Geometrically defined environments direct cell division rate and subcellular YAP localization in single mouse embryonic stem cells

Abstract

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell–cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

Two-Photon Polymerization of 2.5D and 3D Microstructures Fostering a Ramified Resting Phenotype in Primary Microglia

“…The most significant limitation of 2PP is the relatively long printing time hindering the upscaling of the technology ( Moroni et al, 2018 ). Multiple polymeric, hydrogel or composite materials were employed in combination with 2PP to fabricate micro- and nano-patterns as well as 3D structures for in vitro cellular studies involving neuroblastoma, glioblastoma, prostate cancer, murine cerebellar granule, chondrocytes, macrophages, neuronal and stem cells ( Marino et al, 2013 ; Accardo et al, 2017 , 2018 ; Turunen et al, 2017 ; Maciulaitis et al, 2019 ; Fendler et al, 2019 ; Babi et al, 2021 ; Bertels et al, 2021 ; Maciulaitis et al, 2021 ; Nouri-Goushki et al, 2021 ; Akolawala et al, 2022 ; Costa et al, 2022 ).…”

Matrix Stiffness and Architecture Drive Fibro-adipogenic Progenitors’ Activation into Myofibroblasts

“…The increase in YAP nuclear localization was highly correlated with the increase in myofibroblast activation indicating that YAP may be driving this change. Previous studies have shown that YAP nuclear localization is tied to fibroblast activation and proliferation [32][33][34] . Blockage of YAP activity with verteporfin reduced myofibroblast activation on 25 kPa gels and on plastic.…”

Matrix stiffness and architecture drive fibro-adipogenic progenitors’ activation into myofibroblasts

“…The increase in YAP nuclear localization was highly correlated with the increase in myofibroblast activation indicating that YAP may be driving this change. Previous studies have shown that YAP nuclear localization is tied to fibroblast activation and proliferation 32 – 34 . Blockage of YAP activity with verteporfin reduced myofibroblast activation on 25 kPa gels and on plastic.…”