Single-Molecule Nanoinjection: Unveiling Cellular Responses to α-Synuclein Aggregates

The cell is a microscopic chemical reactor, in which thousands of cellular processes run simultaneously in a highly crowded environment.  These processes all must be tightly regulated or this can result in the disruption of important cellular functions and can lead to disease. To study cells in health and disease, scientists manipulate these cellular processes and one way this can be achieved is via the delivery of proteins and DNAs into cells.

Conventional methods of delivering molecules into cells have significant limitations as they often damage the cell, have little control of when the molecules enter the cell and crucially no information on how many molecules are actually delivered. These drawbacks can be overcome with nanoinjection, a method we have developed.

Nanoinjection uses fine needles fitted with electrodes, known as nanopipettes, to deliver molecules into cells.  The small size of the nanopipette relative to the cell means the cell is not harmed by the injection, and molecule delivery only starts when triggered by applying a small voltage to the nanopipette. The key feature of the nanopipette is the small size of the pore at its tip, which is only a bit larger than the molecules it delivers. Crucially when a molecule passes through the pore it changes the current we measure through the nanopipette’s tip, a blip. Therefore, count the number of blips and you know how many molecules are delivered, something other methods can’t do.

In this project we will use nanoinjection to deliver alpha-synuclein into cells. This protein has an important role in Parkinson’s disease, where it clumps together to form various aggregates. These aggregates vary in size and shape, some are small doughnut shaped structures and others are long fibres. Understanding which of these is responsible for making nerve cells sick and die is a key question in Parkinson’s disease. In this project we will inject known numbers of different alpha-synuclein aggregates into nerve cells and look at the cell’s response. We will look at this at different levels, at the level of the cell to see if it gets sick or dies, at the level of gene expression to see if key cellular processes are changed and, using electron microscopy to look at the cell interior at very high magnification to see which compartments in the cell are disrupted.  

Collectively, these different approaches will then tell us which alpha-synuclein aggregates are damaging to the cell and crucially how many of each aggregate are required to make the cells sick. Whilst this project focuses on alpha-synuclein, it will also demonstrate that nanoinjection can be used to deliver molecules into cells to study cellular processes. We therefore see many different applications for nanoinjection, for example looking at protein aggregates linked to other diseases such as Alzheimer's. Likewise, nanoinjection could be used to study viral infection, by injecting viruses into cells and then studying how the virus hijacks cells in order to replicate and how the cells try to stop the infection.