Despite the natural ability of the immune system to recognize cancer and, in some patients, even to eliminate it, cancer cells have acquired numerous evading mechanisms. With the increasing knowledge and focus shifting from targeting rapidly proliferating cells with chemotherapy to modulating the immune system, there have been recent efforts to integrate (e.g., simultaneously or sequentially) various therapeutic approaches. Combining the oncolytic activity of some chemotherapeutics with immunostimulatory molecules, so-called chemoimmunotherapy, is an attractive strategy. An example of such an immunostimulatory molecule is polyinosinic:polycytidylic acid [Poly(I:C)], a synthetic analogue of double-stranded RNA characterized by rapid nuclease degradation hampering its biological activity. This study investigated the possible interactions of tetracycline and anthracycline chemotherapeutics with different commercial Poly(I:C) molecules and protection against nuclease degradation. Fluorescence spectroscopy and circular dichroism revealed an interaction of all of the selected chemotherapeutics with Poly(I:C)s and the ability of doxycycline and minocycline to prolong the resistance to RNase cleavage, respectively. The partial protection was observed in vitro as well.

• Publication type
• Journal Article MeSH

The viscoelastic properties of biological membranes are crucial in controlling cellular functions and are determined primarily by the lipids' composition and structure. This work studies these properties by varying the structure of the constituting lipids in order to influence their interaction with high-density lipoprotein (HDL) particles. Various fluorescence-based techniques were applied to study lipid domains, membrane order, and the overall lateral as well as the molecule-internal glycerol region mobility in HDL-membrane interactions (i.e., binding and/or cargo transfer). The analysis of interactions with HDL particles and various lipid phases revealed that both fully fluid and some gel-phase lipids preferentially interact with HDL particles, although differences were observed in protein binding and cargo exchange. Both interactions were reduced with ordered lipid mixtures containing cholesterol. To investigate the mechanism, membranes were prepared from single-lipid components, enabling step-by-step modification of the lipid building blocks. On a biophysical level, the different mixtures displayed varying stiffness, fluidity, and hydrogen bond network changes. Increased glycerol mobility and a strengthened hydrogen bond network enhanced anchoring interactions, while fluid membranes with a reduced water network facilitated cargo transfer. In summary, the data indicate that different lipid classes are involved depending on the type of interaction, whether anchoring or cargo transfer.

• Keywords
• Laurdan polarity, glycerol region mobility, hydrogen bond network, lipoprotein, membrane order,
• Publication type
• Journal Article MeSH

Biomembranes, important building blocks of living organisms, are often exposed to large local fluctuations of pH and ionic strength. To capture changes in the membrane organization under such harsh conditions, we investigated the mobility and hydration of zwitterionic and anionic lipid bilayers upon elevated H3O+ and Ca2+ content by the time-dependent fluorescence shift (TDFS) technique. While the zwitterionic bilayers remain inert to lower pH and increased calcium concentrations, anionic membranes are responsive. Specifically, both bilayers enriched in phosphatidylserine (PS) and phosphatidylglycerol (PG) become dehydrated and rigidified at pH 4.0 compared to at pH 7.0. However, their reaction to the gradual Ca2+ increase in the acidic environment differs. While the PG bilayers exhibit strong rehydration and mild loosening of the carbonyl region, restoring membrane properties to those observed at pH 7.0, the PS bilayers remain dehydrated with minor bilayer stiffening. Molecular dynamics (MD) simulations support the strong binding of H3O+ to both PS and PG. Compared to PS, PG exhibits a weaker binding of Ca2+ also at a low pH.

• Keywords
• Laurdan, anionic lipids, calcium, headgroup mobility, headgroup organization, lipid hydration, molecular dynamics, phospholipid bilayer, proton, time dependent fluorescence shift,
• MeSH
• Phosphatidylserines MeSH
• Ions MeSH
• Lipid Bilayers * chemistry   MeSH
• Protons * MeSH
• Molecular Dynamics Simulation MeSH
• Calcium chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylserines MeSH
• Ions MeSH
• Lipid Bilayers * MeSH
• Protons * MeSH
• Calcium MeSH

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

• Keywords
• MD simulation, environment-sensitive probes, lipid saturation, model membranes, spectral imaging, time-resolved emission shift,
• MeSH
• Cell Membrane chemistry   MeSH
• Cholesterol MeSH
• Fluorescent Dyes * analysis   chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Fluorescent Dyes * MeSH

Coexisting liquid ordered (Lo) and liquid disordered (Ld) lipid phases in synthetic and plasma membrane-derived vesicles are commonly used to model the heterogeneity of biological membranes, including their putative ordered rafts. However, raft-associated proteins exclusively partition to the Ld and not the Lo phase in these model systems. We believe that the difference stems from the different microscopic structures of the lipid rafts at physiological temperature and the Lo phase studied at room temperature. To probe this structural diversity across temperatures, we performed atomistic molecular dynamics simulations, differential scanning calorimetry, and fluorescence spectroscopy on Lo phase membranes. Our results suggest that raft-associated proteins are excluded from the Lo phase at room temperature due to the presence of a stiff, hexagonally packed lipid structure. This structure melts upon heating, which could lead to the preferential solvation of proteins by order-preferring lipids. This structural transition is manifested as a subtle crossover in membrane properties; yet, both temperature regimes still fulfill the definition of the Lo phase. We postulate that in the compositionally complex plasma membrane and in vesicles derived therefrom, both molecular structures can be present depending on the local lipid composition. These structural differences must be taken into account when using synthetic or plasma membrane-derived vesicles as a model for cellular membrane heterogeneity below the physiological temperature.

• Publication type
• Journal Article MeSH