Light at the end of the tunnel: FRAP assays combined with super resolution microscopy confirm the presence of a tubular vacuole network in meristematic plant cells

David Scheuring, Elena A Minina, Falco Krueger, Upendo Lupanga, Melanie Krebs, Karin Schumacher
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Abstract

Plant vacuoles play key roles in cellular homeostasis, performing catabolic and storage functions, and regulating pH and ion balance. Despite their essential role, there is still no consensus on how vacuoles are established. A model proposing that the endoplasmic reticulum is the main contributor of membrane for growing vacuoles in meristematic cells has been challenged by a study proposing that plant vacuoles are formed de novo by homotypic fusion of multivesicular bodies (MVBs). Here, we use the Arabidopsis thaliana root as a model system to provide a systematic overview of successive vacuole biogenesis stages, starting from the youngest cells proximate to the quiescent center. We combine in vivo high- and super-resolution (STED) microscopy to demonstrate the presence of tubular and connected vacuolar structures in all meristematic cells. Using customized fluorescence recovery after photobleaching (FRAP) assays, we establish different modes of connectivity and demonstrate that thin, tubular vacuoles, as observed in cells near the quiescent center, form an interconnected network. Finally, we argue that a growing body of evidence indicates that vacuolar structures cannot originate from MVBs alone but receive membrane material from different sources simultaneously.
隧道尽头的曙光FRAP 检测与超分辨率显微镜相结合,证实分生植物细胞中存在管状液泡网络
植物液泡在细胞平衡中发挥着关键作用,具有分解代谢和储存功能,并能调节 pH 值和离子平衡。尽管液泡发挥着重要作用,但人们对液泡是如何形成的仍未达成共识。有研究提出,植物液泡是通过多囊体(MVBs)的同型融合从新形成的,这对提出内质网是分生细胞中生长液泡的主要膜贡献者的模型提出了挑战。在这里,我们以拟南芥根为模型系统,从接近静止中心的最年轻细胞开始,系统地概述了液泡的连续生物发生阶段。我们结合体内高分辨和超分辨(STED)显微镜,证明了所有分生组织细胞中都存在管状和相连的液泡结构。利用定制的光漂白后荧光恢复(FRAP)测定,我们建立了不同的连接模式,并证明在静止中心附近的细胞中观察到的细管状液泡形成了一个相互连接的网络。最后,我们认为,越来越多的证据表明,液泡结构不可能仅源于 MVB,而是同时接收来自不同来源的膜材料。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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