The basic principle of conventional atomic force microscopy (AFM) imaging is that a sharp tip attached to the free end of a microcantilever probes the sample surface, while this tip is being scanned ...over the sample surface. HS-AFM, developed around 2008, can capture dynamic images of biomolecules at sub-100 ms temporal and submolecular lateral resolution. Ando et al focus on application studies of HS-AFM, in which dynamics of proteins and live cells are visualized. They also describe the HS-AFM setup, substrate surfaces, and precautions to be considered in HS-AFM imaging experiments.
Living cells are viscoelastic materials, with the elastic response dominating at long timescales (≳1 ms)1. At shorter timescales, the dynamics of individual cytoskeleton filaments are expected to ...emerge, but active microrheology measurements on cells accessing this regime are scarce2. Here, we develop high-frequency microrheology (HF-MR) to probe the viscoelastic response of living cells from 1Hz to 100 kHz. We report the viscoelasticity of different cell types and upon cytoskeletal drug treatments. At previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequency, providing a univocal mechanical fingerprint. Microrheology over a wide dynamic range up to the frequency of action of the molecular components provides a mechanistic understanding of cell mechanics.
•HS-AFM detects conformational changes of unlabeled membrane proteins.•In situ responses to pH, ligands, temperature and light can be visualized.•Developments in techniques now allow microsecond ...temporal resolution.
Recent advances in high-speed atomic force microscopy (HS-AFM) have made it possible to study the conformational dynamics of single unlabeled transmembrane channels and transporters. Improving environmental control with the integration of a non-disturbing buffer exchange system, which in turn allows the gradual change of conditions during HS-AFM operation, has provided a breakthrough toward the performance of structural titration experiments. Further advancements in temporal resolution with the use of line scanning and height spectroscopy techniques show how high-speed atomic force microscopy can measure millisecond to microsecond dynamics, pushing this method beyond current spatial and temporal limits offered by less direct techniques.
Many biological processes in a living cell are consequences of sequential and hierarchical dynamic events of biological macromolecules such as molecular interactions and conformational changes. ...Hence, knowledge of structures, assembly and dynamics of proteins is the foundation for understanding how biological molecules work. Among several techniques to analyze dynamics of proteins, high-speed atomic force microscopy (HS-AFM) is unique to provide direct information about both structure and dynamics of single proteins at work.
The scope of this review is overviewing recent progresses of HS-AFM for studying dynamic processes of biomolecular systems. In the technical descriptions, key developments enabling fast and non-invasive imaging of biological samples are briefly mentioned. Then recent successful applications of HS-AFM are overviewed to showcase the power of HS-AFM in biological research.
We discuss examples where HS-AFM movies captured important dynamic biological processes, including conformational dynamics of membrane proteins, processive movements of enzymes, assembly and disassembly processes of protein supramolecular structures, and dynamics in a two-dimensional protein crystal. These examples demonstrate the usability of HS-AFM to reveal biomolecular processes at high spatiotemporal (nanometer and subsecond) resolution.
Real-time movies of unlabeled proteins at work captured by HS-AFM allowed us to directly gain insights into mechanisms of molecular actions. Together with further functional extensions, HS-AFM will enable researchers to investigate more complex biological systems involving multiple proteins and will become an indispensable technique for life science.
This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” Guest Editor: Dr., Professor Koichi Kato.
In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been ...possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior.
Dynamics are fundamental to the functions of biomolecules and can occur on a wide range of time and length scales. Here we develop and apply high-speed AFM height spectroscopy (HS-AFM-HS), a ...technique whereby we monitor the sensing of a HS-AFM tip at a fixed position to directly detect the motions of unlabeled molecules underneath. This gives Angstrom spatial and microsecond temporal resolutions. In conjunction with HS-AFM imaging modes to precisely locate areas of interest, HS-AFM-HS measures simultaneously surface concentrations, diffusion coefficients and oligomer sizes of annexin-V on model membranes to decipher key kinetics allowing us to describe the entire annexin-V membrane-association and self-assembly process in great detail and quantitatively. This work displays how HS-AFM-HS can assess the dynamics of unlabeled bio-molecules over several orders of magnitude and separate the various dynamic components spatiotemporally.
Receptor–ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, ...detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin–biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and ratedependent induced-fit motions for intermediates, which might be relevant for other receptor–ligand bonds.
Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer ...and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ∼200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (kA) as 106 pN/nm and 199 pN/nm and the bending stiffness (kc) as 18 kBT and 57 kBT for the liquid and gel phases, respectively.
ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates ...membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.
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•Snf7 forms highly flexible filaments that spontaneously curl•Snf7 filaments forms spirals at the surface of lipid membranes•Snf7 spirals are springs as they can deform under lateral compression•Relaxation of compressed Snf7 spirals leads to membrane deformation
A component of the ESCRT-III membrane fission machinery self-organizes into spiral springs that trigger membrane deformation when released.
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