Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

Abstract

Pluripotent embryonic stem cells (ESCs) exert low-traction forces on their niche in vitro whereas specification to definitive endoderm in vivo coincides with force-mediated motility, suggesting a differentiationmediated switch. However, the onset of contractility and extent to which force-mediated integrin signaling regulates fate choices is not understood. To address the requirement of tractions forces for differentiation, we examined mouse embryonic stem cell (ESC) specification towards definitive endoderm on fibrillar fibronectin containing a deformation-sensitive FRET probe. Inhibiting contractility resulted in an increase in the observed fibronectin FRET intensity ratio but also decreased the amount of phosphorylated nuclear SMAD2, leading to reduced expression of the definitive endoderm marker SOX17. By contrast ESCs maintained in pluripotency medium did not exert significant tractions against the fibronectin matrix. When laminin-111 was added to fibrillar matrices to improve the efficiency of definitive endoderm induction, ESCs decreased their fibronectin traction forces in a laminin-dependent manner; blocking the laminin-binding α3-integrin restored fibronectin matrix deformation and reduced SOX17 expression and SMAD2 phosphorylation, probably because of compensation of inhibitory signaling from SMAD7 after 5 days in culture. These data imply that traction forces and integrin signaling are important regulators of early fate decisions in ESCs.

Quantitative proteomics reveal lineage-specific protein profiles in iPSC-derived Marfan syndrome smooth muscle cells

“…Given that these proteins were strongly up-regulated in LM SMCs and subsequently validated in separate human MFS aortic root aneurysms, our initial theory was that each may contribute to focal aneurysm development. Integrins play an important role in cytoskeleton polymer assembly, cell signaling and cytoskeleton attachment to the ECM 37,38,46 . Consistent with our proteomic findings, Parker et al analyzed the ascending aortic proteome within the MFS Fbn1 C1041G/+ mouse model and noted an up-regulation in integrin αVβ3 expression 47 .…”

Haptotaxis is Cell Type Specific and Limited by Substrate Adhesiveness

“…Denatured fibronectin was dual-labeled with Alexa Fluor 488 (donor) and Alexa Fluor 546 (acceptor) fluorophores, as previous described. 34, 35 Briefly, denatured fibronectin was incubated with a 30-fold molar excess of Alexa Fluor 546 C5 Maleimide (Life Technologies) for 2 hours to label cysteine residues within the III7 and III15 domains of fibronectin. The single-labeled fibronectin was buffer exchanged into 0.1 M sodium bicarbonate pH 8.3 and separated from unreacted acceptor fluorophores using a spin desalting column (Thermo Scientific).…”

Zyxin regulates embryonic stem cell fate by modulating mechanical and biochemical signaling interface

“…In specialized cells, zyxin is recruited last to the adhesion sites to promote FA maturation and to facilitate internal force transmission and external force sensing 24 . Since traction stress and substrate sensing are important regulators of early fate decisions in mESCs 7,9 , we hypothesize that zyxin may function as a mechanosensor to regulate stem cell fate through the modulation of actin cytoskeleton-mediated traction stress and substrate rigidity sensing in mESCs.…”

“…Apart from the well-documented biochemical signaling pathways 5 , biophysical cues from the microenvironment also play important roles in ESC maintenance and differentiation 6 . Amongst the biophysical cues, traction stress and substrate stiffness are found to profoundly influence stem cell status [7][8][9][10] . Integrin-focal adhesion mediated modulation of multipotent adult stem cell and mesenchymal stem cell specification is a well-known example of stem cell fate regulation by mechanotransduction pathways [11][12][13] .…”