Advancing Membrane Biology: Single-Molecule Approaches Meet Model Membrane Systems.

Model membrane systems have emerged as essential platforms for investigating membrane-associated processes in controlled environments, mimicking biological membranes without the complexity of cellular systems. However, integrating these model systems with single-molecule techniques remains challenging due to the fluidity of lipid membranes, including undulations and the lateral mobility of lipids and proteins. This mini-review explores the evolution of various model membranes ranging from black lipid membranes to nanodiscs and giant unilamellar vesicles as they adapt to accommodate electrophysiology, force spectroscopy, and fluorescence microscopy.

Force-fluorescence setup for observing protein-DNA interactions under load.

This chapter explores advanced single-molecule techniques for studying protein-DNA interactions, particularly focusing on Replication Protein A (RPA) using a force-fluorescence setup. It combines magnetic tweezers (MT) with total internal reflection fluorescence (TIRF) microscopy, enabling detailed observation of DNA behavior under mechanical stress. The chapter details the use of DNA hairpins and bare DNA to examine RPA's binding dynamics and its influence on DNA's mechanical properties.

High-speed measurements of SNARE-complexin interactions using magnetic tweezers.

In neuroscience, understanding the mechanics of synapses, especially the function of force-sensitive proteins at the molecular level, is essential. This need emphasizes the importance of precise measurement of synaptic protein interactions. Addressing this, we introduce high-resolution magnetic tweezers (MT) as a novel method to probe the mechanics of synapse-related proteins with high precision.

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements.

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes.

Quantitative imaging of vesicle-protein interactions reveals close cooperation among proteins.

Membrane-bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell-surface proteins, but a generalizable technique that can quantitatively observe these vesicle-protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell-surface proteins, either in a surface-tethered or in a membrane-embedded state.

Tension exerted on cells by magnetic nanoparticles regulates differentiation of human mesenchymal stem cells.

Cells can 'sense' physical cues in the surrounding microenvironment and 'react' by changing their function. Previous studies have focused on regulating the physical properties of the matrix, such as stiffness and topography, thus changing the tension 'felt' by the cell as a result. In this study, by directly applying a quantified magnetic force to the cell, a correlation between differentiation and tension was shown.

High-Resolution Single-Molecule Magnetic Tweezers.

Single-molecule magnetic tweezers deliver magnetic force and torque to single target molecules, permitting the study of dynamic changes in biomolecular structures and their interactions. Because the magnetic tweezer setups can generate magnetic fields that vary slowly over tens of millimeters-far larger than the nanometer scale of the single molecule events being observed-this technique can maintain essentially constant force levels during biochemical experiments while generating a biologically meaningful force on the order of 1-100 pN. When using bead-tether constructs to pull on single molecules, smaller magnetic beads and shorter submicrometer tethers improve dynamic response times and measurement precision.

Nano-Precision Tweezers for Mechanosensitive Proteins and Beyond.

Mechanical forces play pivotal roles in regulating cell shape, function, and fate. Key players that govern the mechanobiological interplay are the mechanosensitive proteins found on cell membranes and in cytoskeleton. Their unique nanomechanics can be interrogated using single-molecule tweezers, which can apply controlled forces to the proteins and simultaneously measure the ensuing structural changes.

Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes.

Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly.

Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway.

To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding.

Submicrometer elasticity of double-stranded DNA revealed by precision force-extension measurements with magnetic tweezers.

Submicrometer elasticity of double-stranded DNA (dsDNA) governs nanoscale bending of DNA segments and their interactions with proteins. Single-molecule force spectroscopy, including magnetic tweezers (MTs), is an important tool for studying DNA mechanics. However, its application to short DNAs under 1 μm is limited.

Profiling protein-protein interactions of single cancer cells with in situ lysis and co-immunoprecipitation.

Heterogeneity in a tumor allows a small portion of cancer cells to survive and regrow upon targeted cancer therapy, eventually leading to cancer relapse. Such drug-resistant cells often exhibit dynamic adaptation of their signaling pathways at the level of protein-protein interactions (PPIs). To probe the rewiring of signaling pathways and the heterogeneity across individual cancer cells, we developed a single-cell version of the co-immunoprecipitation (co-IP) analysis that examines the amount and PPIs of target proteins immunoprecipitated from individual cells.

Profiling of protein-protein interactions via single-molecule techniques predicts the dependence of cancers on growth-factor receptors.

The accumulation of genetic and epigenetic alterations in cancer cells rewires cellular signalling pathways through changes in the patterns of protein-protein interactions (PPIs). Understanding these patterns may facilitate the design of tailored cancer therapies. Here, we show that single-molecule pull-down and co-immunoprecipitation techniques can be used to characterize signalling complexes of the human epidermal growth-factor receptor (HER) family in specific cancers.

Focused clamping of a single neuronal SNARE complex by complexin under high mechanical tension.

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze synaptic vesicle fusion with presynaptic membranes through the formation of SNARE complexes. Complexin (Cpx) is the only presynaptic protein that tightly binds to SNAREs and regulates membrane fusion, but how it modulates the energy landscape of SNARE complex assembly, especially under mechanical tension on the complex, remains unclear. Here, using magnetic tweezers, we report how Cpx interacts with single SNARE complexes.

Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy.

Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant.

Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley.

Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a 'DNA pulley' for position-resolved nano-mechanical measurements of protein-DNA interactions.

Mass action at the single-molecule level.

We developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated "dimples". This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry.