Abstract
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.
Highlights
Human embryonic stem cell lines and induced pluripotent stem cells grow in compact colonies at high cell density; while this condition is usually associated in somatic epithelial cells to Yesassociated protein (YAP) inactivation and nuclear exclusion, YAP remains predominantly expressed in PSC nuclei (Supplementary Fig. 1a)
We cultured induced pluripotent stem cells (iPSCs) onto micropatterned substrates that allow precise manipulation of colony size and cell density, and compared YAP subcellular localization to adult human mesenchymal stem cells or dermal fibroblasts grown at a similar density. iPSC density in micropatterned colonies correlated inversely with colony area (Fig. 1a), while YAP appeared mostly expressed in the nucleus and co-localized with pluripotency markers NANOG (Fig. 1b), OCT4 and β-CATENIN. (Supplementary Fig. 1b)
YAP–TEAD transcriptional activity in PSCs was homogeneous throughout the micropatterned colonies regardless the increasing density, as shown by human embryonic stem cell lines (hESCs) reporter line based on the expression of mCherry fluorescent tag under YAP–TEAD promoter (Fig. 1e) [16]
Summary
During cell differentiation and organogenesis, cells encounter a rearrangement in their shape and size which is instrumental to the acquisition of their new identity [1] This process requires the dynamic adjustment of the cytoskeleton. YAP acts downstream of Hippo kinase network and integrates mechanical and biochemical signals arising from the ECM and the surrounding cells to shuttle to the nucleus and activate given genetic programmes, by interacting with cell- and stage-specific transcription factors [11,12,13,14,15]. Whether YAP function is mechanically regulated in human embryos and pluripotent stem cells (PSCs) and if its co-transcriptional activity can be exploited to maintain their potency or drive their specification is still debated. The fine tuning of YAP–TEAD-induced cell tension is required during PSC mesoderm specification in order to allow the timely rearrangement of the cytoskeleton these cells need to acquire a new identity
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