RESUMEN
A distinguishing feature of eukaryotes is the presence of a nuclear envelope (NE) and endomembrane system. The NE is a double-membrane system that surrounds chromatin and is continuous with the endoplasmic reticulum (ER). This interface is crucial in various processes such as calcium signaling and ER-associated degradation. The outer nuclear membrane and ER share a multitude of proteins although some are only functional in one domain, whereas the inner nuclear membrane has its own unique proteome. Until recently, it was not possible to distinguish between the inner and outer nuclear membranes as well as perinuclear ER using light microscopy - only electron microscopy was suitable for this. Now, however, using super-resolution live cell imaging, this can be achieved while still observing protein and membrane dynamics in real time. The protocols described here will allow researchers to determine subcellular localization of potential NE/ER proteins in live plant cells, helping to gain new insights into protein functionality.
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Microscopía , Membrana Nuclear , Señalización del Calcio , Cromatina , Retículo Endoplásmico , Proteínas de la MembranaRESUMEN
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.
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The plant nucleus and the actin cytoskeleton are intimately connected. The actin cytoskeleton is pivotal for nuclear positioning, shape, and dynamics. These properties of the nucleus are important for its functions during normal development and in response to external cues such as biotic and abiotic stresses. Moreover, we know that there is a direct physical connection between the actin cytoskeleton and the nucleus which spans the double-membraned nuclear envelope into the nuclear lamina, and this connection is called the linker of nucleoskeleton and cytoskeleton (LINC) complex. Recently a role for actin in regulating inter-nuclear organization via the control of nuclear invaginations has emerged. Therefore, a detailed understanding of nuclear shape, organization, and dynamics and the techniques used to measure and quantify these metrics will allow us to determine and further understand the contribution made by actin to these parameters. The protocols described here will allow researchers to determine the circularity index of a nucleus, quantify nuclear deformations, and determine dynamics of nuclei within plant cells.
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Actinas , Proteínas Nucleares , Núcleo Celular , Membrana Nuclear , Citoesqueleto , Matriz NuclearRESUMEN
For the past 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 that can be used to translate qualitative images into quantitative parameters. Many tools have been designed to delimit objects in 2D and, eventually, in 3D in order to define their shapes, their number or their position in nuclear space. Today, the field is driven by deep-learning methods, most of which take advantage of convolutional neural networks. These techniques are remarkably adapted to biomedical images when trained using 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 the availability of these methods. We highlight why the 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 fewer than 12 that biologists can use, and we explain why this is the case. Based on this experience, we propose best practices to share deep-learning methods with biologists.
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Aprendizaje Profundo , Núcleo Celular , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional , Microscopía/métodos , Redes Neurales de la ComputaciónRESUMEN
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.
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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. Abbreviations: 3D and 4D: Three and Four dimensional; AI: Artificial Intelligence; ant: antipodal nuclei (ant); CLSM: Confocal Laser Scanning Microscopy; CTs: Chromosome Territories; DL: Deep Learning; DLIm: Dynamic Live Imaging; ecn: egg nucleus; FACS: Fluorescence-Activated Cell Sorting; FISH: Fluorescent In Situ Hybridization; FP: Fluorescent Proteins (GFP, RFP, CFP, YFP, mCherry); FRAP: Fluorescence Recovery After Photobleaching; GPU: Graphics Processing Unit; KEEs: KNOT Engaged Elements; INTACT: Isolation of Nuclei TAgged in specific Cell Types; LADs: Lamin-Associated Domains; ML: Machine Learning; NA: Numerical Aperture; NADs: Nucleolar Associated Domains; PALM: Photo-Activated Localization Microscopy; Pixel: Picture element; pn: polar nuclei; PSF: Point Spread Function; RHF: Relative Heterochromatin Fraction; SIM: Structured Illumination Microscopy; SLIm: Static Live Imaging; SMC: Spore Mother Cell; SNR: Signal to Noise Ratio; SRM: Super-Resolution Microscopy; STED: STimulated Emission Depletion; STORM: STochastic Optical Reconstruction Microscopy; syn: synergid nuclei; TADs: Topologically Associating Domains; Voxel: Volumetric pixel.
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Núcleo Celular , Imagenología Tridimensional , Células Vegetales , Animales , Inteligencia Artificial , Núcleo Celular/química , Humanos , Hibridación Fluorescente in Situ , Microscopía Confocal , Microscopía FluorescenteAsunto(s)
Núcleo Celular/metabolismo , Animales , Austria , Núcleo Celular/química , Humanos , Plantas/química , Plantas/metabolismoRESUMEN
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. 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.
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Actinas/metabolismo , Segregación Cromosómica , Cromosomas de las Plantas/metabolismo , Meiosis , Proteínas Nucleares/metabolismo , Zea mays/citología , Zea mays/metabolismo , Núcleo Celular/metabolismo , Segregación Cromosómica/genética , Cromosomas de las Plantas/genética , Citoesqueleto/metabolismo , Meiosis/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Dominios Proteicos , Zea mays/genéticaRESUMEN
Mitosis and meiosis in higher plants involve 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 characterizing 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 the 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.
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Meiosis , Mitosis , Membrana Nuclear/metabolismo , Células VegetalesRESUMEN
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, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether 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 nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a 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 and 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. This article has an associated First Person interview with the first author of the paper.
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Núcleo Celular/metabolismo , Proteínas de la Membrana/metabolismo , Nicotiana/metabolismo , Membrana Nuclear/metabolismo , Señales de Localización Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Agrobacterium tumefaciens/metabolismo , Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismoRESUMEN
The linker of nucleoskeleton to cytoskeleton (LINC) 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. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 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.This article has an associated First Person interview with the first author of the paper.
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Proteínas Asociadas a Microtúbulos/genética , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Zea mays/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , División Celular , Cromatina/metabolismo , Cromatina/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Ontología de Genes , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Anotación de Secuencia Molecular , Familia de Multigenes , Membrana Nuclear/ultraestructura , Matriz Nuclear/ultraestructura , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Células Vegetales/metabolismo , Células Vegetales/ultraestructura , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Zea mays/metabolismoRESUMEN
The analysis of nuclear envelope components and their function has recently been progressed by the use of computational methods of analysis. The methods in this chapter provided by members of the International Plant Nucleus Consortium address the identification of novel nuclear envelope proteins and the study of structure and mobility of the nucleus. DORY2 is an upgrade of the KASH-finder DORY, and NucleusJ is used to characterize the three-dimensional structure of the nucleus in light microscope images. Finally, a method is provided for analysis of the migration of the nucleus, a key technique for exploring the function of plant nuclear proteins.
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Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Vegetales/metabolismo , Biomarcadores , Núcleo Celular/ultraestructura , Biología Computacional/métodos , Bases de Datos Factuales , Imagen Molecular , Células Vegetales/ultraestructura , Programas InformáticosRESUMEN
In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.
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Citoesqueleto de Actina/metabolismo , Arabidopsis/citología , Pared Celular/metabolismo , Arabidopsis/genética , Benzamidas/farmacología , Membrana Celular/metabolismo , Pared Celular/química , Pared Celular/efectos de los fármacos , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
In this study, we explore the plasticity during evolution of proteins of the higher plant nuclear envelope (NE) from the most ancestral plant species to advanced angiosperms. The higher 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 hypothesized 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.
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Evolución Molecular , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Células Vegetales/metabolismo , Proteínas de Plantas/metabolismo , Lámina Nuclear/metabolismo , FilogeniaRESUMEN
This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.
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Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas de la Membrana/fisiología , Membrana Nuclear/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Western Blotting , Cromatina/metabolismo , Clonación Molecular , Citoesqueleto/metabolismo , Genes de Plantas , Proteínas de la Membrana/genética , Microscopía Confocal , Membrana Nuclear/genética , Filogenia , Técnicas del Sistema de Dos HíbridosRESUMEN
Due to rather limited sequence similarity, targeted identification of plant nuclear envelope and nuclear pore complex proteins has mainly followed two routes: (1) advanced computational identification followed by experimental verification and (2) immunoaffinity purification of complexes followed by mass spectrometry. Following candidate identification, fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) provide powerful tools to verify protein-protein interactions in situ at the NE. Here, we describe these methods for the example of Arabidopsis thaliana nuclear pore and nuclear envelope protein identification.