ABSTRACT

Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4’s capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation–dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous E. coli, demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 – unlike its N-terminal βTrCP degron motif – robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional in vivo experiments showed that although STIL was not required for Plk4’s initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4’s ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4’s autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.

摘要

Polo 样激酶 4 (Plk4) 是中心粒生物发生的关键调节因子。研究表明 Plk4 经历了从中心粒周围的环状模式到原中心粒组装位点的点状形态的动态重新定位，这一事件对于诱导中心粒生物发生至关重要。然而，Plk4 驱动其对称性破坏环到点重定位能力的详细机制在很大程度上仍然未知。在这里，我们表明 Plk4 以依赖于自磷酸化的方式自启动该过程，并且该事件不需要其下游目标 STIL。使用 mEOS 融合光可转换 Plk4 进行的时间相关分析显示，环态 Plk4 的一部分获得了一种能力，大概是通过自磷酸化，在中心粒周围徘徊，最终产生点状形态。大肠杆菌，证明其自磷酸化依赖性自组织能力。在生化水平上，Plk4——与其 N 端 βTrCP degron 基序不同——在其 C 端隐蔽 polo-box 内强力自磷酸化 PC3 SSTT 基序，这是诱导其物理聚类的关键事件。额外的体内实验表明，虽然 Plk4 的初始环到点转换不需要 STIL，但共表达的 STIL 大大增强了 Plk4 生成球形冷凝物和招募 Sas6（中心侧车轮结构的主要组成部分）的能力。我们建议 Plk4 的自磷酸化诱导的聚类足以诱导其环到点的定位转换，随后招募的 STIL 增强了这一过程，以生成对 Plk4 依赖的中心粒生物发生至关重要的原中心粒组装体。