Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
Doctoral Program in Chemical Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
Nat Commun. 2024 Apr 15;15(1):3222. doi: 10.1038/s41467-024-47330-4.
High-resolution imaging of biomolecular condensates in living cells is essential for correlating their properties to those observed through in vitro assays. However, such experiments are limited in bacteria due to resolution limitations. Here we present an experimental framework that probes the formation, reversibility, and dynamics of condensate-forming proteins in Escherichia coli as a means to determine the nature of biomolecular condensates in bacteria. We demonstrate that condensates form after passing a threshold concentration, maintain a soluble fraction, dissolve upon shifts in temperature and concentration, and exhibit dynamics consistent with internal rearrangement and exchange between condensed and soluble fractions. We also discover that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its potential applicability as a reporter to differentiate the two in vivo. Overall, this framework provides a generalizable, accessible, and rigorous set of experiments to probe the nature of biomolecular condensates on the sub-micron scale in bacterial cells.
在活细胞中对生物分子凝聚体进行高分辨率成像对于将其性质与通过体外测定观察到的性质相关联至关重要。然而，由于分辨率的限制，此类实验在细菌中受到限制。在这里，我们提出了一种实验框架，用于探测大肠杆菌中形成凝聚体的蛋白质的形成、可逆性和动力学，以此来确定细菌中生物分子凝聚体的性质。我们证明，凝聚体在超过临界浓度后形成，保持可溶性部分，在温度和浓度变化时溶解，并表现出与内部重排和凝聚相与可溶性相之间交换一致的动力学。我们还发现，一种用于不可溶性蛋白质聚集体的既定标记物 IbpA，与细菌凝聚体和聚集体的共定位模式不同，这表明它作为一种报告蛋白在体内区分两者具有潜在的适用性。总的来说，该框架提供了一组可推广、可访问和严格的实验，可在细菌细胞的亚微米尺度上探测生物分子凝聚体的性质。
