Peter Rosenthal

*Video coming soon*

Structural analysis of organelle structure and dynamics in situ by electron cryomicroscopy

Our group studies the architecture of large protein assemblies in order to understand basic molecular mechanisms that control protein and membrane traffic in the cell and in virus infection. Recently, the technique of cryomicroscopy (cryoEM) has made revolutionary advances in its ability to image biological specimens at high resolution by singl+e particle analysis or tomography. In this project the student will apply cryotomography to study the high-resolution architecture of the cell.

The endothelial cells that line the inner surfaces of blood vessels play roles in hemostasis, thrombosis, and inflammation by secretion of the large, multimeric blood glycoprotein von Willebrand factor (VWF). VWF has multiple ligands and on acute release, functions as an adhesive protein to bind platelets to sites of vascular injury. Defects in VWF and its storage are responsible for bleeding disorders including von Willebrand's disease. VWF is stored for rapid exocytic release in specialized secretory organelles called Weibel-Palade bodies (WPBs). Our studies have focused on two fundamental questions posed by VWF trafficking: How can a long multimeric protein be organized for dense storage in WPBs, and how can the packaging of this protein determine the identity and morphology of this unique secretory granule? Both these questions will be addressed by high-resolution studies of VWF tubule architecture and their interactions in the dense paracrystalline environment of the granule interior. In addition, we are interested in a structural and physical understanding of how VWF is released when granules fuse with the plasma membrane during exocytosis.

The student may also investigate the architecture of filopodia, which are thin, finger-like, projections that extend from the surface of mammalian cells and are thought to have roles in formation of cell-cell contacts and neuronal path-finding. They are ideally suited to high-resolution structural studies by a combination of light and electron microscopy. The central core consists of a cross-linked actin filament bundle along which myosin motor proteins walk, carrying various molecular cargoes out to the extreme tip of the filopodium. Using single molecule, super resolution light microscopy we can visualise and then localize individual proteins with nanometre precision and then, using cryo-electron tomography we can image the same, rapidly frozen, specimen in its native state in order to obtain high-resolution structural information. We wish to solve the structure of an individual filopodium and study the dynamics of individual proteins and protein complexes both in live cells and in rapidly frozen cells.

Please note that only one studentship is available with this group and the precise project will be decided on consultation with the supervisor.

1. Berriman, J. A., Li, S., Hewlett, L. J., Wasilewski, S., Kiskin, F. N., Carter, T., Hannah, M. J. and Rosenthal, P. B. (2009)
Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells.
Proceedings of the National Academy of Sciences of the United States of America  106: 17407-17412. PubMed abstract

2. Sader, K., Stopps, M., Calder, L. J. and Rosenthal, P. B. (2013)
Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electron-optical effects of charging.
Journal of Structural Biology  183: 531-536. PubMed abstract

3. Rosenthal, P. B. (2015)
From high symmetry to high resolution in biological electron microscopy: a commentary on Crowther (1971) 'Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs'.
Philosophical Transactions of the Royal Society B: Biological Sciences370: 20140345. PubMed abstract

4. Calder, L. J. and Rosenthal, P. B. (2016)
Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.
Nature Structural & Molecular Biology  23: 853-858. PubMed abstract