Biophysics

The transmembrane receptor–ligand interactions play a vital role in the physiological and pathological processes of living cells, such as immune cell activation, neural synapse formation, or viral invasion into host cells. Mounting evidence suggests that these processes involve mechanosensing and mechanotransduction, which are directly mediated by the force-dependent transmembrane receptor–ligand interactions. Some single-molecule force spectroscopy techniques have been applied to investigate force-dependent kinetics of receptor–ligand interactions. Among these, the biomembrane force probe (BFP), a unique and powerful technique, can quantitatively and accurately determine the force-dependent parameters of transmembrane receptor–ligand interactions at the single-molecule level on living cells. The stiffness, spatial resolution, force, and bond lifetime range of BFP are 0.1–3 pN/nm, 2–3 nm, 1–10³ pN, and 5 × 10^-4–200 s, respectively. Therefore, this technique is very suitable for studying transient and weak interactions between transmembrane receptors and their ligands. Here, we share in detail the in situ characterization of the single-molecule force-dependent bond lifetime of transmembrane receptor–ligand interactions, based on a force-clamp assay with BFP.

CD4⁺ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4⁺ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.

With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.

Graphical abstract:

Overview of the experimental workflow. Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right.

The Sec translocon, consisting of a heterotrimeric transmembrane channel (SecYEG) and an associated ATPase (SecA), catalyzes the export of unfolded proteins from the cytosol in bacteria. Kinetically resolving protein translocation at high resolution yields mechanistic insight into the process. Translocation is typically followed by measuring the protection of proteins transported into lipid vesicles, which only allows visualization of translocation after it has already been completed and limits time resolution. Here, we describe the implementation of an assay for measuring translocation in real-time. By priming the reconstituted translocon with suitably engineered substrate proteins, the kinetics of the actual translocation process can be resolved at high resolution. To analyze translocation kinetics, we developed a detailed kinetic model of the process that includes on-pathway and off-pathway processes. Together, this experimental protocol and model permit detailed mechanistic analyses of Sec-dependent protein translocation.

Graphic abstract:

Synchronized real-time measurements, combined with a detailed kinetic model, enable a mechanistic analysis of protein transport.

Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with isolating single proteins within a biological system. Here we provide a detailed protocol to conduct ex vivo single molecule imaging where single transmembrane proteins are isolated by rapidly extracting nanovesicles containing receptors of interest from different regions of the brain and subjecting them to single molecule study by using total internal reflection fluorescence (TIRF) microscopy. This protocol discusses the isolation and separation of brain region specific nanovesicles as well as a detailed method to perform TIRF microscopy with those nanovesicles at the single molecule level. This technique can be applied to study trafficking and stoichiometry of various transmembrane proteins from the central nervous system. This approach can be applied to a wide range of animals that are genetically modified to express a membrane protein-fluorescent protein fusion with a wide range of potential applications in many aspects of neurobiology.

Graphic abstract:

EX vivo single molecue imaging of membrane receptors