Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.

Highlights d Determining the polarity of individual actin filaments inside cells d Reconstruction of actin networks from cryo-tomograms d The polarity of actin changes from mixed to uniform along focal adhesions

Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the detailed molecular mechanisms underlying FA mechanosensitivity are still unclear. Besides talin, another key candidate for regulating FA-associated mechanosensing, is vinculin, a prominent FA component, which possesses either closed (“auto-inhibited”) or open (“active”) conformation. A direct experimental demonstration, however, of the conformational transition between the two states is still absent. In this study, we combined multiple structural and biological approaches to probe the transition from the auto-inhibited to the active conformation, and determine its effects on FA structure and dynamics. We further show that the transition from a closed to an open conformation requires two sequential steps that can differentially regulate FA growth and stability.

Understanding the function of cellular machines requires a thorough analysis of the structural elements that underline their function. Electron microscopy (EM) has been pivotal in providing information about cellular ultrastructure, as well as macromolecular organization. Biological materials can be physically fixed by vitrification and imaged with cryo-electron tomography (cryo-ET) in a close-to-native condition. Using this technique, one can acquire three-dimensional (3D) information about the macromolecular architecture of cells, depict unique cellular states and reconstruct molecular networks. Technical advances over the last few years, such as improved sample preparation and electron detection methods, have been instrumental in obtaining data with unprecedented structural details. This presents an exciting opportunity to explore the molecular architecture of both individual cells and multicellular organisms at nanometer to subnanometer resolution. In this Commentary, we focus on the recent developments and in situ applications of cryo-ET to cell and structural biology.

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5–30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s−1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s−1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods – serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362–1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238–246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.