Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.
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.
Evans
E
E
Department of Physics, University of British Columbia, Vancouver, Canada.
Ritchie
K
K
Merkel
R
R
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D006224:Cricetinae; D003599:Cytoskeleton / Q000502:physiology; D004910:Erythrocyte Membrane / Q000502:physiology*; D004912:Erythrocytes / Q000502:physiology*; D007668:Kidney; D008051:Lipid Bilayers; D008322:Mammals; D008857:Microscopy, Interference / Q000379:methods; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D013314:Stress, Mechanical; D013499:Surface Properties
D008051:Lipid Bilayers