Clare M. Waterman

Nanoscale architecture of integrin-based cell adhesions

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell–ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ∼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

Mechanical Integration of Actin and Adhesion Dynamics in Cell Migration

Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation

Focal adhesions undergo myosin-II-mediated maturation wherein they grow and change composition to modulate integrin signalling for cell migration, growth and differentiation. To determine how focal adhesion composition is affected by myosin II activity, we performed proteomic analysis of isolated focal adhesions and compared protein abundance in focal adhesions from cells with and without myosin II inhibition. We identified 905 focal adhesion proteins, 459 of which changed in abundance with myosin II inhibition, defining the myosin-II-responsive focal adhesion proteome. The abundance of 73% of the proteins in the myosin-II-responsive focal adhesion proteome was enhanced by contractility, including proteins involved in Rho-mediated focal adhesion maturation and endocytosis- and calpain-dependent focal adhesion disassembly. During myosin II inhibition, 27% of proteins in the myosin-II-responsive focal adhesion proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in focal adhesions, establishing that focal adhesion protein recruitment is also negatively regulated by contractility. We focused on the Rac guanine nucleotide exchange factor β-Pix, documenting its role in the negative regulation of focal adhesion maturation and the promotion of lamellipodial protrusion and focal adhesion turnover to drive cell migration.

Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

FAK-mediated myosin-dependent paxillin phosphorylation is necessary to bring vinculin to maturing focal adhesions, reinforcing the link between the cytoskeleton and the ECM.

Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

Vinculin functions as a molecular clutch that organizes leading edge F-actin, generates traction, and promotes focal adhesion formation and turnover but not adhesion growth.

A Zyxin-Mediated Mechanism for Actin Stress Fiber Maintenance and Repair

To maintain mechanical homeostasis, cells must recognize and respond to changes in cytoskeletal integrity. By imaging live cells expressing fluorescently tagged cytoskeletal proteins, we observed that actin stress fibers undergo local, acute, force-induced elongation and thinning events that compromise their stress transmission function, followed by stress fiber repair that restores this capability. The LIM protein zyxin rapidly accumulates at sites of strain-induced stress fiber damage and is essential for stress fiber repair and generation of traction force. Zyxin promotes recruitment of the actin regulatory proteins α-actinin and VASP to compromised stress fiber zones. α-Actinin plays a critical role in restoration of actin integrity at sites of local stress fiber damage, whereas both α-actinin and VASP independently contribute to limiting stress fiber elongation at strain sites, thus promoting stabilization of the stress fiber. Our findings demonstrate a mechanism for rapid repair and maintenance of the structural integrity of the actin cytoskeleton.

Integration of actin dynamics and cell adhesion by a three-dimensional, mechanosensitive molecular clutch

During cell migration, the forces generated in the actin cytoskeleton are transmitted across transmembrane receptors to the extracellular matrix or other cells through a series of mechanosensitive, regulable protein–protein interactions termed the molecular clutch. In integrin-based focal adhesions, the proteins forming this linkage are organized into a conserved three-dimensional nano-architecture. Here we discuss how the physical interactions between the actin cytoskeleton and focal-adhesion-associated molecules mediate force transmission from the molecular clutch to the extracellular matrix.

Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy

Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.