confocal-laser-scanning-microscopy-based-quantitative-analysis

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of <em>Aspergillus fumigatus</em> Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

Moscow Institute of Physics and Technology

Forschungszentrum Jülich

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Research on halophilic microorganisms is important due to their relation to fundamental questions of survival of living organisms in a hostile environment. Here we introduce a novel method to stain halophiles with MitoTracker fluorescent dyes in their growth medium. The method is based on membrane-potential sensitive dyes, which were originally used to label mitochondria in eukaryotic cells. We demonstrate that these fluorescent dyes provide high staining efficiency and are beneficial for multi-staining purposes due to the spectral range covered (from orange to deep red). In contrast with other fluorescent dyes used so far, MitoTracker does not affect growth rate, and remains in cells after several washing steps and several generations in cell culture. The suggested dyes were tested on three archaeal (Hbt. salinarum, Haloferax sp., Halorubrum sp.) and two bacterial (Salicola sp., Halomonas sp.) strains of halophilic microorganisms. The new staining approach provides new insights into biology of Hbt. salinarum. We demonstrated the interconversion of rod-shaped cells of Hbt. salinarium to spheroplasts and submicron-sized spheres, as well as the cytoplasmic integrity of giant rod Hbt. salinarum species. By expanding the variety of tools available for halophile detection, MitoTracker dyes overcome long-standing limitations in fluorescence microscopy studies of halophiles.

Efficient non-cytotoxic fluorescent staining of halophiles.

Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall after  conidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1 or Ly6G neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h after  infection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates upon  infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

 Infection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice.

People are constantly exposed to airborne fungal spores, including  conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense  conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the  conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII antigen-presenting cells, including IE-DCs, and CD11b phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of  conidia increased the number of CD11b phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During -Induced Inflammation.

X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules.

XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces.

Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLTR and CysLTR), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing.

Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs.

A Correction to this paper has been published: https://doi.org/10.1038/s41597-020-00759-w.

Author Correction: Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs.

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.

Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis.

Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation.

Article	Year
Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi. PloS one\| PubMed ID: 28837632	2017
Efficient non-cytotoxic fluorescent staining of halophiles. Scientific reports\| PubMed ID: 29416075	2018
Infection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice. Journal of immunology research\| PubMed ID: 29577051	2018
Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During -Induced Inflammation. Frontiers in immunology\| PubMed ID: 32161590	2020
XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces. Materials (Basel, Switzerland)\| PubMed ID: 33080816	2020
Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs. Scientific data\| PubMed ID: 33184270	2020
Author Correction: Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs. Scientific data\| PubMed ID: 33230124	2020
Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis. Journal of molecular biology\| PubMed ID: 33359098	2021
Modulation of Toll-like receptor 1 intracellular domain structure and activity by Zn ions. Communications biology\| PubMed ID: 34429510	2021

Valentin I. Borshchevskiy

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