Single-Molecule Nanoinjection: Unveiling Cellular Responses to a-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...