@article{DraffehnKumke2016, author = {Draffehn, Soeren and Kumke, Michael Uwe}, title = {Monitoring the Collapse of pH-Sensitive Liposomal Nanocarriers and Environmental pH Simultaneously: A Fluorescence-Based Approach}, series = {Molecular pharmaceutics}, volume = {13}, journal = {Molecular pharmaceutics}, publisher = {American Chemical Society}, address = {Washington}, issn = {1543-8384}, doi = {10.1021/acs.molpharmaceut.5b00064}, pages = {1608 -- 1617}, year = {2016}, abstract = {Nowadays, the encapsulation of therapeutic compounds in so-called carrier systems is a very smart method to achieve protection as well as an improvement of their temporal and spatial distribution. After the successful transport to the point of care, the delivery has to be released under controlled conditions. To monitor the triggered release from the carrier, we investigated different fluorescent probes regarding their response to the pH-induced collapse of pH-sensitive liposomes (pHSLip), which occurs when the environmental pH falls below a critical value. Depending on the probe, the fluorescence decay time as well as fluorescence anisotropy can be used equally as key parameters for monitoring the collapse. Especially the application of a fluorescein labeled fatty acid (fPA) enabled the monitoring of the pHSLips collapse and the pH of its microenvironment simultaneously without interference. Varying the pH in the range of 3 < pH < 9, anisotropy data revealed the critical pH value at which the collapse of the pHSLips occurs. Complementary methods, e.g., fluorescence correlation spectroscopy and dynamic light scattering, supported the analysis based on the decay time and anisotropy. Additional experiments with varying incubation times yielded information on the kinetics of the liposomal collapse.}, language = {en} } @article{GhoshCherstvyMetzler2014, author = {Ghosh, Surya K. and Cherstvy, Andrey G. and Metzler, Ralf}, title = {Non-universal tracer diffusion in crowded media of non-inert obstacles}, series = {Physical Chemistry Chemical Physics}, volume = {3}, journal = {Physical Chemistry Chemical Physics}, number = {17}, editor = {Metzler, Ralf}, publisher = {The Royal Society of Chemistry}, address = {Cambridge}, issn = {1463-9076}, pages = {1847 -- 1858}, year = {2014}, abstract = {We study the diffusion of a tracer particle, which moves in continuum space between a lattice of excluded volume, immobile non-inert obstacles. In particular, we analyse how the strength of the tracer-obstacle interactions and the volume occupancy of the crowders alter the diffusive motion of the tracer. From the details of partitioning of the tracer diffusion modes between trapping states when bound to obstacles and bulk diffusion, we examine the degree of localisation of the tracer in the lattice of crowders. We study the properties of the tracer diffusion in terms of the ensemble and time averaged mean squared displacements, the trapping time distributions, the amplitude variation of the time averaged mean squared displacements, and the non-Gaussianity parameter of the diffusing tracer. We conclude that tracer-obstacle adsorption and binding triggers a transient anomalous diffusion. From a very narrow spread of recorded individual time averaged trajectories we exclude continuous type random walk processes as the underlying physical model of the tracer diffusion in our system. For moderate tracer-crowder attraction the motion is found to be fully ergodic, while at stronger attraction strength a transient disparity between ensemble and time averaged mean squared displacements occurs. We also put our results into perspective with findings from experimental single-particle tracking and simulations of the diffusion of tagged tracers in dense crowded suspensions. Our results have implications for the diffusion, transport, and spreading of chemical components in highly crowded environments inside living cells and other structured liquids.}, language = {en} } @article{PetazziKoikkarahAjiTischleretal.2021, author = {Petazzi, Roberto Arturo and Koikkarah Aji, Amit and Tischler, Nicole D. and Chiantia, Salvatore}, title = {Detection of envelope glycoprotein assembly from old world hantaviruses in the Golgi apparatus of living cells}, series = {Journal of virology}, volume = {95}, journal = {Journal of virology}, number = {4}, publisher = {American Society for Microbiology}, address = {Baltimore, Md.}, issn = {1098-5514}, doi = {10.1128/JVI.01238-20}, pages = {18}, year = {2021}, abstract = {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.}, language = {en} } @article{TzonevaStoyanovaPetrichetal.2020, author = {Tzoneva, Rumiana and Stoyanova, Tihomira and Petrich, Annett and Popova, Desislava and Uzunova, Veselina and Momchilova, Albena and Chiantia, Salvatore}, title = {Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models}, series = {Biomolecules}, volume = {10}, journal = {Biomolecules}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2218-273X}, doi = {10.3390/biom10050802}, pages = {17}, year = {2020}, abstract = {Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.}, language = {en} }