Understanding mechanisms of cellular geometry scaling

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

All life on Earth is made of cells. Intricately organized assemblies of different types of cells make up complex multicellular organisms such as humans or plants. Simpler organisms including yeasts or protozoa live as single cells. Cells come in an astonishing diversity of shapes optimized for their specific function. A cell can grow to different size, depending on the amount of nutrients or other signals in the environment. If specific shape is important, how do cells make sure that it remains the same across different sizes? This is an interesting biological and engineering problem that can be solved by understanding how the intrinsic cellular polarity machinery that generates cell shape by restricting growth to certain sites on the cellular membrane, can decrease or increase the sizes of these growth zones in response to changes in cellular volume. We call this property cellular geometry scaling. It is incredibly widespread in biology but we know very little about it.

We will use a simple unicellular fungal organism called Schizosaccharomyces japonicus (S. japonicus) to understand how cells scale their shape. It is a great system for addressing this problem because it grows fast, is easily amenable to genetic manipulations and, of course, exhibits very robust scaling. We previously found that a regulator of the cellular polarity machinery, a protein called Rga4, was critical for scaling. If S. japonicus does not have Rga4, it cannot scale down when nutrients become scarce and dies, demonstrating that the ability to scale is really important. Now we propose to obtain mechanistic explanation for Rga4 function in scaling. Our program will consist of two interrelated objectives. First, we will understand how cellular polarity changes during scaling and how Rga4 contributes to these changes. We will also probe the relationship between Rga4 and a protein called Tea4 that normally functions to landmark growth at specific membrane sites. In the second objective we will investigate how cells regulate Rga4 by phosphorylation to promote scaling when required. Phosphorylation is one of the most important modifications regulating protein function through changes in conformation and we will additionally look at phosphoregulation of many more cellular proteins to reveal hidden connections between different parts of cellular physiology important for scaling.

It is essential to do this type of fundamental research in a simple organism because it provides important insights into molecular underpinnings of polarized growth and scaling in all eukaryotic cells, from humans to plants. The answers we get may open entirely new possibilities in dealing with devastating fungal pathogens, which rely on changing their size and shape to infect their hosts.

Technical Summary

For cells of a specific type, functionally optimal cellular organization is typically maintained across a range of cell sizes. This phenomenon, known as scaling, is widespread in biology but surprisingly poorly understood. We want to understand the molecular mechanisms underlying scaling of cellular polarity, which allows cells to maintain their optimal shape at different cell volumes.

We recently established the fission yeast Schizosaccharomyces japonicus as a genetically tractable model system for understanding both the mechanisms and functional implications of cellular geometry scaling. We discovered that the GTPase activating protein Rga4 was essential for this process. In the proposed work we will test the hypothesis, built on our preliminary data, that scaling requires modulation of Rga4 function by a polarity landmark protein Tea4 and specific phosphoregulation events.

We will pursue two interrelated objectives. First, we will examine contribution of Rga4 to regulating Cdc42 and Rho2 GTPase pathways during scaling and address the molecular function of a cellular polarity landmark Tea4 in regulating Rga4. This set of experiments will allow us to identify the target(s) of Rga4 critical for scaling and interpret phenotypes of mutants obtained in Objective 2. In the second objective, we will focus on identifying specific Rga4 phosphoregulation events key to scaling. To gain insight into the broader regulatory context of scaling and reveal possible epistatic relationships, we will also carry out unbiased phosphoproteomics analyses of scaling. These data will further inform our studies on Rga4 regulation and function.

All in all, the proposed research will lead to discovery of new fundamental mechanisms underlying cell-size dependent morphogenetic plasticity in eukaryotic cells and inform our thinking of the underlying biological and engineering principles of cellular organization.

Planned Impact

Training workforce for the UK economy
The proposed program will provide a framework for professional training of a postdoctoral researcher. She will master a highly desirable set of skills for modern biomedical research, such as proteomics, bioinformatics and microdevice engineering. The postdoc will acquire advanced communication and managerial skills, as well as gain more experience in supervising students. This comprehensive training will maximize her value as a skilled employee capable of making important contributions to the UK academia and industry on completion of her training in the PI's lab.

Education and public engagement
The program will allow us to make a lasting impact in the education sector. We will supervise summer and term research projects of undergraduate students from King's (at least 1 per year). In addition, we will offer one-week placements to children who attend state-funded schools within the borough of Camden (1 per year). The first-hand experience in modern research will raise students' awareness of different science professions and enhance their educational opportunities. Of note, the PI's laboratory has an established track record of hosting undergraduate (~35) and school (5) students both in Singapore and the UK, with most students choosing to study biomedical sciences further.

The concept of scale engages imagination, from Gulliver's Travels to Alice in Wonderland to Kandinsky's exploration of geometric rules for painting. Together with a British designer, researcher, and educator Mike Thompson (Thought Collider), we will develop a performative artwork to explore the relationship between size and shape in biological and man-made systems. Our vision is to engage our audience in the creative process, to maximize learning interactions on both sides. We plan for a two-tier approach starting with engagement at the Crick Manby Gallery to prototype, develop and refine our project, eventually taking it to a wider audience through local community (e.g. Somerstown Festival of Cultures or Cally Fest) or the national festivals. This will provide opportunities for conversation and deeper engagement with the artwork and the underlying scientific questions.

Together, these activities will contribute to scientific education in the UK and make fundamental cell biology a part of a wider cultural landscape.

Medicine and agriculture
Our focus on understanding morphogenetic plasticity in a fungal system should deliver long-term impacts in medicine and agriculture. Diseases caused by ascomycete fungi cause significant morbidity and mortality in humans. In plants, ascomycetes are responsible for devastating crop diseases such as rice blast and grey mould. Fungal diseases claim a huge socioeconomic burden, e.g. human fungal diseases in the European Union cost up to 50,000 EUR per patient, depending on the duration of the follow-up (PMID: 24026863). Rice blast typically causes 10-30% loss of grain, staggering numbers given that ~50% of the world's population relies on rice for their primary caloric intake (PMID: 22471698). Understanding biology of fungal pathogenesis is vital from societal and economic viewpoints. Although S. japonicus has a saprophytic lifestyle, it exhibits major morphogenetic plasticity including cellular geometry scaling and nutrient- and stress-triggered yeast-to-hyphae transitions. Fungal pathogens rely on similar morphogenetic events to infect their hosts and we believe that our results will be of use to researchers working on cellular aspects of fungal pathogenesis. To maximize the impact of our work in this field, we will publish our results as open-access papers in journals with widest readerships possible and reach out to relevant academic and industry scientists in London and elsewhere in the UK to discuss areas of common interest with a view of establishing collaborations, e.g. through BBSRC-funded Industrial CASE Partnerships and London Interdisciplinary Doctoral Programme.
 
Description Related to Aim 1, we established the sequence of Rga4-dependent events during normal growth and scaling. We discovered that in addition to regulating Cdc42, Rga4 regulates the MAP kinase cell wall integrity pathway through Rho2. In line with Rga4 negatively regulating Rho2, S. japonicus cells lacking Rga4 exhibit elevated levels of MAP kinase signalling. To probe this aspect of Rga4 function, we generated and characterized a panel of molecular reagents in the pathway, including pmk1 deletion mutant (lacking MAP kinase), functional Pmk1-sfGFP fusion, the downstream protein kinase C Pck2-sfGFP, an artificial Pck2 activity sensor, functional intramolecular insertional fusion with sfGFP of Rho2, etc. Currently we are evaluating cortical dynamics of Pck2, as a readout for Rho2-MAPK axis, in rga4 and other GAP mutants, and testing biochemically if Rga4 functions as a GAP for Rho2. We further established the architecture of Tea4-Dis2-Pom1 signaling unit in S. japonicus and showed that Rga4 must be able to bind to the phosphatase PP1/Dis2 to execute its function in polarized growth and scaling. We identified a specific PP1/Dis2 binding mutant (L478A, F480A). We showed that Rga4 GAP activity and the regulation by the Tea4-Dis2-Pom1 axis are required for its dynamic behavior and steady state distribution at the cellular cortex.
We made a striking observation that as long as Rga4 localizes to the plasma membrane, it can function both at cell sides and cell tips to execute its functions in normal growth and scaling. Briefly, we constructed a set of targeting tools, including a general plasma membrane tether (ectopic Pom1-KD), equatorial cortex tether (Mid1) and a tip tether (Tea1-mCherry-GBP) and used them to retarget a cytosolic active version of Rga4 to specific locations in the wild type and a panel of Tea4-Dis2-Pom1 signaling unit mutants. Rga4-GAPdead mutant was used as a control. We also constructed nanobody tethers for other cortical domains, including those based on Mtl2, Pma1, Fhn1 and Bud6 (also used in Objective 2.1).

Related to Aim 2, we identified two CDK1 phosphorylation sites (S144, S175) on Rga4, close to the GAP domain. The CDK1-dependent phosphorylation promotes Rga4 activity and mobility at the cortex. Introducing phosphonull mutations disables Rga4. Interestingly, whereas the phosphomimetic Rga4 (S144D, S175D) mutant can function at the lateral cortex, it can no longer support polarized growth when constitutively targeted to the close vicinity of the phosphatase PP1/Dis2. Together with results obtained in Aim 1, these data suggest that Rga4 is regulated by a tug-of-war between CDK1 and PP1/Dis2. Phosphorylation of Rga4 may lead to its conformational changes, modulating the formation of Rga4 complexes with its partners in a spatially distinct manner. In addition, we have identified two further scaling specific Rga4 phosphorylation events. One, a decrease in phosphorylation at positions S385 and S387, occurs at early stages of scaling. The other is an increase in phosphorylation at position S674 in fully scaled (smaller) cells. However, mutating these sites either to phosphonull or phosphomimetic versions was not sufficient to produce defects in scaling or in supporting cellular geometry during normal growth, likely due to the complexity of the system. What is clear, however, is that the CDK1 kinase and PP1/Dis2 phosphatase regulate Rga4 function. Thus, we decided to concentrate our efforts on understanding this regulatory axis.

We are now working on a manuscript describing these results.

Our studies have additionally led us to an exciting series of observations related to the metabolism of cells undergoing scaling due to nutrient restriction. Briefly, we discovered that compartmentalization of the last steps of lysine and histidine biosynthesis in peroxisomes is required for efficient scaling and population growth upon nutrient restriction. Our results suggest that there exist an upper limit on peroxisome size beyond which the competing enzymatic reactions sharing the common NAD+/NADH co-factor cannot function. This work has recently been recently published ( Gu et al, 2023. Peroxisomal compartmentalization of amino acid biosynthesis reactions imposes an upper limit on compartment size. Nature Communications. doi: 10.1038/s41467-023-41347-x). This aspect of our work further highlights the utility of S. japonicus as a model for scaling, beyond cellular polarity changes.

This award has also contributed to other papers from the lab, as outlined in Publications.
Exploitation Route This Wee1 phosphoproteomics dataset will be of high value to scientists working on both cellular polarity and cell cycle. Our recent studies will be of interest to researchers working on cellular metabolism and peroxisomal function.
Sectors Agriculture

Food and Drink

Education

Healthcare

 
Title JaponicusDB 
Description We have deployed the open-source, modular code and tools originally developed for PomBase, the S. pombe model organism database (MOD), to create JaponicusDB (www.japonicusdb.org), a new MOD dedicated to S. japonicus. By providing a central resource with ready access to a growing body of experimental data, ontology-based curation, seamless browsing and querying, and the ability to integrate new data with existing knowledge, JaponicusDB supports fission yeast biologists to a far greater extent than any other source of S. japonicus data. Although the bulk of this work was funded by Wellcome Trust (103741/Z/14/Z), both S. Oliferenko (the PI on this award) and Y. Gu (the postdoctoral researcher funded through this award) have spent substantial time on testing and curating JaponicusDB. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact JaponicusDB enables S. japonicus researchers to realize the full potential of studying a newly emerging model species and illustrates the widely applicable power and utility of harnessing reusable PomBase code to build a comprehensive, community-maintainable repository of species-relevant knowledge. 
URL https://www.japonicusdb.org/
 
Description Nate Goering lab 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Participated in the development of new method for spectral autofluorescence correction
Collaborator Contribution The Goering lab spearheaded the development, whereas we contributed to the validation of the algorithm
Impact Published a paper in Development (2022) 149 (14): dev200545. https://doi.org/10.1242/dev.200545
Start Year 2021
 
Description PomBase team (University of Cambridge) 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been the driving force behind building and maintaining the model organism database for S. japonicus (https://www.japonicusdb.org/). Additionally, we have been responsible for literature and datasets curation.
Collaborator Contribution The PomBase team at the University of Cambridge deployed the open-source, modular code and tools originally developed for PomBase, the S. pombe model organism database (MOD), to create JaponicusDB. Currently, they are helping us to maintain it.
Impact We have published a paper outlining the creation and the utility of JaponicusDB in Genetics: https://doi.org/10.1093/genetics/iyab223
Start Year 2021
 
Description The Bahler lab 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution In the process of working on the process of nutrient-dependent scaling and other physiological processes in S. japonicus, we have realized the extent to which its central carbon and energy metabolism differs from its cousin S. pombe. Metabolism ultimately feeds into every aspect of organismal physiology. We have spearheaded an effort to understand the evolutionary divergence of central carbon metabolism within the fission yeast clade (bioRxiv. doi: https://doi.org/10.1101/2022.12.29.522219, accepted in principle to Current Biology)
Collaborator Contribution Advice and help, in particular on gene expression and bioinformatics.
Impact No outputs yet but we are planning to submit at least one story later this year.
Start Year 2017
 
Description The Balasubramanian lab 
Organisation University of Warwick
Department Warwick Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution We have collaborated with the Balasubramanian lab at the University of Warwick Medical School to address two aspects of actomyosin ring constriction during cytokinesis: 1) the mechanical cues underlying ring disassembly; and 2) the roles of actin and myosin II turnover in this process. We have contributed our expertise in molecular genetics of the fission yeast Schizosaccharomyces japonicus - all S. japonicus strains used in these papers were made by our lab. The use of S. japonicus as an excellent model system for ring constriction has been inspired directly by our work and through our communications with Mohan Balasubramanian. We also contributed to discussing the results, preparing them for publication, editing and revising the papers. We continue collaborating with the Balasubramanian lab on understanding actomyosin ring organization and function - we are now finalizing another collaborative paper.
Collaborator Contribution The Balasubramanian lab has been the driving force for this collaboration - they have performed the bulk of biochemical and cell biological experiments published in these papers.
Impact doi: 10.7554/eLife.21383 doi: 10.1083/jcb.201701104
Start Year 2015
 
Description The Nieduszynski lab 
Organisation Earlham Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have contributed to two papers led by the Nieduszynski lab: 1) on telomere-to-telomere assembly of S. japonicus genome (doi: 10.1002/yea.3912), where we have contributed to functional annotations and interpretation of genome assembly results, and co-wrote the paper; 2) on the assignment of a new fission species, S. versatilis (doi: 10.1002/yea.3919), where we've validated experimentally the existence of a species barrier between S. versatilis and S. japonicus, contributed to functional annotation and interpretation of S. versatilis genome assembly results, and co-wrote the paper
Collaborator Contribution Long read sequencing, assembly and bioinformatic analyses of S. japonicus and S. versatilis genomes
Impact Two papers: doi: 10.1002/yea.3912 and doi: 10.1002/yea.3919
Start Year 2022
 
Description 3 Days of Fat 
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 The PI has participated as an expert in 3 Days of Fat', with a British designer, researcher, and educator Mike Thompson. This was conducted as a series of art-science experiments, and a follow-up podcast. Mike and his artistic collaborator Arne Hendriks explore the nature of fat and our relationship with this material by constructing the Fatberg and using this man-made island of fat as a device to engage and provoke the audiences.

We continue our collaboration.
Year(s) Of Engagement Activity 2019
 
Description Participation in an open day or visit at my research institution - Undergraduate research project 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Participation in an open day or visit at my research institution - Undergraduate research project
Year(s) Of Engagement Activity 2022
 
Description Undergraduate research project 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Undergraduate research project of a King's undergraduate student, Nuru Msallam, supervised by a BBSRC-funded postdoctoral fellow Ying Gu. Nuru's project dealt with investigating the role of the Rho2 GTPase in the geometrical scaling in S. japonicus (Aim 1 of the grant).
Year(s) Of Engagement Activity 2023
 
Description Undergraduate research project 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact We are currently hosting an undergraduate student who is developing automated image analysis to analyze S. japonicus geometry.
Year(s) Of Engagement Activity 2021
 
Description Undergraduate summer research project 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact We hosted a summer student, Alexander Colton, for two months last summer. He worked on understanding the requirements for anaerobic growth of S. japonicus.
Year(s) Of Engagement Activity 2022
 
Description Visits by school children and undergraduates 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We hosted several lab visits of A-level students and undergraduates. We have additionally hosted longer placements for 2 school children and several summer and term projects, mostly for King's undergraduates (16 in the last 5 years). Many students chose to study biomedical sciences further.
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019,2020,2021