Recent advances in bioimaging with high-speed atomic force microscopy

Abstract

Among various microscopic techniques for characterizing protein structures and functions, high-speed atomic force microscopy (HS-AFM) is a unique technique in that it allows direct visualization of structural changes and molecular interactions of proteins without any labeling in a liquid environment. Since the development of the HS-AFM was first reported in 2001, it has been applied to analyze the dynamics of various types of proteins, including motor proteins, membrane proteins, DNA-binding proteins, amyloid proteins, and artificial proteins. This method has now become a versatile tool indispensable for biophysical research. This short review summarizes some bioimaging applications of HS-AFM reported in the last few years and novel applications of HS-AFM utilizing the unique ability of AFM to gain mechanical properties of samples in addition to structural information.

Keywords: Conformational dynamics; High-speed atomic force microscopy; Intermolecular interaction; Mechanical indentation; Single-molecule imaging.

Fig. 1

HS-AFM images capturing conformational dynamics of ring-shaped ATPases. a Diverse oligomeric forms of ΔN-TClpB observed by HS-AFM in 0.5 mM ATP at 25 °C. Scale bar, 20 nm. b Clipped HS-AFM images of aΔN-TClpB hexamer captured at 10 fps in the presence of 10 μM ATP. Scale bar, 5 nm. c Cryo-EM maps of Abo1 in the ATP, ADP, and apo states. d Clipped HS-AFM images of an Abo1 hexamer undergoing conformational change in the presence of 2 mM ATP captured at 5 fps. Scale bar, 5 nm. e Analysis of the position of Abo1 ring opening (gray boxes) sorted by the protomers according to time shows random subunit activation

Fig. 2

HS-AFM monitoring of intermolecular interactions. Binding of KaiA molecules to the CI side of a the KaiC phospho-mimics and b the KaiC dephospho-mimics. Frame rate, 10 fps. Scale bar, 20 nm. c KaiA’s bound state lifetime (τ_bound) depends on KaiC^WT phosphostatus over a 51 h time course of the in vitro cycle of phosphorylation. d Typical AFM images of a KscA channel reconstituted in a DMPC bilayer with (right) or without (left) AgTx2. Height profiles along the white dotted lines in the AFM images are shown below the images. The background illustration behind the height profiles indicates the corresponding structures of the channel and AgTx2. e Time-lapse images of AgTx2 binding to and dissociation from the KcsA channels (top) and time courses of the averaged height h (nm) around the center of the extracellular surface of two corresponding K⁺ channels (bottom). White dotted squares represent regions of interest for visualization of the tetrameric channels. AgTx2 bindings onto the channels are indicated by white arrowheads on the AFM images. Frame rate, 10 fps. Scale bar, 5 nm

Fig. 3

Mechanical indentation with HS-AFM. a HS-AFM images of the extracellular side of PIEZO1 in a lipid bilayer observed at specific applied force (left, about 20 pN; right, about 50 pN). b Top: HS-AFM images of PIEZO1 (dashed circles) during the stepwise increase in the loading force. Bottom: loading force (red), A_set/A_free ratio (blue) from the feedback control and z-piezo displacement (green) as function of frame acquisition time. c Time-lapse HS-AFM images of doublet microtubules with (upper panels) and without (lower panels) inner lumen proteins after increasing the force at a local area indicated by the red circles. In the doublet microtubules with inner lumen proteins present, enlargement of the hole usually stopped within a few seconds, whereas most of the B-tubules without the inner lumen proteins were broken by 40 s. Frame rate, 1 fps. Scale bar, 100 nm. d Creation (upper panels) of a defect in a microtubule using in-line-force-curve HS-AFM and the subsequent growth and complete healing of the defect (lower panels). The insets in the images show the region of the dashed rectangle with a pronounced contrast for the defect. The red star indicates the frame and the position of force application. e HS-AFM images after the local mechanical indentation and force curves during the indentation for different situations (left, fully reversible deformation; middle, single dimer defect; right, 12 dimer defect). The red arrows indicate the downward jumps during approach and the black arrows indicate the upward jumps during retraction