Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young's modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young's modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.

中文翻译：

---

缺乏网格蛋白介导的内吞作用的胚胎干细胞的多能性不能通过恢复细胞硬度来挽救


小鼠胚胎干细胞 (mESC) 显示出独特的机械特性，包括与硬度更高的分化细胞相比，细胞硬度较低。我们之前已经表明，缺乏网格蛋白重链（Cltc）（网格蛋白介导的内吞作用（CME）的重要组成部分）的mESC表现出多能性丧失和分化标记物表达增强。然而，尚不清楚细胞硬度等物理特性是否也会在 Cltc 丢失后发生变化，类似于在分化细胞中看到的情况，如果是这样，这些改变的特性如何具体影响多能性。使用原子力显微镜（AFM），我们证明缺乏 Cltc 的 mESC 表现出更高的杨氏模量，表明与 WT mESC 相比具有更大的细胞刚度。硬度的增加伴随着肌动蛋白应力纤维的存在和无活性、磷酸化的肌动蛋白结合蛋白肌动蛋白丝切蛋白的积累。用肌动蛋白聚合抑制剂处理 Cltc 敲低的 mESC 导致杨氏模量降低至与 WT mESC 获得的值相似的值。然而，多能性因子的表达谱并未得到挽救。此外，虽然 WT 小鼠胚胎成纤维细胞可以被重编程为多能性状态，但在没有 Cltc 的情况下，这种情况会受到抑制。这表明活性 CME 的存在对于胚胎干细胞的多能性至关重要。此外，虽然物理特性可以作为细胞状态的简单读数，但它们可能并不总是忠实地概括潜在的分子命运。