High-Speed Atomic Force Microscopy for Filming Protein Molecules in Dynamic Action.

Structural biology is currently undergoing a transformation into dynamic structural biology, which reveals the dynamic structure of proteins during their functional activity to better elucidate how they function. Among the various approaches in dynamic structural biology, high-speed atomic force microscopy (HS-AFM) is unique in the ability to film individual molecules in dynamic action, although only topographical information is acquirable. This review provides a guide to the use of HS-AFM for biomolecular imaging and showcases several examples, as well as providing information on up-to-date progress in HS-AFM technology. Finally, we discuss the future prospects of HS-AFM in the context of dynamic structural biology in the upcoming era. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Dynamic state of low-Reynolds-number turbulent channel flow.

We numerically study the dynamic state of a low-Reynolds-number turbulent channel flow from the viewpoints of symbolic dynamics and nonlinear forecasting. A low-dimensionally (high-dimensionally) chaotic state of the streamwise velocity fluctuations emerges at a viscous sublayer (logarithmic layer). The possible presence of the chaotic states is clearly identified by orbital instability-based nonlinear forecasting and ordinal partition transition network entropy in combination with the surrogate data method.

Technical advances in high-speed atomic force microscopy.

It has been 30 years since the outset of developing high-speed atomic force microscopy (HS-AFM), and 15 years have passed since its establishment in 2008. This advanced microscopy is capable of directly visualizing individual biological macromolecules in dynamic action and has been widely used to answer important questions that are inaccessible by other approaches. The number of publications on the bioapplications of HS-AFM has rapidly increased in recent years and has already exceeded 350. Although less visible than these biological studies, efforts have been made for further technical developments aimed at enhancing the fundamental performance of HS-AFM, such as imaging speed, low sample disturbance, and scan size, as well as expanding its functionalities, such as correlative microscopy, temperature control, buffer exchange, and sample manipulations. These techniques can expand the range of HS-AFM applications. After summarizing the key technologies underlying HS-AFM, this article focuses on recent technical advances and discusses next-generation HS-AFM.

Faster high-speed atomic force microscopy for imaging of biomolecular processes.

High-speed atomic force microscopy (HS-AFM) has enabled observing protein molecules during their functional activity at rates of 1-12.5 frames per second (fps), depending on the imaging conditions, sample height, and fragility. To meet the increasing demand for the great expansion of observable dynamic molecular processes, faster HS-AFM with less disturbance is imperatively needed. However, even a 50% improvement in the speed performance imposes tremendous challenges, as the optimization of major rate-limiting components for their fast response is nearly matured. This paper proposes an alternative method that can lower the feedback control error and thereby enhance the imaging rate. This method can be implemented in any HS-AFM system by minor modifications of the software and hardware. The resulting faster and less-disturbing imaging capabilities are demonstrated by the imaging of relatively fragile actin filaments and microtubules near the video rate, and of actin polymerization that occurs through weak intermolecular interactions, at ∼8 fps.

Complex network analysis of spatiotemporal dynamics of premixed flame in a Hele-Shaw cell: A transition from chaos to stochastic state.

We study the dynamical state of a noisy nonlinear evolution equation describing flame front dynamics in a Hele-Shaw cell from the viewpoint of complex networks. The high-dimensional chaos of flame front fluctuations at a negative Rayleigh number retains the deterministic nature for sufficiently small additive noise levels. As the strength of the additive noise increases, the flame front fluctuations begin to coexist with stochastic effects, leading to a fully stochastic state. The additive noise significantly promotes the irregular appearance of the merge and divide of small-scale wrinkles of the flame front at a negative Rayleigh number, resulting in the transition of high-dimensional chaos to a fully stochastic state.

Atomic Force Microscopy Visualizes Mobility of Photosynthetic Proteins in Grana Thylakoid Membranes.

Thylakoid membranes in chloroplasts contain photosynthetic protein complexes that convert light energy into chemical energy. Photosynthetic protein complexes are considered to undergo structural reorganization to maintain the efficiency of photochemical reactions. A detailed description of the mobility of photosynthetic complexes in real time is necessary to understand how macromolecular organization of the membrane is altered by environmental fluctuations. Here, we used high-speed atomic force microscopy to visualize and characterize the in situ mobility of individual protein complexes in grana thylakoid membranes isolated from Spinacia oleracea. Our observations reveal that these membranes can harbor complexes with at least two distinctive classes of mobility. A large fraction of grana membranes contained proteins with quasistatic mobility exhibiting molecular displacements smaller than 10 nm2. In the remaining fraction, the protein mobility is variable with molecular displacements of up to 100 nm2. This visualization at high spatiotemporal resolution enabled us to estimate an average diffusion coefficient of ∼1 nm2 s-1. Interestingly, both confined and Brownian diffusion models could describe the protein mobility of the second group of membranes. We also provide the first direct evidence, to our knowledge, of rotational diffusion of photosynthetic complexes. The rotational diffusion of photosynthetic complexes could be an adaptive response to the high protein density in the membrane to guarantee the efficiency of electron transfer reactions. This characterization of the mobility of individual photosynthetic complexes in grana membranes establishes a foundation that could be adapted to study the dynamics of the complexes inside intact and photosynthetically functional thylakoid membranes to be able to understand its structural responses to diverse environmental fluctuations.

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies.

BACKGROUND: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. METHODS: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. RESULTS: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. CONCLUSIONS: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. GENERAL SIGNIFICANCE: We achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription.

The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.

Functional extension of high-speed AFM for wider biological applications.

High-speed atomic force microscopy (HS-AFM) has been established and used, which can visualize biomolecules in dynamic action at high spatiotemporal resolution without disturbing their function. Various studies conducted in the past few years have demonstrated that the dynamic structure and action of biomolecules revealed with HS-AFM can provide greater insights than ever before into how the molecules function. However, this microscopy has still limitations in some regards. Recently, efforts have been carried out to overcome some of the limitations. As a result, it has now become possible to visualize dynamic processes occurring even on live cells and perform simultaneous observations of topographic and fluorescent images at a high rate. In this review, we focus on technical developments for expanding the range of objects and phenomena observable by HS-AFM as well as for granting multiple functionalities to HS-AFM.

Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force microscope.

In tip-scan atomic force microscopy (AFM) that scans a cantilever chip in the three dimensions, the chip body is held on the Z-scanner with a holder. However, this holding is not easy for high-speed (HS) AFM because the holder that should have a small mass has to be able to clamp the cantilever chip firmly without deteriorating the Z-scanner's fast performance, and because repeated exchange of cantilever chips should not damage the Z-scanner. This is one of the reasons that tip-scan HS-AFM has not been established, despite its advantages over sample stage-scan HS-AFM. Here, we present a novel method of cantilever chip holding which meets all conditions required for tip-scan HS-AFM. The superior performance of this novel chip holding mechanism is demonstrated by imaging of the α3ß3 subcomplex of F1-ATPase in dynamic action at ∼7 frames/s.

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.

Effect of ibudilast on the reciprocal inhibitory visual-vestibular interaction closely related to dizziness after cerebral ischemia.

BACKGROUND: Many patients with chronic cerebrovascular diseases suffer dizziness. Our earlier findings suggested that prolonged terms of dizziness episodes may decrease the regional cerebral blood flow (CBF) in the occipital visual cortex via a remote effect from the vestibular cortex. METHODS: We studied 9 patients who suffered episodes of dizziness since the onset of chronic cerebral ischemia. Their at-rest CBF was measured at entry into the study and approximately 3 months after the start of ibudilast therapy when all patients reported the resolution of dizziness. RESULTS: After 3 months of ibudilast their at-rest CBF was significantly increased in the left occipital lobe (P = .02). CBF after acetazolamide (ACZ) loading was significantly increased in the bilateral occipital lobes (right, P = .049; left, P = .02) and in the bilateral parieto-insular vestibular cortex (PIVC; right and left, P = .02). There were no significant CBF changes in any other areas. CONCLUSIONS: Our findings indicate that the occipital cortex and PIVC were implicated in their dizziness after cerebral ischemia. We discuss the underlying mechanism(s) and the relationship between dizziness and reciprocal inhibitory visual-vestibular interactions.

High-speed atomic force microscope combined with single-molecule fluorescence microscope.

High-speed atomic force microscopy (HS-AFM) and total internal reflection fluorescence microscopy (TIRFM) have mutually complementary capabilities. Here, we report techniques to combine these microscopy systems so that both microscopy capabilities can be simultaneously used in the full extent. To combine the two systems, we have developed a tip-scan type HS-AFM instrument equipped with a device by which the laser beam from the optical lever detector can track the cantilever motion in the X- and Y-directions. This stand-alone HS-AFM system is mounted on an inverted optical microscope stage with a wide-area scanner. The capability of this combined system is demonstrated by simultaneous HS-AFM∕TIRFM imaging of chitinase A moving on a chitin crystalline fiber and myosin V walking on an actin filament.