@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} } @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} } @misc{DunsingIrmscherBarbirzetal.2019, author = {Dunsing, Valentin and Irmscher, Tobias and Barbirz, Stefanie and Chiantia, Salvatore}, title = {Microviscosity of bacterial biofilm matrix characterized by fluorescence correlation spectroscopy and single particle tracking}, 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}, doi = {https://doi.org/10.1007/s00249-019-01373-4}, pages = {S115 -- S115}, year = {2019}, language = {en} } @misc{LucknerDunsingChiantiaetal.2017, author = {Luckner, Madlen and Dunsing, Valentin and Chiantia, Salvatore and Herrmann, Andreas}, title = {Influenza virus vRNPs: quantitative investigations via fluorescence cross-correlation spectroscopy}, series = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, volume = {46}, journal = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, publisher = {Springer}, address = {New York}, issn = {0175-7571}, pages = {S368 -- S368}, year = {2017}, language = {en} } @article{PetrichDunsingBoboneetal.2021, author = {Petrich, Annett and Dunsing, Valentin and Bobone, Sara and Chiantia, Salvatore}, title = {Influenza A M2 recruits M1 to the plasma membrane}, series = {Biophysical journal : BJ / ed. by the Biophysical Society}, volume = {120}, journal = {Biophysical journal : BJ / ed. by the Biophysical Society}, number = {24}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2021.11.023}, pages = {5478 -- 5490}, year = {2021}, abstract = {Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin [HA], neuraminidase, matrix protein 2 [M2]) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and number and brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the event that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.}, language = {en} } @misc{DunsingMayerMulthaupetal.2017, author = {Dunsing, Valentin and Mayer, M. and Multhaup, G. and Chiantia, Salvatore}, title = {Direct visualization of APLP1 cell-cell adhesion platforms via fluorescence fluctuation spectroscopy}, series = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, volume = {46}, journal = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, publisher = {Springer}, address = {New York}, issn = {0175-7571}, pages = {S374 -- S374}, year = {2017}, language = {en} } @misc{DunsingMagnusLiebschetal.2018, author = {Dunsing, Valentin and Magnus, Mayer and Liebsch, Filip and Multhaup, Gerhard and Chiantia, Salvatore}, title = {Direct Evidence of APLP1 Trans Interactions in Cell-Cell Adhesion Platforms Investigated via Fluorescence Fluctuation Spectroscopy}, series = {Biophysical journal}, volume = {114}, journal = {Biophysical journal}, number = {3}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2017.11.2067}, pages = {373A -- 373A}, year = {2018}, abstract = {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.}, language = {en} } @article{DunsingMayerLiebschetal.2017, author = {Dunsing, Valentin and Mayer, Magnus and Liebsch, Filip and Multhaup, Gerhard and Chiantia, Salvatore}, title = {Direct evidence of amyloid precursor-like protein 1 trans interactions in cell-cell adhesion platforms investigated via fluorescence fluctuation spectroscopy}, series = {Molecular biology of the cell : the official publication of the American Society for Cell Biology}, volume = {28}, journal = {Molecular biology of the cell : the official publication of the American Society for Cell Biology}, publisher = {American Society for Cell Biology}, address = {Bethesda}, issn = {1059-1524}, doi = {10.1091/mbc.E17-07-0459}, pages = {3609 -- 3620}, year = {2017}, abstract = {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.}, language = {en} } @article{MayerSchauenburgThompsonSteckeletal.2016, author = {Mayer, Magnus C. and Schauenburg, Linda and Thompson-Steckel, Greta and Dunsing, Valentin and Kaden, Daniela and Voigt, Philipp and Schaefer, Michael and Chiantia, Salvatore and Kennedy, Timothy E. and Multhaup, Gerhard}, title = {Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2}, series = {Journal of neurochemistry}, volume = {137}, journal = {Journal of neurochemistry}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0022-3042}, doi = {10.1111/jnc.13540}, pages = {266 -- 276}, year = {2016}, abstract = {The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains.}, language = {en} }