Supra-molecular rules in signalling networks: A single molecule comparative study in cells and tissues

Lead Research Organisation: STFC - Laboratories
Department Name: Central Laser Facility (CLF)

Abstract

Signalling is the means by which proteins orchestrate basic intra-cellular activities and cell-to-cell communication, to regulate cell fate and to allow the development of multi-cellular organisms. To achieve a cohesive cell fate within a multi-cellular tissue, some proteins (receptors) are organised into groups at the cell surface (the plasma membrane) to function as antennas to detect extracellular chemical cues. From their position at the cell surface the receptors detect multiple inputs which they transduce across the plasma membrane to output signals in the cell interior. These signals are decoded, amplified and processed in the cell cytoplasm by intracellular signalling networks; some are subsequently transduced to the nucleus to initiate DNA transcription, replication. Then net effect is the determination of cell fate (growth, differentiation, etc.). Understanding how signal inputs and outputs are organised in protein signalling networks is one of the most fascinating questions in biology. The current dream is to derive methods that would allow the 'watching' of these network proteins in action and at atomic resolution to see details of their structure. This requires the addition of a 'time' dimension to structural biology so that the spatio-temporal parameters of all atoms in each protein can be described in detail. This is a huge challenge that in cell-free systems has begun to be partially addressed through dynamic experiments combined with molecular simulations. However, in cells, the functions of particular structural motifs are not just constraint by Brownian motions, energy landscapes and thermodynamics, but also by the local availability of partners in subcellular compartments and the boundary constraints imposed by cell environments, for example in the plasma membrane, with its 2D dimensionality, local curvature and electric fields. To understand protein function in cells observations have to be made in the only physiologically-relevant 'Laboratory', the cell. This adds many levels of complexity to an already vast challenge. Our programme of work is geared to understanding the intricate network signalling behaviour of cells in their physiological environments within tissues. We aim at describing the basic molecular ingredients, the signalling pathways and the supra-molecular structural and spatio-temporal rules regulating signalling outcomes. Our methods will be based on direct observation, 'watching' the multiple changes in the topology of interactions and its components with time, in conjunction with the modelling of behaviour at atomic resolution within a mathematical framework. Using molecular biology techniques in combination with optical methods, we can now annotate individual genes and gene products, screen for protein-protein, protein-DNA and small molecule interactions, and quantify dynamic changes. However, only single molecule-based imaging currently offers sensitive spatio-temporal detection in cells for low abundance protein interactions. This is beginning to bridge the gap between protein structure and function by allowing real-time quantitative observations of structural details, conformational intermediates, association and dissociation constants, diffusion rates, and rare events. Previous information on complex protein networks has been derived generally from high-throughput screens and/or single cell models using ensemble (averaged) technologies such as biochemical extraction followed by mass spectrometric analysis. The application of this information to understand at the molecular level the dynamic normal physiology in multi-cellular organisms and/or the pathogenetic basis of various disease states, among the heterogeneous human population is therefore limited. The approach we propose offers the means to understand and predict functional properties of cells from the changes in complex interactions between their microscopic molecular components and in response to perturbations.

Technical Summary

The proposed programme of work represents a major technological development because it employs a, thus far unavailable, pioneering technique combination that will, for the first time, provide high-resolution quantitative observations on the topology and composition of signalling complexes and the dynamics of the protein signalling network whilst at work in live cells and tissues. This will place us much closer towards a comprehensive understanding of ErbB signalling - from single molecule to the systems level. Pushing the frontiers of our understanding of signalling networks in the true physiological context requires the type of multidisciplinary approach here proposed. This will deliver in a comprehensive mathematical framework of systems modelling and prediction, integration of: wet-lab biology, several innovative optical/molecular detection techniques multidimensional single molecule microscopy, FLIM, hybrid single molecule-FLIM methods, challenging and novel data analysis algorithms, coarse-grained and molecular dynamics modelling. The methodologies to be employed are state-of-the-art and pioneering in Biology. The synergistic approach we propose will unleash the potential of each of the technologies in their application to the study of signalling networks by placing discrete groups of structure-function relationships in the context of the many thousands of possible combinatorial interactions. This is a step-change in the methods of exploitation of systems biology models because so far the only constraints available to develop systems models have arisen from putative interactions derived from high-throughput cell-free methods. Our approach is unique and will result in high-content conformational, stoichiometric, kinetic and dynamic information at the plasma membrane and the correlation with events inside the cell, being placed at the core of a mathematical systems analysis model that describes and predicts the behaviour of RTK signalling networks. Joint with BB/G007160/1

Publications

10 25 50

 
Description New methods: how to extract macromolecular structure at extreme resolution on cells (~ 6nm).
New methods to measure protein orientation on cells
New single molecule imaging methods.
New methods of analysing single molecule data to extract kinetic and dynamic parameters.
Creating a portfolio of fluorescent drugs to visualise the conformational effects of these drugs on their targets directly on cells
These developments, together with the super-resolution methods funded by BBSRC have been integrated in the Octopus facility at the Research Complex at Harwell through which they are disseminated to non experts at the earliest opportunity.

Key Scientific findings:
Structure of inactive and active oligomers of the Epidermal Growth Factor Receptor (EGFR) on cells at 6 nm resolution
The nature of the conformational change in these oligomers that triggers signal transduction
Structural determinants of EGFR oligomerisation at the plasma membrane
Exploitation Route I am pursuing proof-of-concept finding for STFC to translate my work into a clinical method for stratifying patients.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.thenakedscientists.com/HTML/content/interviews/interview/1687/
 
Description Besides increasing scientific knowledge, the findings and developments achieved in this grant have been combined together to create a multi user national imaging facility called Octopus based at the Research Complex at Harwell and utilised by over 50 groups in the country. In this way, the legacy of the BBSRC funding has been preserved and developed for the benefit of many other scientists, including many non-experts. The facility is continually used by UK academics and industry throughout the year and many non expert users benefit from access to state-of-the-art instrumentation and methods. Update March 2018: The research work and the techniques developed have led directly to two industrial collaborations, one with Oxford Nanoimaging on development of the FLImP technique on a compact instrument, with the ultimate aim of deploying it in the clinic, and the other with AstraZeneca on use of FLImP to characterise EGFR complex structure and its application to drug discovery and development. These collaborations build on an STFC Proof-of-Concept award that aimed to take the developments into the clinic, and both industrial partners are collaborators on an STFC CLASP proposal (outline proposal accepted, awaiting decision on full application). Added in March 2019: The CLASP proposal mentioned above was funded and the work is underway. Publications continue to be produced from this LoLa grant.
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Policy & public services

 
Description An automated macromolecular structure-based approach to personalised anti-cancer therapy
Amount £576,054 (GBP)
Funding ID ST/S000682/1 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 09/2018 
End 09/2021
 
Description STFC Proof of Concept
Amount £250,000 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 09/2017
 
Title Fluorescence Localization Imaging with Photobleaching (FLImP) 
Description A method to image protein complexes on cells (initially 10 nm resolution but now reaching 5 nm). 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Paper in Nat Comm under revision. Applications to use the method in the OCTOPUS facility. Collaboration with David Shaw Research. 
 
Title Octopus facility in the Research Complex at Harwell 
Description The grant funding has provided the basis for establishing the "Octopus" microscopy facility operated by STFC's Central Laser Facility in the Research Complex at Harwell. This facility offers multiple microscopy techniques for users through a peer-review process. BBSRC funding has supported the development of single molecule microscopy within Octopus. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Usage by multiple research groups (50 groups per annum), leading to approximately 100 publications in the last 5 years. 
URL http://www.clf.stfc.ac.uk/CLF/12266.aspx
 
Description Collaboration on correlative microscopy development 
Organisation Diamond Light Source
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Howard Hughes Medical Institute
Department Janelia Research Campus
Country United States 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Rosalind Franklin Institute
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Yale University
Country United States 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration with AstraZeneca on EGFR-drug interactions 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Provision of expertise in FLImP technique and its application to the study of EGFR.
Collaborator Contribution Expertise in drug development and potentially access to drug molecules for proposed CLASP grant programme (see below).
Impact AZ are setting up a single molecule system in house. They are partners on an STFC CLASP proposal that aims to exploit single molecule techniques (FLImP) for drug discovery and development (outline proposal accepted, awaiting decision on grant).
Start Year 2017
 
Description Collaboration with Oxford NanoImaging on Single Molecule techniques 
Organisation Oxford Nanoimaging
PI Contribution Provision of expertise in single molecule techniques (FLImP).
Collaborator Contribution Expertise in development of compact microscope systems.
Impact Joint proposal to STFC CLASP scheme for funding to develop single molecule techniques for targeted cancer therapies. Outline proposal accepted. Awaiting decision on award.
Start Year 2017
 
Description Collaborative work on EGFR mutants 
Organisation University of Washington
Country United States 
Sector Academic/University 
PI Contribution Use of single molecule techniques to study EGFR mutants in cells.
Collaborator Contribution Provision of a range of EGFR mutants.
Impact New insights into EGFR clustering and activation in cells. Manuscript in preparation.
Start Year 2011
 
Description Complementary methods for studying EGFR clustering in cells 
Organisation Swinburne University of Technology
Country Australia 
Sector Academic/University 
PI Contribution Provision of data on EGFR clustering from single molecule techniques.
Collaborator Contribution Provision of data on EGFR clustering using correlation imaging.
Impact New information on EGFR clustering in cells. A publication is in preparation.
Start Year 2010
 
Description MD simulations of EGFR 
Organisation D. E. Shaw Research
Country United States 
Sector Private 
PI Contribution Provision of single molecule data on EGFR to inform and validate molecular dynamics simulations.
Collaborator Contribution Advanced MD simulation of EGFR using "Anton" computer.
Impact Combination of simulation and single molecule data is providing insights into EGFR function in cells. Manuscript in preparation.
Start Year 2014
 
Description Preparation and characterisation of fluorescent tyrosine kinase inhibitors 
Organisation Evotec
Country Germany 
Sector Private 
PI Contribution Access to single molecule microscopy equipment and expertise. Co-supervision of a PhD student.
Collaborator Contribution Provision of chemistry preparation laboratories, expertise, and consumables. Co-supervision of PhD student.
Impact Availability of a new panel of fluorescent tyrosine kinase inhibitors for the investigation of drug activity and receptor function in cells. Training of a PhD student (ongoing).
Start Year 2012
 
Title Receptor tyrosine kinase biomarkers 
Description This patent protects the use of the FLImP method to characterise receptor complex structure in cells, and to use it to direct targeted therapies against cancer, on a patient-by-patient basis. 
IP Reference EP16725883.9 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This work has resulted in a collaboration with AstraZeneca and a joint proposal to the STFC CLASP scheme to further develop the method for use in the clinical setting.
 
Title "Biggles" single particle tracking software. 
Description Algorithm for global spatio-temporal tracking of single molecule data. 
Type Of Technology Software 
Year Produced 2013 
Impact Available for use by users of the Octopus facility, including users specifically requiring this software for analysis of data collected elsewhere. Manuscript in preparation. 
 
Title "Quincy" single molecule analysis software 
Description Bayesian segmentation algorithms for single molecule feature detection and tracking. 
Type Of Technology Software 
Year Produced 2012 
Impact Publication describing the software. Also enabled multiple publications arising from use of the Octopus facility in the Research Complex at Harwell. The software is available for use by facility users. 
 
Title FLImP single molecule analysis software 
Description The FLImP software analyses single molecule photobleaching tracks and determines inter- and intra-molecular separations with ~ 5 nm resolution. 
Type Of Technology Software 
Year Produced 2015 
Impact A number of publications using this technique, including two in Nature Communications. 
 
Description Harwell Campus Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Contribution to Harwell Campus open days, in which the laboratory was visited by 16000 members of the public. Special events were also held for schools and VIPs.

We set up and manned displays explaining our research.
Year(s) Of Engagement Activity 2015
URL http://harwellcampus.com/open-days/
 
Description Multiple school visits to Rutherford APpleton Laboratory and the Research Complex at Harwell 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Discussion with visitors about my research area and optical imaging in general.

Ongoing interest from schools in sending further pupils to the lab.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description Press releases describing aspects of my work 
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 Media (as a channel to the public)
Results and Impact Press releases attracted attention from regional and national media.

Requests for additional information received from a number of interested parties.
Year(s) Of Engagement Activity 2012,2013,2014