A practical guide to bioimaging research data management in core facilities.

Bioimage data are generated in diverse research fields throughout the life and biomedical sciences. Its potential for advancing scientific progress via modern, data-driven discovery approaches reaches beyond disciplinary borders. To fully exploit this potential, it is necessary to make bioimaging data, in general, multidimensional microscopy images and image series, FAIR, that is, findable, accessible, interoperable and reusable. These FAIR principles for research data management are now widely accepted in the scientific community and have been adopted by funding agencies, policymakers and publishers. To remain competitive and at the forefront of research, implementing the FAIR principles into daily routines is an essential but challenging task for researchers and research infrastructures. Imaging core facilities, well-established providers of access to imaging equipment and expertise, are in an excellent position to lead this transformation in bioimaging research data management. They are positioned at the intersection of research groups, IT infrastructure providers, the institution´s administration, and microscope vendors. In the frame of German BioImaging - Society for Microscopy and Image Analysis (GerBI-GMB), cross-institutional working groups and third-party funded projects were initiated in recent years to advance the bioimaging community's capability and capacity for FAIR bioimage data management. Here, we provide an imaging-core-facility-centric perspective outlining the experience and current strategies in Germany to facilitate the practical adoption of the FAIR principles closely aligned with the international bioimaging community. We highlight which tools and services are ready to be implemented and what the future directions for FAIR bioimage data have to offer.

The adhesion GPCR and PCP component flamingo (FMI-1) alters body size and regulates the composition of the extracellular matrix.

The extracellular matrix (ECM) is a network of macromolecules that presents a vital scaffold for cells and enables multiple ways of cellular communication. Thus, it is essential for many physiological processes such as development, tissue morphogenesis, homeostasis, the shape and partially the size of the body and its organs. To ensure these, the composition of the ECM is tissue-specific and highly dynamic. ECM homeostasis is therefore tightly controlled by several mechanisms. Here, we show that FMI-1, the homolog of the Adhesion GPCR Flamingo/CELSR/ADGRC in the nematode Caenorhabditis elegans, modulates the composition of the ECM by controlling the production both of ECM molecules such as collagens and also of ECM modifying enzymes. Thereby, FMI-1 affects the morphology and functionality of the nematode´s cuticle, which is mainly composed of ECM, and also modulates the body size. Mechanistic analyses highlight the fact that FMI-1 exerts its function from neurons non-cell autonomously (trans) solely via its extracellular N terminus. Our data support a model, by which the activity of the receptor, which has a well-described role in the planar cell polarity (PCP) pathway, involves the PCP molecule VANG-1, but seems to be independent of the DBL-1/BMP pathway.

Cristae dynamics is modulated in bioenergetically compromised mitochondria.

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here, we investigated whether and how cristae morphology and dynamics are dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨm), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. CCCP treatment leading to ΔΨm abrogation even enhanced cristae dynamics showing its ΔΨm-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors.

Biochemical processes within the living cell occur in a highly crowded environment, where macromolecules, first of all proteins and nucleic acids, occupy up to 30% of the volume. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein-packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, alters kinetic and thermodynamic properties of biochemical reactions, and modulates the membrane organization. Despite its importance, the non-invasive quantification of the membrane crowding is not trivial. Here, we developed a genetically-encoded fluorescence-based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by flexible linker domains were characterized in vitro, and the procedures for the membrane reconstitution were established. Steric pressure induced by membrane-tethered synthetic and protein crowders altered the sensors' conformation, causing increase in the intramolecular Förster's resonance energy transfer. Notably, the effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge contribute to the crowding via the quinary interactions. Finally, measurements performed in inner membrane vesicles of Escherichia coli validated the crowding-dependent dynamics of the sensors in the physiologically relevant environment. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis.

Recognition of polymorphic Csd proteins determines sex in the honeybee.

Sex in honeybees, Apis mellifera, is genetically determined by heterozygous versus homo/hemizygous genotypes involving numerous alleles at the single complementary sex determination locus. The molecular mechanism of sex determination is however unknown because there are more than 4950 known possible allele combinations, but only two sexes in the species. We show how protein variants expressed from complementary sex determiner (csd) gene determine sex. In females, the amino acid differences between Csd variants at the potential-specifying domain (PSD) direct the selection of a conserved coiled-coil domain for binding and protein complexation. This recognition mechanism activates Csd proteins and, thus, the female pathway. In males, the absence of polymorphisms establishes other binding elements at PSD for binding and complexation of identical Csd proteins. This second recognition mechanism inactivates Csd proteins and commits male development via default pathway. Our results demonstrate that the recognition of different versus identical variants of a single protein is a mechanism to determine sex.

One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells.

BACKGROUND: A commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters. RESULTS: As a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable. CONCLUSIONS: We conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

Streptomyces development is involved in the efficient containment of viral infections.

The formation of plaques represents the hallmark of phage infection visualizing the clearance of the bacterial lawn in structured environments. In this study, we have addressed the impact of cellular development on phage infection in Streptomyces undergoing a complex developmental life cycle. Analysis of plaque dynamics revealed, after a period of plaque size enlargement, a significant regrowth of transiently phage-resistant Streptomyces mycelium into the lysis zone. Analysis of Streptomyces venezuelae mutant strains defective at different stages of cellular development indicated that this regrowth was dependent on the onset of the formation of aerial hyphae and spores at the infection interface. Mutants restricted to vegetative growth (ΔbldN) featured no significant constriction of plaque area. Fluorescence microscopy further confirmed the emergence of a distinct zone of cells/spores with reduced cell permeability towards propidium iodide staining at the plaque periphery. Mature mycelium was further shown to be significantly less susceptible to phage infection, which is less pronounced in strains defective in cellular development. Transcriptome analysis revealed the repression of cellular development at the early stages of phage infection probably facilitating efficient phage propagation. We further observed an induction of the chloramphenicol biosynthetic gene cluster highlighting phage infection as a trigger of cryptic metabolism in Streptomyces. Altogether, our study emphasizes cellular development and the emergence of transient phage resistance as an important layer of Streptomyces antiviral immunity.

Cardiac injection of USSC boosts remuscularization of the infarcted heart by shaping the T-cell response.

Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT+/BrdU+ cardiomyocytes that was paralleled by progressively restored ejection fraction. USSC induced significant T-cell enrichment in ischemic tissue with enhanced expression of T-cell related cytokines. Inhibition of T-cell activation by anti-CD28 monoclonal antibody, fully abolished the regenerative response which was restored by adoptive T-cell transfer. Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors over an extended period of time which boosts a T-cell driven endogenous regenerative response mainly from adult cardiomyocytes.

Research data management for bioimaging: the 2021 NFDI4BIOIMAGE community survey.

Background:  Knowing the needs of the bioimaging community with respect to research data management (RDM) is essential for identifying measures that enable adoption of the FAIR (findable, accessible, interoperable, reusable) principles for microscopy and bioimage analysis data across disciplines. As an initiative within Germany's National Research Data Infrastructure, we conducted this community survey in summer 2021 to assess the state of the art of bioimaging RDM and the community needs. Methods: An online survey was conducted with a mixed question-type design. We created a questionnaire tailored to relevant topics of the bioimaging community, including specific questions on bioimaging methods and bioimage analysis, as well as more general questions on RDM principles and tools. 203 survey entries were included in the analysis covering the perspectives from various life and biomedical science disciplines and from participants at different career levels. Results: The results highlight the importance and value of bioimaging RDM and data sharing. However, the practical implementation of FAIR practices is impeded by technical hurdles, lack of knowledge, and insecurity about the legal aspects of data sharing. The survey participants request metadata guidelines and annotation tools and endorse the usage of image data management platforms. At present, OMERO (Open Microscopy Environment Remote Objects) is the best known and most widely used platform. Most respondents rely on image processing and analysis, which they regard as the most time-consuming step of the bioimage data workflow. While knowledge about and implementation of electronic lab notebooks and data management plans is limited, respondents acknowledge their potential value for data handling and publication. Conclusion: The bioimaging community acknowledges and endorses the value of RDM and data sharing. Still, there is a need for information, guidance, and standardization to foster the adoption of FAIR data handling. This survey may help inspiring targeted measures to close this gap.

Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell.

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

PLETHORA-WOX5 interaction and subnuclear localization control Arabidopsis root stem cell maintenance.

Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion-like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination.

Homo-FRET Imaging to Study Protein-Protein Interaction and Complex Formation in Plants.

Protein-protein interactions in living plant cells can be measured by changes in fluorescence anisotropy due to homo-FRET (Förster Resonance Energy Transfer). Here, the energy transfer between identical fluorophores, e.g., enhanced green fluorescent protein (EGFP) fused to a protein of interest, serves as a read-out for protein interaction and clustering. By applying homo-FRET imaging, not only dimeric complexes, but also bigger homomeric complex formation can be followed in vivo at high spatial and temporal resolution. Therefore, this method provides a powerful tool to investigate changes in complex formation over time in their natural environment with high precision at a subcellular level. Here, we describe the necessary theoretical background and how homo-FRET imaging is practically carried out. We also discuss potential pitfalls and points of consideration.

Quantification and Surface Localization of the Hemolysin A Type I Secretion System at the Endogenous Level and under Conditions of Overexpression.

Secretion systems are essential for Gram-negative bacteria, as these nanomachineries allow communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type I secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli, which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC, and the substrate HlyA, a member of the family of repeats in toxins (RTX) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression [T7 expression system, BL21(DE3)-BD]. The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by superresolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS clusters at the outer membrane, generating domains of so-far-undescribed identity. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide, representing a global burden to the health care system. UPEC strains secrete many virulence factors, among these, the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore, and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the superresolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery.

BACKGROUND: A major task of the endosomal sorting complex required for transport (ESCRT) machinery is the pinching off of cargo-loaded intraluminal vesicles (ILVs) into the lumen of maturing endosomes (MEs), which is essential for the complete degradation of transmembrane proteins in the lysosome. The ESCRT machinery is also required for the termination of signalling through activated signalling receptors, as it separates their intracellular domains from the cytosol. At the heart of the machinery lies the ESCRT-III complex, which is required for an increasing number of processes where membrane regions are abscised away from the cytosol. The core of ESCRT-III, comprising four members of the CHMP protein family, organises the assembly of a homopolymer of CHMP4, Shrub in Drosophila, that is essential for abscission. We and others identified the tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 as a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively. RESULTS: Here, we show that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. This hypomorphic shrub mutant situation causes a slight decrease in the rate of ILV formation that appears to result in incomplete incorporation of Notch into ILVs. We found that the forced incorporation in ILVs of lgd mutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis of Su(dx) lgd double mutants clarifies their relationship and suggests that they are not operating in a linear pathway. We could show that, despite prolonged lifetime, the MEs of lgd mutants have a similar ILV density as wild-type but less than rab7 mutant MEs, suggesting the rate in lgd mutants is slightly reduced. The analysis of the MEs of wild-type and mutant cells in the electron microscope revealed that the ESCRT-containing electron-dense microdomains of ILV formation at the limiting membrane are elongated, indicating a change in ESCRT activity. Since lgd mutants can be rescued to normal adult flies if extra copies of shrub (or its mammalian ortholog CHMP4B) are added into the genome, we conclude that the net activity of Shrub is reduced upon loss of lgd function. Finally, we show that, in solution, CHMP4B/Shrub exists in two conformations. LGD1/Lgd binding does not affect the conformational state of Shrub, suggesting that Lgd is not a chaperone for Shrub/CHMP4B. CONCLUSION: Our results suggest that Lgd is required for the full activity of Shrub/ESCRT-III. In its absence, the activity of the ESCRT machinery is reduced. This reduction causes the escape of a fraction of cargo, among it Notch, from incorporation into ILVs, which in turn leads to an activation of this fraction of Notch after fusion of the ME with the lysosome. Our results highlight the importance of the incorporation of Notch into ILV not only to assure complete degradation, but also to avoid uncontrolled activation of the pathway.

Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis.

Eukaryotic microorganisms use monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. During translation of these peptides the ribosome stalls, the peptide chain is released and the ribosome resumes translation. Thus, two independent polypeptide chains can be encoded from a single mRNA when a 2A peptide sequence is placed inbetween the two open reading frames. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

A Joint Action in Times of Pandemic: The German BioImaging Recommendations for Operating Imaging Core Facilities During the SARS-Cov-2 Emergency.

Operating shared resource laboratories (SRLs) in times of pandemic is a challenge for research institutions. In a multiuser, high-turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could be operated with minimal risk of spreading SARS-CoV-2 between users and staff. Here, we describe the resulting guidelines and explain their rationale, with a focus on separating users in space and time, protective face masks, and keeping surfaces virus-free. These recommendations may prove useful for other types of SRLs. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.