Figure 1: Correlated fluorescence and electron microscopy can be used to locate a defined intermediate stage in endocytosis and extract quantitative information. This can be applied to multiple stages to understand the whole process (Kukulski et al. 2012).
Figure 2: 3D reconstructions of COPI coated vesicles obtained using cryo-electron tomography and sub-tomogram averaging (Faini et al. 2012)
The Briggs group develops and applies cryo-electron microscopy techniques to study the assembly mechanisms of enveloped viruses such as HIV and influenza, as well as coated trafficking vesicles.
Previous and current research
We study the structure and the molecular assembly mechanisms of important, pathogenic, enveloped viruses (e.g. HIV and Influenza virus), and of cellular trafficking vesicles (e.g. clathrin and COPI coated vesicles). Viruses and vesicles are extraordinary self-assembling machines – the protein building blocks interact with one another to bring all the components or cargo together, to reshape the lipid bilayer, and to release a free virus or vesicle. The building blocks will often then undergo an intricate reassembly to prepare for fusion with a target cell or membrane. The level of understanding we aim to achieve could be imagined as a 3D, functionally annotated movie, with molecular resolution, showing the assembly and budding process.
To reach this goal we need to obtain detailed structural information at different stages during assembly, ideally under close-to-native conditions, even within cells. This is a challenge for conventional structural biology tools, so we are also developing and applying new approaches for cryo-electron microscopy and tomography, correlated fluorescence and electron microscopy, and image processing. Members of the group have varied and complementary skills, including biochemistry, cell biology, physics, engineering and computer sciences.
HIV and Influenza viruses
A particular emphasis of our research is the structure and lifecycle of asymmetric membrane viruses such as HIV. Recently we were able to use cryo-tomography methods optimised in the lab to determine the structure of the immature HIV-1 capsid within heterogeneous HIV virus particles. Further details on our research into the structure and inhibition of HIV are available on our Molecular Medicine Partnership Unit webpage. We are also studying the structure and assembly of influenza virus.
We are studying the assembly mechanisms of coated vesicles both within cells, and using in-vitro reconstituted budding reactions. By applying correlative fluorescence and electron microscopy we are able to image defined intermediates during budding in cells. Using in vitro systems we are able to get detailed structural information on the arrangement of the coat proteins in the assembled vesicles. Together these methods are giving us important insights into how the clathrin and COPI machineries mediate vesicle formation.
The development of methods
How can we get detailed 3D structural information about variable membrane-containing systems such as influenza, HIV, or a COPI coated vesicle? These kinds of samples cannot be crystallised or studied using single particle cryo-electron microscopy methods. Instead we have been applying and further developing a combination of cryo-electron tomography and image processing by sub-tomogram averaging. We were recently able to show for the first time that this approach can resolve individual alpha-helices within proteins in heterogeneous systems. We are now applying these developments to a number of projects (see above). We make use of the world class facilities for cryo-electron microscopy at EMBL, and interact closely with companies to assess and apply new technologies.
How can we find and image dynamic intermediate timepoints during vesicle budding within cells? We have developed correlative fluorescence and electron microscopy methods that allow fluorescent signals from transient intermediates to be localised at <100 nm precision within cells and imaged in 3D by cryo-electron tomography. As part of this work, we have interacted with a commercial company to design and build a new cryo-fluorescence microscopy stage.
Future projects and goals
Our overarching biological goal is to understand the interplay between protein complexes, membrane shape and virus/vesicle structure. What kind of protein-protein interactions can drive virus assembly while maintaining structural flexibility? How do structural switches allow viruses and vesicles that have completed the assembly pathway to start disassembling? How do proteins induce the distortion of cellular membranes into vesicles of different dimensions? What are the similarities and differences between the variety of cellular budding events? How do viruses hijack cellular systems for their own use? How does the curvature of a membrane influence its interaction with particular proteins? We will also aim to generate detailed and specific information on the mechanism of HIV and influenza virus assembly. Our technical goal is to develop novel microscopy and image processing approaches that can be used to address these questions, but which can also be widely applied by other research labs.