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

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.

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.

Andrey O. Bogorodskiy

Research Center for Molecular Mechanisms of Aging and Age-Related Diseases

Moscow Institute of Physics and Technology

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

Andrey O. Bogorodskiy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Research Center for Molecular Mechanisms of Aging and Age-Related Diseases

Moscow Institute of Physics and Technology

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

Research Center for Molecular Mechanisms of Aging and Age-Related Diseases