Cytokinesis in Arabidopsis: Cytokinetic vesicles, cargo proteins and fusion machinery

在植物细胞质分裂中，分隔膜(又名细胞板)是通过定向地将分泌小泡沿着动态的细胞骨架阵列(又名成膜体)输送到细胞分裂平面，在那里这些小泡彼此融合，然后与横向扩展的细胞板的边缘融合。细胞板经历成熟，包括新形成的细胞壁的逐步合成和组装。膜融合涉及特定的QA-SNARs(如Knolle或SYP132)，它们与其伴侣形成反式SNARE复合体。胞动囊泡运输的是什么货物蛋白，以及它们在胞质分裂中可能扮演什么角色几乎是未知的。在目前的资助期(JU 179/24-1)，我们已经建立了方法学基础，利用免疫沉淀和质谱仪鉴定细胞动泡上的货运蛋白。在我们在Knolle标记的囊泡、SYP132标记的囊泡或这两组上检测到的250个候选蛋白中，我们选择了17个由G2/M期特异表达的基因编码的膜相关蛋白，用于在转基因植物中进行更详细的分析。(1)主要目的是解决细胞动泡是只有一个群体还是有几个群体的问题。例如，可以想象，带有特定SNARE蛋白复合体的囊泡只运输细胞动力学货物蛋白的特定亚群。另一种可能性是，新合成的货运蛋白在其他囊泡上运输，而不是从质膜内吞并重新定位到细胞分裂平面的蛋白。(2)另一个目标将是分析货运蛋白--既有已知的，也有待鉴定的--对细胞质分裂的功能或结构贡献。我们将优先考虑在细胞周期的G2/M期特异性合成的膜相关蛋白。我们将产生基因敲除突变体，并使用合适的亚细胞标记或超微结构分析，分析它们可能存在的细胞质分裂缺陷。此外，我们还将尝试识别相互作用的蛋白质。(3)我们还将寻求新的策略来识别细胞动力学货物蛋白。沿着这些思路，我们将尝试增加IP-MS分析的起始材料中分裂细胞的比例。此外，我们还将探索一种非常不同的方法。我们将筛选在G2/M期特异表达的基因数据集，以寻找编码膜相关蛋白并在其启动子区域具有所谓的MSA(有丝分裂特异激活剂)元件的基因。再一次，我们将主要关注功能未知的蛋白质，其序列在进化中是保守的。

In plant cytokinesis, the partitioning membrane (aka cell plate) originates through targeted delivery of secretory vesicles along a dynamic cytoskeletal array (aka phragmoplast) to the cell division plane where these vesicles fuse with one another and later with the margin of the laterally expanding cell plate. The cell plate undergoes maturation including the stepwise synthesis and assembly of the newly forming cell wall. The membrane fusions involve specific Qa-SNAREs (such as KNOLLE or SYP132) which form trans-SNARE complexes with their partners. What cargo proteins are transported by the cytokinetic vesicles and what roles they might play in cytokinesis is virtually unknown. In the current funding period (JU 179/24-1), we have established methodological foundations to identify cargo proteins resident on cytokinetic vesicles, using immunoprecipitation followed by mass spectrometry. Of 250 candidates that we have detected on KNOLLE-tagged vesicles, SYP132-tagged vesicles or both groups, we have selected 17 membrane-associated proteins encoded by G2/M-phase specifically expressed genes for more detailed analysis in transgenic plants.(1) A primary aim will be to address the question of whether there is only a single population of cytokinetic vesicles or whether there are several ones. For example, it is conceivable that vesicles bearing specific SNARE protein complexes transport only specific subsets of cytokinetic cargo proteins. Another possibility could be that newly synthesised cargo proteins are transported on other vesicles than proteins that are endocytosed from the plasma membrane and re-targeted to the plane of cell division.(2) Another aim will be to analyse the functional or structural contributions of cargo proteins – both known ones and those to be identified – to cytokinesis. We will give preference to membrane-associated proteins that are specifically synthesised during G2/M phase of the cell cycle. We will generate knockout mutants and analyse them for possible cytokinesis defects, using suitable subcellular markers or ultrastructural analysis. In addition, we will attempt to identify interacting proteins.(3) We will also pursue new strategies with which to identify cytokinetic cargo proteins. Along these lines, we will attempt to increase the proportion of dividing cells in the starting material for IP-MS analysis. In addition, we will explore a very different approach. We will screen available datasets of genes specifically expressed during G2/M phase for genes that encode membrane-associated proteins and have so called MSA ("mitosis-specific activator") elements in their promoter regions. Again, we will predominantly focus on proteins of unknown function whose sequences have been conserved in evolution.