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 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 M 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 F oF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment. fluorescent indicator ͉ FRET ͉ live imaging ͉ oxidative phosphorylation A denosine 5Ј-triphosphate (ATP) is the ubiquitous energy currency of all living organisms. The high phosphatetransfer potential of ATP is used for many biological processes, including muscle contraction, synthesis and degradation of biological molecules, and membrane transport. In addition, it has been suggested that ATP acts as an intracellular or extracellular signaling molecule in cellular processes, such as insulin secretion (1), neurotransmission (2), cell motility (3), and organ development (4). However, it has been difficult to precisely understand how ATP controls cellular processes and how the intracellular ATP level is regulated at the single cell level, because the conventional ATP quantification methods can only provide the averaged ATP level of an ensemble of cells based on cell extract analysis. Moreover, the distribution pattern of ATP between different intracellular compartments is unclear. Several attempts have been made to monitor ATP levels real-time in individual cells; however, these methods present several problems. For example, in chemiluminescence imaging from cells expressing firefly luciferase (5), chemiluminescence by luciferase depends not only on the intracellular ATP level but also on the luciferase concentration, as well as the other substrates, oxygen, and luciferin. Moreover, pH also affects luciferase activity. Another drawback of this method is that the intracellular ATP level could be perturbed because of ATP consumption. Furthermore, the dim luminescence of luciferase requires longer exposure time for image acquisition, making real-time observation cumbersome. Other approaches include measurement of the ion channel activity (6) or conformational change (7) of the ATP-s...

Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.

One-sentence summary:Intrinsic cooperativity engenders cyclical propagation of conformational states in the stator ring of an ATP-driven rotary motor. 2 ABSTRACTF 1 is an ATP-driven motor in which three torque-generating β subunits in the α 3 β 3 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 use high-speed atomic force microscopy to show that the rotor-less F 1 still "rotates"; in the isolated α 3 β 3 stator ring, the three β subunits cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F 1 . The structural basis of unidirectionality is programmed in the stator ring. These findings have implications for cooperative interplay between subunits in other hexameric ATPases.3 F 1 -ATPase, a water-soluble portion of ATP synthase (1), is a rotary motor protein. The α 3 β 3 γ subcomplex (referred to here as F 1 ) suffices as the motor, in which the rotor γ subunit rotates in the stator α 3 β 3 ring upon ATP hydrolysis (2). The concept of the "rotary catalysis" of F 1 was proposed based on biochemical studies (3). It was strongly supported by the first crystal structure (4) and directly proven by observations of rotating single molecules (5). In F 1 , the catalytic sites are located at the α-β interfaces, mainly on the β subunits. In the crystal structure (4), three catalytic sites are in different nucleotide-bound states; one binds to an ATP analog (α TP -β TP in Fig. 1E), another binds to ADP (α DP -β DP ), and the third is unbound (α E -β E ). Both β TP and β DP assume the closed conformation, swinging the C-terminal domain toward γ, whereas β E assumes the open conformation, swinging the domain away from γ. As these two general conformational states appear to push or be pushed by γ, respectively, it was proposed that interactions with γ control the conformational and catalytic states of individual βs to sequentially generate torque (6). In fact, some biochemical studies are thought to suggest that the α 3 β 3 ring alone does not possess intrinsic cooperativity and γ mediates the interplay among βs (7-9). This view was reinforced by studies showing that backward mechanical rotation of γ with external force reverses the chemical reaction toward ATP synthesis (10, 11), whereas forced forward rotation results in accelerated ATP binding (12). 4Recently, however, this contention has been challenged by the finding that even when most interaction sites between β and γ are abolished, F 1 retains catalytic power to rotate γ unidirectionally (13, 14). A few biochemical studies also suggest the intrinsic cooperativity of the α 3 β 3 ring (15, 16). However, as conventional single-molecule optical microscopy requires attachment of a probe onto the rotary shaft for visualization (5), it does not allow direct examin...