Quantifying biomolecular organisation in membranes with brightness-transit statistics

• Published in: Nature communications 2024-08, Vol.15 (1), p.7082-16
• Main Authors: Schneider, Falk, Cespedes, Pablo F., Karedla, Narain, Dustin, Michael L., Fritzsche, Marco
• Format: Article
• Language: English
• Subjects: 13/1; 14/19; 14/34; 14/63; 631/1647/245/2225; 631/1647/527/1819; 631/250/2503; 631/57/2267; Animals; B-Lymphocytes - metabolism; Biomolecules; Brightness; CD40 antigen; Cell Membrane - chemistry; Cell Membrane - metabolism; Computer Simulation; Diffusion; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - metabolism; Homeostasis; Humanities and Social Sciences; Humans; In vitro methods and tests; Ligands; Lipid bilayers; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Lipids; Lymphocytes B; Mathematical models; Membranes; Methods; multidisciplinary; Oligomerization; Protein Multimerization; Science; Science (multidisciplinary); Sensitivity analysis; Spectrometry, Fluorescence - methods; Spectroscopy; Statistical analysis; Statistics; Transit time
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-51435-1

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. Methods to assess diffusion dynamics and oligomerisation of biomolecules remain scarce. Here, the authors present a brightness-transit statistics method to measure diffusion and oligomerisation simultaneously with high sensitivity, and they apply their approach to study the organisation of CD40.