Arp2/3 Complex Activity Enables Nuclear YAP for Naïve Pluripotency of Human Embryonic Stem Cells
Our understanding of transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency that includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, uniform cell mechanics within naïve colonies, nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restored in actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states. Overall design: HUES8 human embryonic stem cells were dedifferentiated to a naïve state of pluripotency over the course of 6 days with or without 80 µM CK666. CK666 is a known Arp2/3 complex activity inhibitor. Samples were submitted for bulk RNAseq in quadruplicate with the following conditions: control primed cells, control D6 naive cells, and cells which were dedifferentiated in naive media with 80 µM CK666 for 6 days. RNA was isolated using RNeasy Plus kits and assessed for quality using Nanodrop and Bioanalyzers prior to submission to ensure quality.