Significant technical advances in imaging, molecular biology and genomics have fueled a revolution in cell biology, in that the molecular and structural processes of the cell are now visualized and measured routinely. Driving much of this recent development has been the advent of computational tools for the acquisition, visualization, analysis and dissemination of these datasets. These tools collectively make up a new subfield of computational biology called bioimage informatics, which is facilitated by open source approaches. We discuss why open source tools for image informatics in cell biology are needed, some of the key general attributes of what make an open source imaging application successful, and point to opportunities for further operability that should greatly accelerate future cell biology discovery.

We report the outcomes of the discussion initiated at the workshop entitled A 3D Cellular Context for the Macromolecular World and propose how data from emerging three-dimensional (3D) cellular imaging techniques—such as electron tomography, 3D scanning electron microscopy and soft X-ray tomography—should be archived, curated, validated and disseminated, to enable their interpretation and reuse by the biomedical community.

Mitotic entry and progression require the activation of several mitotic kinases and the proper regulation and localization of several phosphatases. The activity and localization of each of these enzymes is tightly controlled through a series of specific activators, inhibitors and regulatory subunits. Two proteins, Ensa and Arpp-19, were recently identified as specific inhibitors of PP2A-B55 and are critical for allowing full activity of Cdk1/cyclin B and entry into mitosis. Here we show that Bod1, a protein required for proper chromosome alignment at mitosis, shares sequence similarity with Ensa and Arpp-19 and specifically inhibits the kinetochore-associated PP2A-B56 holoenzyme. PP2A-B56 regulates the stability of kinetochore-microtubule attachments by dephosphorylating several kinetochore proteins. Loss of Bod1 changes the balance of phosphorylation at kinetochores, causing defects in kinetochore function. Bod1, Ensa and Arpp-19 define a family of specific PP2A inhibitors that regulate specific PP2A holoenzymes at distinct locations and points in the cell cycle.

The history and the current state of the PDB and EMDB archives is briefly described, as well as some of the challenges that they face. It seems natural that the role of structural biology archives will change from being a pure repository of historic data into becoming an indispensable resource for the wider biomedical community. As part of this transformation, it will be necessary to validate the biomacromolecular structure data and ensure the highest possible quality for the archive holdings, to combine structural data from different spatial scales into a unified resource and to integrate structural data with functional, genetic and taxonomic data as well as other information available in bioinformatics resources. Some recent developments and plans to address these challenges at PDBe are presented.

PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.

Three-dimensional Electron Microscopy (3DEM) has become a key experimental method in structural biology for a broad spectrum of biological specimens from molecules to cells. The EMDataBank project provides a unified portal for deposition, retrieval and analysis of 3DEM density maps, atomic models and associated metadata (emdatabank.org). We provide here an overview of the rapidly growing 3DEM structural data archives, which include maps in EM Data Bank and map-derived models in the Protein Data Bank. In addition, we describe progress and approaches toward development of validation protocols and methods, working with the scientific community, in order to create a validation pipeline for 3DEM data.

The tumour suppressor Adenomatous Polyposis Coli (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic Xenopus egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted Xenopus egg extract frequently formed tight round or elongated structures. Such abnormally compacted chromatin predominantly formed spindles with low microtubule content. Furthermore, in mitotic HeLa cells expressing GFP- and mCherry-labelled H2B histones, depletion of APC caused a decrease in the donor fluorescence lifetime of neighbouring fluorophores, indicative of excessive chromatin compaction. Profiling the chromatin-associated proteome of sperm chromatin incubated with Xenopus egg extracts revealed temporal APC-dependent changes in the abundance of histones, closely mirrored by chromatin-associated Topoisomerase IIa, condensin I complex and Kif4. In the absence of APC these factors initially accumulated on chromatin, but then decreased faster than in controls. We also found and validated significant APC-dependent changes in chromatin modifiers Set-a and Rbbp7. Both were decreased on chromatin in APC-depleted extract; in addition, the kinetics of association of Set-a with chromatin was altered in the absence of APC.

Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.

For almost 50 years, structural biology has endeavoured to conserve and share its experimental data and their interpretations (usually, atomistic models) through global public archives such as the Protein Data Bank, Electron Microscopy Data Bank and Biological Magnetic Resonance Data Bank (BMRB). These archives are treasure troves of freely accessible data that document our quest for molecular or atomic understanding of biological function and processes in health and disease. They have prepared the field to tackle new archiving challenges as more and more (combinations of) techniques are being utilized to elucidate structure at ever increasing length scales. Furthermore, the field has made substantial efforts to develop validation methods that help users to assess the reliability of structures and to identify the most appropriate data for their needs. In this Review, we present an overview of public data archives in structural biology and discuss the importance of validation for users and producers of structural data. Finally, we sketch our efforts to integrate structural data with bioimaging data and with other sources of biological data. This will make relevant structural information available and more easily discoverable for a wide range of scientists.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

This protocol illustrates the steps necessary to deposit correlated 3D cryo-imaging data from cryo-structured illumination microscopy and cryo-soft X-ray tomography with the BioStudies and EMPIAR deposition databases of the European Bioinformatics Institute. There is currently a real need for a robust method of data deposition to ensure unhindered access to and independent validation of correlative light and X-ray microscopy data to allow use in further comparative studies, educational activities, and data mining. For complete details on the use and execution of this protocol, please refer to Kounatidis et al. (2020).

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself -- OME-Zarr -- along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain -- the file format that underlies so many personal, institutional, and global data management and analysis tasks.

Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery?

Volume Electron Microscopy is a group of techniques that reveal the 3D ultrastructure of cells and tissues through volumes greater than 1 cubic micron. A burgeoning grass roots community effort is fast building the profile, and revealing the impact, of vEM technology in the life sciences and clinical research.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores.

Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores.

BACKGROUND: Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF). To find the optimal formulation of the multi-component IEF rehydration buffer (RB) we applied the Taguchi method, a widely used approach for the robust optimisation of complex industrial processes, to determine optimal concentrations for the detergents, carrier ampholytes and reducing agents in RB for 2DE using commercially supplied immobilised pH gradient (IPG) gel strips. RESULTS: Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins. CONCLUSIONS: While the optimised RB (oRB) is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems.