Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA.
Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA.
J Cell Biol. 2020 Nov 2;219(11). doi: 10.1083/jcb.202006054.
Tau protein in vitro can undergo liquid-liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.
在体外，Tau 蛋白可以发生液-液相分离（LLPS）；然而，在活细胞中观察到这种相转变的情况有限。为了研究活细胞中的蛋白质状态转变，我们将 Cry2 连接到 Tau 上，并研究了驱动 Tau 聚集的每个结构域的贡献。令人惊讶的是，富含脯氨酸的结构域（PRD），而不是微管结合结构域（MTBD），驱动 LLPS，并且这种驱动受其磷酸化状态的控制。可观察到的 PRD 衍生的细胞质凝聚体发生融合，光漂白后的荧光恢复与体外的 PRD LLPS 一致。模拟表明，PRD 的电荷特性预测了相分离。Tau PRD 与 EB1 形成异质凝聚体，EB1 是微管动态不稳定性的正端调节剂。MTBD 和 PRD 的特定结构域特性发挥着不同但互补的作用，它们在细胞环境中利用 LLPS 来实现新的功能，从而将它们与 α-β 微管异二聚体结合的关系扩展到更大比例的微管上。
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