Katanin复合体在植物周质微管动态及细胞形态建成中的调控机制研究

Microtubule severing, which plays a central role in the remodeling of microtubule arrays during cell morphogenesis, is precisely regulated. During the rearrangement of plant cortical microtubule arrays, microtubule severing is specifically triggered either at branching points associated with microtubule nucleation or at microtubule crossover points. However, mechanisms underlying regulation of the specific recruitment and microtubule severing remain largely unknown. In this study, using cortical microtubule arrays in Arabidopsis epidermal cells as a model system, we will investigate the regulatory role of the Katanin (p60/p80) complex in microtubule severing, which substantially influences microtubule dynamics and cell morphogenesis. Firstly, live cell imaging will be performed to reveal the localization of Katanin p80 subunit at cortical microtubules, and to investigate whether the p80 subunit specifically recruits the p60 subunit either to branching nucleation points or to microtubule crossover points, and triggers microtubule severing on site. Moreover, using the state-of-the-art genome editing technique, CRISPR-CAS9, four Katanin p80 encoding genes will be simultaneously disrupted to determine the redundant roles of p80s in microtubule severing. In addition, immunoprecipitation and ultrahigh resolution Mass-Spec will be conducted to identify adaptors associated with precise recruitment of the Katanin complex and subsequent microtubule severing. The predicted outcome will greatly facilitate the elucidation of mechanisms underlying regulation of the specific microtubule severing either at branching nucleation points or at microtubule crossover points, and help us understand how plant cell responds the developmental cues or environmental stimuli via microtubule remodeling during morphogenesis.

微管切割在微管动态重构与细胞形态建成中发挥核心作用，并受到精密地调控。植物周质微管阵列中微管切割特异地发生在微管成核点处或微管交叉点处，但分子调控机制尚不清楚。本研究将以拟南芥表皮细胞周质微管为模型，深入研究微管切割蛋白Katanin（p60/p80）复合体如何调控微管切割、进而影响微管动态重构及细胞形态建成？将运用活细胞显微成像技术，观察Katanin p80亚基是否定位于周质微管、特异地招募Katanin p60亚基到微管成核点处或微管交叉点处，并引发微管切割？将用CRISPR-CAS9基因组编辑技术同时敲除四个编码p80的基因以确定其功能。并将用免疫共沉淀与超高分辨质谱鉴定这种特异性招募及切割是否需要其他衔接蛋白参与？预期结果将阐明Katanin复合体在微管切割中的作用机制，并增进人们理解在形态建成中植物细胞如何响应发育或环境信号而进行微管的动态重构。

微管切割在微管动态重构与细胞形态建成中发挥核心作用，并受到精密地调控。植物周质微管阵列中微管切割特异地发生在微管成核点处或微管交叉点处，但人们对其分子调控机制尚不清楚。本研究将按照计划，深入研究了微管切割蛋白Katanin(p60/p80)复合体在微管交叉点处的精准切割机制。我们首先构建了一系列优化的微管荧光蛋白标签，pTUB6::VisGreen-TUB6、pTUB6::mCherry-TUB6在不同类型的细胞中有着均一稳定且明亮的荧光信号。随后利用构建的双价荧光蛋白标签载体获得了微管与katanin组分带有不同荧光信号的拟南芥株系，并进行了活细胞显微成像观测。结合CRISPR-CAS9基因组编辑技术，我们同时敲除四个编码p80的基因，并进一步确认了katanin p80可以特异定位在微管交叉点处，招募katanin p60亚基引发微管切割。此外，我们也通过活细胞成像观察到微管成核蛋白Augmin可以特异的在微管交叉点处定位，并能够拮抗katanin介导的微管切割作用。进一步通过遗传学和计算机模拟分析，我们解析了augmin和katanin协同调控微管阵列组织的作用机制。上述研究成果为阐明微管切割的精准调控机制提供了关键证据，并为全面解析微管动态重构在植物细胞形态建成中作用机制提供了重要的理论依据。