Nuclear actin tunes ERK activity to promote somatic cell reprogramming into pluripotency

Yamanaka reprogramming is a stochastic process resulting in only a small fraction of somatic cells successfully converting into iPSCs. The molecular and cellular basis underlying this stochasticity remains elusive. Here we demonstrate that this stochasticity can be eliminated when ERK activity is tuned within a narrow range. This tuning can be accomplished by one tenth the concentration of MEK inhibitor in the 2i media. In the absence of pharmacologic ERK inhibition, cells fine tune ERK by morphological changes, growing taller by allocating more actin into their nucleus. A minimal cell height of 10 ?m is required for pluripotency, a cell geometric feature underlying the “dome-shaped” colony morphology of naïve pluripotent stem cells. Nuclear actin tunes ERK activity by binding to and immobilizing TFII-I?, a transcription factor that binds and mediates ERK's nuclear activation. This work uncovers a mechanistic link for how cell morphology controls cell identity. Insights into these fundamentally coupled processes provide effective approaches to overcome the stochasticity in reprogramming into pluripotency. Overall design: We performed bulk RNA-seq of control (EV), Actin overexpressed (Actin) and NLS-actin OE (NLA-Actin) in mouse MEF and day 4 reporgramming cells. We also performed bulk RNA-seq of green positive or red positive reprogramming cells with or without NLS-actin under day 10 and day 14.

山中伸弥重编程（Yamanaka reprogramming）是一种随机过程，仅极少数体细胞可成功转化为诱导多能干细胞（induced pluripotent stem cells, iPSCs）。该随机性背后的分子与细胞机制仍有待阐明。本研究证实，当细胞外调节蛋白激酶（Extracellular signal-regulated kinase, ERK）的活性被调控在狭窄范围内时，该随机性可被消除。在2i培养基中使用十分之一浓度的丝裂原活化蛋白激酶激酶（Mitogen-activated protein kinase kinase, MEK）抑制剂，即可实现这种调控。在不使用药物抑制ERK的情况下，细胞可通过形态变化微调ERK活性：通过向细胞核内募集更多肌动蛋白（actin）使细胞高度增加。多能性的维持需要细胞高度至少达到10微米，这一细胞几何特征正是幼稚态多能干细胞（naïve pluripotent stem cells）形成“穹顶状”集落形态的基础。核内肌动蛋白通过结合并固定转录因子TFII-I（原文标注为TFII-I?）来调控ERK活性，该转录因子可结合并介导ERK的核内激活。本研究揭示了细胞形态调控细胞身份的潜在机制关联。对这些紧密耦合的基础过程的深入理解，为克服多能性重编程中的随机性提供了有效策略。总体实验设计：我们对小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）以及重编程第4天的细胞，分别设置空载对照（empty vector, EV）、肌动蛋白过表达（Actin）以及核定位信号-肌动蛋白过表达（NLS-actin OE）三组，进行批量RNA测序（bulk RNA-seq）。此外，我们还对重编程第10天和第14天、带有或不带有NLS-actin的绿色荧光阳性或红色荧光阳性细胞进行了批量RNA测序。