Integrated microscopy approach to protein assembly on and in membranes

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

Proteins are key building blocks and the working horses of the living cell. They generate energy, make muscles contract, and translate light in our eyes into an image as perceived by our brain, to name but a few of many examples. In many cases, proteins need to form larger assemblies to carry out their biological function. Incorrect assembly causes biological malfunction and disease. Hence there is a general interest to understand how proteins form such larger assemblies. However, though we have an extensive toolkit to determine the structures of proteins and their assemblies, it is much harder to trace the processes by which these assemblies are being built.

In this project, we will therefore develop new methodology to visualise protein assembly at length and time scales that will enable us to determine how such processes take place. Because the therefore required resolution can not be achieved by one technique alone, we will use a combination of different microscopy techniques: Single-molecule fluorescence microscopy, which can track individual labelled proteins as they move about; atomic force microscopy, which can visualise protein assemblies as they are being formed; and electron microscopy, which can provide static snapshots of the structure of proteins and protein assemblies.

To test this correlative and integrated microscopy approach, we will apply it to a protein (named perforin) that is used by the immune system to attack virally infected and cancerous cells in the human body, essentially by perforating the membranes that protect cells from their environment. Perforin drills holes in these membranes by forming assemblies of several tens of proteins that span the membrane.

We aim to understand the mechanisms of membrane pore formation by these proteins, out of a fundamental interest in how this nanometre-scale machinery works, to better understand diseases or enhanced vulnerability to cancer caused by malfunctioning perforin, and because mechanistic understanding can facilitate the design of drugs that prevent such pores from being formed when the immune response needs to be suppressed transplant, for example during organ transplantation.

Technical Summary

While life scientists have an extensive toolkit to determine the structures of proteins and protein assemblies, it remains a significant and fundamental challenge to determine the pathways via which large biomolecular machines are assembled from individual molecular components. To provide a comprehensive time-resolved and molecular-scale view of such assembly processes, we propose here an integrated microscopy approach in which the same sample can be transferred between single-molecule (super-resolution) fluorescence microscopy, atomic force microscopy (AFM), and electron microscopy (EM).

Specifically, we will use novel grid materials to form supported lipid model membranes and use these to image protein assembly on and in membranes via these different microscopy techniques on the same sample. Single-molecule fluorescence microscopy and AFM allow for real-time imaging of protein assembly as it happens, but struggle to image highly mobile proteins and protein assemblies at sufficient spatial resolution for structural analysis (e.g., number of subunits in an assembly). By arresting protein assembly in such measurements at any chosen time point and transferring the sample to EM, we can obtain the required spatial resolution from static snapshots of mobile proteins and protein assemblies.

The power of this approach will be demonstrated by elucidating a crucial assembly process in human immune defence: The transformation of soluble perforin monomers into assemblies that punch holes into target cell membranes, focussing on the yet unknown initial stages of assembly on the membrane and on the pathways from such early assembly into membrane-perforating pores.

Planned Impact

This proposal aims at developing new methodology to study protein assembly on and in membranes. This methodology will be of wide applicability to gain understanding of protein self-assembly, which can facilitate the exploitation of self-assembly processes to develop new biomaterials for healthcare and energy applications (e.g., biomimetic approaches for efficient photosynthetic materials).

A more specific and shorter-term outcome of this project will be a better understanding of membrane pore formation by perforin, as well as the potential to achieve similar understanding for other pore forming proteins. Perforin is the main weapon of natural killer cells. It punches holes in virally infected or cancerous cells that have been detected by the immune system, and delivers lethal granzymes through these holes. Babies born with defective perforin succumb to viral infections or tumours early in life. On the other hand, if perforin is too active, normal cells can be incorrectly killed.

Perforin is part of the superfamily of membrane attack complex/perforin (MACPF) and cholesterol-dependent cytolysin (CDC) proteins, which are of significant medical importance. The CDC perfringolysin O rapidly induces irreversible cellular injury in a deadly form of gangrene that is caused by the bacterium Clostridium perfringens. The CDC pneumolysin is a major virulence factor of Streptococcus pneumoniae, at the root of bacterial pneumonia, still a major cause of death and illness throughout the world despite the widespread use of antibiotics. When released in the lungs, pneumolysin damages the lung tissue and its blood vessels. Antibiotics may exacerbate lung damage because they are designed to kill the bacteria by breaking them open, which leads to the further release of pneumolysin.

A better understanding of MACPF/CDC membrane pore formation can create new opportunities for drug design: The ability to control the activity of MACPF proteins in the human immune system, for example, could be an important means of regulating the immune response during and after tissue and organ transplantation or could alleviate the perforin-dependent cytotoxicity in autoimmune diabetes. The prevention of pore formation by CDC pneumolysin would be a significant advance in the treatment of bacterial pneumonia.

As already emphasised before, the here proposed methodology is not restricted to pore-forming proteins alone. It can be applied to a variety of other medically relevant interactions between membranes and macromolecules. Examples of this are antimicrobial peptides that are currently investigated as new therapies against bacterial infections, as well as pH-sensitive polymers that are used for intracellular drug (e.g., gene and RNA therapies) delivery across the membrane. We anticipate that the development of such novel therapeutic approaches will be enhanced by molecular-scale understanding as can be achieved with the methods outlined in this proposal.

This research will thus benefit pharmaceutical industry and biomedical and biotechnological SMEs developing new therapeutic approaches and biomaterials. On the longer term (taking into account the lead times for drug development), it will have an impact on healthcare practitioners and patients.

Further impact can be achieved on the technology for microscopy. In particular, we closely collaborate with AFM manufacturers, beta-testing new instrumentation and AFM probes. Our research at the cutting edge of AFM technology helps manufacturers to identify current limitations and direct their R&D efforts accordingly. The research in this proposal will thus benefit AFM manufacturers and their representations in the UK.

Publications

10 25 50
 
Description In facilitating the continuation of previous research, this grant has helped us to explain how the immune system kills virus-infected and cancerous cells, involving the drilling of holes in target cells by a protein called perforin.
Exploitation Route We collaborate with the Peter MacCallum Cancer Centre in Melbourne, where they use insights obtained in our work to find new cures against diseases involving deficient perforin.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The methods developed in this grant have been used in a joint project with MedImmune (see MedImmune collaboration), to quantify drug-receptor binding.
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC ALERT
Amount £173,000 (GBP)
Funding ID BB/R000042/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 08/2018
 
Description MRC Research Grant
Amount £442,227 (GBP)
Funding ID MR/R000328/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 06/2021
 
Title Correlative microscopy method 
Description We have developed and applied a method to analyse protein binding to supported lipid bilayers across microscopy platforms 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact We used this method to determine the rate-limiting step in the assembly of the membrane attack complex, one of the molecular machines of the immune system to kill Gram-negative bacteria. We have also determined the pharmacokinetic parameters for drug binding to its cell surface receptor, in collaboration with MedImmune Ltd. Results are expect to be published over the coming year. 
 
Description AstraZeneca collaboration 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Our partner is interested in DNA strand-break repair as is required for the proliferation of tumour cells, in particular when treated by e.g. radio-/chemo-therapy. They have an anticancer drug on the market that suppresses this repair process to the detriment of the tumour cells. We use single-molecule biophysics approaches, as developed refined in various research-council funded projects, to identify the mechanism by which this drug interferes with DNA strand-break repair, with the aim to provide our partner with a better scientific understanding, which can in turn facilitate further optimisation of their drug in terms of efficacy and selectivity for cancer cells.
Collaborator Contribution They provide the background knowledge on the biology and medical context for this research. They provide materials (purified proteins, inhibitor drugs) and expertise.
Impact The collaboration is in a too early stage (only few months on-going).
Start Year 2017
 
Description Crick collaboration 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution I have initiated a joint project with Crick and AstraZeneca (see under AstraZeneca collaboration) and am co-supervising a PhD project.
Collaborator Contribution Scientific expertise. Access to equipment.
Impact Too early to state.
Start Year 2017
 
Description Industrial collaboration with AFM manufacturer Bruker Nano 
Organisation Bruker Corporation
Department Bruker Nano
Country Germany 
Sector Private 
PI Contribution Following successful high-resolution atomic force microscopy (AFM) imaging, we have signed a joint development agreement with world-leading AFM manufacturer Bruker Nano (formerly Veeco, formerly Digital Instruments), on testing and developing protocols on prototype AFM equipment. Bruker Nano contributes in-kind to this project. Our work with their instrumentation and probes has provided Bruker with AFM images of the DNA double helix (and protocols for acquiring these), as well as an assessment of probe tip sharpness (both by benchmarking on DNA and on assemblies of pore forming proteins.
Collaborator Contribution Provision of and access to latest commercial AFM equipment (including beta-version pre market release), provision of AFM proves, all at zero or greatly reduced price.
Impact Multidisciplinary - involving engineering, physics and biology. Outcomes of broad and general use are protocols and instructions for double-helix-resolution imaging of DNA in liquid, see, e.g., a webinar on this (http://www.bruker.com/service/education-training/webinars/afm.html), which helps AFM manufacturer Bruker and its representation in the UK. Technical feedback from our side has helped Bruker to optimise its products before and after market release. Another outcome is the visualisation of membrane lesions and of prepore-state bacterial toxins diffusing on a membrane surface, elucidating the pathways of membrane pore formation by bacterial toxins, as well as understanding of mechanism by which antimicrobial peptides (potential next-generation antibiotics) attack bacteria.
Start Year 2012
 
Description MedImmune 
Organisation MedImmune
Country United States 
Sector Private 
PI Contribution Imaging of cell surface receptors, to help MedImmune (Cambridge) understand the mechanisms by which monoclonal antibody drugs target cell surface receptors.
Collaborator Contribution Technical advise, PhD student supervision, purified proteins, financial contributions (cash) to support research.
Impact The results are largely confidential, but include understanding of the working mechanisms of monoclonal antibody drugs targeting cell surface receptors. We have acquired single-molecule kinetic data of these drugs binding to their receptors embedded in membranes, where the idea is that such kinetic data inform further drug development.
Start Year 2014
 
Description National Physical Laboratory 
Organisation National Physical Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided the technology to visualise how next-generation antibiotics (de-novo designed antimicrobial peptides) attack bacteria.
Collaborator Contribution Design of antimicrobial peptides. Expertise.
Impact Multidisciplinary, involving physicists, chemists, bioengineers, and biologists. This collaboration has helped to put forward various antimicrobial peptides as (potential) next-generation antibiotics.
Start Year 2010
 
Description Peter MacCallum Cancer Centre 
Organisation Peter MacCallum Cancer Centre
Country Australia 
Sector Academic/University 
PI Contribution Insights in the pathways of membrane pore formation by perforin, a component of the human immune system.
Collaborator Contribution Scientific advise and purified proteins.
Impact We have determined the self-assembly mechanism by which the immune protein perforin forms pores in cancerous and virus-infected cells in our bodies (published in Nature Nanotechnology). This has given an indication for explaining how patients with perforin deficiencies show enhanced susceptibility to blood cancer. In addition, on-going research has highlighted reasons why certain cancer cells may be more or less susceptible for immune clearance in latest cancer immunotherapies.
Start Year 2012
 
Description University of Leicester 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Insights in mechanism of bacterial toxins that are subject of drug development programmes in Leicester
Collaborator Contribution Provision of purified proteins.
Impact Insights in pathways of membrane pore formation by the bacterial toxin suilysin (published in eLide).
Start Year 2012
 
Description Utrecht collaboration 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution We carry out nanoscale characterisation of life bacteria as they are attacked by immune proteins (complement) in serum, with the aim to determine which are the mechanism by which the immune system clears our body from harmful bacteria.
Collaborator Contribution Scientific expertise, different strains of bacteria, purified proteins.
Impact We have identified a new role of certain immune enzymes in killing bacteria. This can now be used to guide immune therapies to bacterial infections.
Start Year 2016
 
Description Yale collaboration 
Organisation Yale University
Department School of Medicine
Country United States 
Sector Academic/University 
PI Contribution We have carried out computational and microscopic analysis of nanometre-scale filters that the Yale team build to emulate the super selective properties of the nuclear pore complex, which governs nuclear import/export in our cells, and provided scientific expertise in conceiving experiments.
Collaborator Contribution The Yale team provided the original design and materials, and provided scientific expertise.
Impact This has resulted in a publication in ACS Nano (2018) and generated a platform with which we can now aim to better understand underlying biology.
Start Year 2016
 
Description Cafe Scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact 30 students learned about biological physics in a pub.
Year(s) Of Engagement Activity 2016
 
Description Lecture for school children - UCL Science Centre 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact >100 6-formers and teachers intended my presentation on the use of physics in understanding molecular machinery of life.
Year(s) Of Engagement Activity 2017