Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells.

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

Small peptides derived from the CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) gene family play a key role in various cell-cell communications in land plants. Among them, tracheary element differentiation inhibition factor (TDIF; CLE41/CLE44 peptide) and CLE42 peptide of Arabidopsis have almost identical amino acid sequences and act as inhibitors of tracheary element differentiation. In this study, we report a novel function of TDIF and CLE42. We found by the GUS (β-glucuronidase) reporter gene assay that while CLE41 and CLE44 are expressed preferentially in vascular bundles, CLE42 is expressed strongly in the shoot apical meristem (SAM) and axillary meristems. Overexpression of CLE42 and CLE41 enhanced axillary bud formation in the leaf and cotyledon axils. Before floral transition, the emergence of axillary buds in these plants occurred in an acropetal order. Exogenous supply of either TDIF or CLE42 peptide to the wild type induced similar excess bud emergence. In vascular bundles, the TDIF RECEPTOR (TDR) acts as the main receptor for TDIF. The axillary bud emergence of tdr mutants was little affected by either of the peptides. It was confirmed by scanning electron microscopy that peptide-treated wild-type plants form an axillary meristem-like structure earlier than non-treated plants. SHOOT MERISTEMLESS (STM), a marker gene for meristems, was up-regulated in peptide-treated plants before the axillary meristem becomes morphologically distinguishable. These results indicate that CLE42 peptide and TDIF have an activity to enhance axillary bud formation via the TDR. Judging from its expression pattern, CLE42 may play an important role in the regulation of secondary shoot development.

F o F 1 -ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within F o F 1 -ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single F o F 1 -ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single F o F 1 -ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

Adenosine triphosphate (ATP) is often referred as the energy currency of the cell. Yet, non-invasive, real-time, and quantitative measurement of its concentration in living mammalian cells has been difficult. Here we report an improved fluorescent ATP indicator protein, QUEEN-37C, which is optimized for measuring ATP concentration in living mammalian cells. Absolute value of the ATP concentration can be estimated from the ratiometric fluorescence imaging, and its accuracy was verified by the luciferase assay. Since QUEEN-37C enables the single-cell measurement of ATP concentration, we can not only measure its mean but its distribution in the cell population, which revealed that the ATP concentration is tightly regulated in most cells. We also noted the positive correlations in the ATP concentration among adjacent cells in epithelial cell sheet and mouse embryonic stem cell colonies. Thus, QUEEN-37C would serve as a new tool for the investigation of the single cell heterogeneity of metabolic states.

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TheBiophysicalSociety of Japan General IncorporatedAssociation 2Mt624XeeewD-Fif In this meeting, we examine the free cnergy landscape of the single-headed myosin VI along the actin filaillent using the samc methedology and discuss how it compares to the myosin II and how it relates to the observed difference of binding probability depending on the applied force. 2N1412ATP tz ;,tr-rATeam] eMLIZmsgopmevapa ATP mevaopg eleu7JvsrlrATanmuyu Real-time observation of the ATP concentration inside E eoti cells using ATP biosenser "ATeam" Hideyuki Yaginuma''2, Keisuke Tomiyama2, Kazuhito Tabatai, Hiromj lmamura3, Hiroyuki NojiL'2 CiDepartment of'Alzplied Chemist]y, School of EngineeJ'ing, f7ie [inivensity of Tblyo, i Gi'aduate Sthoot of Fbeontiet' Biosciences, Osaka UhiveFsity, ]7)ie Hakubi 1'rqiect, 19,oto Uhiversity) 2M1636 Keigo lkezakii Nishikawai ' Biosciences, 3Research IhstituteY?)t' Electronic Sbience, :-trvy6o2=opeeeeftm[[6ituwr-7-koprcev Contribution ofthe lever arm to myosin Vl's dual function as a transporter and an anchor . Tomotaka Komorii, Mitsuhiro Sugawa2, yoshiyuki Arai3. So Atsuko Iwanei, Toshio Yanagidai (iGraduate Sehool qffrontier Osaka Ubiive,'sio,, 2FZictfity (ti' Science, Gakushuin blaiversity', Ifokkaido UniveJ'siO') Myosin VI is an ATP molccular motor that functions as both a vesicle transporter and a cytoskeletal anchor. Recentiy, using single molecule imaging techniques we reported that myosin VI generates Targe forward steps by taking a distant binding state in which the inter-head distance is 34 nm and small forward and backwaTd steps by taking an adjacent binding state in which the inter-head distance less than 1O nm, However, how the levcr arm swing, a centTal tenet to myos{nATPase ehemo-mechanical coupling, regutates these steps is unctear, To help ctarify this, we performed simultaneous obsen'ation of the myosin VI head and tail dornain during motility, finding the lever arm stably tiits foiward when myosin Vl is in the adjaeent binding statc. By proposing a mechanism for the typc of stepping needed for thc distani and adjacent binding stutes to occur, we prepose this forward tilt like]y ensures transporter function by prohibiting successive backward steps and promoting anchoring by stab{Iizing the myesin at its position. Thus, along with the transporter function seen in most myosins, myesin Vl also can achieve an anchor function, which is medulated by the for",ard directional bias efthe lever arin domain during the adjaccnL binding state. 2M1648:-XYYVIM70ftz7tJptYFtcomastoprpEttEf'J)t[di 6em=*Jbi-neut Free energy landscape analysis of the movement of myosin Vl alollg actin filament using a coarse-grained model i, Masaki sasai2, Takeshi Sasaki3, Tomoki Terada? (dDqpt. qf' Grad, Sc,h. qf Engineering, Nag(u,a Univ., ! Dept. qf' Science and Engineering, Grad. SZ'h of'EngineeJ'ing, Nagoya of Human bV2)rmatics, Facut4, of Hiiman blformatics, riiehi ATP plays an important role as the encrgy sou'ce in many aspccts of cell growth, such as protein synthesis, genome duplication, cell division and m...

Light is the most important energy source for organisms. The photoactive retinal proteins are responsible for a variety of biological functions, such as ion pumps, ion channels and photosensors. The question arises of how these proteins can function so differently? Although ~75% of amino acid residues differ among retinal proteins, only three mutations convert a proton pump into a photosensor [1]. Recently we succeeded in conversion from a light-driven ion pump into a light-gated ion channel by three mutations [2]. The results reveal that the β-ionone ring of the chromophore is a key element for the functional difference among the active and passive ion transporters.[1] Sudo et al., (2006) PNAS, 103, 16129, (2011 Inoue et al., in preparation. 2SAP-01 分子個性と少数性 Molecular Individuality and Minority in BiologyTamiki Komatsuzaki (Hokkaido Univ., Res. Inst. Electronic Sci.) Complex biological systems inherently possess hierarchies in time and space and biological functions occur across several orders of the scales. However, the question of how "individuality (=structures, dynamics) of the same kind of molecules survives in a single cell has not been addressed. We overview one of our methods, which extracts the underlying reaction scheme that enables us to address the kinetic significance of the diversity of molecular structures from time series data of single molecule experimental measurement. The resultant reaction scheme does not rely on an a priori ansatz such as detailed balance. We demonstrate the potential of our method by applying it to the analysis of, for example, a singlemolecule turnover enzymatic reaction. Cells integrate diverse molecules to keep their reproduction and exhibit a variety of robust functions, taking essential resources from environment. The numbers of specific components are not large, thus, competition for the few components will be relevant and commonly occurring. Understanding the effect in a general catalytic network will clarify how they contribute to the robust cellular behavior. In this talk, I will show two such examples. First results will show that a minority molecule in a catalytic reaction network results in reproductions and growth-division processes of a simple spatial structure, i.e., a protocell. Second will show depletion of and competition for a variety of resources result in reproduction of diverse compositions of protocells. A dendritic spine is a very small structure (~0.1 μm 3 ) of a neuron that processes input timing information. Why are spines so small? Here, we provide following functional reasons obtained by the stochastic simulations of input timing-dependent Ca 2+ increases in a cerebellar Purkinje cell's spine. Spines code input timing information by the probability of Ca 2+ increases, rather than the amplitude, for input timing detection by utilizing the small number of molecules in a spine volume. Probability coding in a spine volume was more robust against input fluctuation and more sensitive to input numbers than amplitude coding in a cell volume. Thus, s...