All structures within living cells must form at the right time and place. This includes condensates such as the nucleolus, Cajal bodies and stress granules, which form via liquid–liquid phase separation of biomolecules, particularly proteins enriched in intrinsically disordered regions (IDRs)^1,2. In non-living systems, the initial stages of nucleated phase separation arise when thermal fluctuations overcome an energy barrier due to surface tension. This phenomenon can be modelled by classical nucleation theory (CNT), which describes how the rate of droplet nucleation depends on the degree of supersaturation, whereas the location at which droplets appear is controlled by interfacial heterogeneities^3,4. However, it remains unknown whether this framework applies in living cells, owing to the multicomponent and highly complex nature of the intracellular environment, including the presence of diverse IDRs, whose specificity of biomolecular interactions is unclear^5,6,7,8. Here we show that despite this complexity, nucleation in living cells occurs through a physical process similar to that in inanimate materials, but the efficacy of nucleation sites can be tuned by their biomolecular features. By quantitatively characterizing the nucleation kinetics of endogenous and biomimetic condensates in living cells, we find that key features of condensate nucleation can be quantitatively understood through a CNT-like theoretical framework. Nucleation rates can be substantially enhanced by compatible biomolecular (IDR) seeds, and the kinetics of cellular processes can impact condensate nucleation rates and specificity of location. This quantitative framework sheds light on the intracellular nucleation landscape, and paves the way for engineering synthetic condensates precisely positioned in space and time.

活细胞内的所有结构都必须在正确的时间和地点形成。这包括凝聚物，例如核仁、Cajal 体和应力颗粒，它们是通过生物分子的液-液相分离形成的，特别是富含固有无序区域 (IDR) 的蛋白质^1,2。在非生命系统中，当热波动克服表面张力引起的能垒时，会出现成核相分离的初始阶段。这种现象可以通过经典成核理论 (CNT) 进行建模，该理论描述了液滴成核速率如何取决于过饱和程度，而液滴出现的位置由界面异质性控制^3,4. 然而，由于细胞内环境的多组分和高度复杂的性质，包括存在多种 IDR，其生物分子相互作用的特异性尚不清楚，因此该框架是否适用于活细胞仍然未知^5,6,7,8. 在这里，我们表明，尽管存在这种复杂性，但活细胞中的成核通过类似于无生命材料的物理过程发生，但成核位点的功效可以通过它们的生物分子特征进行调整。通过定量表征活细胞中内源和仿生凝聚物的成核动力学，我们发现凝聚成核的关键特征可以通过类似 CNT 的理论框架来定量理解。兼容的生物分子 (IDR) 种子可以显着提高成核率，并且细胞过程的动力学可以影响凝聚成核率和位置特异性。这个定量框架揭示了细胞内成核景观，并为在空间和时间中精确定位的工程合成凝聚物铺平了道路。