Biological membranes make life possible. Proteins recruited to the lipid bilayers of cell membranes are pivotal in sensing environmental changes, establishing polarity, and regulating division.

But understanding the dynamic and transient molecular interactions that occur at membranes in real-time without altering the interacting biomolecules, poses major technical challenges. Current methods are restricted to providing static snapshots of membrane protein interactions that involve tagging proteins.

A new method called Mass-Sensitive Particle Tracking (MSPT) developed by scientists at the Max Planck Institute of Biochemistry and published in the Nature Methods article titled “Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle,” overcomes some of these technical hurdles and allows scientists to measure the movements and reactions of un-modified proteins on biological membranes through the measurement of their mass alone.

Petra Schwille, PhD, director at the Max Planck Institute of Biochemistry (left), and Nikolas Hundt, PhD, from the Ludwig-Maximilians-Universität München (right) are corresponding authors of the paper.

The team led by Petra Schwille, PhD, director at the Max Planck Institute of Biochemistry, and Nikolas Hundt, PhD, from the Ludwig-Maximilians-Universität München build upon recent advances in mass photometry that can determine the molecular mass of unlabeled molecules in solution, to develop this new technology.

MSPT identifies individual proteins on membranes by tracking their molecular mass without labeling them. This allows the analysis of rapid interactions of membrane-associated proteins in their natural physiological settings.

Frederik Steiert, PhD, one of the first authors of the publication, says, “We can now track directly on biological membranes what mass individual proteins have, how they move and how they interact. This allows us to study the dynamics of biological systems in greater detail.”

The new technology is based on the principle of light scattering. When light encounters a particle, it scatters in such a way that the intensity of the scattered light depends on the mass of the particle. The authors introduce a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, that enables them to track single unlabeled biomolecules on a lipid bilayer. As part of this method, the team conducts video microscopy of individual proteins on membranes, tracks the intensity of scattered light from these proteins and measures their individual mass with the aid of an analytical software.

The method can analyze proteins with a molecular weight of at least 50 kDa, which includes most known proteins. Labeling proteins to make them visible changes their overall structure and can potentially alter their function. Therefore, a distinct advantage of MSPT is that it does not require proteins to be labelled.

To showcase the abilities of the MSPT method for the detailed analysis of membrane-associated interactions in complex biological systems, the researchers analyze Escherichia coli’s MinDE system that is the focus of study in the Schwille lab. The system consists of three proteins—MinC, MinD and MinE—and is required for the regulation of cell division in these bacteria.

Tamara Heermann, another first author of the paper, says, “The method permits us to characterize properties of dynamical systems that were previously not measurable. This allowed us not only to verify established findings about the Min system, but also to gain new insights.”

Using MSPT, the team shows that the complexes of MinD proteins are larger than previous estimates and offers the additional insight that MinE can connect MinD monomers, enabling the assembly of large oligomers and higher-order heteromeric polymers, and not just MinD dimers as classically thought.

Their dissection of the membrane-associated MinDE reaction cycle through determining the stoichiometry, turnover and diffusion of individual membrane-bound protein complexes of the system, validates the applicability of the new technology.

“We believe our experiments on the Min system demonstrate that MSPT is a powerful, widely applicable tool for the mechanistic analysis of both pro- and eukaryotic membrane-associated systems,” the authors conclude.

The researchers are currently improving the method further so that it can be used to analyze integral membrane proteins and proteins smaller than 50kDa in mass.