Introduction

In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an ‘ultrasound tweezers’-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed “actinin-sstFRET,” to monitor in situ single-cell allostasis in response to transient stimulation in real time.

Methods

The ultrasound tweezers utilize 1 Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca²⁺ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis.

Results

By subjecting a 1 Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30 min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca²⁺ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation.

Conclusions

By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

中文翻译：

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使用超声镊子探测单细胞机械平衡。

介绍

为应对外部压力，细胞会改变其形态、代谢活动和功能，以通过细胞稳态来机械地适应动态的局部环境。为了探索细胞内稳态中的机械转导，我们应用了一个集成的微机械系统，该系统结合了基于“超声波镊子”的机械压力源和基于 Förster 共振能量转移 (FRET) 的分子力生物传感器，称为“肌动蛋白-sstFRET”，以进行原位监测实时响应瞬时刺激的单细胞稳态。

方法

超声镊子利用 1 Hz、10 秒的瞬态超声脉冲声学激发与细胞膜结合的脂质包裹的微泡，并通过 RGD-整合素连接对细胞施加皮-到纳米-牛顿范围的力. 肌动蛋白-sstFRET 分子传感器与活细胞中的肌动蛋白应力纤维结合，用于绘制实时肌动球蛋白力动态。^{然后，通过分析 Ca 2+}流入、肌动球蛋白细胞骨架 (CSK) 活性和 GTPase RhoA 信号传导的动力学来检查机械敏感行为，以定义单细胞机械平衡。

结果

通过承受 1 Hz、10 秒的物理压力，观察到单个血管平滑肌细胞 (VSMC) 在 30 分钟内以双相机械平衡方式重塑自身，从而调整其收缩性和肌动球蛋白活性。强调 CSK 平衡在细胞机械稳态中的重要作用的细胞机制包括 Ca ²⁺流入、肌动球蛋白 CSK 的重塑和收缩以及 GTPase RhoA 信号传导。观察到来自 II 型糖尿病 (T2DM) 患者的 VSMC 的机械平衡受到损害，这可能会加剧平衡失调。

结论

通过集成同时允许局部机械扰动和映射肌动球蛋白力的工具，我们在我们的微机械系统中揭示了不同的细胞机械平衡分布。我们对细胞机械稳态和适应不良的发现为机械表型分型细胞揭示其致病背景及其生物物理介质提供了潜力，这些是糖尿病、高血压或衰老等多病因疾病的基础。