On the organization of self-assembled actin networks in giant vesicles

We studied the formation of actin scaffolds in giant vesicles of dimyristoylphosphatidylcholine (DMPC). Polymerization of actin was induced at low ionic strength through ionophore-mediated influx of Mg$^{2+}$ (2 mM). The spatial organization of the filamentous actin was visualized by confocal and epifluorescence microscopy as a function of the filaments length and membrane composition, by including various amounts of cholesterol or lipids with neutral and positively charged polyethyleneglycol headgroups (PEG lipopolymers). In vesicles of pure DMPC, the newly polymerized actin adsorbs to the membrane and forms a thin shell. In the presence of 2.5 mol% lipopolymers or of cholesterol at a molar fraction $x=0.37$, formation of a thin adsorbed film is impeded. A fuzzy cortex is predominantly formed in vesicles of diameter $d$ smaller than the filament persistence length ($d\leq 15~\mu$m) while for larger vesicles a homogeneous network formation is favoured in the bulk of the vesicle. The fuzzy-cortex formation is interpreted as a consequence of the reduction of the bending energy if the actin filaments accumulate close to the vesicle wall

CD8 co-receptor enhances T-cell activation without any effect on initial attachment

The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to measure at the single molecule level the kinetics of bond formation and rupture between TCR- transfected CD8+ and CD8- Jurkat cells and surfaces coated with five peptide-exposing major histocompatibility antigens (pMHCs) of varying activating power. We also used interference reflection microscopy to image the spreading of these cells dropped on pMHC-exposing surfaces. CD8 did not influence the TCR-pMHC interaction during the first few seconds following cell surface encounter, but it promoted the subsequent spreading responses, suggesting that CD8 was involved in early activation rather than binding. Further, the rate and extent of spreading, but not the lag between contact and spreading initiation, depended on the pMHC. Elucidating T-lymphocyte detection strategy may help unravel underlying signaling networks

Coupling artificial actin cortices to bio-functionalised lipid monolayers

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

TCR–pMHC kinetics under force in a cell-free system show no intrinsic catch bond, but a minimal encounter duration before binding

The T cell receptor (TCR)–peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR–pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR–pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch–slip transitions and, second, 2D TCR–pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR–pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation

Mechanical forces impair antigen discrimination by reducing differences in T-cell receptor/peptide–MHC off-rates

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