Quantifying biomolecular organisation in membranes with brightness-transit statistics.

Autor: Schneider F; Kennedy Institute for Rheumatology, Roosevelt Drive, University of Oxford, Oxford, OX3 7LF, United Kingdom. falkschn@usc.edu.; Translational Imaging Center, University of Southern California, Los Angeles, California, 90089, United States of America. falkschn@usc.edu., Cespedes PF; Kennedy Institute for Rheumatology, Roosevelt Drive, University of Oxford, Oxford, OX3 7LF, United Kingdom., Karedla N; Kennedy Institute for Rheumatology, Roosevelt Drive, University of Oxford, Oxford, OX3 7LF, United Kingdom.; Rosalind Franklin Institute, Harwell Campus, Didcot, OX11 0FA, United Kingdom., Dustin ML; Kennedy Institute for Rheumatology, Roosevelt Drive, University of Oxford, Oxford, OX3 7LF, United Kingdom., Fritzsche M; Kennedy Institute for Rheumatology, Roosevelt Drive, University of Oxford, Oxford, OX3 7LF, United Kingdom. marco.fritzsche@kennedy.ox.ac.uk.; Rosalind Franklin Institute, Harwell Campus, Didcot, OX11 0FA, United Kingdom. marco.fritzsche@kennedy.ox.ac.uk.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2024 Aug 17; Vol. 15 (1), pp. 7082. Date of Electronic Publication: 2024 Aug 17.
DOI: 10.1038/s41467-024-51435-1
Abstrakt: Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.
(© 2024. The Author(s).)
Databáze: MEDLINE
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