Melittin is a model system for the action of antimicrobial peptides which are potential candidates for novel antibiotics. We investigated the membrane lysis effect of melittin on phase-separated supported lipid bilayers (DOPC-DPPC) by atomic force microscopy. AFM images show that the peptide first forms defects at the interface between the two lipid phases and then degrades preferentially the liquid-phase DOPC-enriched domains. Vesicular structures of 10-20 nm radius were observed to form, suggesting a mixed carpet-toroidal model mechanism for the resolved action of melittin.

Integrin-mediated adhesion is essential for metazoan life. Integrin binding to ligand requires an activation step prior to binding ligand that depends on direct binding of talin and kindlin to the β-integrin cytoplasmic tail and the transmission of force from the actomyosin via talin to the integrin–ligand bonds. However, the affinity of talin for integrin tails is low. It is therefore still unclear how such low-affinity bonds are reinforced to transmit forces up to 10 to 40 pN. In this study, we use single-molecule force spectroscopy by optical tweezers to investigate the mechanical stability of the talin•integrin bond in the presence and absence of kindlin. While talin and integrin alone form a weak and highly dynamic slip bond, the addition of kindlin-2 induces a force-independent, ideal talin•integrin bond, which relies on the steric proximity of and the intervening amino acid sequences between the talin- and kindlin-binding sites in the β-integrin tail. Our findings show how kindlin cooperates with talin to enable transmission of high forces required to stabilize cell adhesion.

Integrins are large heterodimeric proteins that play an important role in force transduction across the cell membrane. In order to bind the extracellular matrix integrins have to be in an activated open form. The activation of integrins requires the binding of the talin N-terminal F3 domain to the integrin b-tail and the association with the contractile actomyosin cytoskeleton. Despite its critical role in force transduction, the affinity between integrin b-tail and talin-F3 domain is surprisingly weak for all isoform pairs (K d >10 mM). This raises the question how such a weak bond resists the actomyosin pulling forces and ensures a stable connection across the cell membrane. To address this question we designed fusion constructs between the talin-F3 domain (but also entire talin head domain) and the b1-integrin cytoplasmic tail. Using a dual-beam optical tweezer setup we probed the mechanics of the talinintegrin tail bond at the single molecule level. Our results confirm the dynamic character of the talin-integrin tail bond, which shows an unbinding-rate of z 50/s. The binding energy of the talin-integrin tail bond is weak and ranges around 4-6 k B T, and the bond is mainly in an open state at forces higher than 5 pN. Interestingly, we discovered that the talin-integrin tail bond is stabilized by the presence of kindlin, which is also a FERM-domain protein that synergizes with talin to activate integrins. When measuring the talin-integrin tail interaction with kindlin in solution, the talin-integrin bound states become longer. Surprisingly, the length of the bound states is not influenced by force. This kindlin-induced stabilization effect might be of crucial importance for integrin activation, clustering and development of adhesion site assemblies.