Regulation of focal adhesion in embryonic stem cells

Abstract

Focal adhesions (FAs) are cellular components crucial to mechanotransduction. Changes to FA morphology and distribution in embryonic stem cells (ESCs) have been associated with stem cell differentiation. It has been postulated that mouse ESCs (mESCs) could be a model cell line to study FAs as a developmental baseline. However, the role of FAs in mESCs during differentiation remain unclear. We aimed to study the effects of induced differentiation in D3 and E14 mESCs on cellular geometry and associated mechanotransduction structures, namely FAs and the actin cytoskeleton. All-trans retinoic acid (RA) was used to induce differentiation. Western blot of pluripotency and differentiation markers verified the successful induction of differentiation. Immunostaining of FAs and actin filaments and subsequent quantification revealed significant and consistent changes to cellular geometry, FA characteristics and actin filament abundance. FAs could have undergone maturation following differentiation. Previously, we also showed that during mESC differentiation, the focal adhesion protein (FAP) zyxin was consistently enriched. Thus, we overexpressed zyxin in D3 mESCs via transfection to assess the role of zyxin during differentiation. Preliminary findings via Western blot revealed an intriguing upregulation of pluripotency markers instead. Zyxin overexpression could potentially mediate pluripotency in ESCs, but this is inconclusive without further validation.