Far - Field Optical Nanoscopy Stefan

37. E. Evans, Annu. Rev. Biophys. Biomol. Struct. 30, 105 (2001). 38. G. I. Bell, Science 200, 618 (1978). 39. For adhesive interactions important in soft tissues and organs of eukaryotic cell systems, the average times toff° reported for spontaneous dissociation of ligand/receptor bonds range from a fraction of a second to 100 s or more. 40. Because of thermal activation, the appropriate unit for “bond strength” is the pN. This scale follows from the ratio of thermal energy kBT (~4.1 × 10 −21 J = 4.1 pN/nm at room temperature) to the nanometer length xb gained in surpassing an activation-energy barrier (Fig. 1). A force of 10 pN is close to one-billionth of a gram weight (that is, 1 pN ≈ 10 g wt). 41. C. Wülfing, M. M. Davis, Science 282, 2266 (1998). 42. E. Evans, K. Ritchie, Biophys. J. 72, 1541 (1997). 43. Extending the duration of a ligand-receptor bond by a factor of 100 requires a modest collective increase of 4 to 5 kBT in the height of the activation-energy barrier that impedes dissociation. Yet, the added persistence appears to be accompanied by a concomitant decrease in sensitivity to stress rate (lower slope fb), suggesting that the energy landscape changes to increase the length gained in the direction of force when the bond breaks. Advanced computational methods like the “steered molecular dynamics” described in the companion review by Sotomayor and Schulten (36) provide valuable tools for investigating how variations in chemical structure affect activation energy barriers and pathways governing bond strength. 44. In typical laboratory tests of single adhesion bonds, constructs of the ligand and receptor molecules are chemically immobilized on solid surfaces at very low surface densities, for example, a ligand to the face of an ultrasensitive force probe and its receptor to a solid target held by a feedback-stabilized piezo translator. The target is then repeatedly moved to/from contact to the probe face, during which time the deflection of the probe is tracked at high precision and multiplied by its “spring” constant kf (pN/nm) to report the force history f(t). Bond events are identified by the cycles showing periods of probe stretch ending in precipitous recoil, as sketched in Fig. 2A. 45. E. Evans, A. Leung, V. Heinrich, C. Zhu, Proc. Natl. Acad. Sci. U.S.A. 101, 11281 (2004). 46. E. Evans, K. Kinoshita, in Methods in Cell Biology: Cell Mechanics, Vol. 83, Y. L. Wang, D. E. Discher, Eds. (Elsevier, New York, 2007), chap. 16. 47. E. Perret, A. Leung, H. Feracci, E. Evans, Proc. Natl. Acad. Sci. U.S.A. 101, 16472 (2004). 48. M. V. Bayas, A. Leung, E. Evans, D. Leckband, Biophys. J. 90, 1385 (2006). 49. Acting as a soft spring linked in series with the probe spring kf, the elastic response of the cell cortex kcell reduces the force rate rf relative to the probe rate kfvpull. The ratio crf = rf/(kfvpull) provides a direct assay of the cell cortical stiffness, that is, kcell ≈ kfcrf/(1 – crf). It is important to note that different cell types possess very different levels of interfacial stiffness and that these levels often change with cell activation or spreading on a stiffer substrate. 50. As lipid material flows onto a tether, bilayer-spanning proteins (especially those that interact with the cytoskeleton) are expected to remain behind in the cell membrane. However, the acylated proteins bound weakly to the bilayer could build up at the base of the tether, causing some to be expelled from the surface when approaching the tether-cell junction. 51. V. Heinrich, A. Leung, E. Evans, Biophys. J. 88, 2299 (2005). 52. E. Evans, V. Heinrich, A. Leung, K. Kinoshita, Biophys. J. 88, 2288 (2005). 53. Supported by grants from the National Institutes of Health.