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The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid–lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.
The detection of auto-fluorescence in phytogenic, hydrated amorphous silica depositions (phytoliths) has been found to be a promising approach to verify if phytoliths were burnt or not, especially in archaeological contexts. However, it is unknown so far at what temperature and how auto-fluorescence is induced in phytoliths. We used fluorescence microscopy, scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared spectroscopy to analyze auto-fluorescence in modern phytoliths extracted from plant samples or in intact leaves of winter wheat. Leaves and extracted phytoliths were heated at different temperatures up to 600 degrees C. The aims of our experiments were i) to find out what temperature is needed to induce auto-fluorescence in phytoliths, ii) to detect temperature-dependent changes in the molecular structure of phytoliths related to auto-fluorescence, and iii) to derive a mechanistic understanding of auto-fluorescence in phytoliths. We found organic compounds associated with phytoliths to cause auto-fluorescence in phytoliths treated at temperatures below approx. 400 degrees C. In phytoliths treated at higher temperatures, i.e., 450 and 600 degrees C, phytolith auto-fluorescence was mainly caused by molecular changes of phytolith silica. Based on our results we propose that auto-fluorescence in phytoliths is caused by clusterization-triggered emissions, which are caused by overlapping electron clouds forming non-conventional chromophores. In phytoliths heated at temperatures above about 400 degrees C dihydroxylation and the formation of siloxanes result in oxygen clusters that serve as non-conventional chromophores in fluorescence events. Furthermore, SEM-EDX analyses revealed that extractable phytoliths were dominated by lumen phytoliths (62%) compared to cell wall phytoliths (38%). Our findings might be not only relevant in archaeological phytolith-based examinations, but also for studies on the temperature-dependent release of silicon from phytoliths and the potential of long-term carbon sequestration in phytoliths.
Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, protein-protein interactions leading to the formation of new virions in infected cells are not fully understood. We used quantitative fluorescence microscopy (i.e., number and brightness analysis and fluorescence fluctuation spectroscopy) to monitor the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we quantified protein-protein interactions for the glycoproteins Gn and Gc from Puumala and Hantaan orthohantaviruses in several cellular models. The oligomerization of each protein was analyzed in relation to subcellular localization, concentration, and the concentration of its interaction partner. Our results indicate that, when expressed separately, Gn and Gc form, respectively, homo-tetrameric and homo-dimeric complexes, in a concentration-dependent manner. Site-directed mutations or deletion mutants showed the specificity of their homotypic interactions. When both glycoproteins were coexpressed, we observed in the Golgi apparatus clear indication of GnGc interactions and the formation of Gn-Gc multimeric protein complexes of different sizes, while using various labeling schemes to minimize the influence of the fluorescent tags. Such large glycoprotein multimers may be identified as multiple Gn viral spikes interconnected via Gc-Gc contacts. This observation provides the possible first evidence for the initial assembly steps of the viral envelope within this organelle, and does so directly in living cells. <br /> IMPORTANCE In this work, we investigate protein-protein interactions that drive the assembly of the hantavirus envelope. These emerging pathogens have the potential to cause deadly outbreaks in the human population. Therefore, it is important to improve our quantitative understanding of the viral assembly process in infected cells, from a molecular point of view. By applying advanced fluorescence microscopy methods, we monitored the formation of viral spike complexes in different cell types. Our data support a model for hantavirus assembly according to which viral spikes are formed via the clustering of hetero-dimers of the two viral glycoproteins Gn and Gc. Furthermore, the observation of large Gn-Gc hetero-multimers provide the possible first evidence for the initial assembly steps of the viral envelope, directly in the Golgi apparatus of living cells.
Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid–lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.