TY - JOUR A1 - Puppe, Daniel A1 - Leue, Martin A1 - Sommer, Michael A1 - Schaller, Jörg A1 - Kaczorek, Danuta T1 - Auto-fluorescence in phytoliths BT - a mechanistic understanding derived from microscopic and spectroscopic analyses JF - Frontiers in Environmental Science N2 - 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. KW - fluorescence microscopy KW - FTIR spectroscopy KW - SEM-EDX KW - burnt phytoliths; KW - carbon sequestration Y1 - 2022 U6 - https://doi.org/10.3389/fenvs.2022.915947 SN - 2296-665X VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Petazzi, Roberto Arturo A1 - Koikkarah Aji, Amit A1 - Tischler, Nicole D. A1 - Chiantia, Salvatore T1 - Detection of envelope glycoprotein assembly from old world hantaviruses in the Golgi apparatus of living cells JF - Journal of virology N2 - 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.
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. KW - fluorescence fluctuation microscopy KW - number and brightness KW - virus KW - assembly KW - fluorescence correlation spectroscopy KW - protein-protein KW - interaction KW - fluorescence microscopy KW - fluorescent image analysis Y1 - 2021 U6 - https://doi.org/10.1128/JVI.01238-20 SN - 1098-5514 VL - 95 IS - 4 PB - American Society for Microbiology CY - Baltimore, Md. ER - TY - JOUR A1 - Tzoneva, Rumiana A1 - Stoyanova, Tihomira A1 - Petrich, Annett A1 - Popova, Desislava A1 - Uzunova, Veselina A1 - Momchilova, Albena A1 - Chiantia, Salvatore T1 - Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models JF - Biomolecules N2 - 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. KW - alkylphospholipids KW - fluorescence microscopy KW - fluorescence correlation spectroscopy KW - lipids KW - plasma membrane KW - cancer KW - lipid–lipid interactions KW - membrane microdomains KW - membrane biophysics Y1 - 2020 U6 - https://doi.org/10.3390/biom10050802 SN - 2218-273X VL - 10 IS - 5 PB - MDPI CY - Basel ER -