Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle

Abstract

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.

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

“…The presence of vesicles, however, is clearly distinct due to the vesicles’ large scattering cross-section and enables the observation of vesicle rupture and fusion into homogeneous membranes ( Figure 2b and Supplementary Movie 1 ). When removing the static scattering signal of the glass surface with a ratiometric approach that emphasizes the dynamic elements within the field of view 24,25 , one can uncover unlabeled proteins diffusing on the membrane ( Figure 2d ) while an empty SLB ( Figure 2c ) or glass itself ( Figure 2a ) appear as a noisy image.…”

“…ii) The mass of particles, which is extracted from the amplitude of their corresponding PSF fit, becomes systematically underestimated and mass peaks broaden because it becomes increasingly difficult to separate the static background signal from the dynamic particle signal ( Figure 7c ). Currently, visual assessment of data quality in the process of acquiring MSPT videos is difficult on the available commercial microscopes because the implemented ratiometric view in the acquisition software is using the background removal established for mass photometry 21 instead of the median-based algorithm described here and in references 24, 25 ( Figure 7d ). The mean-based continuous background removal used to visualize landing molecules in mass photometry causes diffusing particles to appear as dark fronts with bright tails which makes the spots appear highly anisotropic and interferes with PSF fitting during the detection procedure.…”

“…The presented protocol extends mass photometry 21 , a technique that analyzes the mass of single biomolecules adsorbing on glass, to an even more versatile tool capable of simultaneously measuring the mass and diffusion of unlabeled membrane-interacting biomolecules. This analysis extension is achieved through the implementation of a modified background removal strategy adapted to the lateral motion of molecules 24, 25 . In general, the background removal is of utmost importance for iSCAT-based approaches, since the strong scattering of the glass surface roughness represents the main analysis impediment, and the accurate determination of each pixel’s local background is essential for quantification of particle mass and location.…”

“…In light of the single-molecule nature of MSPT, it should be avoided to measure at high membrane particle densities as those can interfere with accurate contrast and mass determination. It has been shown that densities below one particle per squared micrometer are favorable for MSPT measurements 24 . An additional consideration is the expected diffusion coefficients in the sample.…”

“…However, for the detection and characterization of mobile molecules diffusing on lipid membranes, a more sophisticated image analysis approach had to be developed. This has meanwhile been successfully implemented and allows to detect, track and determine the molecular mass of single unlabeled biomolecules diffusing on a lipid interface 24, 25 . Referred to as dynamic mass photometry or mass-sensitive particle tracking, this technique now enables the assessment of complex macromolecule interactions by directly recording changes in the molecular mass of the tracked entities and hence opens up new possibilities for the mechanistic analysis of membrane-associated molecular dynamics.…”