Researchers at the Rosalind Franklin Institute, the University of Oxford and the University of Southern California have developed a new method for studying how molecules behave on the cell membrane.
The technique, known as brightness-transit statistics (BTS), will enhance our understanding of important biological processes including immune responses and cell signalling (the way cells communicate with each other). It could also have applications in drug development – for example, by identifying new targets for therapies based on more detailed insights into how molecules behave when subject to diseases such as cancer or neurodegeneration.
To date, accurately measuring these molecular dynamics has been a challenge for scientists. In comparison with existing methods, BTS provides a more comprehensive overview of the clustering (known as oligomerisation) and movement (diffusion) of molecules at the cell membrane. BTS uses a technique called scanning fluorescence fluctuation spectroscopy to gather simultaneous data on how quickly molecules diffuse and how they oligomerise by measuring their brightness across a range of points in space. The BTS pipeline then pulls out relevant measurements from the data and carries out statistical analysis, with results being presented visually in easy-to-read 2D histograms.
The research team validated its method with computer simulations and lab experiments before applying BTS to a real biological scenario by examining CD40 – a protein important in immune responses – and achieving new insights into its behaviour. Additionally, the researchers employ an artificial bilayer system that mimics a cell membrane and enables the study of molecules as if in their natural environment.
The paper’s lead author is Franklin collaborator Dr Falk Schneider, formerly of Oxford’s Kennedy Institute for Rheumatology and now at the University of Southern California. Dr Schneider says: “Broadly speaking, this paper is about quantifying how molecules interact and organise at the cell membrane. We have lacked an accurate way to do this, which is why we set out to develop a new method with increased sensitivity and specificity. What we have developed in BTS isn’t just a technique for gathering the data – it’s a whole pipeline including a simulation platform to produce and analyse data in silico.
“We have placed our simulation and analysis code on GitHub for others to use, and the technique itself can be performed using standard instrumentation. This research began during the COVID-19 pandemic and involved a lot of remote training and communication, which is further evidence that we have developed an easily describable and reproducible method. It also gave us strong motivation to keep going, given viruses like SARS-CoV-2 rely on interactions at the cell membrane to be able to enter and infect.”
Co-author Dr Narain Karedla, Staff Scientist at the Franklin, adds: “One of the fundamental questions in understanding life’s processes is how key molecules organise and behave in space and time. The BTS method published recently holds the potential to provide answers to such essential questions.”
The research team plans to continue its efforts to improve the technique and explore its wider applicability. Senior author Professor Marco Fritzsche, of the Franklin and the University of Oxford, says: “The BTS method exemplifies the experimental power of novel technology in biology. In our paper, we show for the first time after years of experimental confusion that CD40 forms no mobile oligomers in living B cells. I am looking forward to the application of the BTS method in the drug screening space.”
Related publication
F. Schneider et al. - Quantifying biomolecular organisation in membranes with brightness-transit statistics - Nature Communications (2024)