Extraction of Accurate Biomolecular Parameters from Single-Molecule Force Spectroscopy Experiments

The atomic force microscope (AFM) is able to manipulate biomolecules and their complexes with exquisite force sensitivity and distance resolution. This capability, complemented by theoretical models, has greatly improved our understanding of the determinants of mechanical strength in proteins and revealed the diverse effects of directional forces on the energy landscape of biomolecules. In unbinding experiments, the interacting partners are usually immobilized on their respective substrates via extensible linkers. These linkers affect both the force and contour length (Lc) of the complex at rupture. Surprisingly, while the former effect is well understood, the latter is largely neglected, leading to incorrect estimations of Lc, a parameter that is often used as evidence for the detection of specific interactions and remodeling events and for the inference of interaction regions. To address this problem, a model that predicts contour length measurements from single-molecule forced-dissociation experiments is presented that considers attachment position on the AFM tip, geometric effects, and polymer dynamics of the linkers. Modeled data are compared with measured contour length distributions from several different experimental systems, revealing that current methods underestimate contour lengths. The model enables nonspecific interactions to be identified unequivocally, allows accurate determination of Lc, and, by comparing experimental and modeled distributions, enables partial unfolding events before rupture to be identified unequivocally

Germinal center B cells recognize antigen through a specialized immune synapse architecture

B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we show that in contrast to naive and memory B cells, which gathered antigen towards the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β (PKC-β)–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T-cell dependent selection of high-affinity B cells in GCs

Force-induced remodelling of proteins and their complexes

Force can drive conformational changes in proteins, as well as modulate their stability and the affinity of their complexes, allowing a mechanical input to be converted into a biochemical output. These properties have been utilised by nature and force is now recognised to be widely used at the cellular level. The effects of force on the biophysical properties of biological systems can be large and varied. As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results. Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study

Cytoskeletal control of B cell responses to antigens.

The actin cytoskeleton is essential for cell mechanics and has increasingly been implicated in the regulation of cell signalling. In B cells, the actin cytoskeleton is extensively coupled to B cell receptor (BCR) signalling pathways, and defects of the actin cytoskeleton can either promote or suppress B cell activation. Recent insights from studies using single-cell imaging and biophysical techniques suggest that actin orchestrates BCR signalling at the plasma membrane through effects on protein diffusion and that it regulates antigen discrimination through the biomechanics of immune synapses. These mechanical functions also have a role in the adaptation of B cell subsets to specialized tasks during antibody responses