Alan Rick Horwitz

Non-muscle myosin II takes centre stage in cell adhesion and migration

Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signalling pathways, NM II is central in the control of cell adhesion, cell migration and tissue architecture. Recent insight into the role of NM II in these processes has been gained from loss-of-function and mutant approaches, methods that quantitatively measure actin and adhesion dynamics and the discovery of NM II mutations that cause monogenic diseases.

Cell adhesion: integrating cytoskeletal dynamics and cellular tension

Cell migration affects all morphogenetic processes and contributes to numerous diseases, including cancer and cardiovascular disease. For most cells in most environments, movement begins with protrusion of the cell membrane followed by the formation of new adhesions at the cell front that link the actin cytoskeleton to the substratum, generation of traction forces that move the cell forwards and disassembly of adhesions at the cell rear. Adhesion formation and disassembly drive the migration cycle by activating Rho GTPases, which in turn regulate actin polymerization and myosin II activity, and therefore adhesion dynamics.

Actin and |[alpha]|-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner

Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an α-actinin–actin template that elongates centripetally from nascent adhesions. α-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.

Integrins in cell migration – the actin connection

The connection between integrins and actin is driving the field of cell migration in new directions. Integrins and actin are coupled through a physical linkage, which provides traction for migration. Recent studies show the importance of this linkage in regulating adhesion organization and development. Actin polymerization orchestrates adhesion assembly near the leading edge of a migrating cell, and the dynamic cross-linking of actin filaments promotes adhesion maturation. Breaking the linkage between actin and integrins leads to adhesion disassembly. Recent quantitative studies have revealed points of slippage in the linkage between actin and integrins, showing that it is not always efficient. Regulation of the assembly and organization of adhesions and their linkage to actin relies on signaling pathways that converge on components that control actin polymerization and organization.

Paxillin phosphorylation at Ser273 localizes a GIT1–PIX–PAK complex and regulates adhesion and protrusion dynamics

Continuous adhesion formation and disassembly (adhesion turnover) in the protrusions of migrating cells is regulated by unclear mechanisms. We show that p21-activated kinase (PAK)–induced phosphorylation of serine 273 in paxillin is a critical regulator of this turnover. Paxillin-S273 phosphorylation dramatically increases migration, protrusion, and adhesion turnover by increasing paxillin–GIT1 binding and promoting the localization of a GIT1–PIX–PAK signaling module near the leading edge. Mutants that interfere with the formation of this ternary module abrogate the effects of paxillin-S273 phosphorylation. PAK-dependent paxillin-S273 phosphorylation functions in a positive-feedback loop, as active PAK, active Rac, and myosin II activity are all downstream effectors of this turnover pathway. Finally, our studies led us to identify in highly motile cells a class of small adhesions that reside near the leading edge, turnover in 20–30 s, and resemble those seen with paxillin-S273 phosphorylation. These adhesions appear to be regulated by the GIT1–PIX–PAK module near the leading edge.

Probing the integrin-actin linkage using high-resolution protein velocity mapping.

Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins α-actinin, α5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at α-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.

Stoichiometry of molecular complexes at adhesions in living cells

We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels, each detecting a different kind of protein. The number and brightness (N&B) analysis, namely, the use of the ratio between the variance and the average intensity to obtain the brightness of molecules, is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin, and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However, at the adhesions, large aggregates leave, forming a hole, during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the correlated fluctuations we determine the composition of the aggregates leaving the adhesions. These aggregates disassemble rapidly in the cytoplasm because large complexes are found only in very close proximity to the adhesions or at their borders.

Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as ∼ 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin‐enhanced‐green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.

Reducing background fluorescence reveals adhesions in 3D matrices

To the editor: Adhesion complexes in cells growing on planar substrates have been studied for over three decades. From these studies, several classes of adhesions have been described based on size, location, morphology, dynamics or molecular composition, and their dual role as signalling centres and linkages that connect the extracellular matrix (ECM) with the cytoskeleton has also emerged1. In contrast to this large and growing understanding of adhesion on two-dimensional (2D) substrates, little is known about the adhesions formed during three-dimensional (3D) growth of cells, including whether they even exist. Immunostaining and microscopy of fixed samples2–8 and dynamic imaging at low spatiotemporal resolution9 have shown the presence of large, elongated adhesions on cells in various 3D model systems. However, attempts to visualize adhesions in living cells growing in 3D with resolution similar to that routinely used in 2D have not been successful10. This has led to the recent conclusion that the adhesions defined and characterized in 2D cultures either do not exist in cells in 3D, or are too small or short-lived to be observed11. One major limitation of imaging live cells in 3D culture is background fluorescence caused by the overexpression of genetically encoded, fluorescently tagged proteins. Having saturated all available association sites, excess fusion proteins accumulate in the cytoplasm resulting in diffuse background fluorescence. In cells on 2D substrates, this cytoplasmic background is less detrimental because of the thinness of the lamellae in migrating cells. Moreover, background fluorescence can be reduced in 2D studies by using TIRF (total internal reflection fluorescence) microscopy, which excludes fluorescence above approximately 100 nm from the substrate. In contrast, protrusions formed on 3D matrices may be thicker12 and must be visualized using wide-field, confocal or multi-photon microscopy, which section a minimum thickness of 500–800 nm (ref. 13). Therefore, overexpression of an adhesion-specific fluorescently tagged protein can be more detrimental to imaging in 3D than in 2D and result in diffuse cytoplasmic fluorescence that masks the signal of molecules localized to adhesions. To address this issue, we used an EGFP (enhanced green fluorescent protein)–paxillin construct under the control of a truncated CMV (cytomegalovirus) promoter that was originally developed to express GFP–β-actin at very low levels14. The truncated CMV promoter reduces expression from the plasmid and therefore results in lower protein levels than those achievable by simply minimizing plasmid copy number. U2OS osteosarcoma cells were transfected with EGFP–paxillin in this vector, seeded within fibrillar collagen gels, and imaged with a laser scanning confocal microscope between 3–5 h after seeding. The collagen fibres were simultaneously imaged in reflectance mode. All images were taken several millimetres from the lateral edges of the gels. The cells were mostly multi-polar with three or more protrusions of variable size and shape that extended out from the cell body, spanned multiple focal planes, and exhibited numerous adhesions (Fig. 1a and Supplementary Information, Fig. S1a–f). Adhesions were rarely observed in areas more proximal to the cell body; however, as these areas of the cell were generally much thicker than the distal ends of the protrusion, and thus had higher cytoplasmic background fluorescence, we cannot exclude their existence. We observed adhesions in cells at depths up to the limit of the working distance of our objective (approximately 350 µm). Regardless of depth, at least 50% of the cells with a detectable level of the low-expressing EGFP–paxillin exhibited adhesions (Supplementary Information, Fig. S1g, m). Cells in which adhesions were not visible tended to have fluorescence intensities that were either relatively high (high background) or relatively low (low overall signal; Supplementary Information, Fig. S1h, n). Figure 1 Cell-matrix adhesions are detected in 3D collagen gels. (a) Z-projection of a multi-polar U2OS cell expressing EGFP–paxillin under the control of a truncated promoter, with adhesions on the distal sections of the protrusions (arrows). See Supplementary ... High-spatial-resolution imaging of the protrusions revealed distinct adhesions (Fig. 1b, lower panels) with a median length of 1 µm (0.8–1.45 µm, 25th–75th percentiles; n = 72 adhesions). Adhesions were also observed in similar short-term experiments with HT-1080 cells (Fig. 1b; median 1.2 µm; n = 36), with both cell types expressing EGFP–vinculin from the same vector (Fig. 1c), and in overnight cultures of U2OS cells (data not shown). In contrast, U2OS cells transfected with low levels of EGFP–paxillin expressed under the full-length CMV promoter, or even with high levels of the truncated promoter plasmid, had a diffuse cytoplasmic fluorescence similar to that of a fluorescent protein expressed alone (Fig. 1b, upper panels). Finally, we observed adhesions in HT-1080 cells cultured overnight in rat-tail collagen gels (Supplementary Information, Fig. S1i–n). To image adhesion formation, growth, and disassembly, we acquired time-lapse Z-stacks of protrusions from U2OS cells cultured 3–5 h as in Figure 1. As highly dynamic nascent adhesions form and disassemble rapidly with an average lifetime of about 1 min15, we imaged with a 10 s time-interval to be sure of capturing short-lived adhesions. Figure 2 shows frames from a representative time-lapse movie of the protrusion of a U2OS cell transfected with a plasmid encoding the promoter-truncated EGFP–paxillin (Supplementary Information, Video S1). The protrusion undergoes cycles of extension and adhesion formation followed by rearward adhesion movement and matrix fibre deformation. Similar observations were made in six independent experiments and with HT-1080 cells (Supplementary Information, Video S2). Figure 2 Dynamics of cell-matrix adhesions in 3D culture. Frames are taken from Supplementary Information, Video S1. The top row shows Z-projections of a protrusion end from a U2OS cell that was transfected with a plasmid encoding promoter-truncated EGFP–paxillin. ... In general, new adhesions formed along collagen fibres at the leading edge of protrusions and travelled rearward, causing fibre deformation. The adhesions had an initial diameter of 0.4 ± 0.07 µm (mean ± s.d.), placing them in the range of focal complexes, but larger than diffraction-limited nascent adhesions1,15. Over the course of the movies, most translocating adhesions grew in intensity and length; adhesions that did not translocate were stable, apparently maintaining isometric force on the matrix. Treatment with a combination of the Rho kinase (ROCK) inhibitor Y-27632 and the myosin light chain kinase (MLCK) inhibitor ML-7 decreased adhesion size similar to their effect in 2D (ref. 1). Our observations demonstrate that cell-matrix adhesions can form in 3D collagen matrices, show that their dynamics can be studied in living cells, and support previous observations by immunostaining and microscopy in collagen2,4,5,8, fibrin7,8, and cell-derived3,8 3D ECMs. In addition to reducing background fluorescence, factors that affect adhesion size (and therefore signal intensity) may affect adhesion detectability. For example, microenvironment stiffness will affect cell contractility and thus adhesion size and composition2,3,16–18. Therefore, more-pliable regions of collagen gels may have smaller and consequently less readily detected adhesions; if so, it would point to pliability, in addition to dimensionality, as the determining factor. In addition, adhesions are difficult to visualize in certain cell types19. For example, leukocytes do not show prominent adhesions on ICAM-1 coated substrates20 and can migrate in an integrin-independent manner21. The effects of cell contractility on adhesion phenotype have been well-studied in 2D systems; future work will need to determine how these interactions occur in 3D. Reducing background fluorescence overcomes an initial barrier to adhesion visualization in 3D and allows studies of the influence of 3D topography, pliability, matrix protein composition, and cell contractility on adhesion phenotype.

Paxillin Dynamics Measured during Adhesion Assembly and Disassembly by Correlation Spectroscopy

Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.