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Microrheology of cells suggests that the dynamics of single filaments in the cytoskeleton dominate at high frequencies. This response can be used to detect differences between cell types and states — including benign and malignant cancer cells. Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond 1 . On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce 2 . Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On 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 frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range—up to the frequency characterizing the molecular components—provides a mechanistic understanding of cell mechanics.

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. The dynamics of biomolecules can occur over a wide range of time and length scales. Here the authors develop a high-speed AFM height spectroscopy method to directly detect the motion of unlabeled molecules at Angstrom spatial and microsecond temporal resolution.

Cryogenic electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR) contribute structural data that are interchangeable, cross-verifiable, and visualizable on common platforms, making them powerful tools for our understanding of protein structures. Unfortunately, atomic force microscopy (AFM) has so far failed to interface with these structural biology methods, despite the recent development of localization AFM (LAFM) that allows extracting high-resolution structural information from AFM data. Here, we build on LAFM and develop a pipeline that transforms AFM data into 3D-density files (.afm) that are readable by programs commonly used to visualize, analyze, and interpret structural data. We show that 3D-LAFM densities can serve as force fields to steer molecular dynamics flexible fitting (MDFF) to obtain structural models of previously unresolved states based on AFM observations in close-to-native environment. Besides, the .afm format enables direct 3D or 2D visualization and analysis of conventional AFM images. We anticipate that the file format will find wide usage and embed AFM in the repertoire of methods routinely used by the structural biology community, allowing AFM researchers to deposit data in repositories in a format that allows comparison and cross-verification with data from other techniques. Here, the authors developed a pipeline to transform AFM data into 3D-density files for cross-methodology structural biology analysis. Combined with molecular dynamics flexible fitting, the approach yields structural information on protein dynamics.

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.

The cantilever mediates tip-sample interaction detection in all atomic force microscopes (AFMs). Canonical cantilevers are beams, where length, width, and thickness define the physical properties such as stiffness and resonant frequency, that also mediate laser-reflection to report on cantilever deflection. High-speed AFM (HS-AFM) demands miniaturized cantilevers that are soft and fast, but miniaturized beams reduce laser signal quality. Here, we present a seesaw cantilever with a rigid reflective board oscillating over torsional hinges separating the laser-reflective and mechanical functions. Finite element analysis verified the seesaw mechanism. The board can be optimized for laser-reflection and the shortened distance between tip and hinges enhances the angular sensitivity, while the stiffness is tunable via the hinge dimensions. We detail seesaw cantilever design, fabrication, tip addition, physical equations, and sub-molecular imaging of biological samples. We propose that seesaw cantilevers offer a promising alternative to traditional beam cantilevers for diverse AFM applications. The authors present AFM seesaw cantilevers (SSCs) that separate the cantilever’s mechanical and laser-reflective functions to a rigid board and torsional hinges, respectively. The SSCs readily allow biomolecular high-speed AFM imaging.

Annexins are abundant cytoplasmic proteins, which bind to membranes that expose negatively charged phospholipids in a Ca 2+ -dependent manner. During cell injuries, the entry of extracellular Ca 2+ activates the annexin membrane-binding ability, subsequently initiating membrane repair processes. However, the mechanistic action of annexins in membrane repair remains largely unknown. Here, we use high-speed atomic force microscopy (HS-AFM), fluorescence recovery after photobleaching (FRAP), confocal laser scanning microscopy (CLSM) and molecular dynamics simulations (MDSs) to analyze how annexin-V (A5) binds to phosphatidylserine (PS)-rich membranes leading to high Ca 2+ -concentrations at membrane, and then to changes in the dynamics and organization of lipids, eventually to a membrane phase transition. A5 self-assembly into lattices further stabilizes and likely structures the membrane into a gel phase. Our findings are compatible with the patch resealing through vesicle fusion mechanism in membrane repair and indicate that A5 retains negatively charged lipids in the inner leaflet in an injured cell. Annexins are cytoplasmic proteins, which bind to membranes exposing negatively charged phospholipids in a Ca 2+ -dependent manner. Here the authors use high-speed atomic force microscopy and other techniques to show that annexin-V self-assembles into highly structured lattices that lead to a membrane phase transition on PS-rich membranes.

Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s −1 . The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended. Here, the authors use high-speed atomic force microscopy (HS-AFM) methods to characterize the single molecule kinetics of wild-type bacteriorhodopsin (bR) with millisecond temporal resolution, providing new insights into the bR conformational cycle.

The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small β-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.

Bcs1, a homo-heptameric transmembrane AAA-ATPase, facilitates folded Rieske iron-sulfur protein translocation across the inner mitochondrial membrane. Structures in different nucleotide states (ATPγS, ADP, apo) provided conformational snapshots, but the kinetics and structural transitions of the ATPase cycle remain elusive. Here, using high-speed atomic force microscopy (HS-AFM) and line scanning (HS-AFM-LS), we characterized single-molecule Bcs1 ATPase cycling. While the ATP conformation had ~5600 ms lifetime, independent of the ATP-concentration, the ADP/apo conformation lifetime was ATP-concentration dependent and reached ~320 ms at saturating ATP-concentration, giving a maximum turnover rate of 0.17 s −1 . Importantly, Bcs1 ATPase cycle conformational changes occurred in concert. Furthermore, we propose that the transport mechanism involves opening the IMS gate through energetically costly straightening of the transmembrane helices, potentially driving rapid gate resealing. Overall, our results establish a concerted ATPase cycle mechanism in Bcs1, distinct from other AAA-ATPases that use a hand-over-hand mechanism. Bcs1, a transmembrane AAA-ATPase, facilitates the translocation of folded ISP across the inner mitochondrial membrane. This study shows that the Bcs1 ATPase cycle conformational changes are highly concerted, unlike the canonical hand-over-hand mechanism.