Quantifying biomolecular organisation in membranes with brightness-transit statistics

Area of Science:

• Cell biology
• Biophysics
• Molecular dynamics

Background:

• Cellular homeostasis depends on plasma membrane biomolecular interactions.
• Quantifying biomolecular organization, diffusion, and oligomerization remains a challenge.

Purpose of the Study:

• Introduce and validate the brightness-transit statistics (BTS) method.
• Elucidate biomolecular diffusion and oligomerization dynamics.
• Investigate CD40 ectodomain and endogenous CD40 oligomerization.

Main Methods:

• Utilized fluorescence fluctuation spectroscopy.
• Combined brightness and transit time analysis.
• Validated with in silico simulations, supported lipid bilayers, and primary B cells.

Main Results:

• The BTS method quantifies diffusion and oligomerization.
• CD40 ectodomain shows potential for concentration/ligand-dependent oligomerization in vitro.
• No mobile CD40 oligomers were observed on primary B cells.

Conclusions:

• BTS provides sensitive, quantitative, and visual analysis of biomolecular organization.
• CD40 oligomerization dynamics differ between in vitro and cell-surface conditions.
• The method offers a powerful tool for studying membrane protein organization.