Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces

Abstract

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.

Contribution of cellular contractility to spatial and temporal variations in cellular stiffness

“…The mechanical properties of the cellular cortex have been measured by several techniques such as cell poking [12,13], micropipette aspiration [14,15], optical tweezers [16][17][18] and magnetic beads twisting [19][20][21]. However, the temporal and/or spatial resolutions of these techniques do not suffice for a thorough understanding of the variations in mechanical properties caused by cellular functions.…”

Correlation Force Spectroscopy for Single Molecule Measurements

“…The technique has a force range > 0.1 pN and bandwidth < 10 Hz. (3) The Biomembrane force probe is used in studies of molecular adhesion and structural linkage at biological interfaces [11]. This technique has a force range of 0.5 to 1000 pN and bandwidth < 1 kHz.…”

“…From the equipartition theorem, it is known that the area under the first resonant peak in 11 G or 22 G is equal to . Thus we can normalize each of and by dividing through by their respective areas under the first resonant peak, to obtain ii G .…”

“…This contribution is also insignificant and can be approximated from the radial fluid velocity field around an oscillating cylinder (cylinder is the theoretical model for an oscillating cantilever [119]). At 30 µm separation, the fluid velocity drops to less than 20% of Likewise, the friction coefficient for the motion of a single particle, 11  , includes the effect of the cantilever, and I wish to subtract this contribution. I estimate the cantilever contribution using the expression for the drag on an infinite cylinder in a viscous fluid [43,94]:…”

Long reach cantilevers for sub-cellular force measurements

“…The force on the AFM cantilever is then measured as the cantilever-cell unit is retracted from the substrate. This method has shed light on a wide range of ligand-receptor adhesive interactions [20][21][22], as have the conceptually similar biomembrane force probe [23] and laser trap [24] methods. A challenge for such cell-as-probe techniques lies in minimizing the contact area to a single, cell-initiated adhesive contact: that between a pseudopod tip and a substrate, for example.…”