U-Net is a convolutional neural network model developed in 2015 and has proven to be one of the most inspiring deep-learning models for image segmentation. Numerous U-Net-based applications have since emerged, constituting a heterogeneous set of tools that illustrate the current reproducibility crisis in the deep-learning field. Here we propose a solution in the form of Biom3d, a modular framework for deep learning facilitating the integration and development of novel models, metrics, or training schemes for 3D image segmentation. The new development philosophy of Biom3D provides an improved code sustainability and reproducibility in line with the FAIR principles and is available as a graphical user interface and an open-source deep-learning framework to target a large community of users, from end users to deep learning developers. ### Competing Interest Statement The authors have declared no competing interest.

Bioimage frameworks based on artificial intelligence (AI) offer powerful tools for image segmentation, but their technical overhead often creates a gap between developers and the broader bioimaging community. Biom3d addresses this challenge by providing a modular, PyTorch-based architecture designed to enable reproducible and interoperable 2D and 3D segmentation pipelines while maintaining strict adherence to FAIR principles. The framework's engineering quality is demonstrated through low intra-module complexity, high modularity, and seamless interoperability, exemplified by the successful substitution of transformer-based MONAI models. Its architecture is organized around seven core module types, allowing fine-grained control over data handling, model configuration, optimization, and evaluation. Its default configuration, nnCore, autoconfigures optimal pipelines based on new datasets and competes with state-of-the-art 3D segmentation methods, outperforming classical tools such as NucleusJ/NODeJ and matching the robustness of nnU-Net across diverse modalities. Biom3d is accessible through multiple interfaces including a graphical user interface, a Jupyter Notebook, a command-line tool, a Docker image and as a Python library. Biom3d is compatible with OMERO (Open Microscopy Environment Remote Objects) for streamlined data management and reproducible computation, including execution on HPC servers. Collectively, these results position Biom3d as a sustainable and extensible framework for building, sharing, and reusing advanced bioimage analysis solutions.

The membrane-intrinsic nuclear pore complex component PNET1 is specifically found in proliferating tissue, where it regulates breakdown and reassembly of the nuclear pores and is essential for promoting cell division and tissue maintenance. These dynamics are driven by phosphorylation events that alter PNET1 interactions.

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.

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.

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.

EDITORIAL article Front. Plant Sci., 03 August 2022Sec. Plant Cell Biology https://doi.org/10.3389/fpls.2022.998823

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.

This article comments on: Masuda K, Hikida R, Fujino K. 2021. The plant nuclear lamina proteins NMCP1 and NMCP2 form a filamentous network with lateral filament associations. Journal of Experimental Botany 72, 6190–6204.

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.

The continuous development of microscopy has led biologists to have access to large high-resolution 2D and 3D image datasets. Automatic analysis of cellular and nuclear images has become an important challenge in the bioimaging field. To help biologists extract information from these images, tools have been designed to count objects in the image, study object type, their localization or morphology. The current state-of-the-art is led by deep learning methods. Their development relies on the availability of large data sets, on enhancing graphical processing units (GPU) of computers and on developments of new methodologies such as convolutional neural networks (CNN). However, non-IT users may experience difficulties when trying to use these on their own images. This short paper contains our first results after reviewing the state-of-the-art methods in the domain. It first introduces the current difficulties when working with bioimages, then lists the existing datasets for nuclear images analysis. It then exposes some of the easy-to-use tools for bioimaging and points out the different problems related to their use. It finally presents a new dataset for 3D images of plant nuclei that is designed for benchmarking purposes. This results should shortly be published in an journal of biology.

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.

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.

This special issue comprises papers based in ameeting of theNuclear Dynamics Special Interest Group of the Society for Experimental Biology. The session was entitled ‘Functional organisation of the nuclear periphery’ and was held at the Society’s Annual Main Meeting in the beautiful city of Firenze (Florence) in July 2018. Organized by Katja Graumann and David Evans from Oxford Brookes University, Oxford UK and Roland Foisner, Medical University Vienna, Austria, the session highlighted novel research in plant, animal and fungal nuclear biology since the previous meeting of the group in Brighton, UK in 2016[1]. Themeeting was supported by attendance ofmembers of the INDEPTH (Impact of Nuclear Domains On Gene Expression and Plant Traits) COST Action CA16212 (https://www.cost.eu/actions/CA16212/). A review article in this special issue of Nucleus by members of Workgroup 1 of the COST Action provides insights into microscopy methods for studying 3D nuclear architecture in plants and accompanying challenges being addressed in the consortium[2]. Other papers highlight progress in understanding mechanisms for the dynamic organisation of the nuclear periphery, both in interphase and in dividing cells, including the role of lamina-associated domains (LADs) in spatial chromatin organization[3], the role of the nucleolus in anchoring chromatin structures [4], the contribution of structural maintenance of chromatin (SMC) complexes to chromatin organisation[5], and the function of plant KASH domain proteins in attaching the nucleus to the actin cystoskeleton[6]. The structural role of the nuclear envelope in plant mitosis and meiosis is reviewed [7] with a further paper presenting evidence for the role of lamins in meiotic chromosome movements at the nuclear periphery in C. elegans[8]. The final paper in the special issue [9] provides a comprehensive exploration of the plant nuclear proteome using a sequential extractionmethodwhich has significantly increased the number of plant nuclear proteins identified. As editors, we are grateful to all the contributors, both to the meeting and to this special issue. Their enthusiasm and willingness to share their insights and expertise with others was reflected in their presentations and is evident in the resulting papers. The cross-kingdom format of these meetings provides a fertile environment for generating research ideas and for development of collaborations and this is evidenced in the progress made by the members of the Group.

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.

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.

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.