Supra-molecular rules in signalling networks: A single molecule comparative study in cells and tissues
Lead Research Organisation:
Science and Technology Facilities Council
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
Organisations
- Science and Technology Facilities Council (Lead Research Organisation)
- Francis Crick Institute (Collaboration)
- Oxford NanoImaging (Collaboration)
- Swinburne University of Technology (Collaboration)
- D. E. Shaw Research (Collaboration)
- DIAMOND LIGHT SOURCE (Collaboration)
- UNIVERSITY OF OXFORD (Collaboration)
- AstraZeneca (Collaboration)
- EVOTEC (Collaboration)
- Howard Hughes Medical Institute (Collaboration)
- Yale University (Collaboration)
- Rosalind Franklin Institute (Collaboration)
- University of Washington (Collaboration)
Publications
Abd Halim KB
(2015)
Interactions of the EGFR juxtamembrane domain with PIP2-containing lipid bilayers: Insights from multiscale molecular dynamics simulations.
in Biochimica et biophysica acta
Clarke DT
(2019)
A Brief History of Single-Particle Tracking of the Epidermal Growth Factor Receptor.
in Methods and protocols
Clarke DT
(2011)
Optics clustered to output unique solutions: a multi-laser facility for combined single molecule and ensemble microscopy.
in The Review of scientific instruments
Coban O
(2015)
Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.
in Biophysical journal
Hirsch M
(2019)
A global sampler of single particle tracking solutions for single molecule microscopy.
in PloS one
Hirsch M
(2013)
A stochastic model for electron multiplication charge-coupled devices--from theory to practice.
in PloS one
Ho J
(2019)
Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
in Nature communications
Hussain F
(2011)
CAR modulates E-cadherin dynamics in the presence of adenovirus type 5.
in PloS one
Kiuchi T
(2014)
The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility.
in Science signaling
Lee R
(2013)
Gene expression profiling of endobronchial ultrasound (EBUS)-derived cytological fine needle aspirates from hilar and mediastinal lymph nodes in non-small cell lung cancer.
in Cytopathology : official journal of the British Society for Clinical Cytology
Loeffler HH
(2013)
Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain.
in Proteins
Martin-Fernandez M
(2018)
Fluorescence Localisation Imaging with Photobleaching at 5 nm Resolution Reveals the Architecture of Basal EGFR Complexes and Mechanisms of Autoinhibition and Activation
in Biophysical Journal
Martin-Fernandez ML
(2021)
A brief history of the octopus imaging facility to celebrate its 10th anniversary.
in Journal of microscopy
Martin-Fernandez ML
(2013)
A 'pocket guide' to total internal reflection fluorescence.
in Journal of microscopy
Martin-Fernandez ML
(2023)
A perspective of fluorescence microscopy for cellular structural biology with EGFR as witness.
in Journal of microscopy
Martin-Fernandez ML
(2012)
Single molecule fluorescence detection and tracking in mammalian cells: the state-of-the-art and future perspectives.
in International journal of molecular sciences
Martin-Fernandez ML
(2012)
Human epidermal growth factor receptor (HER1) aligned on the plasma membrane adopts key features of Drosophila EGFR asymmetry.
in Biochemical Society transactions
Martinière A
(2012)
Cell wall constrains lateral diffusion of plant plasma-membrane proteins.
in Proceedings of the National Academy of Sciences of the United States of America
Neat MJ
(2013)
ALK rearrangements in EBUS-derived transbronchial needle aspiration cytology in lung cancer.
in Cytopathology : official journal of the British Society for Clinical Cytology
Needham SR
(2014)
Structure-function relationships and supramolecular organization of the EGFR (epidermal growth factor receptor) on the cell surface.
in Biochemical Society transactions
Needham SR
(2015)
Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution.
in Biochemical Society transactions
Needham SR
(2013)
Measuring EGFR separations on cells with ~10 nm resolution via fluorophore localization imaging with photobleaching.
in PloS one
Needham SR
(2016)
EGFR oligomerization organizes kinase-active dimers into competent signalling platforms.
in Nature communications
Roberts SK
(2012)
Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy.
in Biochemical Society transactions
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 | Public |
Country | United Kingdom |
Start | 08/2018 |
End | 09/2021 |
Description | STFC Proof of Concept |
Amount | £250,000 (GBP) |
Organisation | Science and Technologies Facilities Council (STFC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2015 |
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 | Private |
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). The addition of a FIB-SEM through a BBSRC award has enhanced this collaboration. Although access has been limited due to COVID-19, the microscope was used for a correlative EM project on COVID, in collaboration with eBIC as part of the wider correlative initiative. |
Start Year | 2017 |
Description | Collaboration on correlative microscopy development |
Organisation | Francis Crick Institute |
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). The addition of a FIB-SEM through a BBSRC award has enhanced this collaboration. Although access has been limited due to COVID-19, the microscope was used for a correlative EM project on COVID, in collaboration with eBIC as part of the wider correlative initiative. |
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). The addition of a FIB-SEM through a BBSRC award has enhanced this collaboration. Although access has been limited due to COVID-19, the microscope was used for a correlative EM project on COVID, in collaboration with eBIC as part of the wider correlative initiative. |
Start Year | 2017 |
Description | Collaboration on correlative microscopy development |
Organisation | Rosalind Franklin 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). The addition of a FIB-SEM through a BBSRC award has enhanced this collaboration. Although access has been limited due to COVID-19, the microscope was used for a correlative EM project on COVID, in collaboration with eBIC as part of the wider correlative initiative. |
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). The addition of a FIB-SEM through a BBSRC award has enhanced this collaboration. Although access has been limited due to COVID-19, the microscope was used for a correlative EM project on COVID, in collaboration with eBIC as part of the wider correlative initiative. |
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). The addition of a FIB-SEM through a BBSRC award has enhanced this collaboration. Although access has been limited due to COVID-19, the microscope was used for a correlative EM project on COVID, in collaboration with eBIC as part of the wider correlative initiative. |
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 |
Country | United Kingdom |
Sector | Private |
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 | US20180120313 |
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 | 2010,2011,2012,2013,2014,2015,2016,2017,2018,2019,2021,2022 |
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 |