Atomic force microscopy (AFM) has been used to measure the strength of bonds between biological receptor molecules and their ligands. But for weak noncovalent bonds, a dynamic spectrum of bond strengths is predicted as the loading rate is altered, with the measured strength being governed by the prominent barriers traversed in the energy landscape along the force-driven bond-dissociation pathway. In other words, the pioneering early AFM measurements represent only a single point in a continuous spectrum of bond strengths, because theory predicts that these will depend on the rate at which the load is applied. Here we report the strength spectra for the bonds between streptavidin (or avidin) and biotins-the prototype of receptor-ligand interactions used in earlier AFM studies, and which have been modelled by molecular dynamics. We have probed bond formation over six orders of magnitude in loading rate, and find that the bond survival time diminished from about 1 min to 0.001 s with increasing loading rate over this range. The bond strength, meanwhile, increased from about 5 pN to 170 pN. Thus, although they are among the strongest noncovalent linkages in biology (affinity of 10(13) to 10(15) M(-1)), these bonds in fact appear strong or weak depending on how fast they are loaded. We are also able to relate the activation barriers derived from our strength spectra to the shape of the energy landscape derived from simulations of the biotin-avidin complex.

No abstract

Highlights d Stretch triggers amplitude-dependent supracellular and nuclear mechanoresponses d H3K9me3 heterochromatin mediates nuclear stiffness and membrane tension d Nuclear deformation-triggered Ca 2+ alters chromatin rheology to prevent DNA damage d Supracellular alignment redistributes stress to restore chromatin state

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure. Subsequent loading of an attachment tests mechanical response of the receptor-substrate linkage throughout the force-driven process of detachment.

No abstract

Microfluorescence methods were used to examine monolayer-monolayer and bilayer-substrate coupling in bilayers deposited on glass substrates. In the first part, lateral diffusion of lipid probes in individual lipid layers was measured by the fluorescence recovery after photobleach technique. The aim was to evaluate viscous molecular friction (i) between monolayers that form a single bilayer and (ii) between a bilayer and an adjacent substrate based on a recent phenomenological theory for particle mobility in substrate-coupled membranes (Evans and Sackmann, J. Fluid Mech. 194 (1988) 553 [1]). To obtain coefficients for friction between monolayers, a bilayer was formed with the first (proximal) monolayer fixed to the glass substrate by Si-O-bonds (using silanes) or by ion bridges (using cadmium arachidate) ; then, probe diffusion was measured in the second (distal) monolayer formed by phospholipids. The coefficient for viscous friction (defined by b s-interfacial shear stress/interfacial « slip » velocity) between monolayers with fluid chains (DMPC or DOPC on silane) was calculated to be in the range bs = 106-107 dyn-sec/cm3 ; between a fluid and a solid monolayer (DOPC on Cd-arachidate), the frictional coefficient was much larger, i.e. bs = 1-5 × 108 dyn-sec/cm3. For two opposing monolayers with liquid chains, it was found that bs increased with the degree of interdigitation between the hydrocarbon chains. To investigate the effect of lubrication by a water film between a fluid bilayer and the substrate, the substrate was first Argon sputtered which acted to separate the proximal monolayer from the substrate by a thin lubricating water film (thickness in nm region). The frictional coefficient between the bilayer and the substrate was measured to be in the range bs = 2 x 103-3 × 105 dyn-sec/cm3 which implied that the film thickness was from 1-50 nm. In the second part, we studied the effect of monolayer-monolayer and bilayer-substrate coupling on the acyl-chain crystallization transitions in monolayers of supported bilayers. Symmetric bilayers (separated from the substrate by a water film) exhibited sharp phase transitions at about the same transition temperature as the free bilayers. The transition temperature for asymmetric bilayers was between the transition temperatures for the individual monolayer components. Bilayers formed by phospholipid monolayers on silanes showed a concerted but continuous phase transition which appeared to be due to the constraint of fixed total area and interdigitation of the two monoalayers. By doping the monolayers with different fluorescent probes (NBD and Texas Red lipid analogs), it was demonstrated that, when the proximal layer was in a solid or liquid-solid coexistence state, a phase transition was induced in the superficial monolayer even if this layer was deposited from the fluid state. It was also observed that the patterns of fluid and solid domains in both layers were in complete register. On the other hand, a fluid monolayer of unsaturated lipids on tightly -packed crystall...

Recognition of external mechanical signals is vital for mammalian cells. Cyclic stretch, e.g. around blood vessels, is one such signal that induces cell reorientation from parallel to almost perpendicular to the direction of stretch. Here, we present quantitative analyses of both, cell and cytoskeletal reorientation of umbilical cord fibroblasts. Cyclic strain of preset amplitudes was applied at mHz frequencies. Elastomeric chambers were specifically designed and characterized to distinguish between zero strain and minimal stress directions and to allow accurate theoretical modeling. Reorientation was only induced when the applied stretch exceeded a specific amplitude, suggesting a non-linear response. However, on very soft substrates no mechanoresponse occurs even for high strain. For all stretch amplitudes, the angular distributions of reoriented cells are in very good agreement with a theory modeling stretched cells as active force dipoles. Cyclic stretch increases the number of stress fibers and the coupling to adhesions. We show that changes in cell shape follow cytoskeletal reorientation with a significant temporal delay. Our data identify the importance of environmental stiffness for cell reorientation, here in direction of zero strain. These in vitro experiments on cultured cells argue for the necessity of rather stiff environmental conditions to induce cellular reorientation in mammalian tissues.

Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.