Investigating the structure of CHC22 clathrin and its functional role in GLUT4 storage and glucose homeostasis

This project is available through the MIBTP programme on a competition basis. The successful applicant will join the MIBTP cohort and will take part in all of the training offered by the programme. For further details please visit the MIBTP website - https://warwick.ac.uk/fac/cross_fac/mibtp/

Regulation of blood glucose levels is vital for human health, and, where the body fails to achieve this, diseases such as diabetes result. The human body regulates blood glucose levels by transporting the glucose transporter 4 (GLUT4) to the surface of muscle and adipose tissue in response to postprandial insulin. Upon insulin signalling, GLUT4 is released by vesicle trafficking to the surface of muscle and fat cells to facilitate glucose import. Generation of an intracellular store of GLUT4 that can be rapidly mobilised in response to insulin and efficiently down-regulated is the key to ensuring that glucose is available for brain and muscle function during fasting but does not reach toxic levels after a meal. This joint PhD project aims to understand how the cellular transport protein clathrin (CHC22) functions to regulate the availability of the glucose transporter 4 (GLUT4) and ensure optimal glucose homeostasis.

The Brodsky lab previously observed that the CHC22 form of clathrin accumulates at sites of GLUT4 retention in the muscle of insulin-resistant patients with type two diabetes (Vassilopoulos et al., 2009) in addition to its active role in membrane traffic of GLUT4. Recent studies from the Brodsky laboratory suggested that variation in CHC22 clathrin assembly affects storage of GLUT4 and its availability for insulin-stimulated release (Fumagalli et al 2019). In a key advance, cryo-electron microscopy (cryo-EM) data from the Smith laboratory produced the highest resolution structure to date of a different type of assembled clathrin (CHC17) (Morris et al 2019) with sufficient detail to reveal molecular contacts within the clathrin lattice. This breakthrough provides the opportunity to investigate the structure of the CHC22 clathrin lattice which in turn can tell us how assembly contacts in CHC22 function in cellular GLUT4 membrane traffic. With respect to insulin-dependent glucose metabolism this has direct significance for processes that could increase or decrease glucose clearance.

In this project you will use high resolution 3D cryo-electron microscopy to determine the structure of clathrin lattices both in cells using cryo-tomography and as individual cages using single particle cryo-EM. You will also use biophysical approaches such as dynamic light scattering, time-resolved fluorescence anisotropy, surface plasmon resonance (SPR, Biacore) and isothermal titrating calorimetry to investigate how clathrin interactions result in formation of functional coated vesicles. You will have access to the Gatan K2 Summit direct detector and Jeol 2200FS 200kV transmission electron microscope provided by the Advanced Bioimaging Research Technology Platform, the Titan Krios microscope with Gatan K3 detector available in Leicester through the Midlands Regional Cryo-EM Facility 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 assembly affects glucose regulation in health and disease.

BBSRC Strategic Research Priority: Understanding the Rules of LifeStructural Biology. Integrated Understanding of Health: Diet and Health.

Techniques that will be undertaken during the project:
• 3D imaging of structural data
• High resolution cryo-electron microscopy and single particle analysis
• Image analysis of large data sets
• Biophysical analysis including DLS, fluorescence spectroscopy, stopped flow kinetics
• Protein biochemistry including protein expression and purification, SDS-PAGE and Western blotting