The role of lipids in mediating the spatiotemporal coordination of cytokinesis in Arabidopsis

在植物中，细胞分裂的模式依赖于分裂平面的建立、称为细胞板的膜室的生物发生以及细胞板在分裂部位的插入。一般认为，前期在质膜上有一个标志，在细胞质分裂结束时，扩大的细胞板的前缘被引导到这个标志。这种位置线索的性质仍然难以捉摸，细胞板的引导和插入过程也知之甚少。脂质越来越被认为是重要的膜标志物，但是否有特定的脂质标记分裂位点还有待研究。有趣的是，抑制磷酸肌苷、甾醇或长链脂肪酸（鞘脂的重要组成部分）的合成会导致细胞分裂缺陷。初步数据表明，PI(4,5)P2磷酸肌苷及其磷酸酶SAC9参与确定细胞板插入的空间位置。PI(4,5)P2分布似乎是由稳态脂质相互作用驱动的。事实上，我们发现鞘脂的酰基链长度参与调节PI(4,5)P2的水平，鞘脂定位于细胞板。在动物中，磷酸肌苷和Rab gtpase的特定组合负责维持和协调细胞中交叉的运输途径。我们的初步数据表明，在植物中，Rab-E gtpase可能在细胞板插入中起作用。此外，我们提出Rab-E GTPases受trappi系聚复合体（一种假定的鸟嘌呤核苷酸交换因子）的调节。TRAPPII在植物细胞分裂的时空调控中起着关键作用。在这个项目中，我们将通过部署遗传，细胞生物学和生化工具的独特组合来解决脂质和关键运输蛋白如何介导细胞板插入。这些包括脂质生物传感器、脂质点击化学、光遗传学诱导脂质耗竭、超分辨率显微镜、膜复合物的靶向免疫分离以及尖端的蛋白质组学和脂质组学。我们的项目是围绕三个主要工作包构建的，每个工作包都涉及到所有的合作伙伴。这些解决了以下问题：1)脂质是如何在扩大的细胞板或分裂部位分选的？ii)脂质分选是否需要TRAPPII-Rab-E GTPase模块？iii)相反，脂质标志物是否调节TRAPPII/Rab-E GTPase功能？总而言之，我们的项目旨在汇集国际公认的尖端植物细胞生物学和脂膜生物学和生物化学方面的独特专业知识。这个项目的结果将在解决植物如何通过细胞分裂实现模式的问题上开辟新的领域，细胞分裂是植物生长和发育的关键过程。此外，该项目框架内的新工具和遗传资源的生产将对整个细胞生物学社区有用。

In plants, patterning by cell division relies on division plane establishment, on the biogenesis of a membrane compartment called the cell plate, and on cell plate insertion at the division site. It is generally accepted that a landmark is laid down at the plasma membrane before prophase, and that the leading edge of the expanding cell plate is guided towards this cue at the end of cytokinesis. The nature of this positional cue remains elusive and the processes of cell plate guidance and insertion are poorly understood. Lipids are increasingly recognized as important membrane landmarks, but whether specific lipids mark the division site has yet to be investigated. Interestingly, inhibiting the synthesis of phosphoinositides, sterols or very-long chain fatty acids, an essential component of sphingolipids, results in cytokinesis defects. Preliminary data indicate that PI(4,5)P2 phosphoinositides and its phosphatase SAC9 are involved in defining the spatial site of cell plate insertion. PI(4,5)P2 distribution appears to be driven by shomeostatic lipid-lipid interactions. Indeed, we found that the acyl-chain length of sphingolipids is involved in regulating the level of PI(4,5)P2 and that sphingolipids localize at the cell plate. In animals, specific combinations of phosphoinositides and Rab GTPases are responsible for maintaining and co-ordinating intersecting trafficking pathways in the cell. Our preliminary data suggest that in plants, Rab-E GTPases might play a role in cell plate insertion. Furthermore, we propose that Rab-E GTPases are regulated by the TRAPPII tethering complex, a putative Guanine nucleotide Exchange Factor. TRAPPII plays a pivotal role in the spatiotemporal control of plant cytokinesis. In this project, we will address how lipids and key trafficking proteins mediate cell plate insertion by deploying a unique combination of genetic, cell biological and biochemical tools. These include lipid biosensors, lipid click-chemistry, inducible depletion of lipids by optogenetics, super-resolution microscopy, targeted immuno-isolation of membrane complexes and cutting-edge proteomics and lipidomics. Our project is structured around three main work packages, each of which involves all partners. These address the following questions: i) how are lipids sorted at the expanding cell plate or division site? ii) does lipid sorting require a TRAPPII-Rab-E GTPase module? iii) conversely, do lipid landmarks regulate TRAPPII/Rab-E GTPase function? Altogether, our project proposes to gather internationally recognized and unique expertise in cutting-edge plant cell biology and lipid membrane biology and biochemistry. The outcome of this project will break new ground in tackling the question of how plants achieve patterning by cell division, a crucial process for plant growth and development. Additionally, the production of new tools and genetic resources within the frame of this project will be useful for the entire cell biology community.