Lamellipodium is a myosin-independent mechanosensor.

The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here, we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young's modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced myosin-II independent nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading-edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimental observations. Together, these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which, in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness-sensitive processes.

Attenuation of lipopolysaccharide-induced lung vascular stiffening by lipoxin reduces lung inflammation.

Reversible changes in lung microstructure accompany lung inflammation, although alterations in tissue micromechanics and their impact on inflammation remain unknown. This study investigated changes in extracellular matrix (ECM) remodeling and tissue stiffness in a model of LPS-induced inflammation and examined the role of lipoxin analog 15-epi-lipoxin A4 (eLXA4) in the reduction of stiffness-dependent exacerbation of the inflammatory process. Atomic force microscopy measurements of live lung slices were used to directly measure local tissue stiffness changes induced by intratracheal injection of LPS. Effects of LPS on ECM properties and inflammatory response were evaluated in an animal model of LPS-induced lung injury, live lung tissue slices, and pulmonary endothelial cell (EC) culture. In vivo, LPS increased perivascular stiffness in lung slices monitored by atomic force microscopy and stimulated expression of ECM proteins fibronectin, collagen I, and ECM crosslinker enzyme, lysyl oxidase. Increased stiffness and ECM remodeling escalated LPS-induced VCAM1 and ICAM1 expression and IL-8 production by lung ECs. Stiffness-dependent exacerbation of inflammatory signaling was confirmed in pulmonary ECs grown on substrates with high and low stiffness. eLXA4 inhibited LPS-increased stiffness in lung cross sections, attenuated stiffness-dependent enhancement of EC inflammatory activation, and restored lung compliance in vivo. This study shows that increased local vascular stiffness exacerbates lung inflammation. Attenuation of local stiffening of lung vasculature represents a novel mechanism of lipoxin antiinflammatory action.

Endothelial barrier disruption and recovery is controlled by substrate stiffness.

Circulating barrier disruptive agonists bind specific cell membrane receptors and trigger signal transduction pathways leading to the activation of cell contractility and endothelial cell (EC) permeability. Although all cells in tissues including vascular EC are surrounded by compliant extracellular matrix, the impact of matrix stiffness on agonist-induced signaling, cytoskeletal remodeling and EC barrier regulation is not well understood. This study examined agonist-induced cytoskeletal and signaling changes associated with EC barrier disruption and recovery using pulmonary EC grown on compliant substrates of physiologically relevant (8.6 kPa) stiffness, very low (0.55 kPa) and very high (42 kPa) stiffness. Human pulmonary microvascular and macrovascular EC grown on 0.55 kPa substrate contained a few actin stress fibers, while stress fiber amount increased with increasing matrix stiffness. Thrombin-induced stress fiber formation was maximal in EC grown on 42 kPa substrate, diminished on 8.6 kPa substrate, and was minimal on 0.55 kPa substrate. These effects were linked to a stiffness-dependent increase in thrombin-induced phosphorylation of the Rho kinase target, myosin light chain phosphatase (MYPT1), and regulatory myosin light chains (MLC). Surprisingly, EC barrier recovery and activation of Rac GTPase-dependent barrier protective signaling reached maximal levels in EC grown on 8.6 kPa, but not on 0.55 kPa substrate. In conclusion, these data show a critical role of extracellular matrix stiffness in the regulation of the Rac/Rho signaling balance during onset and resolution of agonist-induced EC permeability. The optimal conditions for the Rho/Rac signaling switch, which provides an effective and reversible EC cytoskeletal and permeability response to agonist, are reached in cells grown on the matrix of physiologically relevant stiffness.

Dia1-dependent adhesions are required by epithelial tissues to initiate invasion.

Developing tissues change shape and tumors initiate spreading through collective cell motility. Conserved mechanisms by which tissues initiate motility into their surroundings are not known. We investigated cytoskeletal regulators during collective invasion by mouse tumor organoids and epithelial Madin-Darby canine kidney (MDCK) acini undergoing branching morphogenesis in collagen. Use of the broad-spectrum formin inhibitor SMIFH2 prevented the formation of migrating cell fronts in both cell types. Focusing on the role of the formin Dia1 in branching morphogenesis, we found that its depletion in MDCK cells does not alter planar cell motility either within the acinus or in two-dimensional scattering assays. However, Dia1 was required to stabilize protrusions extending into the collagen matrix. Live imaging of actin, myosin, and collagen in control acini revealed adhesions that deformed individual collagen fibrils and generated large traction forces, whereas Dia1-depleted acini exhibited unstable adhesions with minimal collagen deformation and lower force generation. This work identifies Dia1 as an essential regulator of tissue shape changes through its role in stabilizing focal adhesions.

Development in frogs with large eggs and the origin of amniotes.

The origin of the amniote egg is one of the most significant events in the evolution of terrestrial vertebrates. This innovation was probably driven by increased egg size, and to find potential parallels, we can examine the derived development of extant amphibians with large eggs. The embryo of the Puerto Rican tree frog, Eleutherodactylus coqui, exhibits an alteration of its fate map and a secondary coverage of its yolky cells, reflecting the large 3.5 mm egg. Comparable changes may have occurred with the derivation of an amniote pattern of development. Future investigations should focus on the molecular organization of the egg. In the model amphibian for development, Xenopus laevis, information for embryonic germ layers, the dorsal axis, and germ cells is stored mainly as localized RNAs at the vegetal pole of the egg. These localizations would likely be changed with increased egg size. A review of the orthologues of the key X. laevis genes raises the possibility that their activities are not conserved in other vertebrates.

Arp2/3 inhibition induces amoeboid-like protrusions in MCF10A epithelial cells by reduced cytoskeletal-membrane coupling and focal adhesion assembly.

Here we demonstrate that Arp2/3 regulates a transition between mesenchymal and amoeboid protrusions in MCF10A epithelial cells. Using genetic and pharmacological means, we first show Arp2/3 inhibition impairs directed cell migration. Arp2/3 inhibition results in a dramatically impaired cell adhesion, causing deficient cell attachment and spreading to ECM as well as an 8-fold decrease in nascent adhesion assembly at the leading edge. While Arp2/3 does not play a significant role in myosin-dependent adhesion growth, mature focal adhesions undergo large scale movements against the ECM suggesting reduced coupling to the ECM. Cell edge protrusions occur at similar rates when Arp2/3 is inhibited but their morphology is dramatically altered. Persistent lamellipodia are abrogated and we observe a markedly increased incidence of blebbing and unstable pseuodopods. Micropipette-aspiration assays indicate that Arp2/3-inhibited cells have a weak coupling between the cell cortex and the plasma membrane, and suggest a potential mechanism for increased pseudopod and bleb formation. Pseudopods are not sensitive to reduced in formin or myosin II activity. Collectively, these results indicate that Arp2/3 is not necessary for rapid protrusion of the cell edge but plays a crucial role in assembling focal adhesions required for its stabilization.

Myosin II-mediated focal adhesion maturation is tension insensitive.

Myosin II motors drive changes in focal adhesion morphology and composition in a "maturation process" that is crucial for regulating adhesion dynamics and signaling guiding cell adhesion, migration and fate. The underlying mechanisms of maturation, however, have been obscured by the intermingled effects of myosin II on lamellar actin architecture, dynamics and force transmission. Here, we show that focal adhesion growth rate stays constant even when cellular tension is reduced by 75%. Focal adhesion growth halts only when myosin stresses are sufficiently low to impair actin retrograde flow. Focal adhesion lifetime is reduced at low levels of cellular tension, but adhesion stability can be rescued at low levels of force by over-expression of α-actinin or constitutively active Dia1. Our work identifies a minimal myosin activity threshold that is necessary to drive lamellar actin retrograde flow is sufficient to permit focal adhesion elongation. Above this nominal threshold, myosin-mediated actin organization and dynamics regulate focal adhesion growth and stability in a force-insensitive fashion.

Tension is required but not sufficient for focal adhesion maturation without a stress fiber template.

Focal adhesion composition and size are modulated in a myosin II-dependent maturation process that controls adhesion, migration, and matrix remodeling. As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown. We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions. Despite relatively unperturbed adhesion dynamics and force transmission, impaired stress fiber assembly impeded focal adhesion compositional maturation and fibronectin remodeling. Finally, we show that compositional maturation of focal adhesions could occur even when myosin II-dependent cellular tension was reduced by 80%. We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions. This work identifies the essential role of lamellar actin architecture in adhesion maturation.

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui.

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.

Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevis.

Eleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3' untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui.