Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with pico-Newton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ~2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET. Fluorescence resonance energy transfer (FRET) has found wide use in structural biology, biochemistry, and polymer science for measuring distances in the 10-to 80-A range (1-5).In FRET, energy is transferred from a donor molecule to an acceptor molecule via an induced-dipole, induced-dipole interaction, with the transfer efficiency E depending on the inverse-sixth-power of the distance R between the donor and acceptor: E = 1/(1+[R/R0]6), where Ro is the distance at which 50% of the energy is transferred. Ro is a function of the properties of the dyes and the relative orientation of their dipole moments: R. = [8.79 x 10-5 J(A) OD n-4 K2]116 [A]. J(A) is the spectral overlap of donor emission and acceptor absorption [nm4_M-1.cm-1], OD is the donor quantum yield, n is the index of refraction of the medium, and K2 is a geometrical factor which accounts for the relative orientation of the two dipoles.Near-field scanning optical microscopy (NSOM) (6-8) is a relatively new technique that allows optical measurements with sub-wavelength resolution. It is based on a probe consisting of a very small (sub-wavelength) aperture that is placed in close proximity (in the near field; <10 nm) to the sample under study. By using the probe as an excitation source, fluorescence of a single molecule has been detected (9). The emission spectra (10) and excited state lifetime (11-13) of a single molecule have also been measured. Another important aspect of near-field detection is that the optical radiation in the near field has an electric field component along its direction of propagation (in contrast to far-field radiation). This allows mapping the transition dipole moment orientation of a single fluorescent molecule in three dimensions (9).The marriage between FRET and NSOM offers many potential advantages when distance and orientation information is required on a molecular level. Here we list several. (i) Because the orientation of donor and acceptor can potentially be measured, the uncertainty in K2, which is often a large source of uncertainty i...

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.