Integrated microscopy approach to protein assembly on and in membranes

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.

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.

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.