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.
- Publikační typ
- časopisecké články MeSH
The aim of the study is to investigate the differences in the interaction of three structurally diverse anthocyanidins, namely peonidin, petunidin, and delphinidin, as well as their glucosides with model biological membranes, human albumin, and plasmid DNA in order to look into their structure-activity relationships. Fluorimetric studies, as well as ATR-FTIR analyses, were jointly used in order to determine the changes observed in both the hydrophilic and hydrophobic layers of cell-mimic membranes (MM) which reflected the membrane lipid composition of tumour cells and red blood cell membranes (RBCM). Our results showed that anthocyanins and anthocyanidins can cause an increase in the packing order of the polar heads of lipids, as well as interact with their deeper layers by reducing the fluidity of lipid chains. The results presented here indicate that all compounds tested here possessed the ability to bind to human serum albumin (HSA) and the presence of a glucose molecule within the structures formed by anthocyanidin reduces their ability to bind to proteins. Using fluorescence correlation spectroscopy, it was demonstrated that the compounds tested here were capable of forming stable complexes with plasmid DNA and, particularly, strong DNA conformational changes were observed in the presence of petunidin and corresponding glucoside, as well as delphinidin. The results we obtained can be useful in comprehending the anthocyanins therapeutic action as molecular antioxidants and provide a valuable insight into their mechanism of action.
Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at the cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. It has been proposed that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we investigate the magnetic field sensitivity of an endogenous autofluorescence in HeLa cells. Under the experimental conditions used, magnetic field sensitivity of an endogenous autofluorescence was not observed in HeLa cells. We present a number of arguments indicating why this is the case in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level.
- MeSH
- elektromagnetická pole * MeSH
- HeLa buňky MeSH
- lidé MeSH
- magnetické pole * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.
FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.
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.
Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.
The aim of the study was to broadly determine the biological activities of purple potato ethanolic extract of the Blue Congo variety (BCE). The antioxidant activity of BCE was determined in relation to liposome membranes, and peroxidation was induced by UVB and AAPH. To clarify the antioxidant activity of BCE, we investigated its interactions with hydrophilic and hydrophobic regions of a membrane using fluorimetric and FTIR methods. Next, we investigated the cytotoxicity and pro-apoptotic activities of BCE in two human colon cancer cell lines (HT-29 and Caco-2) and in normal cells (IPEC-J2). In addition, the ability to inhibit enzymes that are involved in pro-inflammatory reactions was examined. Furthermore, BCE interactions with serum albumin and plasmid DNA were investigated using steady state fluorescence spectroscopy and a single molecule fluorescence technique (TCSPC-FCS). We proved that BCE effectively protects lipid membranes against the process of peroxidation and successfully inhibits the cyclooxygenase and lipoxygenase enzymes. Furthermore, it interacts with the hydrophilic and hydrophobic parts of lipid membranes as well as with albumin and plasmid DNA. It was observed that BCE is more cytotoxic against colon cancer cell lines than normal IPEC-J2 cells; it also induces apoptosis in cancer cell lines, but does not induce cell death in normal cells.
- MeSH
- albuminy MeSH
- antioxidancia chemie farmakologie MeSH
- antitumorózní látky fytogenní chemie farmakologie MeSH
- inhibitory cyklooxygenasy chemie farmakologie MeSH
- inhibitory lipoxygenas chemie farmakologie MeSH
- lidé MeSH
- lipidy chemie MeSH
- liposomy MeSH
- nádorové buněčné linie MeSH
- plazmidy MeSH
- reaktivní formy kyslíku MeSH
- rostlinné extrakty chemie farmakologie MeSH
- sérový albumin chemie metabolismus MeSH
- Solanum tuberosum chemie MeSH
- vazba proteinů MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Recent studies undoubtedly show that the mammalian target of rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are important mediators of mechanical cues. The crosstalk between these pathways as well as de-regulation of their signaling has been implicated in multiple tumor types, including liver tumors. Additionally, physical cues from 3D microenvironments have been identified to alter gene expression and differentiation of different cell lineages. However, it remains incompletely understood how physical constraints originated in 3D cultures affect cell plasticity and what the key mediators are of such process. In this work, we use collagen scaffolds as a model of a soft 3D microenvironment to alter cellular size and study the mechanotransduction that regulates that process. We show that the YAP-mTOR axis is a downstream effector of 3D cellular culture-driven mechanotransduction. Indeed, we found that cell mechanics, dictated by the physical constraints of 3D collagen scaffolds, profoundly affect cellular proliferation in a YAP-mTOR-mediated manner. Functionally, the YAP-mTOR connection is key to mediate cell plasticity in hepatic tumor cell lines. These findings expand the role of YAP-mTOR-driven mechanotransduction to the control hepatic tumor cellular responses under physical constraints in 3D cultures. We suggest a tentative mechanism, which coordinates signaling rewiring with cytoplasmic restructuring during cell growth in 3D microenvironments.
- Publikační typ
- časopisecké články MeSH
Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.
- MeSH
- antibakteriální látky chemie izolace a purifikace farmakologie MeSH
- buněčná membrána chemie účinky léků MeSH
- ethanolaminy chemie MeSH
- fixní kombinace léků MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie MeSH
- fosfatidylcholiny chemie MeSH
- fosfatidylethanolaminy chemie MeSH
- fosfatidylglyceroly chemie MeSH
- kationické antimikrobiální peptidy chemie izolace a purifikace farmakologie MeSH
- kůže chemie MeSH
- lipidové dvojvrstvy chemie MeSH
- magaininy chemie izolace a purifikace farmakologie MeSH
- proteiny Xenopus chemie izolace a purifikace farmakologie MeSH
- sloučeniny boru chemie MeSH
- synergismus léků MeSH
- vazba proteinů MeSH
- Xenopus laevis MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
