Directed control of secretory vesicle fusion

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Control of volume and osmolarity - and of turgor in plants and fungi - lies at the very core of cellular homeostasis in all eukaryotes. In plants and fungi, strongly electrogenic H+-ATPases, and the substantial membrane voltages they foster, drive solute accumulation to generate steep osmotic gradients and turgor pressure for cell growth. Vesicle traffic adds surface area for cell expansion and contributes to wall remodelling as the cell grows. The transport of solutes (especially of K+ ions) must be controlled in concert with secretion for survival and to determine organismal form. Despite their fundamental importance we know little of how cells coordinate the rates membrane traffic and solute transport.

This proposal builds on the discovery of new subsets of secretory and transport proteins that occur in the genomes of all plants described to date and, in the few species examined, are known to interact with one another. In Arabidopsis these interactions contribute to transport regulation, osmotic solute uptake and affect growth; in tobacco uncoupling these processes leads to hypotrophic cell growth and uncontrolled tissue expansion similar to that of a number of plant diseases (e.g. clubroot in Brassicas, scab disease in potato). The findings point to a basal level of coordination between secretion and transport for co-regulation of the two processes.

The findings also indicate a potential mechanism by which secretion may be controlled. The transporter binding partners - a subset of ion channels - include semi-autonomous voltage-sensor domains (VSDs) that move in response to voltage. This movement is known to activate/deactivate the channels, coordinating their activity with all other transporters in the membrane. Secretory protein binding occurs at a conserved site on the VSDs, suggesting that voltage may affect secretion directly. Coupling to membrane voltage is especially significant, because voltage reports on the activity of all solute transport across the plant plasma membrane while governing solute accumulation and, hence, cell turgor and expansion.

I am very excited by these findings. They offer critical evidence of a molecular mechanism that clearly will help unravel the connection between ion transport and secretion in plant growth. Furthermore, they support an entirely new model for regulated secretory traffic that will rewrite the textbooks on membrane traffic in eukaryotic cells. Until recently VSDs were thought unique as components and modulators of a few, well-studied families of ion channels in prokaryotes and eukaryotes, and of a small group of voltage-sensitive and membrane-bound phosphatases in marine tunicates. In each of these instances, however, the VSDs are incorporated as an integral part of the native protein structure; no examples of voltage-related control through direct, VSD binding have surfaced until now. Our evidence to date suggests that voltage-driven movement of the VSDs have been 'hijacked' to function as voltage sensors for this subset of plant SNAREs. Thus my working hypothesis is that the VSDs, through their binding to the secretory protein partners, govern vesicle traffic much as they do the activity of the channels. I now propose to test key elements of this hypothesis. This project will fully characterize the voltage-dependence of VSD binding in order to assess its association with voltage-dependent channel activity and secretion. I also propose selective manipulation and analysis of the interactions between the proteins, modifying VSD movement and secretory protein binding to determine the effects on secretion. Not only will the the results further our understanding of the link between osmotic solute transport and control of cell turgor and growth in plants, but they will also yield crucial information about what is clearly an entirely new mechanism linking membrane traffic with other physiological and pathological processes in plants.

Technical Summary

This proposal builds on our discovery of a novel set of protein-protein interactions between conserved subsets of vesicle trafficking and ion channel proteins. We have shown that the interaction is essential for channel activity, K+ uptake and growth in Arabidopsis; we have since uncovered a complementary role in facilitating secretion. Thus the activities of these proteins appear to be coordinately regulated through interaction. We have localised the binding sites on both sets of proteins, demonstrating binding in Arabidopsis and tobacco, and have confirmed that these sites are highly conserved among the orthologous proteins of land plants. For the channels - the inward-rectifying subfamily of Kv-like K+ channels of plants - the binding site localises to the voltage sensor domains (VSDs) that are known to move within the membrane in response to voltage.

Coupling to membrane voltage is especially important, because the voltage both drives and reports back on the activity of all transporters at the membrane, hence governing solute accumulation and turgor. It is an obvious signal for tempering secretory traffic such as during cell expansion. We know that secretion is affected by mutant VSDs with altered voltage sensitivities. What we do not know is whether the effects on traffic depend on binding/debinding with voltage or whether, when bound, VSD conformation affects secretory activity through the partner trafficking (so-called SNARE) proteins. I am also intent on bridging the gap in knowledge between these molecular events and the effects on cell and tissue growth that we have demonstrated by uncoupling the coordination between ion transport and secretory traffic. Here I propose a combination of molecular, cell biological and physiological studies to address these issues. Regardless of the outcomes, it is clear that understanding the dynamics of these interactions will inform on the mechanisms underpinning cell turgor and volume control in plants.

Planned Impact

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international plant and cell biology communities. The research will stimulate thinking about the interface between traffic and transport in cell growth, plant development and pathology, and it should facilitate a paradigm shift in approach. These studies will also extend recent developments by the MRB laboratory of novel assays and imaging tools for molecular interaction analyses. Thus, the research is expected to benefit fundamental researchers as well as industry through conceptual developments as well as the introduction of new technologies for the analysis of multicomponent systems. The research will feed into higher education training programmes through capacity building at the postgraduate and postdoctoral levels. Additional impact is proposed through public displays and the development of schools resources building on the background work for this proposal. Finally the research will help guide future efforts in applications to agricultural/industrial systems. MRB has established links with industrial/technology transfer partners (Agrisera, Dualsystems, Plant Bioscience) and research institutes (JHI and JIC) to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the attached Impact Pathways.

Publications

10 25 50
 
Description This project has yielded a number of 'firsts'. Most important, it has uncovered (1) an entirely new mechanism at the cell level for coupling between secretion and solute accumulation. These findings address a century-old problem in cell biology of how plants regulate cell volume and turgor. In addition (2), the work has yielded an unexpected role for a regulatory (so-called SM) protein in the secretory cycle. These proteins have long been recognised to regulate the 'out-going' steps in secretion, but we can now show that they also play a role in the 'in-going' or recycling steps as well. Both findings are clearly set to rewrite the textbooks in cell biology.
Exploitation Route This is fundamental research, but could well help in guiding future efforts towards improved crop production as well as disease control.
Sectors Agriculture, Food and Drink,Other

 
Title 2in1 vector systems 
Description Synthetic biology vectors for transient and stable transformation with quantitative visual reporting on cell-by-cell basis 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Computer software tool for quantitative measurement and analysis of root growth/development 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software for electrophysiology and imaging data aquisition and analysis 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Multicistronic vector systems 
Description Synthetic biology vector systems for transient and stable transformation for expressing multiple, tagged proteins and for quantitative analysis of membrane traffic and transport 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title OnGuard 
Description Systems biology software for quantitative modelling of cellular transport and homeostasis 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SUS vector systems 
Description Synthetic biological vector systems for protein-protein interaction screening 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title Software tools for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Software for quantitative trait analysis and acquisition for root growth/development 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software package for electrophysiology and imaging data acquisition and analysis 
Type Of Material Data handling & control 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title OnGuard 
Description Quantitative systems biology modelling of cellular transport and homeostasis 
Type Of Material Computer model/algorithm 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SDM-assist 
Description Software for molecular primer design that enables introduction of silent markers for molecular cloning 
Type Of Material Data analysis technique 
Year Produced 2013 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Description PBL 
Organisation Plant Bioscience Limited Technology
Country United Kingdom 
Sector Private 
PI Contribution IPR on ABA receptor technology and ABA signalling
Collaborator Contribution Funding related to IPR on ABA receptor technology and ABA signalling
Impact Multiple outcomes in publications and industrial contacts
 
Description PSG 
Organisation POSCO - South Korea
Country Korea, Republic of 
Sector Private 
PI Contribution Base support for meetings and exchange of materials
Collaborator Contribution Base support for meetings and exchange of materials
Impact Base support for meetings and exchange of materials
 
Title Software tools and packages for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Technology Software 
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Description International online services 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Prof. Blatt and members of his laboratory have contributed to various media events over the years, including online interview contributions (e.g. People behind the Science, a US-based media program)
Year(s) Of Engagement Activity Pre-2006,2006,2008,2011,2015,2016,2017,2018
 
Description Invited presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I regularly speak to audiences, from small groups (5-20) to large audiences (>1000) in a variety of settings. In addition to teaching and extramural activities associated with the university, I also speak on invitation to national and international groups a number of times each year and in a variety of settings, academic as well as public. I also reach audiences through short video presentations mounted on the web, these primarily via my laboratory website and the ASPB websites. Anyone reading this entry is welcome to visit these sites to learn more.

The impacts arising from my presentations are varied. For example, a common consequence of my speaking in academic settings is to attract potential researchers to visit my laboratory and, frequently, to interest potential collaborators and students/postdocs to my research group. At scientific meetings, my talks often attract interest also from researchers interested in the various tools and materials that my research has produced, including the various vector systems and software packages that I
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Schools and displays 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact As these were multiple events, this question is not informative or useful. Participants varied from numbers in the tens to several thousands

Extensive training of participating laboratory members as well as broad scope reach to schools and communities, in the case of the GCC science days to the west of Scotland and in the case of the IFPD activities to audiences within and outside the UK
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk