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Image made by Dr Narain Karedla
Researchers in the Biophysical Immunology (BPI) Lab led by Professor Marco Fritzsche, in collaboration with the Immunological Synapse Lab led by Professor Mike Dustin, have developed an innovative methodology to study interactions between biomolecules in the plasma membrane of live cells. This new brightness-transit statistics (BTS) approach overcomes several outstanding shortcomings of existing methods, such as lack of accessibility and low sensitivity to oligomerisation. It has the potential to become an important tool for studying protein dynamics, with exciting applications for the development of antibodies and drugs which target receptors within the plasma membrane.
The membrane of a cell forms a semipermeable and active barrier, with a complex network of embedded receptors and channels that not only control which molecules move in and out but also trigger several vital signalling cascades. Many therapeutic antibodies target proteins within the cell membrane, enabling drugs to reach or fine-tune their precise subcellular targets. Live-cell microscopy methods for studying the dynamics of cell surface molecules are important for our understanding of how these complex signalling networks function, and for screening novel drugs designed to target specific membrane proteins.
Decades of ground-breaking fluorescence microscopy and spectroscopy work have given researchers tools for studying dynamic processes within the plasma membrane. These tools include approaches based on fluorescence fluctuation spectroscopy and fluorescence recovery after photobleaching. Current methods do, however, have their limitations. Firstly, many methods are built on specialised hardware which is only accessible to expert microscopists, and require specialised data analysis software, limiting their accessibility. Secondly, they often have a limited ability to detect how molecules of the same type bind to each other – a process known as oligomerisation – which is crucial for understanding how cells signal and interact with their environment.
Researchers in the BPI Lab at the Kennedy Institute of Rheumatology established a new method to overcome these limitations. Their method uses scanning fluorescence correlation spectroscopy (sFCS) acquisitions which can be performed on any commercial laser-scanning microscope. It comes with a straightforward analysis pipeline and intuitive representation of the results. Despite its ease of use, the method can provide extraordinary detail of a molecule’s diffusion dynamics and oligomerisation.
The new BTS method simultaneously gathers statistics on molecules’ ‘transit time’ (how quickly they move through a confocal area) and brightness (at what rate they emit photons on average). The method relies on generating population-level statistics from multiple acquisitions of a study condition, creating intuitive histograms from this data, and comparing against histograms from different conditions. The method has been published in the peer-reviewed journal Nature Communications, where the researchers provide detailed guidance for how to perform the BTS method, along with some tips to optimise measurements.
The researchers successfully validated BTS in silico by simulating various types of molecular motion on cell membranes, and in vitro using fluorescent proteins tethered to supported lipid bilayers. They then used the method to gain novel insights into the oligomerisation of the protein CD40 within the membrane of primary, live B cells. Interaction between CD40 and its ligand is crucial for T cells to respond to antigens and to generate antibody responses. CD40 has been therapeutically targeted in the treatment of multiple cancer types. Through their validation, the researchers found a potential for CD40 to oligomerise, but did not observe mobile oligomers on B cells. This novel finding demonstrates that BTS can be used to answer biological questions that are otherwise hard to answer and provides an example workflow for researchers who wish to use the method for their own studies.
About the study, Dr Falk Schneider, a former Postdoctoral Researcher in the BPI Lab who is currently working with Prof Scott E. Fraser at the University of Southern California, said: ‘The new BTS method will make a crucial contribution to the quantification of oligomers in cells, tissues, and organisms’. Dr Narain Karedla, a senior postdoctoral researcher in the BPI Lab and staff scientist at the Rosalind Franklin Institute, highlights the significance of BTS, stating, ‘What is also unique here is the ability to extract large statistical information in a short time window, enabling the interrogation of spatiotemporal dynamics and organization of biomolecules on live, dynamic immune cells.’
‘In the future, BTS could be used to study the highly dynamic corolla of the immunological synapse, which integrates key targets for immunotherapy such at PD-1 and LAG-3’, said Professor Dustin. ‘It will not be restricted to surface receptors, but can also be applied to transcription factors, kinases and their adapters that operate in part through liquid-liquid phase separation’. Professor Fritzsche enthusiastically said that ‘the BTS method exemplifies the experimental power of novel technology in biology. In our paper, we could show for the first time after years of experimental confusion that CD40 forms no mobile oligomers in living B cells’.
The researchers are grateful for the generous funds from the EPSRC and the support from the Kennedy Trust for Rheumatology Research and the British Heart Foundation for the development of the Oxford-ZEISS Centre of Excellence.