TY - JOUR A1 - Sperber, Hannah Sabeth A1 - Welke, Robert-William A1 - Petazzi, Roberto Arturo A1 - Bergmann, Ronny A1 - Schade, Matthias A1 - Shai, Yechiel A1 - Chiantia, Salvatore A1 - Herrmann, Andreas A1 - Schwarzer, Roland T1 - Self-association and subcellular localization of Puumala hantavirus envelope proteins JF - Scientific reports N2 - Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential. Y1 - 2019 U6 - https://doi.org/10.1038/s41598-018-36879-y SN - 2045-2322 VL - 9 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Dunsing, Valentin A1 - Luckner, Madlen A1 - Zuehlke, Boris A1 - Petazzi, Roberto Arturo A1 - Herrmann, Andreas A1 - Chiantia, Salvatore T1 - Optimal fluorescent protein tags for quantifying protein oligomerization in living cells JF - Scientific reports N2 - Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization. Y1 - 2018 U6 - https://doi.org/10.1038/s41598-018-28858-0 SN - 2045-2322 VL - 8 PB - Nature Publ. Group CY - London ER - TY - GEN A1 - Luckner, Madlen A1 - Dunsing, Valentin A1 - Drüke, Markus A1 - Zuehlke, B. A1 - Petazzi, Roberto Arturo A1 - Chiantia, Salvatore A1 - Herrmann, A. T1 - Quantifying protein oligomerization directly in living cells BT - a systematic comparison of fluorescent proteins and application to Influenza A virus infection T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2019 SN - 0175-7571 SN - 1432-1017 VL - 48 SP - S183 EP - S183 PB - Springer CY - New York ER - TY - GEN A1 - Sperber, Hannah Sabeth A1 - Welke, Robert-William A1 - Petazzi, Roberto Arturo A1 - Bergmann, Ronny A1 - Schade, Matthias A1 - Shai, Yechiel A1 - Chiantia, Salvatore A1 - Herrmann, Andreas A1 - Schwarzer, Roland T1 - Self-association and subcellular localization of Puumala hantavirus envelope proteins T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 648 KW - Sin-Nombre-Virus KW - nucleocapsid protein KW - cytoplasmic tails KW - electron cryotomography KW - autophagic clearance KW - glycoprotein KW - Gn KW - G1 KW - brightness KW - fever Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-425040 SN - 1866-8372 IS - 648 ER - TY - JOUR A1 - Petazzi, Roberto Arturo A1 - Koikkarah Aji, Amit A1 - Chiantia, Salvatore T1 - Fluorescence microscopy methods for the study of protein oligomerization JF - Progress in Molecular Biology and Translational Science N2 - Protein-protein interactions (PPIs) are of fundamental importance in several cellular processes. While "classical" biochemical methods are commonly used to monitor protein multimerization in biological samples, fluorescence microscopy offers the possibility to investigate PPIs directly in living cells, even distinguishing among different cellular compartments. In this chapter, we shortly describe the most common procedures used to label proteins with fluorescent probes. Furthermore, we discuss a variety of fluorescence microscopy techniques that can be used to obtain quantitative information about protein multimerization. Special emphasis is given to fluorescence fluctuation techniques and their applications in the context of, e.g., receptor multimerization and virus assembly. Y1 - 2020 SN - 978-0-12-817929-1 U6 - https://doi.org/10.1016/bs.pmbts.2019.12.001 SN - 1877-1173 SN - 1878-0814 VL - 169 SP - 1 EP - 41 PB - Elsevier CY - Amsterdam 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 -