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ERC

MEMBRANEATTACK Report Summary

Project ID: 294408
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Final Report Summary - MEMBRANEATTACK (Molecular and cellular imaging of membrane interactions in pathogen attack and immune defense)

We have studied the molecular and cellular mechanisms of action of membrane pore-forming proteins. These proteins are used by pathogens to modify or attack their host cells, and by the immune system for defence against invaders. They are produced as soluble, individual proteins, but then bind their target membrane and assemble into rings that punch holes through the membrane. Four substantial papers, jointly with collaborators at Birkbeck, in Melbourne and at the London Centre for Nanotechnology, reveal structures and assembly pathways for the largest family of pore-forming proteins, comprising the membrane attack complex/perforin and cholesterol-dependent cytolysin (MACPF/CDC) proteins. For the bacterial CDC toxin suilysin, cryo electron microscopy (EM) has been used to determine the three-dimensional (3D) structures of two membrane-bound forms of the toxin, a pre-pore intermediate state that is assembled on the membrane surface but has not yet formed a hole, and the final, pore form that has punched a hole through the membrane. The images revealed new details of the dramatic conformational changes upon pore formation. To complement these structural snapshots, real time atomic force microscopy was used to video this process, by scanning the membrane as the toxin molecules bind, assemble into arcs and rings, and then punch holes. Combined with modelling, these studies showed that CDC complexes insert into the membrane in a concerted mechanism and that membrane lipid is ejected when the holes are formed. In the second paper, we report structures of the fungal MACPF protein pleurotolysin. The monomeric, water soluble structures were determined by X-ray crystallography in Melbourne, and we did cryo EM of pre-pore and pore assemblies on membranes. The EM maps reveal new details of the membrane bound forms and computational modelling was used to trace a trajectory for the conformational change involved in the dramatic conversion from soluble protein to the oligomeric, membrane-inserted pore. This change entails the opening of an originally bent beta sheet, which extends to form the membrane-piercing barrel. The third paper revealed a higher resolution structure of complement C9 pore rings, a model for the membrane attack complex, used by the immune system to attack bacterial pathogens invading the bloodstream. The detailed structure determined corroborates our conclusions about the control of beta sheet opening during pore formation. The final main study of the molecular work was a dynamic study of the assembly of the major immune mediator perforin, showing a sequential mechanism for this MACPF protein, rather than concerted insertion seen for bacterial CDCs. In that study we also imaged early, loosely packed intermediates in perforin assembly.

In the later part of the project we determined 3D cellular structures in physiological systems that deploy pore forming proteins to puncture cell membranes. Papers reporting these studies are in preparation. We imaged cells infected with the intracellular pathogen Toxoplasma gondii, which must break through two enclosing membranes to infect new host cells as part of its life cycle. We directly visualized the membrane damage caused by the Toxoplasma perforin-like protein PLP1 and quantified its distribution by immuno-gold labelling. The second cellular project is on perforin mediated immune cell killing of infected or cancerous target cells. After training in the Melbourne lab, we set up the co-culture system of cytotoxic cells and target cells in order to visualise the immune synapses. Using electron and X-ray tomography, we obtained detailed 3D images of these transient intercellular contacts. X-ray cryo tomography is only available at a few sites world wide and has recently been developed at the Diamond Light Source. Although it gives lower resolution than EM, it enables 3D visualisation of intact, frozen-hydrated cells with good membrane contrast, and has provided a unique overview of the membranes and organelles at the immune synapse.

Reported by

BIRKBECK COLLEGE - UNIVERSITY OF LONDON
United Kingdom
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