Linking organelles by actin tracks to establish membrane contact sites

植物液泡对细胞有许多重要的功能。我们的初步工作表明，液泡和细胞骨架之间的连接是至关重要的液泡的形态和大小的调节。限制液泡扩展导致细胞伸长的限制，并最终减慢植物生长。我们的数据表明，NETWORKED（NET）4蛋白家族参与肌动蛋白细胞骨架的组织，从而控制膜融合在液泡。然而，鉴定和确认的四个相互作用的合作伙伴与部分发散的本地化和部分本地化的NET4核膜表明额外的功能。似乎可以想象，NET 4具有比最初预期的更少的特异性作用，并且可能参与其他过程，例如膜接触位点的建立。这些接触位点使细胞器紧密靠近，并允许在细胞器不融合的情况下进行物质交换。这一过程在植物中还没有很好的理解，液泡在膜接触部位的建立中的参与还没有被证明。该提案的目的是调查如何NET4招募肌动蛋白丝到不同的细胞器连同其相互作用的合作伙伴，这是否会导致膜接触点的形成。为此，高分辨率（STED）显微镜将与蛋白质相互作用研究相结合，以表征先前建立的标记系和NET4的基因缺失突变体和相互作用伙伴。此外，还将分析特定基因缺失对接触位点建立的影响，并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.