The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

The centromere is a specific chromosomal region where the kinetochore assembles to ensure the faithful segregation of sister chromatids during mitosis and meiosis. Centromeres are defined by a local enrichment of the specific histone variant CenH3 mostly at repetitive satellite sequences. A larger pericentromeric region containing repetitive sequences and transposable elements surrounds the centromere that adopts a particular chromatin state characterized by specific histone variants and post-translational modifications and forms a transcriptionally repressive chromosomal environment. In the model organism Arabidopsis thaliana centromeric and pericentromeric domains form conspicuous heterochromatin clusters called chromocenters in interphase. Here we discuss, using Arabidopsis as example, recent insight into mechanisms involved in maintenance and establishment of centromeric and pericentromeric chromatin signatures as well as in chromocenter formation.

The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear morphology and gene expression. To understand how the Arabidopsis nucleoskeleton physically connects to the nuclear periphery, we investigated the nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. Computational predictions identified three evolutionary conserved peptide motifs within the N-terminal region of KAKU4. Functional analysis revealed that these motifs are required for homomerization of KAKU4, interaction with the nucleoskeleton proteins CROWDED NUCLEI (CRWN) and localization at the nuclear periphery. We find that similar protein motifs are present in NUP82 and NUP136, two plant specific nucleoporins from the Nuclear Pore Complex (NPC) basket. These conserved motifs allow the two nucleoporins to bind CRWN proteins, thus revealing a physical link between the nucleoskeleton and nuclear pores in plants. Finally, whilst NUP82, NUP136 and KAKU4 have a common evolutionary history predating non-vascular land plants, KAKU4 mainly localizes outside the NPC suggesting neofunctionalization of an ancient nucleoporin into a new nucleoskeleton component.

Identification of membrane protein interactomes is a key issue to better understand how these molecules carry out their functions. However, protein-protein interactions using conventional interaction assays are particularly challenging for integral membrane proteins, because of their hydrophobic nature. Here we describe the membrane yeast two-hybrid (MbY2H) system, a powerful tool for identifying the interactors of membrane and membrane-associated proteins.

Mid-SUN proteins are a neglected family of conserved type III membrane proteins of ancient origin with representatives in plants, animals, and fungi. Previous higher plant studies have associated them with functions at the nuclear envelope and the endoplasmic reticulum (ER). In this study, high-resolution confocal light microscopy is used to explore the localisation of SUN3 and SUN4 in the perinuclear region, to explore topology, and to study the role of mid-SUNs on endoplasmic reticulum morphology. The role of SUN3 in the ER is reinforced by the identification of a protein interaction between SUN3 and the ER membrane-bound transcription factor maMYB. The results highlight the importance of mid-SUNs as functional components of the ER and outer nuclear membrane.