The aim of this project is to use nanoinjection to perform the quantitative delivery of alpha-synuclein fibrils or oligomers into the cytoplasm of neurons and then determine how many of each is required to induce cellular stress and death.
Intraneuronal inclusions known as Lewy bodies are a hallmark of the neurodegenerative movement disorder Parkinson’s. Amyloid fibrils formed by the misfolding of alpha-synuclein are the principal component of Lewy Bodies. However, the role of these fibrils and their oligomeric assembly intermediates in neuronal death is poorly understood. This is because alpha-synuclein misfolds into amyloid fibrils in the cytoplasm, whereas experimental studies have typically involved adding fibrils and oligomers to the cell culture medium. Although this enables biophysical characterisation of the fibrils and oligomers before addition to the cells, their access to the cytoplasm is limited.
Nanoinjection uses quartz needles (≤50nm pore diameter), known as nanopipettes, to deliver individual molecules into cells. The nanopipettes incorporate electrodes and application of a voltage drives the transport of molecules through the pore. When a single protein passes through the nanopipette’s pore there is a corresponding disruption in the ion flow, thus the number of proteins delivered into a cell can be quantified. This project will use nanoinjection to deliver alpha-synuclein fibrils and oligomers into the cytoplasm of neurons. Crucially, this will enable us to not only determine whether alpha-synuclein fibrils or oligomers are toxic in the cytoplasm, but for the first time we will quantify the number of each required to kill a neuron.
Amyloid fibrils and oligomers will be made from recombinant alpha-synuclein, characterised using an array of biophysical techniques. The fibrils and oligomers will then nanoinjected into neuronal cell lines and primary neurons. To inject cells the nanopipette will be integrated into a scanning ion conductance microscope (SICM), which enables the insertion of the nanopipette into the cell at a pre-defined depth and location. Application of a voltage will drive the delivery of the fibrils or oligomers into the cell. By monitoring the corresponding disruptions in ion flow, a defined number of oligomers or fibrils will be delivered. The cellular effects of the nanoinjected fibrils and oligomers on cell stress and viability will then be analysed using microscopy-based assays.
Supervisory team and training
This is an interdisciplinary project that combines the expertise of Dr Eric Hewitt in cell biology, Dr Paolo Actis in nanotechnology and Professor Sheena Radford in structural biology. The successful applicant will work in the laboratories of all three supervisors and will receive training in the array of techniques used in the project including protein expression and purification, cell culture, cell viability assays, fluorescence microscopy, scanning ion conductance microscopy and nanoinjection. Further information about the supervisors’ research interests can be found on their websites: https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/84/dr-eric-hewitt https://eps.leeds.ac.uk/electronic-engineering/staff/806/dr-paolo-actis https://biologicalsciences.leeds.ac.uk/biological-sciences/staff/127/professor-sheena-radford
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/
Iadanza MG, Jackson MP, Hewitt EW, Ranson NA, Radford SE. (2018). A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol. 19:755-773.
Karamanos TK, Jackson MP, Calabrese AN, Goodchild SC, Cawood EE, Thompson GS, Kalverda AP, Hewitt EW, Radford SE. (2019). Structural mapping of oligomeric intermediates in an amyloid assembly pathway. Elife 8: e46574
Ivanov AP, Actis P, Jönsson P, Klenerman D, Korchev Y, Edel JB. (2015) On-demand delivery of single DNA molecules using nanopipets. ACS Nano. 9:3587-95