Want to do a PhD in my group? See below for details
Apply to the MIBTP PhD Programme or the MRC DTP in Interdisciplinary Biomedical Reseach for PhD projects on the structural biology of clathrin-mediated endocytosis
PhD Project 1
Gathering cargo for endocytosis using TPLATEs
Principal Supervisor: Professor Richard Napier, School of Life Sciences
Co-supervisors: Dr Corinne Smith, Dr Alex Jones, Professor Tim Dafforn
The formation of clathrin-coated vesicles at the plasma membrane of plant cells proceeds via either an adaptor protein 2 (AP2) complex or the early TPLATE complex (Zhang et al., 2015) which may later recruit AP2. The TPLATE complex is plant kingdom specific (Gadeyne et al., 2014), although some protists such as Dictyostelium spp. retain an ancestral version (Hirst et al, 2014). TPLATE is a mixed octamer and downregulation of any of its subunits blocks endocytosis, and mutants give rise to defective plant growth and development. It is clearly a fundamentally important complex.
When discovered, the TPLATE complex was shown to include some cargo proteins, including PIN1 and PIN2 (Gadeyne et al., 2014). PIN proteins are auxin transport proteins, and they are involved in establishing and maintaining morphogenic hormone gradients (Bennett, 2015).
We will prepare membrane-enriched samples using differential centrifugation and then optimise the use of SMALPs to solubilise intact and functional complexes in collaboration with Prof Tim Dafforn, Birmingham (Lee et al., 2016). TPLATE complexes will be purified using pull-down experiments. We will use proteomics to identify which proteins co-purify and gel to grid methods to collect TPLATE complexes ready for cryo-electron microscopy and structural determination. SMALP solubilisation is preferred over detergent-based solubilisation methods because it preserves a shell of native lipids around the target complex.
The project will not only define structures for the important TPLATE complex, but it will identify the interactions sites between TPLATE and PINs to help us understand how cargo proteins are selected for clathrin-mediated endocytosis.
PhD Project 2
High resolution cryo-electron microscopy of clathrin cage networks
In this project you will use high resolution 3D cryo-electron microscopy to visualise adaptor proteins binding to clathrin cages and biophysical approaches such as dynamic light scattering, time-resolved fluorescence anisotropy and isothermal titrating calorimetry to investigate how clathrin-adaptor interactions result in formation of a functional coated vesicle network. You will have access to the new Gatan K2 Summit direct detector and Jeol 2200FS 200kV transmission electron microscope provided by the Advanced Bioimaging Research Technology Platform and excellent facilities for biophysical analysis available within Warwick School of Life Sciences. This is a fabulous opportunity to apply cutting edge techniques to discovering how clathrin and its adaptor proteins drive clathrin mediated endocytosis.
Want to know more? Send me an e-mail on corinne dot smith at warwick dot ac dot uk to discuss your research interests
Why are we interested in clathrin-mediated endocytosis?
Clathrin-mediated endocytosis is a fascinating mechanical phenomenon which drives the selective internalisation of molecules into cells. It requires accurate and timely assembly of a clathrin lattice and coordination with a network of more than 20 adaptor proteins to form a coated vesicle which will be able to select molecules from the outside of the cell for delivery to specific destinations.This mechanism acts at many levels within eukaryotic organisms because it supports diverse functions such as nutrient uptake, synaptic vesicle recycling, signalling, determination of cell polarity and development. It is also exploited by viral and bacterial pathogens to gain entry into cells and malfunctions lead to tumour formation, neurodegeneration and heart disease.
We are using high resolution 3D cryo-electron microscopy and dynamic biophysical techniques to understand how the proteins involved in the network of clathrin and its adaptor proteins interact to achieve coated vesicle formation. The superior signal sensitivity of new direct electron detectors has revolutionised the field of structure determination by cryo-electron microscopy, allowing sub-4Å structures of challenging targets such as membrane proteins and ribosomes to be obtained without using X-ray crystallography. We are exploiting this improvement in capability to carry out high resolution structural analysis of clathrin cage complexes.
To find out more about our cryo-electron microscopy facilities visit Advanced Bioimaging Research Technology Platform
Dr Sarah Smith
Imaging Facility Manager
Current PhD students
Dr Alice Rothnie has now become a lecturer at Aston University
Dr Anna Young
Dr Sarah Batson
Dr Daniel Beck