About the Project
The aim of this project is to use a nanoinjection platform for the quantitative and targeted delivery of amyloid fibrils and oligomers into cells for functional analysis.
Diseases such as Parkinson’s disease and Huntington’s disease are associated with the aggregation of proteins into amyloid fibrils, which form the intracellular inclusions that are a hallmark of these disorders. Whilst, significant progress has been made in the determining the structure of amyloid fibrils and their oligomeric intermediates, to understand how amyloid formation results in cell death and tissue destruction these findings need to be integrated with studies of their biological properties. This is a particularly challenging for proteins, such as alpha-synuclein (Parkinson’s) and huntingtin (Huntington’s), which assemble into amyloid fibrils inside cells.
The nanoinjection platform uses quartz needles with ≤50nm diameter pores, known as nanopipettes, to inject macromolecules into cells. Due to the small size of the pore individual macromolecules can be detected when they are delivered into cells, thus cellular delivery can be quantified. We will use the nanoinjection platform to deliver amyloid fibrils and their oligomeric assembly intermediates into cells. A defined number of structurally characterised amyloid fibrils and oligomers will be delivered by nanoinjection into the cytoplasm and nuclei of cells. The effect of these protein complexes on cells will be determined using microscopy-based assays for cellular stress and viability. Thus for the first time will be able to determine not only which amyloid fibrils and oligomers are toxic inside cells, but also how many of each it takes to make a cell sick and die.
This project is highly collaborative and multidisciplinary and the supervisory team’s labs combine expertise in cell biology (Eric Hewitt), amyloid structure and assembly (Sheena Radford) and nanotechnology (Paolo Actis)
Please contact Dr Eric Hewitt ([Email Address Removed]) if you have any questions about the project
Actis P et al (2014). Electrochemical nanoprobes for single-cell analysis. ACS Nano. 8: 875-84
Jakhria et al (2014). β2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes. J Biol Chem 289: 35781-94
Tipping et al (2015). pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers. Proc Natl Acad Sci U S A. 112: 5691-6
Iadanza et al (2018). The structure of a β2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism.Nat Commun. 9: 4517.
Karamanos et al (2019). Structural mapping of oligomeric intermediates in an amyloid assembly pathway. Elife. 8: e46574.
Nadappuram et al (2019) Nanoscale Tweezers for Single-Cell Biopsies Nature Nanotechnology 14: 80-88
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