YAP regulates cell mechanics by controlling focal adhesion assembly

Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.Peer reviewe

YAP regulates cell mechanics by controlling focal adhesion assembly

Hippo effectors YAP/TAZ act as on–off mechanosensing switches by sensing modifications inextracellular matrix (ECM) composition and mechanics. The regulation of their activity hasbeen described by a hierarchical model in which elements of Hippo pathway are under thecontrol of focal adhesions (FAs). Here we unveil the molecular mechanism by which cellspreading and RhoA GTPase activity control FA formation through YAP to stabilize theanchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAPco-transcriptional function and involves the activation of genes encoding for integrins and FAdocking proteins. Tuning YAP transcriptional activity leads to the modification of cellmechanics, force development and adhesion strength, and determines cell shape, migrationand differentiation. These results provide new insights into the mechanism of YAPmechanosensing activity and qualify this Hippo effector as the key determinant of cellmechanics in response to ECM cues.</p

YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype

siRNA–Mediated Methylation of Arabidopsis Telomeres

Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA–based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat–containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)n repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA–dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA–directed mechanism of chromatin maintenance at telomeres in Arabidopsis

MEK and TGF-beta Inhibition Promotes Reprogramming without the Use of Transcription Factor

<div><p>The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages <i>in vitro</i> nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells.</p></div

Comparison of microleakage under orthodontic brackets bonded with five different adhesive systems: in vitro study

Abstract Background Orthodontic treatment is associated with numerous adverse side effects, such as enamel discoloration, demineralization or even caries. The presence of microleakage between the enamel and the adhesive and between the adhesive and the base of the orthodontic bracket allows penetration of the bacteria, molecules, and liquids into the enamel and can lead to unpleasant “white spot lesions” or secondary caries beneath and around the brackets. The aim of this in vitro study was to evaluate microleakage in five adhesive systems commonly used in orthodontic practice for bonding brackets. Methods One hundred extracted premolars were divided into five groups of twenty teeth. Stainless steel Legend medium metal brackets were bonded to teeth using five adhesive systems: resin-reinforced glass ionomer cement GC Fuji Ortho LC (GCF) and composite materials Light Bond (LB), Transbond XT (TB), Trulock™ Light Activated Adhesive (TL), and GC Ortho Connect (GCO). The specimens were subjected to thermal cycling, stained with 2% methylene blue, sectioned with low–speed diamond saw Isomet and evaluated under a digital microscope. Microleakage was detected at the enamel-adhesive and adhesive-bracket interfaces from occlusal and gingival margins. Statistical analysis was performed using generalized linear mixed models with beta error distribution. Results Microleakage was observed in all materials, with GCF showing the highest amount of microleakage. Composite materials GCO, TB, and LB exhibited the lowest amount of microleakage with no statistical difference between them, while TL showed a statistically significantly higher amount of microleakage (p < 0.001). The enamel–adhesive interface had more microleakage in all composite materials (GCO, LB, TB, and TL) than the adhesive bracket–interface (p < 0.001). The highest amount of microleakage occurred in the gingival region in all materials. Conclusion Composite materials showed better adhesive properties than a resin-reinforced glass ionomer cement. The presence of microleakage at the enamel-adhesive interface facilitates the penetration of various substances into enamel surfaces, causing enamel demineralization and the development of dental caries

Timescale of the reprogramming experiment and combinations of inhibitors.

<p><b>[A]</b> Cell culture was seeded at concentration of 25,000–33,000 cells/cm² at day 0 (1.7 x 10^6 cells per 10cm dish). On days 1, 2 and 4 the inhibitors PD0325901 and SB431542 were applied at concentrations 0.5 μM and 2 μM. Cultures were three times replated (1:3 ratio) as indicated by a triple arrow. At day 25, the first colonies positive for alkaline phosphatase were detected (brown bar) and MEF culture medium was replaced by LIF–containing ESM medium (blue bar). In parallel experiments, fibroblast cultures were treated with mir294 mimics (days 1, 3 and 7) and p53 inhibitor (days 4 and 6). <b>[B]</b> Combinations of inhibitors for chiPSc reprogramming as applied on individual plates (experimental setup 1–9) with the number of resulting morphologically distinguishable and phosphatase positive iPSc-like colonies from four independent experiments in each setup.</p

Expression of pluripotency markers in chiPSc colonies.

<p>Left column: staining of nuclei using DAPI (blue). Right column: the same colony stained with antibodies against several pluripotency markers including, Oct4, Sox2, Ssea1 and Nanog. Oct4- and Nanog-positive colonies are enlarged to show specificity of the staining in comparison to surrounding fibroblasts. 100x magnification was used for Oct4, Ssea1 and Nanog, 200x magnification for Sox2.</p

Histological characterization of chiPSc-derived teratomas.

<p>Two independent chiPSc lines (two replicas of each line) were tested in nude mice. Histological sections of hematoxylin-and-eosin-stained sections of teratomas from both lines consist of tissues from all three germ layers. A) Adipose-like tissue [Ad], muscle [Mu]; B) bone; C,D) gut-like epithelial structures with mature goblet cells; E) neuroblastic tissue with neuronal rosettes; F,G) epidermis; and H) cartilage. Magnified 100x.</p