Ryota Iino

Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

Adenosine 5′-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7–10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

Single Molecule Imaging of Green Fluorescent Proteins in Living Cells: E-Cadherin Forms Oligomers on the Free Cell Surface

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions).

High-Speed Atomic Force Microscopy Reveals Rotary Catalysis of Rotorless F1-ATPase

Intrinsic cooperativity drives cyclic propagation of conformational states in the stator ring of an adenosine triphosphate–driven rotary motor. F1 is an adenosine triphosphate (ATP)–driven motor in which three torque-generating β subunits in the α3β3 stator ring sequentially undergo conformational changes upon ATP hydrolysis to rotate the central shaft γ unidirectionally. Although extensive experimental and theoretical work has been done, the structural basis of cooperative torque generation to realize the unidirectional rotation remains elusive. We used high-speed atomic force microscopy to show that the rotorless F1 still “rotates”; in the isolated α3β3 stator ring, the three β subunits cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F1. The structural basis of unidirectionality is programmed in the stator ring. These findings have implications for cooperative interplay between subunits in other hexameric ATPases.

The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques (Review)

Models of the organization of the plasma membrane of live cells as discovered through diffusion measurements of integral membrane molecules (transmembrane and GPI-anchored proteins, and lipid) at the single molecule level are discussed. Diffusion of transmembrane protein and, indeed, even lipid is anomalous in that the molecules tend to diffuse freely in limited size compartments, with infrequent intercompartment transitions. This average residency time in a compartment is dependent on the diffusing species and on its state of oligomerization, becoming completely confined to a single compartment upon sufficient oligomerization. This will be of great importance in determining cellular mechanisms for controlling the random diffusive motion of membrane molecules and in understanding signalling processes.

Phosphate release in F1-ATPase catalytic cycle follows ADP release

F(1)-ATPase is an ATP-driven rotary motor protein in which the γ-subunit rotates against the catalytic stator ring. Although the reaction scheme of F(1) has mostly been revealed, the timing of inorganic phosphate (P(i)) release remains controversial. Here we addressed this issue by verifying the reversibility of ATP hydrolysis on arrested F(1) with magnetic tweezers. ATP hydrolysis was found to be essentially reversible, implying that P(i) is released after the γ rotation and ADP release, although extremely slow P(i) release was found at the ATP hydrolysis angle as an uncoupling side reaction. On the basis of this finding, we deduced the chemomechanical coupling scheme of F(1). We found that the affinity for P(i) was strongly angle dependent, implying a large contribution by P(i) release to torque generation. These findings imply that under ATP synthesis conditions, P(i) binds to an empty catalytic site, preventing solution ATP (though not ADP) from binding. Thus, this supports the concept of selective ADP binding for efficient ATP synthesis.

Rotation and structure of FoF1-ATP synthase.

F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

Simple Dark-Field Microscopy with Nanometer Spatial Precision and Microsecond Temporal Resolution

Molecular motors such as kinesin, myosin, and F(1)-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems. Here, we describe a simple dark-field imaging system that employs objective-type evanescent illumination to selectively illuminate a thin layer on the coverslip and thus yield images with high signal/noise ratios. Only by substituting the dichroic mirror in conventional objective-type total internal reflection fluorescence microscope with a perforated mirror, were nanometer spatial precision and microsecond temporal resolution simultaneously achieved. This system was applied to the study of the rotary mechanism of F(1)-ATPase. The fluctuation of a gold nanoparticle attached to the gamma-subunit during catalytic dwell and the stepping motion during torque generation were successfully visualized with 9.1-mus temporal resolution. Because of the simple optics, this system will be applicable to various biophysical studies requiring high spatial and temporal resolution in vitro and also in vivo.

A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 10(6) dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young-Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme β-galactosidase and rotary motor protein F(1)-ATPase enclosed in a droplet.

Large-scale femtoliter droplet array for digital counting of single biomolecules

We present a novel device employing one million femtoliter droplets immobilized on a substrate for the quantitative detection of extremely low concentrations of biomolecules in a sample. Surface-modified polystyrene beads carrying either zero or a single biomolecule-reporter enzyme complex are efficiently isolated into femtoliter droplets formed on hydrophilic-in-hydrophobic surfaces. Using a conventional micropipette, this is achieved by sequential injection first with an aqueous solution containing beads, and then with fluorinated oil. The concentration of target biomolecules is estimated from the ratio of the number of signal-emitting droplets to the total number of trapped beads (digital counting). The performance of our digital counting device was demonstrated by detecting a streptavidin-β-galactosidase conjugate with a limit of detection (LOD) of 10 zM. The sensitivity of our device was >20-fold higher than that noted in previous studies where a smaller number of reactors (fifty thousand reactors) were used. Such a low LOD was achieved because of the large number of droplets in an array, allowing simultaneous examination of a large number of beads. When combined with bead-based enzyme-linked immunosorbent assay (digital ELISA), the LOD for the detection of prostate specific antigen reached 2 aM. This value, again, was improved over that noted in a previous study, because of the decreased coefficient of variance of the background measurement determined by the Poisson noise. Our digital counting device using one million droplets has great potential as a highly sensitive, portable immunoassay device that could be used to diagnose diseases.