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 - JOUR A1 - Schweighöfer, F. A1 - Moreno, J. A1 - Bobone, Sara A1 - Chiantia, Salvatore A1 - Herrmann, A. A1 - Hecht, S. A1 - Wachtveitl, Josef T1 - Connectivity pattern modifies excited state relaxation dynamics of fluorophore-photoswitch molecular dyads JF - Physical chemistry, chemical physics : a journal of European Chemical Societies N2 - In order to modulate the emission of BODIPY fluorophores, they were connected to a diarylethene (DAE) photoswitch via phenylene-ethynylene linkers of different lengths and orientations. The latter allowed for modulation of the electronic coupling in the prepared four BODIPY-DAE dyads, which were compared also to appropriate BODIPY and DAE model compounds by steady state as well as time-resolved spectroscopies. In their open isomers, all dyads show comparable luminescence behavior indicative of an unperturbed BODIPY fluorophore. In strong contrast, in the closed isomers the BODIPY emission is efficiently quenched but the deactivation mechanism depends on the nature of the linker. The most promising dyad was rendered water-soluble by means of micellar encapsulation and aqueous suspensions were investigated by fluorescence spectroscopy and microscopy. Our results (i) illustrate that the electronic communication between the BODIPY and DAE units can indeed be fine-tuned by the nature of the linker to achieve fluorescence modulation while maintaining photoswitchability and (ii) highlight potential applications to image and control biological processes with high spatio-temporal resolution. Y1 - 2016 U6 - https://doi.org/10.1039/c6cp07112k SN - 1463-9076 SN - 1463-9084 VL - 19 SP - 4010 EP - 4018 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Whiteley, Liam A1 - Haug, Maria A1 - Klein, Kristina A1 - Willmann, Matthias A1 - Bohn, Erwin A1 - Chiantia, Salvatore A1 - Schwarz, Sandra T1 - Cholesterol and host cell surface proteins contribute to cell-cell fusion induced by the Burkholderia type VI secretion system 5 JF - PLoS one N2 - Following escape into the cytoplasm of host cells, Burkholderia pseudomallei and the related species Burkholderia thailandensis employ the type VI secretion system 5 ( T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by B. thailandensis. Acute protease treatment of RAW264.7 macrophages during infection with B. thailandensis followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl-beta-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of B. thailandensis to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by B. pseudomallei and B. thailandensis involves a complex interplay between the T6SS-5 and the host cell. Y1 - 2017 U6 - https://doi.org/10.1371/journal.pone.0185715 SN - 1932-6203 VL - 12 PB - PLoS CY - San Fransisco 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 - GEN A1 - Luckner, Madlen A1 - Dunsing, Valentin A1 - Chiantia, Salvatore A1 - Herrmann, Andreas T1 - Influenza virus vRNPs: quantitative investigations via fluorescence cross-correlation spectroscopy T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S368 EP - S368 PB - Springer CY - New York 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 -