Cellular and molecular mechanics by atomic force microscopy: Capturing the exocytotic fusion pore in vivo ?

The atomic force microscope (AFM) is a remarkably simple instrument that can measure forces down to piconewtons and can resolve force changes caused by the displacement of its probe by a fraction of a nanometer. Furthermore, these measurementscanbedoneunderwater,allowingforthestudy of biological material under conditions that resemble thosein vivo.TheAFMfunctionslikeaminiaturephonograph.Asharp tip,mountedonacantilever,interactswiththesample,causing minutedeflectionsthatcanbecalibratedasaforce.TheAFM, in its force-measuring mode, has been applied to the study of molecular mechanics. For example, the laboratory of Hermann Gaub in Munich succeeded in the remarkable feat of measuring the adhesion force between single avidin‐biotin pairs (’160 pN; ref. 1). These measurements were independently confirmed by molecular dynamic simulations of the avidin‐biotininteractionthatrevealedtheatomictrajectoryof a rupture and predicted the associated forces (2). Hence, the techniquesofmolecularmechanicsnowrivalthecapabilitiesof patch-clamp techniques (3) in describing the activity of single proteins. The patch-clamp technique seals a lipid membrane, containingionchannelproteins,ontoaglasspipetteelectrode. Ioniccurrentsflowingthroughasingleionchannelproteinare then easily measured, reporting on the conformational states of the transmembrane protein. In contrast to the patch-clamp technique, useful mainly for the characterization of ion channels, molecular mechanics studies can be done on single proteins or more complex structures that do not carry an associated electrical activity. ThemostfamiliaruseoftheAFMisincreatingtopographic imagesofasample.ThisisdonebyscanningtheAFMtipover the sample and recording the z axis displacement required to maintain a constant contact force. The nanometer resolution of the AFM imaging technique has been used to examine the structural features of proteins, producing data that is comparable to that obtained by x-ray crystallography (4). However, the biggest promise of the AFM technique is in the measurement of the activity of single protein structuresin vivoduring signaling events or during other types of cellular activities. Indeed, we may be getting close. A recent example demonstratedthatitispossibletoobserveconformationalchangesin the structure of a single nuclear pore in Xenopus oocyte nuclear envelopes (5). However, these measurements were doneinfixedtissue.RecentworkfromthelaboratoryofBhanu