Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculinnull cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V H ) and tail domains. V H enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with V H , implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V H has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V H , correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.cell adhesion | fibronectin I ntegrin-mediated adhesion to ECM provides mechanical anchorage and signals that direct cell migration, proliferation, and differentiation (1, 2), processes central to tissue organization, maintenance, and repair. After ligand binding, integrins cluster into focal adhesion (FA) complexes that transmit adhesive and traction forces (3-6). FAs consist of integrins and actins separated by a ∼40 nm core that includes cytoskeleton (CSK) elements, such as vinculin and talin, and signaling molecules, including focal adhesion kinase and paxillin (7). FAs mediate responses to internal and external stresses by modulating force transfer between integrins and the CSK (8-10). This function has been likened to a "mechanical clutch" between an engine and transmission (11).On the basis of its structure and binding partners, vinculin represents an attractive candidate for orchestrator of clutch function. Vinculin consists of a globular head (V H ) linked to a tail domain (V T ) by a proline-rich strap (12). V H contains talin, α-actinin, and α-and β-catenin binding sites; actin, paxillin, and phosphatidylinositol 4,5-bisphosphate (PIP2) binding sites are in V T ; and vasodilator-stimulated phosphoprotein (VASP), actinrelated protein 2/3 (Arp2/3), and vinexin binding sites reside in the proline-rich region. Interactions with these partners are regulated by an autoinhibited conformation arising from high-affinity intramolecular head-tail binding (13,14). Activation of vinculin can occur by simultaneous binding to talin and actin or α-catenin and actin (15,16). Vinculin is activated when localized t...

We demonstrate a new method for creating synthetic tissue that has the potential to capture the three-dimensional (3D) complexity of a multi-cellular organism with submicron precision. Using multiple laminar fluid flows in a microfluidic network, we convey cells to an assembly area where multiple, time-shared optical tweezers are used to organize them into a complex array. The cells are then encapsulated in a 30 microm x 30 microm x 45 microm volume of photopolymerizable hydrogel that mimicks an extra-cellular matrix. To extend the size, shape and constituency of the array without loss of viability, we then step to an adjacent location while maintaining registration with the reference array, and repeat the process. Using this step-and-repeat method, we formed a heterogeneous array of E. coli genetically engineered with a lac switch that is functionally linked to fluorescence reporters. We then induced the array using ligands through a microfluidic network and followed the space-time development of the fluorescence to evaluate viability and metabolic activity.

The demonstration of a practical technology for 3D optical microfabrication is a vital step in the development of photonic-crystal-based optical signal processing.[1] However, the extension of the optical methods that dominate integrated electronic circuit fabrication to three dimensions is a formidable materials-processing challenge: such a process must be capable not only of sub-micrometer pattern definition in three dimensions, but also of the transfer of this pattern into a homogeneous dielectric with an appropriately high refractive index. In a companion paper, [2] we show that two optical methods, holographic lithography [3] and direct two-photon laser writing, [4][5][6] can be combined to create a rapid and flexible method for the definition of photonic crystal device structures in photoresist. In this communication, we report a further essential step towards the creation of devices operating within a full photonic bandgap: we have used atomic layer deposition (ALD), itself an established semiconductor processing technique, to create high-index TiO 2 inverted replicas of holographically defined photonic crystals, followed by removal of the polymeric template by plasma etching. A range of techniques for 3D optical lithography has been demonstrated. A 3D photonic crystal structure can be written by holographic lithography, [3] which makes use of a periodic interference pattern generated by a multiple-beam interferometer to expose a thick layer of photoresist. 3D microstructures, both periodic and aperiodic, can also be generated by point-by-point exposure of the resist by two-photon absorption at a laser focus. [4][5][6][7] Two-photon laser writing is a serial process; point-by-point fabrication of a 3D photonic crystal is necessarily slower than holographic lithography, which is capable of defining the entire periodic structure in a single laser pulse.[3] The two techniques are complementary: two-photon laser writing can be used to modify a holographic exposure.[8]We have shown that, by imaging the distribution of photochemical change induced by holographic exposure, it is possible to align a subsequent two-photon exposure with the 3D photonic crystal lattice to achieve the precise registration that is required of a device structure embedded in a 3D photonic crystal. [2] This hybrid technique is rapid and flexible, but the polymeric resists used for 3D microfabrication have refractive indices n in the range 1.4-1.6, which is too low for most device applications. Devices based on waveguides and microcavities embedded within a photonic crystal [1] are designed to operate at frequencies within a complete (omnidirectional) photonic bandgap in order to suppress radiative loss; [9] to create a complete photonic bandgap, even in an optimized air-dielectric structure, a refractive contrast of at least 1.9 is necessary.