The ability of cells to respond to physical forces is fundamental to development and physiology, including regulation of blood pressure, cell adhesion and migration. A major limitation to the study of these phenomena has been the difficulty of measuring molecular forces in cells in vivo. Grashoff et al. now report the development of a genetically encoded, fluorescent tension-sensing module capable of measuring mechanical forces across specific proteins in vivo. The sensor was tested on vinculin, a membrane-cytoskeletal protein that is recruited to focal adhesions and connects cell-adhesion molecules (integrins) to actin filaments. The data reveal a regulatory mechanism in which the ability of vinculin to bear force determines whether focal adhesions assemble or disassemble under force. This new biosensor should be applicable to other proteins involved in mechanotransduction. The ability of cells to respond to physical forces is central to development and physiology, but until now it has been difficult to directly measure forces across proteins in vivo. Here, however, a calibrated biosensor is described that can measure forces with high sensitivity across specific proteins in cells. This is applied to the vinculin protein, and a regulatory mechanism is revealed in which the force applied to vinculin determines whether focal adhesions assemble or disassemble. 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 piconewton (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.

Super-resolution microscopy has overcome a long-held resolution barrier—the diffraction limit—in light microscopy and enabled visualization of previously invisible molecular details in biological systems. Since their conception, super-resolution imaging methods have continually evolved and can now be used to image cellular structures in three dimensions, multiple colors, and living systems with nanometer-scale resolution. These methods have been applied to answer questions involving the organization, interaction, stoichiometry, and dynamics of individual molecular building blocks and their integration into functional machineries in cells and tissues. In this Review, we provide an overview of super-resolution methods, their state-of-the-art capabilities, and their constantly expanding applications to biology, with a focus on the latter. We will also describe the current technical challenges and future advances anticipated in super-resolution imaging.

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites.

Despite the recent advances in single-molecule manipulation techniques, purely mechanical approaches cannot detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence that allowed us to examine the effect of subpiconewton forces on the nanometer scale motion of the Holliday junction (HJ) at 100-hertz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer-length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two-dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.