Linking organelles by actin tracks to establish membrane contact sites

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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.