Many proteins with intrinsically disordered regions undergo liquid-liquid phase separation under specific conditions in vitro and in vivo. These complex biopolymers form a metastable phase with distinct mechanical properties defining the timescale of their biological functions. However, determining these properties is nontrivial, even in vitro, and often requires multiple techniques. Here we report the measurement of both viscosity and surface tension of biomolecular condensates via correlative fluorescence microscopy and atomic force microscopy (AFM) in a single experiment (fluorescence recovery after probe-induced dewetting, FRAP-ID). Upon surface tension evaluation via regular AFM-force spectroscopy, controlled AFM indentations induce dry spots in fluorescent condensates on a glass coverslip. The subsequent rewetting exhibits a contact line velocity that is used to quantify the condensed-phase viscosity. Therefore, in contrast with fluorescence recovery after photobleaching (FRAP), where molecular diffusion is observed, in FRAP-ID fluorescence recovery is obtained through fluid rewetting and the subsequent morphological relaxation. We show that the latter can be used to cross-validate viscosity values determined during the rewetting regime. Making use of fluid mechanics, FRAP-ID is a valuable tool to evaluate the mechanical properties that govern the dynamics of biomolecular condensates and determine how these properties impact the temporal aspects of condensate functionality.

中文翻译：

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通过 FRAP-ID 量化生物分子凝聚物中的表面张力和粘度


许多具有固有无序区域的蛋白质在体外和体内的特定条件下进行液-液相分离。这些复杂的生物聚合物形成一个亚稳相，具有不同的机械特性，决定了它们的生物功能的时间尺度。然而，即使在体外，确定这些特性也并非易事，并且通常需要多种技术。在这里，我们报告了在单个实验中通过相关荧光显微镜和原子力显微镜 （AFM） 测量生物分子凝聚物的粘度和表面张力（探针诱导去湿后的荧光恢复，FRAP-ID）。通过常规 AFM 力谱进行表面张力评估后，受控的 AFM 压痕会在玻璃盖玻片上的荧光冷凝物中产生干点。随后的再润湿表现出用于量化凝聚相粘度的接触线速度。因此，与观察到分子扩散的光漂白后荧光恢复 （FRAP） 相比，FRAP-ID 荧光恢复是通过液体再润湿和随后的形态弛豫获得的。我们表明，后者可用于交叉验证在再润湿状态下确定的粘度值。利用流体力学，FRAP-ID 是一种有价值的工具，用于评估控制生物分子凝聚物动力学的机械特性，并确定这些特性如何影响凝聚态功能的时间方面。