@article{HoeferDiLellaDahmanietal.2019, author = {H{\"o}fer, C. T. and Di Lella, S. and Dahmani, Ismail and Jungnick, N. and Bordag, N. and Bobone, Sara and Huang, Q. and Keller, S. and Herrmann, A. and Chiantia, Salvatore}, title = {Structural determinants of the interaction between influenza A virus matrix protein M1 and lipid membranes}, series = {Biochimica et biophysica acta : Biomembranes}, volume = {1861}, journal = {Biochimica et biophysica acta : Biomembranes}, number = {6}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0005-2736}, doi = {10.1016/j.bbamem.2019.03.013}, pages = {1123 -- 1134}, year = {2019}, abstract = {Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1 protein interactions and multimerization have not been clarified, yet. In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.}, language = {en} } @inproceedings{DunsingPetrichChiantia2021, author = {Dunsing, Valentin and Petrich, Annett and Chiantia, Salvatore}, title = {Spectral detection enables multi-color fluorescence fluctuation spectroscopy studies in living cells}, series = {Biophysical journal : BJ / ed. by the Biophysical Society}, volume = {120}, booktitle = {Biophysical journal : BJ / ed. by the Biophysical Society}, number = {3, Suppl. 1}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2020.11.2206}, pages = {356A -- 356A}, year = {2021}, language = {en} } @article{SperberWelkePetazzietal.2019, author = {Sperber, Hannah Sabeth and Welke, Robert-William and Petazzi, Roberto Arturo and Bergmann, Ronny and Schade, Matthias and Shai, Yechiel and Chiantia, Salvatore and Herrmann, Andreas and Schwarzer, Roland}, title = {Self-association and subcellular localization of Puumala hantavirus envelope proteins}, series = {Scientific reports}, volume = {9}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-018-36879-y}, pages = {15}, year = {2019}, abstract = {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.}, language = {en} } @misc{SperberWelkePetazzietal.2019, author = {Sperber, Hannah Sabeth and Welke, Robert-William and Petazzi, Roberto Arturo and Bergmann, Ronny and Schade, Matthias and Shai, Yechiel and Chiantia, Salvatore and Herrmann, Andreas and Schwarzer, Roland}, title = {Self-association and subcellular localization of Puumala hantavirus envelope proteins}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {648}, issn = {1866-8372}, doi = {10.25932/publishup-42504}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-425040}, pages = {15}, year = {2019}, abstract = {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.}, language = {en} } @misc{LucknerDunsingDruekeetal.2019, author = {Luckner, Madlen and Dunsing, Valentin and Dr{\"u}ke, Markus and Zuehlke, B. and Petazzi, Roberto Arturo and Chiantia, Salvatore and Herrmann, A.}, title = {Quantifying protein oligomerization directly in living cells}, series = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, volume = {48}, journal = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, publisher = {Springer}, address = {New York}, issn = {0175-7571}, pages = {S183 -- S183}, year = {2019}, language = {en} } @article{DunsingIrmscherBarbirzetal.2019, author = {Dunsing, Valentin and Irmscher, Tobias and Barbirz, Stefanie and Chiantia, Salvatore}, title = {Purely Polysaccharide-Based Biofilm Matrix Provides Size-Selective Diffusion Barriers for Nanoparticles and Bacteriophages}, series = {Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences}, volume = {20}, journal = {Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences}, number = {10}, publisher = {American Chemical Society}, address = {Washington}, issn = {1525-7797}, doi = {10.1021/acs.biomac.9b00938}, pages = {3842 -- 3854}, year = {2019}, abstract = {Biofilms are complex mixtures of proteins, DNA, and polysaccharides surrounding bacterial communities as protective barriers that can be biochemically modified during the bacterial life cycle. However, their compositional heterogeneity impedes a precise analysis of the contributions of individual matrix components to the biofilm structural organization. To investigate the structural properties of glycan-based biofilms, we analyzed the diffusion dynamics of nanometer-sized objects in matrices of the megadalton-sized anionic polysaccharide, stewartan, the major biofilm component of the plant pathogen, Pantoea stewartii. Fluorescence correlation spectroscopy and single-particle tracking of nanobeads and bacteriophages indicated notable subdiffusive dynamics dependent on probe size and stewartan concentration, in contrast to free diffusion of small molecules. Stewartan enzymatic depolymerization by bacteriophage tailspike proteins rapidly restored unhindered diffusion. We, thus, hypothesize that the glycan polymer stewartan determines the major physicochemical properties of the biofilm, which acts as a selective diffusion barrier for nanometer-sized objects and can be controlled by enzymes.}, language = {en} } @article{BoboneHilschStormetal.2017, author = {Bobone, Sara and Hilsch, Malte and Storm, Julian and Dunsing, Valentin and Herrmann, Andreas and Chiantia, Salvatore}, title = {Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes}, series = {Journal of virology}, volume = {91}, journal = {Journal of virology}, publisher = {American Society for Microbiology}, address = {Washington}, issn = {0022-538X}, doi = {10.1128/JVI.00267-17}, pages = {15}, year = {2017}, abstract = {Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Forster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N\&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N\&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.}, language = {en} } @article{DunsingLucknerZuehlkeetal.2018, author = {Dunsing, Valentin and Luckner, Madlen and Zuehlke, Boris and Petazzi, Roberto Arturo and Herrmann, Andreas and Chiantia, Salvatore}, title = {Optimal fluorescent protein tags for quantifying protein oligomerization in living cells}, series = {Scientific reports}, volume = {8}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-018-28858-0}, pages = {12}, year = {2018}, abstract = {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.}, language = {en} } @misc{LucknerDunsingChiantiaetal.2018, author = {Luckner, Madlen and Dunsing, Valentin and Chiantia, Salvatore and Hermann, Andreas}, title = {Oligomerization and nuclear shuttling dynamics of viral proteins studied by quantitative molecular brightness analysis using fluorescence correlation spectroscopy}, series = {Biophysical journal}, volume = {114}, journal = {Biophysical journal}, number = {3}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2017.11.1951}, pages = {350A -- 350A}, year = {2018}, language = {en} } @article{DunsingPetrichChiantia2021, author = {Dunsing, Valentin and Petrich, Annett and Chiantia, Salvatore}, title = {Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection}, series = {eLife}, volume = {10}, journal = {eLife}, publisher = {eLife Sciences Publications}, address = {Cambridge}, issn = {2050-084X}, doi = {10.7554/eLife.69687}, pages = {33}, year = {2021}, abstract = {Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.}, language = {en} }