Project Outline

Endocytosis via clathrin coated vesicles is a process fundamental to eukaryotic cell health and vitality, and the core proteins as well as the beautiful geometry of clathrin cages are highly conserved. The Smith group has used world-leading cryo-electron microscopy and structural biology to publish the highest resolution images of clathrin coated vesicles (CCVs) and their constituent proteins to date. In this project you will extend this work on animal CCVs to the plant kingdom, allowing you to compare and contrast the small structural details which have evolved to deliver the same cell biology in very different contexts.

In cryo-electron microscopy, the superior signal sensitivity of direct electron detectors has revolutionised the field of structure determination allowing sub-4Å structures of challenging targets. We are exploiting this improvement in capability to carry out high resolution structural analysis of clathrin cage complexes (Morris et al., 2019)

Clathrin-mediated endocytosis is a fascinating mechanical phenomenon that drives the selective internalisation of molecules into cells. In order to work properly, clathrin-mediated endocytosis requires accurate and timely assembly of a clathrin lattice. The cage complexes imaged so far have been from animals. The core proteins are highly conserved, and yet endocytosis in animals works in isotonic conditions whereas plant cells are under high turgor pressure. You will purify and examine in detail the structure of plant cages using high resolution 3D cryo-electron microscopy. You will visualise adaptor proteins binding to clathrin cages and use biophysical approaches such as dynamic light scattering, time-resolved fluorescence anisotropy and surface plasmon resonance (SPR, Biacore). This is a fabulous opportunity to apply cutting edge techniques to discover how clathrin and its adaptor proteins have evolved to drive clathrin mediated endocytosis in very different environments.

References

Techniques

• Protein expression and purification
• High resolution electron microscopy
• Image analysis of large data sets
• Structural biology
• Kinetic analysis using light scattering, fluorescence and single molecule methods.