Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts, together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and molecular dynamics simulations prove unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. The lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM-membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but calcium binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role-it not only forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer increasing the exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.
- Klíčová slova
- calcium, calmodulin, lipid membrane, phosphatidylethanolamine, phosphatidylserine,
- MeSH
- buněčná membrána * metabolismus MeSH
- cytosol * metabolismus MeSH
- fosfatidylethanolaminy metabolismus MeSH
- fosfatidylseriny * metabolismus MeSH
- HeLa buňky MeSH
- kalmodulin * metabolismus chemie MeSH
- lidé MeSH
- lipidové dvojvrstvy * metabolismus MeSH
- simulace molekulární dynamiky * MeSH
- vápník * metabolismus MeSH
- vazba proteinů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfatidylethanolaminy MeSH
- fosfatidylseriny * MeSH
- kalmodulin * MeSH
- lipidové dvojvrstvy * MeSH
- phosphatidylethanolamine MeSH Prohlížeč
- vápník * MeSH
Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior - electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane.
- Klíčová slova
- AFM imaging, AFM nanoindentation, FRET-GP, Integral membrane protein, MD simulation,
- MeSH
- lipidové dvojvrstvy * chemie MeSH
- membránové lipidy chemie MeSH
- membránové proteiny chemie MeSH
- peptidy * chemie MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- lipidové dvojvrstvy * MeSH
- membránové lipidy MeSH
- membránové proteiny MeSH
- peptidy * MeSH
Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters but the local effects were considerably stronger (GPFRET ≫ GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby enhancing the depth of information obtained from the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.
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
Benzalkonium chloride (BAK) compounds are commonly used in topical ophthalmic products as preservatives and stabilizers. BAK mixtures containing several compounds with different alkyl chain lengths are typically used. However, in chronic eye conditions, such as dry eye disease and glaucoma, the accumulation of adverse effects of BAKs was observed. Hence, preservative-free eye drops formulations are preferred. On the other hand, selected long-chain BAKs, particularly cetalkonium chloride, exhibit therapeutic functions, promoting epithelium wound healing and tear film stability. Nevertheless, the mechanism of BAKs influence on the tear film is not fully understood. By employing in vitro experimental and in silico simulation techniques, we elucidate the action of BAKs and demonstrate that long-chain BAKs accumulate in the lipid layer of the tear film model, stabilizing it in a concentration-dependent fashion. In contrast, short-chain BAKs interacting with the lipid layer compromise the tear film model stability. These findings are relevant for topical ophthalmic drug formulation and delivery in the context of selecting proper BAK species and understanding the dose dependency for tear film stability.
- Klíčová slova
- Benzalkonium chloride, Lipid films, Molecular dynamics, Tear film, Tear film lipid layer, Topical ophthalmic formulations,
- MeSH
- benzalkoniové sloučeniny škodlivé účinky MeSH
- konzervační prostředky farmaceutické * farmakologie MeSH
- lidé MeSH
- lipidy farmakologie MeSH
- oční roztoky MeSH
- slzy MeSH
- syndromy suchého oka * farmakoterapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- benzalkoniové sloučeniny MeSH
- konzervační prostředky farmaceutické * MeSH
- lipidy MeSH
- oční roztoky 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.
- Klíčová slova
- Laurdan, anionic lipids, calcium, headgroup mobility, headgroup organization, lipid hydration, molecular dynamics, phospholipid bilayer, proton, time dependent fluorescence shift,
- MeSH
- fosfatidylseriny MeSH
- ionty MeSH
- lipidové dvojvrstvy * chemie MeSH
- protony * MeSH
- simulace molekulární dynamiky MeSH
- vápník chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfatidylseriny MeSH
- ionty MeSH
- lipidové dvojvrstvy * MeSH
- protony * MeSH
- vápník 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.
- Publikační typ
- časopisecké články MeSH
The organization of biomolecules and bioassemblies is highly governed by the nature and extent of their interactions with water. These interactions are of high intricacy and a broad range of methods based on various principles have been introduced to characterize them. As these methods view the hydration phenomena differently (e.g., in terms of time and length scales), a detailed insight in each particular technique is to promote the overall understanding of the stunning "hydration world." In this prospective mini-review we therefore critically examine time-dependent fluorescence shift (TDFS)-an experimental method with a high potential for studying the hydration in the biological systems. We demonstrate that TDFS is very useful especially for phospholipid bilayers for mapping the interfacial region formed by the hydrated lipid headgroups. TDFS, when properly applied, reports on the degree of hydration and mobility of the hydrated phospholipid segments in the close vicinity of the fluorophore embedded in the bilayer. Here, the interpretation of the recorded TDFS parameters are thoroughly discussed, also in the context of the findings obtained by other experimental techniques addressing the hydration phenomena (e.g., molecular dynamics simulations, NMR spectroscopy, scattering techniques, etc.). The differences in the interpretations of TDFS outputs between phospholipid biomembranes and proteins are also addressed. Additionally, prerequisites for the successful TDFS application are presented (i.e., the proper choice of fluorescence dye for TDFS studies, and TDFS instrumentation). Finally, the effects of ions and oxidized phospholipids on the bilayer organization and headgroup packing viewed from TDFS perspective are presented as application examples.
- Klíčová slova
- biomembranes, calcium, cholesterol, hydration, lipid headgroups, membrane dynamics, oxidized phosholipids, time-dependent fluorescence shift,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
DNA nanostructures (DNs) can be designed in a controlled and programmable manner, and these structures are increasingly used in a variety of biomedical applications, such as the delivery of therapeutic agents. When exposed to biological liquids, most nanomaterials become covered by a protein corona, which in turn modulates their cellular uptake and the biological response they elicit. However, the interplay between living cells and designed DNs are still not well established. Namely, there are very limited studies that assess protein corona impact on DN biological activity. Here, we analyzed the uptake of functionalized DNs in three distinct hepatic cell lines. Our analysis indicates that cellular uptake is linearly dependent on the cell size. Further, we show that the protein corona determines the endolysosomal vesicle escape efficiency of DNs coated with an endosome escape peptide. Our study offers an important basis for future optimization of DNs as delivery systems for various biomedical applications.
- Klíčová slova
- DNA nanotechnology, bionano interactions, cellular uptake, endolysosomal escape, nanotechnology, protein corona,
- MeSH
- adsorpce MeSH
- DNA chemie metabolismus MeSH
- endozomy metabolismus MeSH
- kationické antimikrobiální peptidy chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- nádorové buněčné linie MeSH
- nanostruktury chemie MeSH
- proteinová korona chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aurein 1.2 peptide MeSH Prohlížeč
- DNA MeSH
- kationické antimikrobiální peptidy MeSH
- proteinová korona 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.
- Klíčová slova
- 3D cultures, YAP, autophagy, cell plasticity, cytoskeleton, mTOR, mechanotransduction,
- Publikační typ
- časopisecké články MeSH
