Real-time fluorescence visualization of slow tautomerization of single free-base phthalocyanines under ambient conditions.

The emission transition dipole moments of single-molecule free-base phthalocyanines at an air/glass interface were visualized using defocused wide-field fluorescence microscopy at a temporal resolution of 100-200 ms. Isolated molecules showed slow proton tautomerization, which is consistent with previous theoretical calculations in the gas phase, which predicted large activation energies.

F1-ATPase: a highly coupled reversible rotary motor.

F1 (F1-ATPase) is a highly coupled rotary molecular motor and hydrolyses three ATP molecules per turn (3 ATP/turn). Recently, we have developed femtolitre reaction chamber arrays for highly sensitive measurement of biological reactions. By combining this technique with the rotating magnetic tweezers, the coupling ratio of the reverse reaction, ATP synthesis catalysed by single F1 molecules, has been investigated. The low coupling ratio of 10% (0.3 ATP/turn), catalysed by the alpha3beta3gamma subcomplex of F1, was significantly improved to 77% (2.3 ATP/turn) after reconstitution of the epsilon subunit. This result revealed the novel function of the epsilon subunit as a coupling factor of ATP synthesis catalysed by F1. The possible mechanism for highly coupled ATP synthesis supported by the epsilon subunit is discussed.

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).