Size Matters: A systems approach to understanding cell size control in a developing multicellular tissue

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

Cells are the building blocks of tissues, but how 3D structures are built from cells is a fundamental unsolved problem in biology. It is even more complex when we consider a growing tissue, in which the physical properties of the building blocks (cells) change constantly as they grow and divide.

We study the growing shoot tip or shoot meristem of the model plant Arabidopsis. This structure is required to produce new leaves and flowers as the plant grows. The early stages of organ development require efficient tissue growth and an increase in cell size is normally observed at this time. Over several years, we have developed techniques allowing us to image the meristem over extended periods in the confocal microscope. We can follow individual cells over time and determine their growth and division. In order to divide, a cell goes through a defined series of processes known as the cell cycle. Using fluorescent reporters we have developed, we can for the first time simultaneously determine the position of all cells in the cell cycle during imaging time courses of growing plant tissues.

Our recently published study showed that the size cells reach when they divide is on average consistent for a given tissue and set of environmental conditions, but is highly plastic when these change. For example we found that cells were smaller, and tissue growth slower, when plants were grown under environmental conditions that restrict photosynthesis. Variation in cell size arises through unequal division and has to be removed. This is done by establishing a balance between growth and division on a cell by cell basis. The plasticity of the system leads us to consider that cell size at division is not determined by a direct "cellular ruler" but is instead determined as a consequence (or "emergent property") of the contributing processes of growth and division. This mechanism appears to be conserved from unicellular organisms which can also achieve a larger cell size and higher absolute growth rate under plentiful conditions. Regulation of cell size in these simple cells is achieved by balancing cell growth and division via the protein synthetic capacity of the cell.

We have developed a model that can predict accurately the size of plant cells based on the rate of growth and the accumulation of activity of two regulatory proteins required for cell division (called CDKs) as the cell grows. We tested this extensively using different mutants and growth conditions and identified the key processes that lead to cell size control. These processes appear to be the production and thresholding of CDK activity. In this proposal we will identify the "sizer" molecules involved in these processes that establish the link between growth and division of cells and analyse their function using our state-of-the-art imaging and analysis techniques. We have a number of candidate sizers, with known roles in CDK regulation, but we will also carry out experiments to identify new candidates in an unbiased manner using a genome-wide approach based on identifying the rate at which proteins are being synthesized under different conditions. We will use a combination of experiments and mathematics to develop a model that will allow us to understand how these sizer molecules are regulated and what effect this has on cell size control.

Technical Summary

The shoot apical meristem (SAM) is the source of all above-ground plant growth and hence central to plant development and productivity. It is a shallow dome of continuously dividing cells that initiates rapidly growing organ primordia on its flanks that develop into flowers or leaves. Cell size is tightly regulated in all organisms, and in mitotically active tissues is determined by the opposing forces of cell growth and division. However, until recently was cell size control was poorly investigated in the important SAM tissue due to technical challenges. By combining time-lapse confocal imaging of live SAMs carrying a new reporter of cell cycle phase, we have recently shown that cell size in the SAM is dependent on developmental stage, genotype and environmental signals (Jones et al., Nature Comms 8:15060; 2017). Notably we found that low light conditions, which limit photosynthesis, led to a reduction in cell size across the SAM. We furthermore demonstrated that cell size at division could be accurately predicted by a minimal two-stage cell cycle model in which either the production or threshold of CYCLIN DEPENDENT KINASE (CDK) is cell size dependent. Here we seek to identify the "sizer" molecules that link cell growth to division and determine cell size. We have identified candidates which we will test, and use to build predictive models of cell size at division based on ordinary differential equations. We will test these predictions by manipulating sizer levels and changing environmental conditions. We will also use ribosomal footprinting to identify potential new sizers in an unbiased way and use cell level determination of protein synthesis rates to understand the link to cell growth parameters.

Planned Impact

Key beneficiaries are identified as:
- Academic researchers and scientists, particularly plant scientists, but also the broader community of cell and developmental biologists, as well as systems biologists interested in predictive modelling of biological systems
- Agronomists and crop breeders
- Industrial researchers, including life scientists, mathematicians and computer scientists
- School pupils
- Members of the public.
Beneficiaries will be engaged throughout the project in order to deliver a range of economic and societal impacts.

1. Economic Impacts
Underpinning Knowledge: This project will advance understanding of plant growth and underpin the development of strategies allowing fuller exploitation of plant metabolism for sustainable agriculture. This impact is likely to be realised beyond the grant period through the integration of cell growth and division into models of plant development at different scales. To ensure this takes place, we will actively engage with life scientists, mathematicians and computer scientists from academia and industry and use knowledge gained from such interactions to inform implementation of our models with future compatibility in mind. These interactions will also generate awareness of our work and allow us to explore future collaborations to apply our models. Professional interactions will primarily be through academic conferences and publications, including regional and national meetings at which commercial researchers are brought together (e.g. meetings of UK Plant Sciences Federation, and those organised by Innovate UK and Welsh Government).
Enhancing research capacity: The fundamental nature of the question addressed means that we do not anticipate that there will be any immediate opportunities to commercialise our work. We will however develop techniques and tools that will contribute to the knowledge and skills of public and private research groups. For example, we will develop a protocol for measuring protein synthesis in plants using a proprietary product of ThermoScientific with their support, which will allow a broader range of researchers to access this tool. Models, transgenic lines and gene constructs generated by the work may similarly be of use to third parties and upon publication we will be freely available.
Skilled staff: Staff employed in this project will receive valuable interdisciplinary training contributing to the highly skilled workforce required to build and sustain the knowledge based economy. Notably the project will use advanced mathematical modelling, live cell imaging and next generation sequencing techniques, all skills in high demand in the biosciences sector. Furthermore all the researchers will gain experience of collaborative, multidisciplinary research and will be encouraged to develop project management and communication skills valuable for work in a variety of settings.

2. Societal Impacts
Improved teaching and learning: We will use the interdisciplinary nature of the team as an opportunity to develop an activity that can be used to promote both plant biology and mathematics/computer programming in secondary schools with the aim of increasing uptake of these subjects at university and filling skills gaps. We will target schools where fewer than average students go on to further education.
Public awareness and understanding of science: Improved public understanding of plant biology is necessary to allow members of the public to participate in an informed manner in ongoing debates including the use of genetically modified plants. Public opinion regarding genetic modification is likely to determine how fundamental knowledge is translated into improved agricultural sustainability. We will use the project as a means to engage with the general public and promote a greater awareness of current plant research. All team members will participate in public engagement events and so aid with the dissemination of scientific knowledge.

Publications

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Jones AR (2019) Double or Nothing? Cell Division and Cell Size Control. in Trends in plant science

 
Description Progress has been made into quantifying the expression patterns of eight candidate sizer molecules by confocal microscopy. Initial datasets have been collected, but further data will be collected once the initial data has been analysed by the modelling PDRA to be appointed in April 2020.
Crosses between fluorescent reporters of candidate sizer and cell cycle markers have been started and are being analysed for homozygous lines.
A Golden Gate system for producing GR-fusion proteins has been developed. The first construct made using this system have been transformed into plants and selected. Induction of subcellular changes in localisation are now being tested in these lines.
Large quantities of plant material are required for transcriptomics and ribosomal profiling in this project. Seeds have been bulked up and pilot experiments have been run to optimise the harvesting conditions. Large scale growth and collection will begin in the next months.
Exploitation Route Too early to say.
Sectors Agriculture, Food and Drink

 
Description At this early stage of the project, the objectives of the project have been communicated by the PI (Murray) and R-CoI (Jones) to the academic community through departmental visits and conference attendance. As the work progresses, particularly with the development of the mathematical model, these contacts will be used to identify possibilities to link our models with existing models of plant growth and metabolism. We have also focused on communicating our research to the public and to school pupils. We have particularly focused on using the interdisciplinary nature of this project to encourage engagement from school pupils and undergraduate students. We have prioritised the development of resources that combine computer programming and plant physiology through the creation of time-lapse movies of plant development. These resources are now ready for use on visits that are planned in the remaining years of the project.
First Year Of Impact 2020
 
Description Cardiff University Research Opportunity Placement - Size Matters: tracking cell size changes in growing roots
Amount £1,200 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 06/2019 
End 09/2019
 
Title GFP-GR fusions for quantifying protein movement 
Description A set of plant transformation vectors containing fusions of the GR domain to GFP and mCherry proteins have been produced. Both N and C terminal fusions have been produced, with a range of linker regions designed to allow further cloning of genes of interest into the plasmid. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact This tool is allowing us to make a quantitative characterisation of protein movement following the application of DEX. Although the GR inducible system has been well described as a system for inducing gene transcription, we are interested in using the system to activity of proteins other than transcription factors. This requires a more detailed understanding of the localisation and concentration of the GR fusion proteins. This requires live cell imaging and the fluorescent protein lines we have created are essential to collecting this data. These vectors will be available following publication to other labs to whom they will be useful as controls and as the basis for cloning and studying genes of interest. We will also make the vectors available before publication to interested groups upon request. 
 
Title GR-GFP plant lines 
Description Wild type Arabidopsis (Col-0) plants stably transformed with GR-GFP vectors (see GR-GFP vectors record). 
Type Of Material Cell line 
Year Produced 2022 
Provided To Others? No  
Impact We are currently analysing these lines to quantify cytoplasmic to nuclear movement of protein upon the application of dexamthasone. This data will be used to optimise induction protocols and interpret phenotypic data. The lines will also serve as a useful control that will identify any non-specific effects of the induction system. 
 
Title Golden Gate GR system 
Description In order to manipulate gene expression in plant cells we are using the GR system. This system has previously been used in plants, but to make construction of the gene fusions required to use the system, we have cloned the GR domain using the golden gate system. This means that the GR domain can be easily fused to any gene of interest, with or without a fluorescent tag. We have cloned 4 genes of interest so far using this system and are currently evaluating the constructs and lines before publishing the clones and making them available. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact This tool has allowed us to make multiple inducible gene constructs and means that we will be able to rapidly test any further genes of interest identified in our project. We have shared the tool locally (within the department) and will make the golden gate plasmids generated for this system available to the community upon publication. 
 
Title Multicolour reporter lines (cell membrane, cell cycle, sizer) 
Description We have made genetic crosses between reporters for 6 of the potential sizer proteins being studied in this project (RBR, CYCD, CDKA, MYB3R3, MYB3R4, CYCB) and lines previously produced by the group that combine cell cycle markers (YFP or mCherry) and cell membrane markers (GFP or YFP). 
Type Of Material Cell line 
Year Produced 2021 
Provided To Others? No  
Impact We are currently using the triple reporter lines to quantify changes in expression and concentration of the potential sizer proteins during the cell cycle. The triple reporter lines are important as they allow dynamic data to be collected over timecourses and for protein concentration, cell size and cell cycle phase to be determined simultaneously. These lines will be useful to other researchers studying other tissues. We will make lines available upon publication or by request prior to publication. 
 
Description Plants at the Movies 
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 are developing a resource using Raspberry Pi computers to record plant growth in schools. The activity combines plant physiology with programming to look at dynamic processes, so is a good way for us to introduce the type of interdisciplinary work that we are doing in this research project with the next generation of scientists.
So far we have had two A-level students carry out a summer project in the lab to set up the equipment and test its use. The two pupils were funded through the Nuffield Summer studentship Scheme and both reported that their perceptions of plant science have changed since carrying out the project. The technology has now been used by two undergraduate students to create resources that they took into two A-level classrooms. Surveys that were carried out in the classroom identified that the activities using Raspberry Pi computers to create time-lapse movies of plants did increase students interest in plants, with some evidence that the use of this type of technology was more effective than carrying out other types of experiment. We plan to continue developing this resource during the remaining course of the project.
Year(s) Of Engagement Activity 2019,2020
 
Description Public Engagement - "Super Science Saturday" at the National Museum of Wales 
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 An interactive display on plant genetics was prepared and presented at "Super Science Saturday" an event organised by Cardiff University at the National Museum of Wales to showcase research. Participants were given the opportunity to extract DNA from strawberries. This gave us an opportunity to discuss plant genetics (both non-GMO and GMO) with members of the public. Having carried out the experiment many participants asked about the work that we do in the lab, were the techniques we use the same and what do we do with the DNA once it is extracted. This gave us the opportunity for us to discuss how we are interested in how changes in genes can affect the size of cells and tissues and how this might be important for agriculture - for example, many people were interested to know that during domestication of strawberries, the amount of DNA per cell has increased leading to an increase in size.
Year(s) Of Engagement Activity 2019
 
Description Public Engagement - "Venom" event at National Museum Cardiff 
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 An interactive display showing the scope of plant science research was prepared, including a hands on activity using microscopes to look at specialised plant structures including trichomes, spines and thorns. Members of the public (mainly families) were asked to look at the different structures and discuss how they are different. For example, trichomes as seen on nettle leaves are single cells, while thorns and spines are multicellular. This opened an opportunity for us to show pictures that have been taken as part of our research project in which we are interested in how cell growth and division is used to build structures.
The display was part of the National Museum of Wales' event focused on "Venom" and was therefore a good opportunity to ensure that plants were represented as an often overlooked area of biology. The Museum reported over 1000 visitors on the day, and received positive feedback about our contribution. We hope to work with the museum again in the future to organise a plant-focused event - perhaps to tie in with the Fascination of Plants Day in 2021
Year(s) Of Engagement Activity 2019