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.

We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front–back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front–back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.

Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical-basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGFindependent modes of angiogenesis. Like VEGF, sphingosine-1-phosphate also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.A ngiogenesis, the process by which new capillary vessels sprout from existing vasculature, plays a critical role in embryonic development and wound healing, and its dysregulation can contribute to cancer progression as well as numerous inflammatory and ischemic diseases (1, 2). Consequently, therapeutic strategies to suppress, enhance, or normalize angiogenesis are widely sought to treat a broad spectrum of diseases (1, 2). The most mature among these approaches targets the activity of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), to modulate relevant signaling pathways and control the angiogenesis process. Indeed, inhibitors of such pathways have emerged as a mainstay therapy for some cancers and diabetic retinopathy (3-5). However, it is still unclear how the endothelial cells (ECs) lining blood vessels form new vessels, or how angiogenic factors regulate such a dynamic, multicellular process.Examining the physical process of angiogenesis requires experimental systems in which the formation of new capillary vessels can be easily observed and manipulated. Commonly used in vivo models such as the mouse dorsal window chamber, chick chorioallantoic membrane, and mouse corneal micro...

The density and architecture of capillary beds that form within a tissue depend on many factors, including local metabolic demand and blood flow. Here, using microfluidic control of local fluid mechanics, we show the existence of a previously unappreciated flowinduced shear stress threshold that triggers angiogenic sprouting. Both intraluminal shear stress over the endothelium and transmural flow through the endothelium above 10 dyn/cm 2 triggered endothelial cells to sprout and invade into the underlying matrix, and this threshold is not impacted by the maturation of cell-cell junctions or pressure gradient across the monolayer. Antagonizing VEcadherin widened cell-cell junctions and reduced the applied shear stress for a given transmural flow rate, but did not affect the shear threshold for sprouting. Furthermore, both transmural and luminal flow induced expression of matrix metalloproteinase 1, and this up-regulation was required for the flow-induced sprouting. Once sprouting was initiated, continuous flow was needed to both sustain sprouting and prevent retraction. To explore the potential ramifications of a shear threshold on the spatial patterning of new sprouts, we used finite-element modeling to predict fluid shear in a variety of geometric settings and then experimentally demonstrated that transmural flow guided preferential sprouting toward paths of draining interstitial fluid flow as might occur to connect capillary beds to venules or lymphatics. In addition, we show that luminal shear increases in local narrowings of vessels to trigger sprouting, perhaps ultimately to normalize shear stress across the vasculature. Together, these studies highlight the role of shear stress in controlling angiogenic sprouting and offer a potential homeostatic mechanism for regulating vascular density.angiogenesis | force | mechanotransduction | migration | morphogenesis T he density of capillary blood vessels varies widely across different organs and tissues and is determined by the ability of unmet local metabolic needs to trigger angiogenesis. Perhaps most well characterized is the induction of VEGF expression by parenchymal hypoxia, leading to angiogenesis that persists until the hypoxia is relieved by subsequent enhanced tissue perfusion (1). Indeed, significant advances have been made in understanding the mechanisms by which numerous biochemical stimuli induce endothelial sprouting (2). Importantly, excess metabolic demand also triggers enhanced local circulation by relaxation of upstream arterioles (3) and results in increased blood flow to these regions. In addition to enhanced delivery of nutrients, the increased blood flow also increases shear stress on the luminal surface of the endothelium, which in some studies has been shown to induce capillary growth in skeletal muscle (4, 5), whereas others showed shear stress enhances endothelial barrier function and inhibits sprouting (6-9).Unlike luminal shear, transmural flow, or fluid flow exiting the wall of the vessel, is universally accepted to induce sprouting o...

There was an error published in J. Cell Sci. 122,[199][200][201][202][203][204][205][206] In the first paragraph of the section entitled 'The integrin-actin linkage functions as a molecular clutch', the protrusion rate was inadvertently reported to increase rather than decrease. The correct paragraph is shown in full below.The clutch hypothesis integrates adhesion, retrograde flow and actin polymerization. The retrograde flow of actin, generated by either myosin II contraction or membrane resistance to actin polymerization at the leading edge, counteracts the protrusive effect of polarized actin polymerization in protrusions. Thus, the net extension rate of a protrusion is the difference between the actin polymerization and the retrograde flow rates. Adhesions can modulate the protrusion mechanism by linking actin filaments to a fixed substratum, thereby creating traction points that inhibit actin retrograde flow (Alexandrova et al., 2008). This linkage would, therefore, increase the protrusion rate by shunting the forces that drive retrograde flow to the substratum and thereby inhibiting the retrograde flow ( Fig. 3; supplementary material Movies 1 and 2). The weakening of such a substratum-integrin-actin linkage would increase the retrograde flow rate and decrease the protrusion rate, whereas decreasing the retrograde flow, by inhibiting myosin II, would increase the overall protrusion rate.The authors apologise for this error and for any confusion caused.

Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a highspatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.cell mechanics | mechanotransduction | migration | actin U nderstanding how cells generate and respond to mechanical forces is critical in cell biology. In anchorage-dependent cells, myosin-II cross-links and contracts actin filaments to generate tension, which is transmitted to the extracellular matrix (ECM) via integrin-mediated adhesions (1-4). The traction stresses (force per area) exerted between adhesions and the ECM drive cell spreading and migration in morphogenesis (5, 6), wound healing (7), and tumor metastasis (8, 9). In addition, these stresses induce changes in adhesion signaling, cytoskeletal reorganization, and gene expression (4, 10-13), thereby regulating functions such as proliferation (14, 15) and differentiation (16,17).Measurements of cellular traction stresses have advanced our understanding of mechanotransduction and enabled quantitative modeling of cellular interactions with the ECM (18-20). These measurements reveal that cells exert inwardly oriented tractions at their periphery, where focal adhesions grow centripetally (3,4,21). However, the vast majority of methods (collectively termed traction force microscopy, TFM) have assumed that cells exert only shear forces (parallel to the plane of the substrate). Interestingly, recent studies have demonstrated that cells on planar substrata exert significant vertical (normal) tractions, indicating that patterns of cellular force generation...

A major challenge in tissue engineering is the development of materials that can support angiogenesis, wherein endothelial cells from existing vasculature invade the surrounding matrix to form new vascular structures. To identify material properties that impact angiogenesis, here we have developed an in vitro model whereby molded tubular channels inside a synthetic hydrogel are seeded with endothelial cells and subjected to chemokine gradients within a microfluidic device. To accomplish precision molding of hydrogels and successful integration with microfluidics, we developed a class of hydrogels that could be macromolded and micromolded with high shape and size fidelity by eliminating swelling after polymerization. Using this material, we demonstrate that matrix degradability switches three-dimensional endothelial cell invasion between two distinct modes: single-cell migration and the multicellular, strand-like invasion required for angiogenesis. The ability to incorporate these tunable hydrogels into geometrically constrained settings will enable a wide range of previously inaccessible biomedical applications.

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.