Steiert, F. | bioRxiv Science

bioRxiv2022

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step towards a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented. SUMMARYThis protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided along-side directions to prevent potential pitfalls.

biophysics↗

bioRxiv2021

Mass-sensitive particle tracking (MSPT) to elucidate the membrane-associated MinDE reactioncycle

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. So far, it has been consensus that their observation with sufficient sensitivity and time resolution requires the introduction of - predominantly fluorescent-labels to the system. However, the recent advent of mass photometry to quantify mass distributions of unlabelled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, we introduce mass-sensitive particle tracking (MSPT), enabling simultaneous label-free tracking and monitoring of molecular masses of single biomolecules diffusing on lipid membranes. We applied this approach to the highly non-linear reaction cycles underlying MinDE protein self-organisation. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. We found that MinD assembles into complexes larger than the commonly postulated dimer, through lateral interactions of membrane-bound complexes and subunit recruitment from solution. Furthermore, the ATPase-activating protein MinE interconnects MinD into high-molecular-weight heteromeric complexes and affects their subunit turnover and concerted membrane release. This study demonstrates the potential of MSPT to enhance our quantitative understanding of both prokaryotic and eukaryotic membrane-associated biological systems.

biophysics↗