TY - JOUR A1 - Bobone, Sara A1 - Hilsch, Malte A1 - Storm, Julian A1 - Dunsing, Valentin A1 - Herrmann, Andreas A1 - Chiantia, Salvatore T1 - Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes JF - Journal of virology N2 - 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. KW - influenza KW - assembly KW - confocal microscopy KW - fluorescence image analysis KW - lipid rafts KW - matrix protein KW - model membranes KW - phosphatidylserine KW - plasma membrane Y1 - 2017 U6 - https://doi.org/10.1128/JVI.00267-17 SN - 0022-538X SN - 1098-5514 VL - 91 PB - American Society for Microbiology CY - Washington ER - TY - GEN A1 - Dahmani, Ismail A1 - Ludwig, Kai A1 - Chiantia, Salvatore T1 - Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1–M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context,M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains). T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 768 KW - confocal microscopy KW - influenza KW - lipid membranes KW - membranes KW - protein-protein interactions KW - viral matrix proteins Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-438689 SN - 1866-8372 IS - 768 ER - TY - JOUR A1 - Dahmani, Ismail A1 - Ludwig, Kai A1 - Chiantia, Salvatore T1 - Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization JF - Bioscience Reports N2 - The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1–M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context,M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains). KW - confocal microscopy KW - influenza KW - lipid membranes KW - membranes KW - protein-protein interactions KW - viral matrix proteins Y1 - 2019 U6 - https://doi.org/10.1042/BSR20191024 SN - 0144-8463 SN - 1573-4935 VL - 39 IS - 8 PB - Portland Press CY - Colchester ER - TY - JOUR A1 - Dunsing, Valentin A1 - Irmscher, Tobias A1 - Barbirz, Stefanie A1 - Chiantia, Salvatore T1 - Purely Polysaccharide-Based Biofilm Matrix Provides Size-Selective Diffusion Barriers for Nanoparticles and Bacteriophages JF - Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences N2 - 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. Y1 - 2019 U6 - https://doi.org/10.1021/acs.biomac.9b00938 SN - 1525-7797 SN - 1526-4602 VL - 20 IS - 10 SP - 3842 EP - 3854 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Dunsing, Valentin A1 - Irmscher, Tobias A1 - Barbirz, Stefanie A1 - Chiantia, Salvatore T1 - Microviscosity of bacterial biofilm matrix characterized by fluorescence correlation spectroscopy and single particle tracking T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2019 U6 - https://doi.org/https://doi.org/10.1007/s00249-019-01373-4 SN - 0175-7571 SN - 1432-1017 VL - 48 SP - S115 EP - S115 PB - Springer CY - New York 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 - Dunsing, Valentin A1 - Magnus, Mayer A1 - Liebsch, Filip A1 - Multhaup, Gerhard A1 - Chiantia, Salvatore T1 - Direct Evidence of APLP1 Trans Interactions in Cell-Cell Adhesion Platforms Investigated via Fluorescence Fluctuation Spectroscopy T2 - Biophysical journal N2 - The Amyloid-precursor-like protein 1 (APLP1) is a neuronal type I transmembrane protein which plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigate APLP1 interactions and dynamics directly in living human embryonic kidney (HEK) cells, using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy (sFCS) and Number&Brightness (N&B). Our results show that APLP1 forms homotypic trans complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from Giant Plasma Membrane Vesicles and live cell actin staining suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1 direct trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and provide a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms. Further, they show that fluorescence fluctuation spectroscopy techniques are useful tools for the investigation of protein-protein interactions at cell-cell adhesion sites. Y1 - 2018 U6 - https://doi.org/10.1016/j.bpj.2017.11.2067 SN - 0006-3495 SN - 1542-0086 VL - 114 IS - 3 SP - 373A EP - 373A PB - Cell Press CY - Cambridge ER - TY - GEN A1 - Dunsing, Valentin A1 - Mayer, M. A1 - Multhaup, G. A1 - Chiantia, Salvatore T1 - Direct visualization of APLP1 cell-cell adhesion platforms via fluorescence fluctuation spectroscopy T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S374 EP - S374 PB - Springer CY - New York ER - TY - JOUR A1 - Dunsing, Valentin A1 - Mayer, Magnus A1 - Liebsch, Filip A1 - Multhaup, Gerhard A1 - Chiantia, Salvatore T1 - Direct evidence of amyloid precursor-like protein 1 trans interactions in cell-cell adhesion platforms investigated via fluorescence fluctuation spectroscopy JF - Molecular biology of the cell : the official publication of the American Society for Cell Biology N2 - The amyloid precursor-like protein 1 (APLP1) is a type I transmembrane protein that plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigated APLP1 interactions and dynamics directly in living human embryonic kidney cells using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy and number and brightness analysis. Our results show that APLP1 forms homotypic trans complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from giant plasma membrane vesicles suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1 trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and allow a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms. Y1 - 2017 U6 - https://doi.org/10.1091/mbc.E17-07-0459 SN - 1059-1524 SN - 1939-4586 VL - 28 SP - 3609 EP - 3620 PB - American Society for Cell Biology CY - Bethesda ER - TY - JOUR A1 - Dunsing, Valentin A1 - Petrich, Annett A1 - Chiantia, Salvatore T1 - Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection JF - eLife N2 - 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. KW - fluorescence KW - optical microscopy KW - virus assembly KW - protein-protein KW - interactions KW - diffusion KW - Viruses Y1 - 2021 U6 - https://doi.org/10.7554/eLife.69687 SN - 2050-084X VL - 10 PB - eLife Sciences Publications CY - Cambridge ER -