Aspergillus(A.)fumigatusis an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells andA.fumigatuswe used SEM, CLSM anddSTORM techniques, which clearly demonstrated that NK cells directly interact withA.fumigatusvia CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells andA.fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide‐rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super‐resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide‐rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells withBacillus cereussphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C₆and long-chain C₁₆-ceramides and azido-functionalized ceramide analogs against pathogenicNeisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C₆-ceramide were active againstNeisseria meningitidisandN. gonorrhoeae, whereas they were inactive againstEscherichia coliandStaphylococcus aureus. Kinetic assays showed that killing ofN.meningitidisoccurred within 2 h with ω–azido-C₆-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C₆-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging bydirectstochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenicNeisseriae.

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution, and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined the fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as the degree of lipid saturation, cholesterol content, double bond position and configuration, and phospholipid headgroup. As a comparison, we investigated the lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g., cholesterol). While the lifetime of Flipper is particularly sensitive to the membrane cholesterol content, the NR12S and NR12A lifetimes are moderately sensitive to both the cholesterol content and lipid acyl chains. Moreover, all of the probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission wavelength dependency results in varying lifetime resolutions at different spectral regions, which are highly relevant for FLIM data acquisition. Our data provide valuable insights on how to perform FLIM with these probes and highlight both their potential and limitations.