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Quantifying biomolecular organisation in membranes with brightness-transit statistics

Author

Listed:
  • Falk Schneider

    (University of Oxford
    University of Southern California)

  • Pablo F. Cespedes

    (University of Oxford)

  • Narain Karedla

    (University of Oxford
    Harwell Campus)

  • Michael L. Dustin

    (University of Oxford)

  • Marco Fritzsche

    (University of Oxford
    Harwell Campus)

Abstract

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.

Suggested Citation

  • Falk Schneider & Pablo F. Cespedes & Narain Karedla & Michael L. Dustin & Marco Fritzsche, 2024. "Quantifying biomolecular organisation in membranes with brightness-transit statistics," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-51435-1
    DOI: 10.1038/s41467-024-51435-1
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    References listed on IDEAS

    as
    1. Christian Eggeling & Christian Ringemann & Rebecca Medda & Günter Schwarzmann & Konrad Sandhoff & Svetlana Polyakova & Vladimir N. Belov & Birka Hein & Claas von Middendorff & Andreas Schönle & Stefan, 2009. "Direct observation of the nanoscale dynamics of membrane lipids in a living cell," Nature, Nature, vol. 457(7233), pages 1159-1162, February.
    2. Elizabeth Hinde & Elvis Pandžić & Zhengmin Yang & Ivan H. W. Ng & David A. Jans & Marie A. Bogoyevitch & Enrico Gratton & Katharina Gaus, 2016. "Quantifying the dynamics of the oligomeric transcription factor STAT3 by pair correlation of molecular brightness," Nature Communications, Nature, vol. 7(1), pages 1-14, April.
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