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The functional organization of the plant nuclear envelope is gaining increasing attention through new connections made between nuclear envelope-associated proteins and important plant biological processes. Animal nuclear envelope proteins play roles in nuclear morphology, nuclear anchoring and movement, chromatin tethering and mechanical signaling. However, how these roles translate to functionality in a broader biological context is often not well understood. A surprising number of plant nuclear envelope-associated proteins are plant-unique, suggesting that separate functionalities evolved after the split of Opisthokonta and Streptophyta. Significant progress has now been made in discovering broader biological roles of plant nuclear envelope proteins, increasing the number of known plant nuclear envelope proteins, and connecting known proteins to chromatin organization, gene expression, and the regulation of nuclear calcium. The interaction of viruses with the plant nuclear envelope is another emerging theme. Here, we survey the recent developments in this still relatively new, yet rapidly advancing field.

Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of wit12 and wifi, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ peaks in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.

In this review, we explore recent advances in knowledge of the structure and dynamics of the plant nuclear envelope. As a paradigm, we focused our attention on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, a structurally conserved bridging complex comprising SUN domain proteins in the inner nuclear membrane and KASH domain proteins in the outer nuclear membrane. Studies have revealed that this bridging complex has multiple functions with structural roles in positioning the nucleus within the cell, conveying signals across the membrane and organizing chromatin in the 3D nuclear space with impact on gene transcription. We also provide an up-to-date survey in nuclear dynamics research achieved so far in the model plant Arabidopsis thaliana that highlights its potential impact on several key plant functions such as growth, seed maturation and germination, reproduction and response to biotic and abiotic stress. Finally, we bring evidences that most of the constituents of the LINC Complex and associated components are, with some specificities, conserved in monocot and dicot crop species and are displaying very similar functions to those described for Arabidopsis. This leads us to suggest that a better knowledge of this system and a better account of its potential applications will in the future enhance the resilience and productivity of crop plants.

In S. cerevisiae prophase meiotic chromosomes move by forces generated in the cytoplasm and transduced to the telomere via a protein complex located in the nuclear membrane. We know that chromosome movements require actin cytoskeleton [13,31] and the proteins Ndj1, Mps3, and Csm4. Until recently, the identity of the protein connecting Ndj1-Mps3 with the cytoskeleton components was missing. It was also not known the identity of a cytoplasmic motor responsible for interacting with the actin cytoskeleton and a protein at the outer nuclear envelope. Our recent work [36] identified Mps2 as the protein connecting Ndj1-Mps3 with cytoskeleton components; Myo2 as the cytoplasmic motor that interacts with Mps2; and Cms4 as a regulator of Mps2 and Myo2 interaction and activities (Figure 1). Below we present a model for how Mps2, Csm4, and Myo2 promote chromosome movements by providing the primary connections joining telomeres to the actin cytoskeleton through the LINC complex. Fil: Lee, C. Y.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Bisig, Carlos Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina Fil: Conrad, M. N.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Ditamo, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina Fil: Almeida, L. Previato de. Oklahoma Medical Research Foundation; Estados Unidos Fil: Dresser, M.E.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Pezza, Roberto. Oklahoma Medical Research Foundation; Estados Unidos

During meiosis, homologous chromosomes undergo a dramatic movement in order to correctly align. This is a critical meiotic event but the molecular properties of this ‘chromosomal dance’ still remainunclear. We identified DEB-1 – an orthologue of mammalian vinculin – as a new component of the mechanistic modules responsible for attaching the chromosomes to the nuclear envelope as apart of the LINC complex. In early meiotic nuclei of C. elegans, DEB-1 is localized to the nuclear periphery and alongside the synaptonemal complex of paired homologues. Upon DEB-1 depletion, chromosomes attached to SUN-1 foci remain highly motile until late pachytene. Although the initiation of homologue pairing started normally, irregularities in the formation of the synaptonemal complex occur, and these results in meiotic defects such as increased number of univalents at diakinesis and high embryonic lethality. Our data identify DEB-1 as a new player regulating chromosome dynamics and pairing during meiotic prophase I.

Exogenous mechanical forces are transmitted through the cell and to the nucleus, initiating mechanotransductive signaling cascades with profound effects on cellular function and stem cell fate. A growing body of evidence has shown that the force sensing and force-responsive elements of the nucleus adapt to these mechanotransductive events, tuning their response to future mechanical input. The mechanisms underlying this “mechano-adaptation” are only just beginning to be elucidated, and it remains poorly understood how these components act and adapt in tandem to drive stem cell differentiation. Here, we review the evidence on how the stem cell nucleus responds and adapts to physical forces, and provide a perspective on how this mechano-adaptation may function to drive and enforce stem cell differentiation.

Nuclear architecture, a function of both chromatin and nucleoskeleton structure, is known to change with stem cell differentiation and differs between various somatic cell types. These changes in nuclear architecture are associated with the regulation of gene expression and genome function in a cell-type specific manner. Biophysical stimuli are known effectors of differentiation and also elicit stimuli-specific changes in nuclear architecture. This occurs via the process of mechanotransduction whereby extracellular mechanical forces activate several well characterized signaling cascades of cytoplasmic origin, and potentially some recently elucidated signaling cascades originating in the nucleus. Recent work has demonstrated changes in nuclear mechanics both with pluripotency state in embryonic stem cells, and with differentiation progression in adult mesenchymal stem cells. This review explores the interplay between cytoplasmic and nuclear mechanosensitivity, highlighting a role for the nucleus as a rheostat in tuning the cellular mechano-response.

The last decade has seen rapid advances in our understanding of the proteins of the nuclear envelope, which have multiple roles including positioning the nucleus, maintaining its structural organization, and in events ranging from mitosis and meiosis to chromatin positioning and gene expression. Diverse new and stimulating results relating to nuclear organization and genome function from across kingdoms were presented in a session stream entitled “Dynamic Organization of the Nucleus” at this year's Society of Experimental Biology (SEB) meeting in Brighton, UK (July 2016). This was the first session stream run by the Nuclear Dynamics Special Interest Group, which was organized by David Evans, Katja Graumann (both Oxford Brookes University, UK) and Iris Meier (Ohio State University, USA). The session featured presentations on areas relating to nuclear organization across kingdoms including the nuclear envelope, chromatin organization, and genome function.

A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells.

Nuclear motions like rotation, translation and deformation suggest that the nucleus is acted upon by mechanical forces. Molecular linkages with the cytoskeleton are thought to transfer forces to the nuclear surface. We developed an approach to apply reproducible, known mechanical forces to the nucleus in spread adherent cells and quantified the elastic response of the mechanically integrated nucleus-cell. The method is sensitive to molecular perturbations and revealed new insight into the function of the LINC complex. While these experiments revealed elastic behaviors, turnover of the cytoskeleton by assembly/disassembly and binding/unbinding of linkages are expected to dissipate any stored mechanical energy in the nucleus or the cytoskeleton. Experiments investigating nuclear forces over longer time scales demonstrated the mechanical principle that expansive/compressive stresses on the nuclear surface arise from the movement of the cell boundaries to shape and position the nucleus. Such forces can shape the nucleus to conform to cell shapes during cell movements with or without myosin activity.

Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis.

© Natalie E Cain and Daniel A Starr. The nuclear envelope consists of 2 membranes separated by 30–50 nm, but how the 2 membranes are evenly spaced has been an open question in the field. Nuclear envelope bridges composed of inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins have been proposed to set and regulate nuclear envelope spacing. We tested this hypothesis directly by examining nuclear envelope spacing in Caenorhabditis elegans animals lacking UNC-84, the sole somatic SUN protein. SUN/KASH bridges are not required to maintain even nuclear envelope spacing in most tissues. However, UNC-84 is required for even spacing in body wall muscle nuclei. Shortening UNC-84 by 300 amino acids did not narrow the nuclear envelope space. While SUN proteins may play a role in maintaining nuclear envelope spacing in cells experiencing forces, our data suggest they are dispensable in most cells.

Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals. Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation.

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

Pairing of homologous chromosome is a unique event in meiosis that is essential for both haploidization of the genome and genetic recombination. Rapid chromosome movements during meiotic prophase are a key feature of the pairing process. This is usually telomere-led, and in metazoans is dependent upon microtubules and dynein. Chromosome movements culminate in the formation of a meiotic "bouquet" in which nuclear envelope-associated telomeres are clustered at the centrosomal pole of the nucleus. Bouquet formation is thought to facilitate homolog pairing. Recent studies reveal that coupling of telomeres to cytoplasmic dynein is mediated by SUN1 in the inner nuclear membrane (INM) and KASH5 a novel protein of the outer nuclear membrane (ONM). Together SUN1 and KASH5 assemble to form a transluminal LINC (linker of the nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes. SUN1 forms attachment sites for telomeres at the INM while KASH5 functions as a dynein adaptor at the ONM. In mice deficient in KASH5, homologous chromosome pairing does not occur. The result is that meiosis is arrested at the leptotene/zygotene stage of meiotic prophase 1, and as a consequence both male and female mice are infertile. This study demonstrates an essential role for dynein directed telomere movement during meiotic prophase.

Linkers of the nucleoskeleton to the cytoskeleton (LINC) complexes formed by SUN and KASH proteins are conserved eukaryotic protein complexes that bridge the nuclear envelope (NE) via protein-protein interactions in the NE lumen. Revealed by opisthokont studies, LINC complexes are key players in multiple cellular processes, such as nuclear and chromosomal positioning and nuclear shape determination, which in turn influence the generation of gametes and several aspects of development. Although comparable processes have long been known in plants, the first plant nuclear envelope bridging complexes were only recently identified. WPP domain-interacting proteins at the outer NE have little homology to known opisthokont KASH proteins, but form complexes with SUN proteins at the inner NE that have plant-specific properties and functions. In this review, we will address the importance of LINC complex-regulated processes, describe the plant NE bridging complexes and compare them to opisthokont LINC complexes.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell.

Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

Nuclear position is actively controlled and can be adjusted according to the needs of a cell by nuclear movement. Microtubules mediate the majority of nuclear movements studied to date, although examples of nuclear movements mediated by the actin cytoskeleton have been described. One such actin-dependent nuclear movement occurs during centrosome orientation in fibroblasts polarizing for migration. Here, the centrosome is maintained at the cell center while the nucleus is moved to the cell rear by actin retrograde flow thus positioning the centrosome between the nucleus and the leading edge of the cell. We have explored the molecular mechanism for actin dependent movement of the nucleus during centrosome centration. We found that a novel linear array of nuclear envelope membrane proteins composed of nesprin-2G and SUN2, called transmembrane actin-associated nuclear (TAN) lines, couple the nucleus to moving actin cables resulting in the nucleus being positioned toward the cell rear. TAN lines are anchored by A-type lamins and this allows the forces generated by the actin cytoskeleton to be transmitted across the nuclear envelope to move the nucleus. Here we review the data supporting this mechanism for nuclear movement, discuss questions remaining to be addressed and consider how this new mechanism of nuclear movement may shed light on human disease.

Active meiotic chromosome movements are a universally conserved feature. They occur at the early stages of prophase of the first meiotic division and support the chromosome pairing process by (1) efficiently installing the synaptonemal complex between homologous chromosomes, (2) discouraging inadvertent chromosome interactions and (3) bringing homologous chromosomes into proximity. Chromosome movements are driven by forces in the cytoplasm, which are passed on to chromosome ends attached to the nuclear periphery by nuclear-membrane-spanning protein modules. In this extra view, we highlight our recent studies into the role of the nuclear lamina during this process to emphasize that it is a highly conserved structure in metazoans. The nuclear lamina forms a rigid proteinaceous network that underlies the inner nuclear membrane to provide stability to the nucleus. Misdemeanors of the nuclear lamina during meiosis has deleterious consequences for the viability and health of the offspring, highlighting the importance of a functional nuclear lamina during this cell cycle stage. Abbreviations: DSB: DNA double strand break; LEM: LAP2, Emerin, MAN1; LINC: LInker of the Nucleoskeleton and Cytoskeleton; RPM: rapid prophase movement; SUN/KASH: Sad1p, UNC-84/Klarsicht, ANC-1, Syne Homology.

The regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a...

Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and duri ...