Integrative imaging and electron cryo-tomography of viral transport mechanisms

To comprehensively understand a biological process or mechanism from molecule to cell it requires the combination of multiple techniques across a range of resolutions and scales. We currently rely on a variety of different approaches to highlight given aspects of biology that are then interlinked through interpretation. Intracellular transport is a process whereby vesicles and organelles are transported to different parts of cells with a precise spatial and temporal accuracy. This process is often hijacked by viruses, which utilize cellular machineries for active transport to and from the nucleus during their entry and egress. In this thesis I focus on the intracellular transport of adenoviruses, which are able to recruit and bind to the cytoskeletal motor, dynein, and use it to travel to the nucleus. I apply electron cryo-microscopy and fluorescence microscopy to elucidate the basis for this process, and link this to the behaviour of adenoviruses as they are recruited to microtubules. I then apply electron cryo-tomography to directly observe proteins in their native cellular environment. I discuss the ability of this technique to be utilized in the in situ determination of the structure of viruses as they infect a cell. I show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features and (ii) that novel, transient processes can be imaged with high levels of detail. As a second example of transport in cells, I study the nuclear egress of herpesvirus capsids, utilizing a multi-modal approach in an attempt to characterize this process from cellular to molecular level, and ask how herpesviruses modulate the nuclear membrane during capsid egress into the cytosol. Finally, I discuss an emergent technique in the field of electron cryo-microscopy, the Volta phase plate, its potential application in molecular tomography, and how it may enhance our current ability to discern contextual molecular mechanism.