Characterisation of Nuclear EnvelopeAssociated Proteins (NEAP) in Arabidopsis thaliana
During evolution, eukaryotic cells have acquired a nuclear envelope (NE) enclosing and protecting the genome, which is organized in chromatin, a structure wrapping DNA around histone proteins. The NE is composed of two membranes: on the nucleoplasmic side, the Inner Nuclear Membrane (INM) and on the cytoplasmic side, the Outer Nuclear Membrane. The NE allows communication between both compartments through Nuclear Pore Complexes and bridges the cytoskeleton to the nucleoskeleton through the LInker of Nucleoskeleton to Cytoskeleton complex. Thus, the nucleoskeleton associated with the INM is needed to transmit signals to the nucleus and induce changes in chromatin organisation and ultimately gene expression. A novel family of NUCLEAR ENVELOPE ASSOCIATED PROTEINS (NEAPs) proposed to be new components of the plant nucleoskeleton has been recently evidenced in the model plant Arabidopsis thaliana. AtNEAP proteins are encoded by a small gene family composed of three genes and are targeted through a nuclear localisation signal to the nucleus where they are anchored at the INM through their C-terminal transmembrane domain. AtNEAPs also possess several long coiled-coil domains reminiscent of the lamin structure in animals. This thesis aimed at performing a functional analysis of AtNEAPs using T-DNA insertion and CRISPR/Cas9 mutant lines. The AtNEAP interactome was investigated by molecular approaches (Yeast Two Hybrid), which indicated AtNEAP interactions with each other to form homo or hetero-dimers; as well as in vivo localisation and co-localisation coupled to image analyses (apFRET, acceptor photobleaching Fluorescence Resonance Energy Transfer), which confirmed interactions with the transcription factor (TF) AtbZIP18. AtNEAP specific antibodies generated during this study were used to confirm expression in vivo. Altogether, results indicated that AtNEAPs are part of the nucleoskeleton, with a role in anchoring TFs at the INM to maintain nuclear morphology and chromatin organisation.
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