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 ...

Max Planck Institute of Biochemistry

As part of the βγ-superfamily, βB2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three βB2-mutant lines Crybb2, Crybb2, and Crybb2 included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.

Crybb2 Mutations Consistently Affect Schizophrenia Endophenotypes in Mice.

Single-particle plasmon spectroscopy has become a standard technique to detect and quantify the presence of unlabeled macromolecules. Here, we extend this method to determine their exact distance from the plasmon sensors with sub-nanometer resolution by systematically varying the sensing range into the surrounding by adjusting the size of the plasmonic nanoparticles. We improved current single-particle plasmon spectroscopy to record continuously for hours the scattering spectra of thousands of nanoparticles of different sizes simultaneously with 1.8 s time resolution. We apply this technique to study the interaction dynamics of bacterial Min proteins with supported lipid membranes of different composition. Our experiments reveal a surprisingly flexible operating mode of the Min proteins: In the presence of cardiolipin and membrane curvature induced by nanoparticles, the protein oscillation occurs on top of a stationary MinD patch. Our results reveal the need to consider membrane composition and local curvature as important parameters to quantitatively understand the Min protein system and could be extrapolated to other macromolecular systems. Our label-free method is generally easily implementable and well suited to measure distances of interacting biological macromolecules.

Plasmonic Nanosensors Reveal a Height Dependence of MinDE Protein Oscillations on Membrane Features.

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.

The E. coli MinCDE system in the regulation of protein patterns and gradients.

The proteins MinD, MinE and MinC are constitutive for the spatiotemporal organization of cell division in Escherichia coli, in particular, for positioning the division machinery at mid-cell. To achieve this function, the ATPase MinD and the ATPase-activating protein MinE undergo coordinated pole-to-pole oscillations and have thus become a paradigm for protein pattern formation in biology. The exact molecular mechanisms enabling MinDE self-organization, and particularly the role of cooperativity in the membrane binding of MinD, thought to be a key requirement, have remained poorly understood. However, for bottom-up synthetic biology aiming at a de novo design of key cellular features, elucidating these mechanisms is of great relevance. By combining in vitro reconstitution with rationally guided mutagenesis of MinD, we found that when bound to membranes, MinD displays new interfaces for multimerization, which are distinct from the canonical MinD dimerization site. We propose that these additional transient interactions contribute to the local self-enhancement of MinD at the membrane, while their relative lability maintains the structural plasticity required for MinDE wave propagation. This could represent a powerful structural regulation feature not reported so far for self-organizing proteins.

Local Self-Enhancement of MinD Membrane Binding in Min Protein Pattern Formation.

As a key mechanism underpinning many biological processes, protein self-organization has been extensively studied. However, the potential to apply the distinctive, nonlinear biochemical properties of such self-organizing systems to biotechnological problems such as the facile detection and characterization of biomolecular interactions has not yet been explored. Here, we describe an  assay in a 96-well plate format that harnesses the emergent behavior of the  Min system to provide a readout of biomolecular interactions. Crucial for the development of our approach is a minimal MinE-derived peptide that stimulates MinD ATPase activity only when dimerized. We found that this behavior could be induced via any pair of foreign, mutually binding molecular entities fused to the minimal MinE peptide. The resulting MinD ATPase activity and the spatiotemporal nature of the produced protein patterns quantitatively correlate with the affinity of the fused binding partners, thereby enabling a highly sensitive assay for biomolecular interactions. Our assay thus provides a unique means of quantitatively visualizing biomolecular interactions and may prove useful for the assessment of domain interactions within protein libraries and for the facile investigation of potential inhibitors of protein-protein interactions.

Probing Biomolecular Interactions by a Pattern-Forming Peptide-Conjugate Sensor.

Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a 'cargo' molecule reversibly to a permanent membrane 'anchor'; and (ii) creating a 'membrane-avidity switch' that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

De novo design of a reversible phosphorylation-dependent switch for membrane targeting.

The formation of large-scale patterns through molecular self-organization is a basic principle of life. Accordingly, the engineering of protein patterns and gradients is of prime relevance for synthetic biology. As a paradigm for such pattern formation, the bacterial MinDE protein system is based on self-organization of the ATPase MinD and ATPase-activating protein MinE on lipid membranes. Min patterns can be tightly regulated by tuning physical or biochemical parameters. Among the biochemically engineerable modules, MinD's membrane targeting sequence, despite being a key regulating element, has received little attention. Here we attempt to engineer patterns by modulating the membrane affinity of MinD. Unlike the traveling waves or stationary patterns commonly observed  on flat supported membranes, standing-wave oscillations emerge upon elongating MinD's membrane targeting sequence via rationally guided mutagenesis. These patterns are capable of forming gradients and thereby spatially target co-reconstituted downstream proteins, highlighting their functional potential in designing new life-like systems.

Increasing MinD's Membrane Affinity Yields Standing Wave Oscillations and Functional Gradients on Flat Membranes.

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.

Tamara Heermann

Department of Cellular and Molecular Biophysics

Max Planck Institute of Biochemistry

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

Tamara Heermann

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cellular and Molecular Biophysics

Max Planck Institute of Biochemistry

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

Department of Cellular and Molecular Biophysics