Linker Of Nucleoskeleton Research Articles

Cytoskeletal control of nuclear morphology and stiffness are required for OPN-induced bone-marrow-derived mesenchymal stem cell migration.

During cell migration, the movement of the nucleus must be coordinated with the cytoskeletal dynamics that influence the efficiency of cell migration. Our previous study demonstrated that osteopontin (OPN) significantly promotes the migration of bone-marrow-derived mesenchymal stem cells (BMSCs). However, the mechanism that regulates nuclear mechanics of the cytoskeleton during OPN-promoted BMSC migration remains unclear. In this study, we investigated how the actin cytoskeleton influences nuclear mechanics in BMSCs. We assessed the morphology and mechanics of the nuclei in the OPN-treated BMSCs subjected to disruption or polymerization of the actin cytoskeleton. We found that disruption of actin organization by cytochalasin D (Cyto D) resulted in a decrease in the nuclear projected area and nuclear stiffness. Stabilizing the actin assembly with jasplakinolide (JASP) resulted in an increase in the nuclear projected area and nuclear stiffness. SUN1 (Sad-1/UNC-84 1) is a component of the LINC (linker of nucleoskeleton and cytoskeleton) complex involved in the connections between the nucleus and the cytoskeleton. We found that SUN1 depletion by RNAi decreased the nuclear stiffness and OPN-promoted BMSC migration. Thus, the F-actin cytoskeleton plays an important role in determining the morphology and mechanical properties of the nucleus. We suggest that the cytoskeletal-nuclear interconnectivity through SUN1 proteins plays an important role in OPN-promoted BMSC migration.

MLKS2 is an ARM domain and F-actin-associated KASH protein that functions in stomatal complex development and meiotic chromosome segregation

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) complex is an essential multi-protein structure spanning the eukaryotic nuclear envelope. The LINC complex functions to maintain nuclear architecture, positioning, and mobility, along with specialized functions in meiotic prophase and chromosome segregation. Members of the LINC complex were recently identified in maize, an important scientific and agricultural grass species. Here we characterized Maize LINC KASH AtSINE-like2, MLKS2, which encodes a highly conserved SINE-group plant KASH protein with characteristic N-terminal armadillo repeats (ARM). Using a heterologous expression system, we showed that actively expressed GFP-MLKS2 is targeted to the nuclear periphery and colocalizes with F-actin and the endoplasmic reticulum, but not microtubules in the cell cortex. Expression of GFP-MLKS2, but not GFP-MLKS2ΔARM, resulted in nuclear anchoring. Genetic analysis of transposon-insertion mutations, mlks2-1 and mlks2-2, showed that the mutant phenotypes were pleiotropic, affecting root hair nuclear morphology, stomatal complex development, multiple aspects of meiosis, and pollen viability. In male meiosis, the mutants showed defects for bouquet-stage telomere clustering, nuclear repositioning, perinuclear actin accumulation, dispersal of late prophase bivalents, and meiotic chromosome segregation. These findings support a model in which the nucleus is connected to cytoskeletal F-actin through the ARM-domain, predicted alpha solenoid structure of MLKS2. Functional conservation of MLKS2 was demonstrated through genetic rescue of the misshapen nuclear phenotype of an Arabidopsis (triple-WIP) KASH mutant. This study establishes a role for the SINE-type KASH proteins in affecting the dynamic nuclear phenomena required for normal plant growth and fertility.Abbreviations: FRAP: Fluorescence recovery after photobleaching; DPI: Days post infiltration; OD: Optical density; MLKS2: Maize LINC KASH AtSINE-like2; LINC: Linker of nucleoskeleton and cytoskeleton; NE: Nuclear envelope; INM: Inner nuclear membrane; ONM: Outer nuclear membrane

Medicago LINC Complexes Function in Nuclear Morphology, Nuclear Movement, and Root Nodule Symbiosis

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.

A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly.

The linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

Sad1 Spatiotemporally Regulates Kinetochore Clustering To Ensure High-Fidelity Chromosome Segregation in the Human Fungal Pathogen Cryptococcus neoformans.

Kinetochore clustering, frequently observed in yeasts, plays a key role in genome organization and chromosome segregation. In the absence of the metaphase plate arrangement, kinetochore clustering in yeast species is believed to facilitate timely kinetochore-microtubule interactions to achieve bivalent attachments of chromosomes during metaphase. The factors determining the dynamics of kinetochore clustering remain largely unknown. We previously reported that kinetochores oscillate between an unclustered and a clustered state during the mitotic cell cycle in the basidiomycetous yeast Cryptococcus neoformans Based on tubulin localization patterns, while kinetochore clustering appears to be microtubule dependent, an indirect interaction of microtubules with kinetochores is expected in C.neoformans In this study, we sought to examine possible roles of the SUN-KASH protein complex, known to form a bridge across the nuclear envelope, in regulating kinetochore clustering in C.neoformans We show that the SUN domain protein Sad1 localizes close to kinetochores in interphase as well as in mitotic cells. Sad1 is nonessential for viability in C.neoformans but is required for proper growth and high-fidelity chromosome segregation. Further, we demonstrate that the onset of kinetochore clustering is significantly delayed in cells lacking Sad1 compared to wild-type cells. Taken together, this study identifies a novel role of the SUN domain protein Sad1 in spatiotemporal regulation of kinetochore clustering during the mitotic cell cycle in C.neoformansIMPORTANCE The linker of nucleoskeleton and cytoskeleton (LINC) complex is present in fungi, animals, and plants. It performs diverse functions in animals, and its role(s) have recently been explored in plants. In ascomycetous yeast species, the role of the LINC complex in spindle pole body function and telomere clustering during meiosis has been determined. However, nothing is known about the LINC complex in the fungal phylum of Basidiomycota. In this study, we identified the role of the LINC complex in kinetochore dynamics as well as in nuclear migration in a basidiomycetous yeast, Cryptococcus neoformans, a human pathogen. Unlike most other yeast species, kinetochores remain unclustered during interphase but gradually cluster during mitosis in C.neoformans We report that the LINC complex is required for timely onset of kinetochore clustering and high-fidelity chromosome segregation in C.neoformans Thus, our study identifies a novel factor required for kinetochore clustering during mitosis in yeast species.

LINC complex-Lis1 interplay controls MT1-MMP matrix digest-on-demand response for confined tumor cell migration

Cancer cells’ ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.

Differential incorporation of SUN-domain proteins into LINC complexes is coupled to gene expression

LInkers of Nucleoskeleton and Cytoskeleton (LINC) complexes, composed of SUN and KASH-domain proteins, span the nuclear envelope and physically connect the nuclear interior to cytoskeletal elements. Most human cells contain two SUN proteins, Sun1 and Sun2, and several KASH-proteins suggesting that multiple functionally distinct LINC complexes co-exist in the nuclear envelope. We show here, however, that while Sun1 and Sun2 in HeLa cells are each able to bind KASH-domains, Sun1 is more efficiently incorporated into LINC complexes under normal growth conditions. Furthermore, the balance of Sun1 and Sun2 incorporated into LINC complexes is cell type-specific and is correlated with SRF/Mkl1-dependent gene expression. In addition, we found that Sun1 has a LINC complex-independent role in transcriptional control, possibly by regulating the SRF/Mkl1 pathway. Together, these data reveal novel insights into the mechanisms of LINC complex regulation and demonstrate that Sun1 modulates gene expression independently of its incorporation into LINC complexes.

Ari-1 Regulates Myonuclear Organization Together with Parkin and Is Associated with Aortic Aneurysms

Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. We identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINCcomplex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. We show that Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, we discovered functional redundancy between Parkin and Ari-1: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. We further show that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, SMCs obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton.

Identifying the Axial Location of Proteins at the Nuclear Envelope with Nanometer Resolution

The nuclear envelope (NE) separates the cytoplasm and nucleus by a double-layered membrane consisting of the inner nuclear membrane (INM) and outer nuclear membrane (ONM). The INM and ONM are separated by tens of nanometers. Specific INM and ONM proteins form LINC (linker of nucleoskeleton and cytoskeleton) complexes, which span the nuclear envelope and mechanically couple the nucleoskeleton and cytoskeleton. It has been suggested that LINC complexes define the spacing of the INM and ONM, which might be modulated by a wide range of diseases associated with LINC complexes. To test this hypothesis in future work we are seeking to directly measure the distance between the INM and ONM in live cells. We axially scan the two-photon point spread function through a cell with a fluorescently-labeled protein residing on a nuclear membrane. The scan generates an intensity profile that reveals the spatial location of the proteins along the scan path. To measure the axial distance separating two protein systems we label each with a distinct color and perform a dual color z-scan, which simultaneously determines the location of each protein species. We experimentally verify that dual-color z-scan is capable of identifying the axial separation of membrane proteins with a few nanometer uncertainty using model systems. We also perform direct measurements of the distance between the INM and ONM on individual cells. In addition, we extend our studies to the translocation of transmembrane proteins from the ONM to the INM. This work has been supported by a grant from the National Institutes of Health (R01 GM064589).

Nuclear Envelope-Associated Chromosome Dynamics during Meiotic Prophase I.

Chromosome dynamics during meiotic prophase I are associated with a series of major events such as chromosomal reorganization and condensation, pairing/synapsis and recombination of the homologs, and chromosome movements at the nuclear envelope (NE). The NE is the barrier separating the nucleus from the cytoplasm and thus plays a central role in NE-associated chromosomal movements during meiosis. Previous studies have shown in various species that NE-linked chromosome dynamics are actually driven by the cytoskeleton. The linker of nucleoskeleton and cytoskeleton (LINC) complexes are important constituents of the NE that facilitate in the transfer of cytoskeletal forces across the NE to individual chromosomes. The LINCs consist of the inner and outer NE proteins Sad1/UNC-84 (SUN), and Klarsicht/Anc-1/Syne (KASH) domain proteins. Meiosis-specific adaptations of the LINC components and unique modifications of the NE are required during chromosomal movements. Nonetheless, the actual role of the NE in chromosomic dynamic movements in plants remains elusive. This review summarizes the findings of recent studies on meiosis-specific constituents and modifications of the NE and corresponding nucleoplasmic/cytoplasmic adaptors being involved in NE-associated movement of meiotic chromosomes, as well as describes the potential molecular network of transferring cytoplasm-derived forces into meiotic chromosomes in model organisms. It helps to gain a better understanding of the NE-associated meiotic chromosomal movements in plants.

Generation and Analysis of Striated Muscle Selective LINC Complex Protein Mutant Mice.

The linker of nucleoskeleton and cytoskeleton (LINC) complex mediates intracellular cross talk between the nucleus and the cytoplasm. In striated muscle, the LINC complex provides structural support to the myocyte nucleus and plays an essential role in regulating gene expression and mechanotransduction. A wide range of cardiac and skeletal myopathies have been linked to mutations in LINC complex proteins. Studies utilizing tissue-specific knockout and mutant mouse models have revealed important insights into the roles of the LINC complex in striated muscle. In this chapter, we describe several feasible approaches for generating striated muscle-specific gene knockout and mutant mouse models to study LINC complex protein function in cardiac and skeletal muscle. The experimental procedures used for phenotyping and analysis of LINC complex knockout mice are also described.

Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy.

Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins. Since the homo-oligomerization of SUN2 is critical for its ability to interact with nesprins within the perinuclear space, the knowledge obtained through quantitative brightness experiments reveals important insights into the in vivo mechanisms of LINC complex assembly. Here we describe the procedure we use to determine the brightness of proteins in the NE of living cells. In addition to the measurement procedure, we discuss the instrumentation requirements and present the results of applying this procedure to measure the brightness of nesprin-2 and SUN2.

Immunolabeling Protocols for Studying Meiosis in Plant Mutants Defective for Nuclear Envelope Components.

The nuclear envelope (NE) is a dynamic boundary that allows the communication between nuclear and cytoplasmic components. It has essential roles in a variety of physiological processes including cell division. The linker of nucleoskeleton and cytoskeleton (LINC) complexes span the NE and are important during meiosis, the specialized cell division needed for sexual reproduction. During this division, the LINC complex proteins AtSUN1 and AtSUN2, located in the inner nuclear membrane (INM), are involved in tethering telomeres to the NE. This attachment promotes chromosome movements by the forces that are generated in the cytoplasmic face. In Arabidopsis, the double mutant Atsun1 Atsun2 exhibits a delayed prophase I meiotic progression, partial synapsis, and recombination defects that lead to the formation of unbalanced gametes and sterility. In meiocytes from these mutants, immunolabeling can be applied to analyze possible changes in the dynamics of different meiotic proteins. In addition, if the specific antibodies are available, this technique is an easy and useful tool to determine the spatial distribution of NE proteins.

Dynamic Interaction Between Microtubules and the Nucleus Regulates Nuclear Movement During Neuronal Migration.

Fine structures of the mammalian brain are formed by neuronal migration during development. Newborn neurons migrate long distances from the germinal zone to individual sites of function by squeezing their largest cargo, the nucleus, through the crowded neural tissue. Nuclear translocation is thought to be orchestrated by microtubules, actin, and their associated motor proteins, dynein and myosin. However, where and how the cytoskeletal forces are converted to actual nuclear movement remains unclear. Using high-resolution confocal imaging of live migrating neurons, we demonstrated that microtubule-dependent forces are directly applied to the nucleus via the linker of nucleoskeleton and cytoskeleton complex, and that they induce dynamic nuclear movement, including translocation, rotation, and local peaking. Microtubules bind to small points on the nuclear envelope via the minus- and plus-oriented motor proteins, dynein and kinesin-1, and generate a point force independent of the actin-dependent force. Dynamic binding of microtubule motors might cause a continuously changing net force vector acting on the nucleus and results in a stochastic and inconsistent movement of the nucleus, which are seen in crowded neural tissues.

Detection of SUN1 Splicing Variants at the mRNA and Protein Levels in Cancer.

The linker of nucleoskeleton and cytoskeleton (LINC) complex, containing the proteins SUN and nesprin, is the fundamental structural unit of the nuclear envelope. The neoplastic-based regulation of the LINC complex in cancer tissues has become increasingly recognized in recent years, including the altered expression, somatic mutation, and methylation of genes. However, precisely how mutations and deregulated expression of the LINC complex contribute to the pathogenic mechanisms of tumorigenesis remain to be elucidated, mainly because of several technical difficulties. First, both the SUN and SYNE (encoding nesprin) genes give rise to a vast number of splicing variants. Second, immunoprecipitation experiments of endogenous SUN and nesprin proteins are difficult owing to the lack of suitable reagents as well as the limited solubility of these proteins in mild extraction conditions. Here, we describe three protocols to investigate these aspects: (1) immunohistochemistry to determine the expression levels and localization of the LINC complex in cancer tissue, (2) detection of SUN1 splicing variants at the mRNA level, and (3) detection of SUN1 splicing variants and binding partners at the protein level.

Luma is not essential for murine cardiac development and function.

Luma is a recently discovered, evolutionarily conserved protein expressed in mammalian heart, which is associated with the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex structurally integrates the nucleus and the cytoplasm and plays a critical role in mechanotransduction across the nuclear envelope. Mutations in several LINC components in both humans and mice result in various cardiomyopathies, implying they play essential, non-redundant roles. A single amino acid substitution of serine 358 to leucine (S358L) in Luma is the unequivocal cause of a distinct form of arrhythmogenic cardiomyopathy. However, the role of Luma in heart has remained obscure. In addition, it also remains to be determined how the S358L mutation in Luma leads to cardiomyopathy. To determine the role of Luma in the heart, we first determined the expression pattern of Luma in mouse heart. Luma was sporadically expressed in cardiomyocytes throughout the heart, but was highly and uniformly expressed in cardiac fibroblasts and vascular smooth muscle cells. We also generated germline null Luma mice and discovered that germline null mutants were viable and exhibited normal cardiac function. Luma null mice also responded normally to pressure overload induced by transverse aortic constriction. In addition, localization and expression of other LINC complex components in both cardiac myocytes and fibroblasts was unaffected by global loss of Luma. Furthermore, we also generated and characterized Luma S358L knock-in mice, which displayed normal cardiac function and morphology. Our data suggest that Luma is dispensable for murine cardiac development and function and that the Luma S358L mutation alone may not cause cardiomyopathy in mice.

Centrifugal Displacement of Nuclei Reveals Multiple LINC Complex Mechanisms for Homeostatic Nuclear Positioning

Nuclear movement is critical for developmental events, cell polarity, and migration and is usually mediated by linker of nucleoskeleton and cytoskeleton (LINC) complexes connecting the nucleus to cytoskeletal elements. Compared to active nuclear movement, relatively little is known about homeostatic positioning of nuclei, including whether it is an active process. To explore homeostatic nuclear positioning, we developed a method to displace nuclei in adherent cells using centrifugal force. Nuclei displaced by centrifugation rapidly recentered by mechanisms that depended on cell context. In cellmonolayers with wounds oriented orthogonal to the force, nuclei were displaced toward the frontand back of the cells on the two sides of the wound. Nuclei recentered from both positions, but at different rates and with different cytoskeletal linkage mechanisms. Rearward recentering was actomyosin, nesprin-2G, and SUN2 dependent, whereas forward recentering was microtubule, dynein, nesprin-2G, and SUN1 dependent. Nesprin-2G engaged actin through its N terminus and microtubules through a novel dynein interacting site near its C terminus. Both activities were necessary to maintain nuclear position in uncentrifuged cells. Thus, even when not moving, nuclei are actively maintained in position by engaging the cytoskeleton through the LINC complex.

The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation

SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture.

Study of the role of plant nuclear envelope and lamina-like components in nuclear and chromatin organisation using 3D imaging

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 supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The first aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphology and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with different nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromocentres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC complex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes advantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional nuclear envelope. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes.