Directional control of extreme polar growth in filamentous fungi
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
A key aspect of growth and development is the control of cell shape. The basis of this is the ability for a cell to establish and maintain polarity, each cell having a defined orientation. An extreme example are neurons, which are able to extend in a linear fashion over the long distances necessary for their role in signal transduction throughout an organism. Similarly filamentous fungi produce linear hyphae, which extend with branching, to form a network. The processes by which fungal cells and other Eukaryotes polarise to form a front are highly conserved, as such fungi represent a good model for understanding the fundamental biological processes that determine polarity.
In addition to providing a good model organism for other cells, the study of fungi is important in its own right. Fungi are the major cause of plant disease and food spoilage, being responsible for the loss of 10-20% of crop production worldwide. They are also important clinical pathogens, causing more deaths than either malaria or tuberculosis. However, fungi are of major benefit in industrial biotechnology, food production, as symbionts increasing crop yields and are fundamental to our ecosystem through biomass degradation and nutrient cycling. Hyphal growth is the means by which fungi explore and enter host tissue, and secretory enzymes can be released from hyphal tips during growth. As such understanding how hyphal growth is controlled has potential impact all the areas outlined above.
Polarity in all Eukaryotic cells is controlled by the same set of proteins, the Rho-GTPases. The action of these proteins is very versatile; in neuronal cells, for example, they create one front during axon formation, and multiple fronts during dendritic spine formation. The same set of proteins can orchestrate varying numbers of fronts for varying periods of time because of the differences between the interaction networks they are embedded within. In this project we will look at one such case, present at the hyphal tip, to understand how these very important Rho-GTPases are tuned to direct the extreme polarised growth. When fungi are grown in favourable conditions they tend have straight hyphae. A set of proteins has been found that is necessary for the straight hyphal morphology, termed 'cell-end-marker proteins'. The function of the cell-end-marker proteins and the Rho-GTPases, in directing growth, overlap. However, it is not yet known how the two sets of proteins interact and how the cell-end-marker proteins tune the Rho-GTPases to ensure persistent unidirectional growth.
In addition to providing a good model organism for other cells, the study of fungi is important in its own right. Fungi are the major cause of plant disease and food spoilage, being responsible for the loss of 10-20% of crop production worldwide. They are also important clinical pathogens, causing more deaths than either malaria or tuberculosis. However, fungi are of major benefit in industrial biotechnology, food production, as symbionts increasing crop yields and are fundamental to our ecosystem through biomass degradation and nutrient cycling. Hyphal growth is the means by which fungi explore and enter host tissue, and secretory enzymes can be released from hyphal tips during growth. As such understanding how hyphal growth is controlled has potential impact all the areas outlined above.
Polarity in all Eukaryotic cells is controlled by the same set of proteins, the Rho-GTPases. The action of these proteins is very versatile; in neuronal cells, for example, they create one front during axon formation, and multiple fronts during dendritic spine formation. The same set of proteins can orchestrate varying numbers of fronts for varying periods of time because of the differences between the interaction networks they are embedded within. In this project we will look at one such case, present at the hyphal tip, to understand how these very important Rho-GTPases are tuned to direct the extreme polarised growth. When fungi are grown in favourable conditions they tend have straight hyphae. A set of proteins has been found that is necessary for the straight hyphal morphology, termed 'cell-end-marker proteins'. The function of the cell-end-marker proteins and the Rho-GTPases, in directing growth, overlap. However, it is not yet known how the two sets of proteins interact and how the cell-end-marker proteins tune the Rho-GTPases to ensure persistent unidirectional growth.
Technical Summary
Cell polarity is fundamental to most biological systems and processes. In the case of filamentous fungi the highly polar growth of hyphae underpins many aspects of fungal biology, including development, colonisation of habitats, invasion of hosts, fungal interactions, reproduction and the secretion of proteins and chemicals. Currently, work on polar growth has focused primarily on the Cdc42 polar module which is conserved across eukaryotes. Less well characterised is the fungal specific TeaR/Mod5 polar module. In filamentous fungi both modules are present but to date no research has focused on how they work together. Another central and broadly significant question is how these modules can be modulated to produce distinct readouts. For example, transitioning from a single point of polar growth to multiple points, as is seen in neuronal development. A specific example of this is tip splitting which we will explore. Similarly, by understanding the attributes of specific polarity mechanisms, their biological consequences can provide new insights into important processes, as we have shown previously with respect to Cdc42 characteristics underpinning chemotropism in yeast.
Central to our work will be the creation of a 3D model describing growth as a direct consequence of protein dynamics and membrane insertion at the hyphal tip. This model will then be used as a tool to test and develop hypotheses, being refined by incorporating new data as it emerges. To facilitate phenotypic analysis we will use mathematical tools to measure hyphal morphologies, allowing quantitative characterisation and comparison of real and in-silico morphologies. Laboratory based work will include the identification and characterisation of proteins that interact with the polarity modules and a detailed assessment of module components with respect to localisation and dynamics within the cell and how these relate to phenotype.
Central to our work will be the creation of a 3D model describing growth as a direct consequence of protein dynamics and membrane insertion at the hyphal tip. This model will then be used as a tool to test and develop hypotheses, being refined by incorporating new data as it emerges. To facilitate phenotypic analysis we will use mathematical tools to measure hyphal morphologies, allowing quantitative characterisation and comparison of real and in-silico morphologies. Laboratory based work will include the identification and characterisation of proteins that interact with the polarity modules and a detailed assessment of module components with respect to localisation and dynamics within the cell and how these relate to phenotype.
Planned Impact
This project has potential impacts in health, agriculture & biotechnology & indirect impacts as a model for mammalian or plant cells. The technology, methodologies, computational tools, strains & data developed & produced during the course of this will be made freely available to the scientific community. Potential areas for impact are described below.
1.) Agriculture
1.1) Pathogenicity
Each year pathogenic fungi destroy crops that could feed 600 million people. The main defence is the use of fungicides. Good practice involves the rotation of fungicides but options are very limited. Excessive use leads to resistance which also impacts on their clinical use. Current fungicides focus on compromising cell wall maintenance or interfere with energy production. An under explored target is to disrupt polarity & filamentous growth. Filamentous growth is the route by which; pathogenic fungi enter host cells, secret virulence factors, obtain nutrients & invade new tissue. Developing fungicides to target filamentous growth could be a valuable strategy.
1.2) Symbiosis
Nitrogen, phosphorous, potassium & magnesium are important nutrients for plant fitness & crop production. Most worked soils lack sufficient nutrients. To improve nutrient levels large amounts of fertilizer are added, with commensurate environmental implications. Mycorrhizal fungi live symbiotically with the roots of numerous plants. They improve nutrient & water uptake, release nutrients to the plants via degradation of organic matter & improve disease resistance. As the plant/ fungal interaction is via fungal hyphae, a better underst & ing of hyphal growth could contribute to the development of more efficient symbiotic strains.
3) Health
3.1) Fungal infection
300 million people, worldwide, suffer from fungal infections every year, with a morbidity rate of 1.35 million. This death toll is comparable to diseases such as malaria & tuberculosis (1.2 & 1.4 million deaths/ year, respectively, www.gaffi.org). Invasive fungal infections are difficult to treat because of the conservation between fungal & host cells. By understanding better the mechanisms of polar growth it will be possible to inform production of drugs to be used.
3.2) Fungi as model organism
Mathematical modelling is a tool already proven to benefit drug development & it is commonly used in the drugs industry. Polarity & morphology defects are implicated in many diseases. For example, Alzheimer's is characterised by a build-up of amyloid plaques in the brain plasma of sufferers. These plaques result in an abundance of active Rho-GTPase within neuronal cells causing morphological defects in, & loss of, dendritic spines. The mathematical tools developed in this project can be used to investigate the morphological effects of drug-induced changes to underlying cell biological mechanisms controlling dendritic spine plasticity, for example.
4) Biotechnology
Filamentous fungi are of major importance in industrial biotechnology. They are used extensively in the production of bulk chemicals (eg 98% of citric acid production) & specialist high value products (eg antibiotics & statins), enzyme production both as a source & production organism (eg biomass degradation), heterologous protein production (eg pharmaceuticals) & directly in the food industry for the production of various fermented products. Hyphal morphology is of direct relevance: The morphology of the fungus is critical when developing high volume production in fermenters. Protein & metabolite secretion is often linked to the morphology, most secretion takes place at the hyphal tip. Therefore an understanding of how the tip is controlled & can be manipulated is of significant potential.
4) Education & outreach
The group will host & maintain a project website which will be linked from all members' University webpages. Material to promote biology, mathematics, & computer science to year 9, 10 & 11, students.
1.) Agriculture
1.1) Pathogenicity
Each year pathogenic fungi destroy crops that could feed 600 million people. The main defence is the use of fungicides. Good practice involves the rotation of fungicides but options are very limited. Excessive use leads to resistance which also impacts on their clinical use. Current fungicides focus on compromising cell wall maintenance or interfere with energy production. An under explored target is to disrupt polarity & filamentous growth. Filamentous growth is the route by which; pathogenic fungi enter host cells, secret virulence factors, obtain nutrients & invade new tissue. Developing fungicides to target filamentous growth could be a valuable strategy.
1.2) Symbiosis
Nitrogen, phosphorous, potassium & magnesium are important nutrients for plant fitness & crop production. Most worked soils lack sufficient nutrients. To improve nutrient levels large amounts of fertilizer are added, with commensurate environmental implications. Mycorrhizal fungi live symbiotically with the roots of numerous plants. They improve nutrient & water uptake, release nutrients to the plants via degradation of organic matter & improve disease resistance. As the plant/ fungal interaction is via fungal hyphae, a better underst & ing of hyphal growth could contribute to the development of more efficient symbiotic strains.
3) Health
3.1) Fungal infection
300 million people, worldwide, suffer from fungal infections every year, with a morbidity rate of 1.35 million. This death toll is comparable to diseases such as malaria & tuberculosis (1.2 & 1.4 million deaths/ year, respectively, www.gaffi.org). Invasive fungal infections are difficult to treat because of the conservation between fungal & host cells. By understanding better the mechanisms of polar growth it will be possible to inform production of drugs to be used.
3.2) Fungi as model organism
Mathematical modelling is a tool already proven to benefit drug development & it is commonly used in the drugs industry. Polarity & morphology defects are implicated in many diseases. For example, Alzheimer's is characterised by a build-up of amyloid plaques in the brain plasma of sufferers. These plaques result in an abundance of active Rho-GTPase within neuronal cells causing morphological defects in, & loss of, dendritic spines. The mathematical tools developed in this project can be used to investigate the morphological effects of drug-induced changes to underlying cell biological mechanisms controlling dendritic spine plasticity, for example.
4) Biotechnology
Filamentous fungi are of major importance in industrial biotechnology. They are used extensively in the production of bulk chemicals (eg 98% of citric acid production) & specialist high value products (eg antibiotics & statins), enzyme production both as a source & production organism (eg biomass degradation), heterologous protein production (eg pharmaceuticals) & directly in the food industry for the production of various fermented products. Hyphal morphology is of direct relevance: The morphology of the fungus is critical when developing high volume production in fermenters. Protein & metabolite secretion is often linked to the morphology, most secretion takes place at the hyphal tip. Therefore an understanding of how the tip is controlled & can be manipulated is of significant potential.
4) Education & outreach
The group will host & maintain a project website which will be linked from all members' University webpages. Material to promote biology, mathematics, & computer science to year 9, 10 & 11, students.
Organisations
Publications
Savage NS
(2021)
Describing the movement of molecules in reduced-dimension models.
in Communications biology
Title | A. nidulans strains |
Description | Numerous florescent tagged protein strains. Over 10 strains containing inducible promotors for adjusting the expression of various genes. Using both Tet and niiA promotors. |
Type Of Material | Cell line |
Year Produced | 2019 |
Provided To Others? | No |
Impact | We are hoping to publish in 2021. The strains will then be available to the entire community. |
Title | Many new Aspergillus nidulans strains |
Description | A number of split YFP strains which can be used to show co-localisation. Numerous florescent tagged protein strains. Over 10 strains containing inducible promotors for adjusting the expression of various genes. Using both Tet and niiA promotors. GEF inactive strains. |
Type Of Material | Cell line |
Year Produced | 2020 |
Provided To Others? | No |
Impact | We are hoping to publish in 2021. The tools will then be available to the entire community. |
Title | Method to model diffusion in reduced dimensions. |
Description | When addressing spatial biological questions using mathematical models, symmetries within the system are often exploited to simplify the problem by reducing its physical dimension. In a reduced-dimension model molecular movement is restricted to the reduced dimension, changing the nature of molecular movement. This change in molecular movement can lead to quantitatively and even qualitatively different results in the full and reduced systems. Within this manuscript we discuss the condition under which restricted molecular movement in reduced-dimension models accurately approximates molecular movement in the full system. For those systems which do not satisfy the condition, we present a general method for approximating unrestricted molecular movement in reduced-dimension models. We will derive a mathematically robust, finite difference method for solving the 2D diffusion equation within a 1D reduced-dimension model. The methods described here can be used to improve the accuracy of many reduced-dimension models while retaining benefits of system simplification. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Providing a tool to the modelling community which enables more accurate models of biological systems, while keeping the models simple. |
URL | http://nature.com/articles/s42003-021-02200-3 |
Title | strains containing florescent tags |
Description | Numerous florescent tagged protein strains. |
Type Of Material | Cell line |
Year Produced | 2017 |
Provided To Others? | No |
Impact | these will be available to the community once the work is published (2021) |
Title | Estimating diffusion in a lower dimension |
Description | This work is now under review. preprint: http://arxiv.org/abs/2011.01069v3 |
Type Of Material | Computer model/algorithm |
Year Produced | 2020 |
Provided To Others? | No |
Impact | This work will have impact once it is published. It will provide researchers with a way to accurately model diffusion in a larger number of cases. |
URL | http://arxiv.org/abs/2011.01069v3 |
Title | Two dimensional 'growth via secretion' model |
Description | In-silico Model of Cell Growth. I have developed a mathematical/computational model of cell growth. Within the model growth occurs when plasma membrane is added to the membrane of the cell during exocytosis. Exocytosis is directed by GTPase activity (and hence cytoskeletan nucleation). GTPase activation and localisation is modelled within the in-silico cell and thus an emergent property of the model. We are using the tool to understand the molecular control of cell morphology. |
Type Of Material | Computer model/algorithm |
Year Produced | 2017 |
Provided To Others? | No |
Impact | This is the first cell growth model of this type (that I am aware of). Because growth occurs at the point of secretion, which is itself under molecular control, this tool enables a direct link between molecular processes and morphology. The tool is innovative because growth is a result of membrane addition. Previous growth models have simply stretched (or expanded) the in-silico cell membrane at sights of intense GTPase activity (polarity patches). Such models do not take into account the interplay between the polarity patch and secretion itself. This interplay has been shown to be important for cell morphology. |
Title | growth model parallelized |
Description | The code for the 2D growth model has now been parallelized, and thus runs a lot faster - in 1 hour rather than 17. This is enables the use of the growth model for research as we run 10s and/or 100s of simulations, simulation number depending on the research question. |
Type Of Material | Computer model/algorithm |
Year Produced | 2017 |
Provided To Others? | No |
Impact | The code for the 2D growth model has now been parallelized, and thus runs a lot faster - in 1 hour rather than 17. This is enables the use of the growth model for research as we run 10s and/or 100s of simulations, simulation number depending on the research question. |
Title | wild type model of molecular control of fungal growth |
Description | Have generated a model of the molecular control of fungal growth. This model incorporates the two polarity mechanisms under question (Cdc42-BemA & TeaA-TeaR), and is thus the first of its kind. The model is now being used to test/generate hypotheses. |
Type Of Material | Computer model/algorithm |
Year Produced | 2018 |
Provided To Others? | No |
Impact | We are now generating/testing hypothesis using this model. |
Title | Method to model diffusion in reduced dimensions. |
Description | Code which enables the user to accurately model diffusion of molecules in 2D space within a 1D model. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2021 |
Open Source License? | Yes |
Impact | Code which enables the user to accurately model diffusion of molecules in 2D space within a 1D model. |
URL | https://www.nature.com/articles/s42003-021-02200-3 |
Description | One Week, full time, Work Placement |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Study participants or study members |
Results and Impact | A mature student (aged 21) shadowed, and worked alongside me for a week. They were be introduced the the biological theory behind the research, modelling and experimentation. They tested hypothesis via simulations. They also did some basic lab and microscope work. |
Year(s) Of Engagement Activity | 2018 |
Description | Speed Date Scientist 2019 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Peter Gould was the scientist this year (Natasha Savage did it last year). School students (from 'under privileged' schools) came to me in groups of 6. They would stay with me for five minutes. After five minutes a new group would arrive. Hence the event title, speed dating a scientist. During the five minutes I would introduce myself and tell them about my work (2 minutes). Then the student would lead the conversation (3 minutes). It was a fabulous event. The purpose of the event was to let the students know that it is entirely possible to be a scientist when you leave school. Plus, let them know what the day to day life of a scientist is, job satisfaction, etc. |
Year(s) Of Engagement Activity | 2019 |
Description | Speed Date a Scientist (2018) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | School students (from 'under privileged' schools) came to me in groups of 6. They would stay with me for five minutes. After five minutes a new group would arrive. Hence the event title, speed dating a scientist. During the five minutes I would introduce myself and tell them about my work (2 minutes). Then the student would lead the conversation (3 minutes). It was a fabulous event. The purpose of the event was to let the students know that it is entirely possible to be a scientist when you leave school. Plus, let them know what the day to day life of a scientist is, job satisfaction, etc. |
Year(s) Of Engagement Activity | 2018 |
Description | Speed Date a Scientist (2020) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | School students (from 'widening participation' schools) came to me in groups of 6. They would stay with me for five minutes. After five minutes a new group would arrive. Hence the event title, speed dating a scientist. During the five minutes I would introduce myself and tell them about my work (2 minutes). Then the student would lead the conversation (3 minutes). It was a fabulous event. The purpose of the event was to let the students know that it is entirely possible to be a scientist when you leave school. Plus, let them know what the day to day life of a scientist is, job satisfaction, etc. |
Year(s) Of Engagement Activity | 2020 |
Description | Visit to North Liverpool Academy |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | I was invited to visit North Liverpool Academy and talk to a group of their 6th form students about my journey (I am myself from a 'widening participation' background), and the science I am now directing/undertaking. |
Year(s) Of Engagement Activity | 2018 |
Description | Work Placement Student (Warrington Collegiate Institute) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | 1 pupil (aged 17) will shadow, and work alongside, a PDRA employed on this grant and myself for a week. They will be introduced the the biological theory behind the research, modelling and experimentation. My mother dies on the student's first day so the work placement did not take place. |
Year(s) Of Engagement Activity | 2017 |