Linking organelles by actin tracks to establish membrane contact sites

植物液泡对细胞具有过多的关键功能。我们的初步工作表明，液泡和细胞骨架之间的连接对于液泡的形态和大小的调节是至关重要的。限制液泡的扩张会限制细胞的伸长，最终减缓植物的生长。我们的数据显示，网络(Net)4蛋白家族参与了肌动蛋白细胞骨架的组织，从而控制了液泡膜的融合。然而，识别和确认四个部分发散定位的相互作用伙伴和部分定位于核膜的net4表明了额外的功能。似乎可以想象，net4的作用没有最初预期的那么具体，并可能参与其他过程，如建立膜接触位点。这些接触部位使细胞器非常接近，允许物质交换，而不需要细胞器的融合。这一过程在植物中还不是很清楚，液泡在膜接触部位的建立中的作用也尚未被证明。这项提议的目的是研究net4如何与其相互作用伙伴一起将肌动蛋白细丝招募到不同的细胞器中，以及这是否导致膜接触部位的形成。为此，高分辨率(STED)显微镜将与蛋白质相互作用研究相结合，以表征先前建立的net4及其相互作用伙伴的标记系和基因缺失突变。此外，还将分析特定基因缺失对接触点建立的影响，最终确定对植物生长发育的影响。

The plant vacuole has a plethora of critical functions for the cell. Our preliminary work has shown that the connection between the vacuole and the cytoskeleton is critical for the regulation of vacuolar morphology and size. Restricting vacuole expansion leads to limitations in cell elongation and eventually slower plant growth. Our data show that the NETWORKED (NET) 4 protein family is involved in the organization of the actin cytoskeleton and thus controls membrane fusions at the vacuole. However, the identification and confirmation of four interaction partners with partially divergent localization and the partial localization of NET4 to the nuclear envelope indicates additional functions. It seems conceivable that NET4 has a less specific role than originally expected and might participate in other processes such as the establishment of membrane contact sites. These contact sites bring organelles into close proximity and allow material exchange without fusion of the organelles. This process is not well understood in plants and the involvement of the vacuole in the establishment of membrane contact sites has not yet been shown. The aim of this proposal is to investigate how NET4 recruits actin filaments to different organelles together with its interaction partners and whether this leads to the formation of membrane contact sites. To this end, high-resolution (STED) microscopy will be combined with protein interaction studies to characterize previously established marker lines and gene deletion mutants of NET4 and interaction partners. In addition, the impact of specific gene deletions on the establishment of contact sites will be analyzed and finally the effect on plant growth and development will be determined.