We report the assembly of protein supramolecular structures at an air−water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include ...electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin−biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.
Significance Statement T cells are the first responders of the adaptive immune system via the recognition of a non-self peptide via the T cell receptor (TCR). Generating force by actomyosin ...machinery, T cells test the TCR to antigen binding. This force needs to be transmitted via a chain of molecules where the receptor-to-cytoskeleton interaction is currently the missing link. We devised a method based on pulling membrane tubes via optical tweezers and a physical model to extract viscoelastic parameters, separable into cell or molecular scales, to probe the mechanics of the receptor to cytoskeleton link. Our analysis suggests that the link stiffness depends on the identity of the receptor being sollicitated. These findings have implications for understanding of T cell mechanotransduction.
Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are ...still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.
Receptors at the membrane of immune cells are the central players of innate and adaptative immunity, providing effective defence mechanisms against pathogens or cancer cells. Their function is ...intimately linked to their position at and within the membrane which provides accessibility, mobility as well as membrane proximal cytoskeleton anchoring, all of these elements playing important roles in the final function and links to cellular actions. Understanding how immune cells integrate the specific signals received at their membrane to take a decision remains an immense challenge and a very active field of fundamental and applied research. Recent progress in imaging and micromanipulation techniques have led to an unprecedented refinement in the description of molecular structures and supramolecular assemblies at the immune cell membrane, and provided a glimpse into their dynamics and regulation by force. Several key elements have been scrutinized such as the roles of relative sizes of molecules, lateral organisation, motion in the membrane of the receptors, but also physical cues such as forces, mediated by cellular substrates of different rigidities or applied by the cell itself, in conjunction with its partner cell. We review here these recent discoveries associated with a description of the biophysical methods used. While a conclusive picture integrating all of these components is still lacking, mainly due to the implication of diverse and different mechanisms and spatio-temporal scales involved, the amount of quantitative data available opens the way for physical modelling and numerical simulations and new avenues for experimental research.
T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It ...is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains unclear. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds), and this was confirmed by molecular dynamic simulations. Consistent with these findings, we show that the best characterized catch-bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination and we suggest this new force-shielding role for the adhesion receptors CD2 and LFA-1. One sentence summary Mechanical forces disproportionately accelerate the off-rates of higher-affinity antigens reducing T cell antigen discrimination
T cells use their T‐cell receptors (TCRs) to discriminate between lower‐affinity self and higher‐affinity foreign peptide major‐histocompatibility‐complexes (pMHCs) based on the TCR/pMHC off‐rate. It ...is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off‐rate remains debated. Here, we measured the effect of mechanical force on the off‐rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower‐affinity TCR/pMHCs with faster solution off‐rates were more resistant to mechanical force (weak slip or catch bonds) than higher‐affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best‐characterized catch bond, involving the OT‐I TCR, has a low affinity and an exceptionally fast solution off‐rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA‐1 in force‐shielding the TCR/pMHC interaction.
Synopsis
The T‐cell antigen receptor (TCR) needs to discriminate between low‐affinity self‐peptide MHC antigens (pMHCs) and higher affinity foreign pMHCs. This study shows that mechanical force likely impairs TCR discrimination, supporting a role for adhesion receptors in shielding the TCR/pMHC interaction from mechanical forces.
A cell‐free assay is used to systematically measure the off‐rate of 13 different TCR/pMHC interactions under different applied forces.
In contrast to studies using intact T cells, we found that applied force increased the off‐rate of high‐affinity TCR/pMHC interactions by more than it did for low‐affinity interactions.
Structured‐based molecular dynamics simulations confirmed these experimental findings.
A kinetic proofreading model incorporating the observed effects of mechanical forces predicts that reducing the force on the TCR/pMHC interaction improves antigen discrimination and sensitivity.
The ability of CD2 and LFA‐1 to improve antigen discrimination and sensitivity supports a role for these adhesion receptors in force‐shielding the TCR/pMHC interaction.
Low‐affinity TCR‐pMHC interactions are less susceptible to disruption by mechanical force as compared to high‐affinity ones implicating a role for cell adhesion molecules in shielding TCR‐pMHC from applied force to promote antigen discrimination.
Living cells sense the physical and chemical nature of their micro/nano environment with exquisite sensitivity. In this context, there is a growing need to functionalize soft materials with ...micro/nanoscale biochemical patterns for applications in mechanobiology. This, however, is still an engineering challenge. Here a new method is proposed, where submicronic protein-patterns are first formed on glass and are then printed on to an elastomer. The degree of transfer is shown to be governed mainly by hydrophobic interactions and to be influenced by grafting an appropriate fluorophore onto the core protein of interest. The transfer mechanism is probed by measuring the forces of adhesion/cohesion using atomic force microscopy. The transfer of functional arrays of dots with size down to about 400 nm, on elastomers with stiffness ranging from 3 kPa to 7 MPa, is demonstrated. Pilot studies on adhesion of T lymphocytes on such soft patterned substrates are reported.
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