are grown in the presence of analog, Sty1 activity can be inhibited even if Sty1 is phosphorylated and "poised" to be active. Upon removal of analog, Sty1 can be activated. The third and fourth mutations are in protein tyrosine phosphatases that are substrates of Sty1 and act in a negative feedback loop to attenuate Sty1 activity after Sty1 activation. We deleted the genes encoding both phosphatases, so that there is little or no negative feedback to limit Sty1 activation. The end result is that when SISA cells are grown in the presence of analog, Sty1 is essentially completely inactive; when analog is removed, Sty1 is rapidly and specifically activated to very high levels. The SISA strain can be used both in live-cell microscopy and large-scale biochemistry contexts. 
Type Of Material Cell line 
Year Produced 2016 
Provided To Others? Yes  
Impact Thus far the SISA strain has been used by a lab at Dartmouth Medical School in the USA, resulting in the publication "Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress" J Biol Chem 2017, doi: 10.1074/jbc.M117.793034 
URL https://www.sciencedirect.com/science/article/pii/S0960982216309927
Title NDR kinase Orb6 phosphoproteomics 
Description We generated a global phosphoproteomics dataset of fission yeast protein phosphorylation sites that decrease in vivo upon inhibition of the conserved NDR kinase Orb6. The purpose was to identify candidate Orb6 substrates, as our work had implicated Orb6 as a key regulator of exocytosis. We used the SILAC (Stable isotope Labeling with Amino acids in Culture) method and cells bearing an ATP-analog-sensitive allele of Orb6 to quantitatively compare the phosphoproteome of untreated cells with cells treated with ATP-analog to inhibit Orb6 activity. The dataset contains over 10,000 identified peptides, of which over 8,000 could be quantified. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact This work led to our discovery that Orb6 positively regulates the exocyst, a multiprotein complex involved in later stages of exocytosis. Additional protein targets involved in membrane trafficking were also identified as very likely Orb6 targets. This work was published in Cell Reports (PMID: 30726745). 
URL https://www.ebi.ac.uk/pride/archive/projects/PXD009408
Description Glass Life exhibition at Royal Botanic Garden Edinburgh 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I served as a representative "researcher" at the Wellcome Trust Centre for Cell Biology "Glass Life" exhibition at the Royal Botanical Gardens, Edinburgh. The exhibition used fused glass art and sculpture to illustrate fundamental aspects of cell biology, as well as, more abstractly, the nature of experimentation. My role was to engage with the public and help to interpret the artwork as they made their way through the exhibition. More than 1400 visitors attended the exhibition over four days in July/August 2016, and another 68 visitors were involved in a specific knowledge and inference-based activity, "Escape the Cell", in which they had to answer questions about cell and molecular biology as quickly as possible, to "beat the clock". The feedback from visitors was generally excellent, and because this was the height of summer, visitors came from a wide range of geographic locations. Visitors--both adults and some children--generally left with a much more heightened awareness of the questions and research activities involved in cell biology, as well as its relevance to medicine and the human body.
Year(s) Of Engagement Activity 2016
URL http://www.rbge.org.uk/whats-on/event-details/4292
Description Life through a Lens (Royal Botanic Garden Edinburgh) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact PI Prof. Kenneth Sawin and co-PI Dr. Andrew Goryachev participated in the public engagement/outreach event "Life through a Lens", which was coordinated by the Wellcome Trust Centre for Cell Biology and took place at the Visitor Centre of the Royal Botanic Garden, Edinburgh on two successive weekends in November 2015. The event was a walk-in, "hands-on" session, open to all members of the public, on how we use light microscopy in scientific research, with educational components and activities for all ages from young children to adults. Participants were also able to ask about our ongoing research. Participants reported a greatly increased understanding and appreciation after the session.
Year(s) Of Engagement Activity 2015
URL http://outreachwcb.bio.ed.ac.uk/wcb/Life_Through_a_Lens.html
Description Life through a Lense (Royal Botanic Garden of Edinburgh) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The even is an annual public engagement show of cell biology connected to various types of microscopy
Year(s) Of Engagement Activity 2016
URL http://outreachwcb.bio.ed.ac.uk/wcb/Life_Through_a_Lens.html