Dr Katja Graumann

Abstract: 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.

The Nuclear Envelope is a hallmark of eukaryotic cells. Despite its emerging role as a key structural and signaling platform, the plant NE remains one of the least understood membrane systems. This Frontiers Research Topic aims to highlight recent advances made to examine the role of the nuclear envelope (NE) as the “key border” in plants. Exploring this border is now proving hugely rewarding, with implications for many aspects of cell function and plant responses to the environment. Better understanding of this membrane system involves understanding its physical connections and its signaling and transport functions. Together they underpin its many functions- as an anchor and shaper; as a transport hub; and as a signaling center. The papers in this Topic provide current knowledge on all these aspects and a valuable basis for further advances.

For a century, the nucleus has been the focus of extensive investigations in cell biology. However, many questions remain about how its shape and size are regulated during development, in different tissues or during disease and aging. To track these changes, microscopy has long been the tool of choice. Image analysis has revolutionized this field of research by providing computational tools, translating qualitative images into quantitative parameters. Many tools were designed to delimit objects in 2D and eventually in 3D, to define their shapes, their number or position in nuclear space. Today, the field is driven by deep-learning methods, most taking advantage of convolutional neural networks. These techniques are remarkably adapted to biomedical images when trained on large datasets and powerful computer graphics cards. To promote these innovative and promising methods to cell biologists, this Review summarizes the main concepts and terminologies of deep learning. Special emphasis is placed on their availability. We highlight why quality and characteristics of training image datasets are important and where to find them, as well as how to create, store and share image datasets. Finally, we describe deep-learning methods well-suited for 3D analysis of nuclei and classify them according to their level of usability for biologists. Out of more than 150 published methods, we identify less than twelve that a biologist can use and explain why. Based on this experience, we propose best practices to share deep learning methods with biologists.

The composition of the plant nuclear lamina has long been of interest; while its overall network-like structure is similar to that of other eukaryotes, homologues of the key building blocks—lamins—are not present in plants. Although plant-specific lamin-like proteins have been characterized, it has remained unknown whether these are indeed present in the plant lamina. Using superresolution microscopy, Masuda et  al. (2021) have now solved this puzzle. They show beautifully how the wellstudied Nuclear Matrix Constituent Protein 1 (NMCP1) and NMCP2 form filaments and bundles and are present in a filamentous network at the nuclear periphery—the plant nuclear lamina.

In eukaryotes, the nuclear envelope (NE) encloses chromatin and separates it from the rest of the cell. The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex physically bridges across the NE, linking nuclear and cytoplasmic components. In plants, these LINC complexes are beginning to be ascribed roles in cellular and nuclear functions, including chromatin organization, regulation of nuclei shape and movement, and cell division. Homologs of core LINC components, KASH and SUN proteins, have previously been identified in maize. Here, we characterized the presumed LINC-associated maize nucleoskeletal proteins NCH1 and NCH2, homologous to members of the plant NMCP/CRWN family, and MKAKU41, homologous to AtKAKU4. All three proteins localized to the nuclear periphery when transiently and heterologously expressed as fluorescent protein fusions in Nicotiana benthamiana. Overexpression of MKAKU41 caused dramatic changes in the organization of the nuclear periphery, including nuclear invaginations that stained positive for non-nucleoplasmic markers of the inner and outer NE membranes, and the ER. The severity of these invaginations was altered by changes in LINC connections and the actin cytoskeleton. In maize, MKAKU41 appeared to share genetic functions with other LINC components, including control of nuclei shape, stomatal complex development, and pollen viability. Overall, our data show that NCH1, NCH2, and MKAKU41 have characteristic properties of LINC-associated plant nucleoskeletal proteins, including interactions with NE components suggestive of functions at the nuclear periphery that impact the overall nuclear architecture.

Identifying protein components of the nuclear envelope is a slow and challenging process. Now a proximity labelling technique adapted for plants reveals novel protein components in this under-researched membrane.

The eukaryotic cell nucleus is a central organelle whose architecture determines genome function at multiple levels. Deciphering nuclear organizing principles influencing cellular responses and identity is a timely challenge. Despite many similarities between plant and animal nuclei, plant nuclei present intriguing specificities. Complementary to molecular and biochemical approaches, 3D microscopy is indispensable for resolving nuclear architecture. However, novel solutions are required for capturing cell-specific, sub-nuclear and dynamic processes. We provide a pointer for utilising high-to-super-resolution microscopy and image processing to probe plant nuclear architecture in 3D at the best possible spatial and temporal resolution and at quantitative and cell-specific levels. High-end imaging and image-processing solutions allow the community now to transcend conventional practices and benefit from continuously improving approaches. These promise to deliver a comprehensive, 3D view of plant nuclear architecture and to capture spatial dynamics of the nuclear compartment in relation to cellular states and responses. 

The 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.

Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism is transport-factor mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore if this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the NE of leaf epidermal cells. Airyscan sub-diffraction limited confocal microscopy showed that it displays localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment and both monopartite or bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery.

Mitosis and meiosis in higher plants involves significant reconfiguration of the nuclear envelope and the proteins that interact with it. The dynamic series of events involves a range of interactions, movement, breakdown and reformation of this complex system. Recently, progress has been made in identifying and characterising the protein and membrane interactome that performs these complex tasks, including constituents of the nuclear envelope, the cytoskeleton, nucleoskeleton and chromatin. This review will present current understanding of these interactions and advances in knowledge of the processes for the breakdown and reformation of the nuclear envelope during cell divisions in plants. 

The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture, and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. Using bioinformatic, biochemical, and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with ZmSUN2 in vivo. FRAP experiments using domain-deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research. 

plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins. Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesised to be associated with it including Crowded Nuclei (CRWN; also termed LINC and NMCP), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. SUN domain proteins appear throughout with a key role for mid-SUN proteins suggested. Evolution of KASH domain proteins has resulted in increasing complexity, with some appearing in all species considered, while other KASH proteins are progressively gained during evolution. Failure to identify CRWN homologs in unicellular organisms included in the study and their presence in plants leads us to speculate that convergent evolution may have occurred in the formation of the lamina with each kingdom having new proteins such as the Lamin B receptor (LBR) and Lamin-Emerin-Man1 (LEM) domain proteins (animals) or NEAPs and KAKU4 (plants). Our data support a model in which increasing complexity at the nuclear envelope occurred through the plant lineage and suggest a key role for mid-SUN proteins as an early and essential component of the nuclear envelope. 

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localise at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants.

The nuclear envelope (NE) is a double membrane system enclosing the genome of eukaryotes. Besides nuclear pore proteins, which form channels at the NE, nuclear membranes are populated by a collection of NE proteins that perform various cellular functions. However, in contrast to well-conserved nuclear pore proteins, known NE proteins share little homology between opisthokonts and plants. Recent studies on NE protein complexes formed by Sad1/UNC-84 (SUN) and Klarsicht/ANC-1/Syne-1 Homology (KASH) proteins have advanced our understanding of plant NE proteins and revealed their function in anchoring other proteins at the NE, nuclear shape determination, nuclear positioning, anti-pathogen defence, root development, and meiotic chromosome organization. In this review, we discuss the current understanding of plant SUN, KASH, and other related NE proteins, and compare their function with the opisthokont counterparts.

The movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC-84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force-generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified.
Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1-1 Atsun2-2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock-like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.

Following the description of SAD1/UNC84 (SUN) domain proteins in higher plants, evidence has rapidly increased that plants contain a functional linker of nucleoskeleton and cytoskeleton (LINC) complex bridging the nuclear envelope (NE). While the SUN domain proteins appear to be highly conserved across kingdoms, other elements of the complex are not and some key components and interactions remain to be identified. This mini review examines components of the LINC complex, including proteins of the SUN domain family and recently identified plant Klarsicht/Anc/Syne-1 homology (KASH) domain proteins. First of these to be described were WIPs (WPP domain interacting proteins), which act as protein anchors in the outer NE. The plant KASH homologs are C-terminally anchored membrane proteins with the extreme C-terminus located in the nuclear periplasm; AtWIPs contain a highly conserved X-VPT motif at the C-terminus in contrast to PPPX in opisthokonts. The role of the LINC complex in organisms with a cell wall, and description of further LINC complex components will be considered, together with other potential plant-specific functions.

SUN-domain proteins belong to a gene family including classical Cter-SUN and mid-SUN subfamilies differentiated by the position of the SUN domain within the protein. Although present in animal and plant species, mid-SUN proteins have so far remained poorly described. Here, we used a combination of genetics, yeast two-hybrid and in planta transient expression methods to better characterize the SUN family in Arabidopsis thaliana. First, we validated the mid-SUN protein subfamily as a monophyletic group conserved from yeast to plant. Arabidopsis Cter-SUN (AtSUN1 and AtSUN2) and mid-SUN (AtSUN3 and AtSUN4) proteins expressed as fluorescent protein fusions are membrane-associated and localize to the nuclear envelope (NE) and endoplasmic reticulum. However, only the Cter-SUN subfamily is enriched at the NE. We investigated interactions in and between members of the two subfamilies and identified the coiled-coil domain as necessary for mediating interactions. The functional significance of the mid-SUN subfamily was further confirmed in mutant plants as essential for early seed development and involved in nuclear morphology. Finally, we demonstrated that both subfamilies interact with the KASH domain of AtWIP1 and identified a new root-specific KASH-domain protein, AtTIK. AtTIK localizes to the NE and affects nuclear morphology. Our study indicates that Arabidopsis Cter-SUN and mid-SUN proteins are involved in a complex protein network at the nuclear membranes, reminiscent of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex found in other kingdoms.

Sad1/UNC84 (SUN) domain proteins are a highly conserved family of inner nuclear membrane localised proteins in eukaryotes. One of their main functions is as key components of nucleo-cytoskeletal bridging complexes, in which SUN proteins associate with nucleoskeletal elements. In metazoans these are the lamins, which form a supportive structural network termed the lamina. Plants lack sequence homologs of lamins but have a similar nucleoplasmic structural network to support the plant NE. Putative components of this plant lamina-like structure are Little Nuclei (LINC) proteins, which bear structural resemblance to lamins and fulfil similar functions. This work explores the associations between AtLINC1, AtSUN1 and AtSUN2. AtLINC1 is recruited to the NE by SUN proteins and is immobilised therein. This recruitment and the immobile properties are likely due to AtSUN1/2-AtLINC1 protein interactions occurring in planta. In addition, the SUN N-terminus appears to play an important role in mediating these interactions. The associations between AtLINC1 and plant SUN proteins are a first indicator of how the nucleoskeleton may be anchored to the nuclear membrane in plants. Building on the previous characterisation of Klarsicht/Anc1/Syne1 homology (KASH) like proteins in plants, this study advances the identification and characterisation of nucleo-cytoskeletal bridging complexes in plants.

Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain–interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions.

The Company of Biologists Workshop entitled 'Mitosis and Nuclear Structure' was held at Wiston House, West Sussex in June 2013. It provided a unique and timely opportunity for leading experts from different fields to discuss not only their own work but also its broader context. Here we present the proceedings of this meeting and several major themes that emerged from the crosstalk between the two, as it turns out, not so disparate fields of mitosis and nuclear structure. Co-chaired by Katherine Wilson (Johns Hopkins School of Medicine, Baltimore, MD), Timothy Mitchison (Harvard University, Cambridge, MA) and Michael Rout (Rockefeller University, New York, NY), this workshop brought together a small group of scientists from a range of disciplines to discuss recent advances and connections between the areas of mitosis and nuclear structure research. Several early-career researchers (students, postdoctoral researchers, junior faculty) participated along with 20 senior scientists, including the venerable and affable Nobel Laureate Tim Hunt. Participants were encouraged to embrace unconventional thinking in the 'scientific sandbox' created by this unusual combination of researchers in the inspiring, isolated setting of the 16th-century Wiston House.

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.

The nuclear periphery is a dynamic, structured environment, whose precise functions are essential for global processes-from nuclear, to cellular, to organismal. Its main components-the nuclear envelope (NE) with inner and outer nuclear membranes (INM and ONM), nuclear pore complexes (NPC), associated cytoskeletal and nucleoskeletal components as well as chromatin are conserved across eukaryotes (Fig. 1). In metazoans in particular, the structure and functions of nuclear periphery components are intensely researched partly because of their involvement in various human diseases. While far less is known about these in plants, the last few years have seen a significant increase in research activity in this area. Plant biologists are not only catching up with the animal field, but recent findings are pushing our advances in this field globally. In recognition of this developing field, the Annual Society of Experimental Biology Meeting in Salzburg kindly hosted a session co-organized by Katja Graumann and David E. Evans (Oxford Brookes University) highlighting new insights into plant nuclear envelope proteins and their interactions. This session brought together leading researchers with expertise in topics such as epigenetics, meiosis, nuclear pore structure and functions, nucleoskeleton and nuclear envelope composition. An open and friendly exchange of ideas was fundamental to the success of the meeting, which resulted in founding the International Plant Nucleus Consortium. This review highlights new developments in plant nuclear envelope research presented at the conference and their importance for the wider understanding of metazoan, yeast and plant nuclear envelope functions and properties.

Inner nuclear membrane Sad1/UNC-84 (SUN) proteins interact with outer nuclear membrane (ONM) Klarsicht/ANC-1/Syne homology (KASH) proteins, forming linkers of nucleoskeleton to cytoskeleton conserved from yeast to human and involved in positioning of nuclei and chromosomes. Defects in SUN-KASH bridges are linked to muscular dystrophy, progeria, and cancer. SUN proteins were recently identified in plants, but their ONM KASH partners are unknown. Arabidopsis WPP domain interacting proteins (AtWIPs) are plant-specific ONM proteins that redundantly anchor Arabidopsis RanGTPase-activating protein 1 (AtRanGAP1) to the nuclear envelope (NE). In this paper, we report that AtWIPs are plant-specific KASH proteins interacting with Arabidopsis SUN proteins (AtSUNs). The interaction is required for both AtWIP1 and AtRanGAP1 NE localization. AtWIPs and AtSUNs are necessary for maintaining the elongated nuclear shape of Arabidopsis epidermal cells. Together, our data identify the first KASH members in the plant kingdom and provide a novel function of SUN-KASH complexes, suggesting that a functionally diverged SUN-KASH bridge is conserved beyond the opisthokonts.

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)-GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.

Scope. Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events.

Conclusion. Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.

Sad1/UNC-84 (SUN)-domain proteins are inner nuclear membrane (INM) proteins that are part of bridging complexes linking cytoskeletal elements with the nucleoskeleton, and have been shown to be conserved in non-plant systems. In this paper, we report the presence of members of this family in the plant kingdom, and investigate the two Arabidopsis SUN-domain proteins, AtSUN1 and AtSUN2. Our results indicate they contain the highly conserved C-terminal SUN domain, and share similar structural features with animal and fungal SUN-domain proteins including a functional coiled-coil domain and nuclear localization signal. Both are expressed in various tissues with AtSUN2 expression levels relatively low but upregulated in proliferating tissues. Further, we found AtSUN1 and AtSUN2 expressed as fluorescent protein fusions, to localize to and show low mobility in the nuclear envelope (NE), particularly in the INM. Deletion of various functional domains including the N terminus and coiled-coil domain affect the localization and increase the mobility of AtSUN1 and AtSUN2. Finally, we present evidence that AtSUN1 and AtSUN2 are present as homomers and heteromers in vivo, and that the coiled-coil domains are required for this. The study provides evidence suggesting the existence of cytoskeletal-nucleoskeletal bridging complexes at the plant NE.

Controlled movement Of the nucleus is important in a wide variety of plant cellular events Positioning involving intact nuclei occurs in cell division, development, tip growing systems such as the root hair and in response to stimuli, including light, touch and infection. Positioning is also essential in the division and replication of nuclear components, ranging from chromosome attachment to the breakdown and reformation of the nuclear envelope. Although description and understanding of the processes involved have advanced rapidly in recent years, significant gaps remain in our knowledge, especially concerning nuclear proteins involved in anchoring and interacting with cytoskeletal and nucleoskeletal elements involved in movement. In the present review, processes involving the movement and positioning of nuclei and nuclear components are described together with novel proteins implicated in nucleoskeletal and cytoskeletal interactions.

A GFP fusion to the N-terminal 238 amino acids of the mammalian lamin B receptor (LBR) localises to the nuclear envelope (NE) when expressed in Nicotiana tabacum plants, showing properties expected of a native plant NE protein. In this study, we have used this chimaeric construct to explore evidence for common mechanisms of NE targeting and retention between plants and animals, given there is no plant homologue of the mammalian LBR or of one of its binding partners, lamin B. Binding mutants of LBR-GFP were created and fluorescence recovery after photobleaching of mutant and wild type constructs employed to examine their retention in the plant NE. Unmutated LBR-GFP was significantly less mobile in the NE than the lamin binding domain deletion mutant, which was also localised to theER and punctate structures in some cells. Mutation of the chromatin binding domain resulted in localisation of the protein in nuclear inclusions, in which it was immobile. Our findings, that expression of truncated LBR-GFP in plant cells results in altered targeting and retention relative to wt LBR-GFP, suggest that plant cells can recognize the INM-targeting motif of LBR. Altered mobility of the truncated probe indicates that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.

The nuclear envelope (NE) is a double membrane system consisting of the inner nuclear envelope (INE), the outer nuclear envelope (ONE) and nuclear pore complexes (NPCs). Most of our knowledge about the NE proteome comes from studies in animal systems. Recent investigations in plant systems have shown that plants do not have homologues for the majority of animal NE proteins. In a previous study in our laboratory, a construct consisting of the N-terminus of the human lamin B receptor (LBR) fused to GFP was shown to target the plant INE. In mammalian cells, LBR is an intrinsic INE protein, whose targeting to the INE is facilitated by a nuclear localization signal and retention in the INE is achieved by LBR binding mainly to chromatin and lamins. In this study the targeting and retention of LBR–GFP in the plant NE has been investigated by introducing mutations in key domains of LBR and employing fluorescence recovery after photobleaching experiments. Mutation of the chromatin binding domain caused LBR to accumulate in nuclear inclusions in which it was immobile. Deletion of the lamin binding domain resulted in the construct being localized not only to the NE but also ER and to be significantly more mobile then the wild type LBR–GFP in the NE. In the case of both the lamin binding deletion and wild type LBR–GFP, mobility was found to be much greater than previously described in mammalian cells. (Abstracts of the Annual Main Meeting of the Society for Experimental Biology, Glasgow, Scotland, 31st March - 4th April, 2007)