Monitoring biomolecular interactions by time-lapse atomic force microscopy

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effec ters and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an a queous environment. This review explores how time-lapse AFM has been used t o learn more about normal and disease-associated biological processes. Thre e specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, a nd undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and m aintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to exa mine and manipulate some of these events in vitro, in order to learn more a bout how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheime r's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn, how to interfere with f ibril formation. One application of time-lapse AFM in this area has been th e direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic dru gs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofi brils) a given active compound will interfere. Nuclear pore complexes (NPCs ) are large supramolecular assemblies embedded in the nuclear envelope. Tra nsport of ions, small molecules, proteins, RNAs, and RNP particles in and o ut of the nucleus occurs via NPCs, Time-lapse AFM has been used to structur ally visualize the response of individual NPC particles to various chemical and physical effecters known to interfere with nucleocytoplasmic transport . Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under bot h normal and pathological conditions at the single molecule level. (C) 2000 Academic Press.