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High resolution cryo-electron microscopy of clathrin cage networks

  • Full or part time
  • Application Deadline
    Applications accepted all year round
  • Self-Funded PhD Students Only
    Self-Funded PhD Students Only

About This PhD Project

Project Description

In cryo-electron microscopy, the superior signal sensitivity of new direct electron detectors has revolutionised the field of structure determination 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.

Our aim is to understand how the proteins involved in the network of clathrin and its adaptor proteins interact to achieve clathrin-coated vesicle formation. Clathrin-mediated endocytosis is a fascinating mechanical phenomenon that drives the selective internalisation of molecules into cells. Nutrient uptake, synaptic vesicle recycling, signalling, determination of cell polarity and development all rely on endocytic mechanisms. In disease, viral and bacterial pathogens exploit endocytosis to gain entry into cells and malfunctions lead to tumour formation, neurodegeneration and heart disease. In order to work properly, clathrin-mediated endocytosis 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.

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.

Techniques that will be undertaken during the project:
◾High resolution electron microscopy
◾Image analysis of large data sets
◾3D imaging of structural data
◾Kinetic analysis using light scattering, fluorescence and single molecule methods.
◾Protein mutagenesis, expression and purification

References

The revolution will not be crystallized: a new method sweeps through structural biology.
Callaway E.
Nature. 2015 Sep 10;525(7568):172-4. doi: 10.1038/525172a.

A method for detergent-free isolation of membrane proteins in their local lipid environment.
Lee SC, Knowles TJ, Postis VL, Jamshad M, Parslow RA, Lin YP, Goldman A, Sridhar P, Overduin M, Muench SP, Dafforn TR.
Nat Protoc. 2016 Jul;11(7):1149-62. doi: 10.1038/nprot.2016.070.

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly. Young A, Stoilova-McPhie S, Rothnie A, Vallis Y, Harvey-Smith P, Ranson N, Kent H, Brodsky FM, Pearse BM, Roseman A, Smith CJ. Traffic (2013) Sep;14(9):987-96.

Rothnie A, Clarke AR, Kuzmic P, Cameron A, Smith CJ. A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin. Proc Natl Acad Sci (2011) 108 pp 6927-32.

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