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The unique functions of intrinsically disordered proteins (IDPs) depend on their dynamic protean structure that often eludes analysis. High-speed atomic force microscopy (HS-AFM) can conduct this difficult analysis by directly visualizing individual IDP molecules in dynamic motion at sub-molecular resolution. After brief descriptions of the microscopy technique, this review first shows that the intermittent tip–sample contact does not alter the dynamic structure of IDPs and then describes how the number of amino acids contained in a fully disordered region can be estimated from its HS-AFM images. Next, the functional relevance of a dumbbell-like structure that has often been observed on IDPs is discussed. Finally, the dynamic structural information of two measles virus IDPs acquired from their HS-AFM and NMR analyses is described together with its functional implications.

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3–20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy. Understanding antibody-antigen interactions is important to deepen the understanding of immunology. Here, the authors report on the application of DNA origami structures for the controlled presentation of antigens to study antibody binding behaviours at room temperature.

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein–nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F 1 -ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10–15 h, depending mainly on the substrate surface to be used.

This Review Article examines the principles, advantages and limitations of emerging bioimaging modes of atomic force microscopy, including multiparametric, molecular recognition, multifrequency and high-speed imaging. Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative ‘visual evidence’ for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action. Walking myosin V caught on video Motor proteins of the myosin family serve many important functions in eukaryotic cells and are central to actin-based motility. Our understanding of the mechanism of myosin action has been hampered by the lack of technology for simultaneously observing the structure and dynamics of biomolecules, but now Kodera et al . have used high-speed atomic force microscopy (HS-AFM) to directly observe myosin V moving along actin filaments with unprecedented time resolution. They obtain high-resolution movies that provide visual evidence supporting the 'swinging lever-arm' model for myosin motility. The high-resolution imaging possible using HS-AFM should make the technique broadly applicable in the fields of structural biology and single-molecule biology. High-speed atomic force microscopy can be used to record the structure and dynamics of biomolecules simultaneously. These authors use this method to directly observe the dynamics of the motor protein myosin V moving along actin filaments, with unprecedented time resolution. The high-resolution movies provide evidence supporting the 'swinging lever-arm' model of myosin motility, and provide important insights into the mechanism of motor movement.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs. High-speed AFM imaging enables a semiquantitative, realistic description of the dynamic structure of intrinsically disordered proteins.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas . These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes. Microtubules in cilia are sufficiently stable to withstand the beating motion, but how they are stabilized while serving as tracks for intraflagellar transport and axonemal dyneins remains unknown. Here authors identify two microtubule inner proteins, FAP45 and FAP52, which stabilize the ciliary axonemes in Chlamydomonas .

A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL–GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL–GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL : GroES2 and asymmetric GroEL : GroES1 complexes, and GroES–unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL : GroES1 complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation and endocytosis in COS-7, HeLa cells and hippocampal neurons.

High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y -scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y -positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20–100 nm/pixel), complicating single-molecule analysis. Thus, AFM systems able to image large sample areas at high speeds and with nanometer resolution have still been missing. Here, we describe a HS-AFM sample-scanner system able to record large topographic images (≤ 36 × 36 µm 2 ) containing up to 16 megapixels, providing molecular resolution throughout the image frame. Despite its large size, the flexure-based scanner features a high resonance frequency (> 2 kHz) and delivers stable operation even at high scans speeds of up to 7.2 mm/s, minimizing the time required for recording megapixel scans. We furthermore demonstrate that operating this high-speed scanner in time-lapse mode can simultaneously identify areas of spontaneous 2-dimensional Annexin A5 crystal growth, resolve the angular orientation of large crystalline domains, and even detect rare crystal lattice defects, all without changing scan frame size or resolution. Dynamic processes first identified from overview scans can then be further imaged at increased frame rates in reduced scan areas after switching to conventional HS-AFM scanning. The added ability to collect large-area, high-resolution images of complex samples within biological-relevant time frames extends the capabilities of HS-AFM from single-molecule imaging to the study of large dynamic molecular arrays. Moreover, large-area HS-AFM scanning can generate detailed structural data sets from a single scan, aiding the quantitative analysis of structurally heterogenous samples, including cellular surfaces.