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

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.

• MeSH
• časové faktory MeSH
• chemické techniky analytické metody   MeSH
• difuze MeSH
• fluorescence * MeSH
• fluorescenční barviva chemie   MeSH
• fosfatidylcholiny chemie   MeSH
• fosfatidylethanolaminy chemie   MeSH
• fosfatidylseriny chemie   MeSH
• fosfolipidy chemie   MeSH
• kinetika MeSH
• lipidové dvojvrstvy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-dielaidoylphosphatidylethanolamine MeSH Prohlížeč
• 1,2-dioleoylphosphatidylserine MeSH Prohlížeč
• 1,2-oleoylphosphatidylcholine MeSH Prohlížeč
• fluorescenční barviva MeSH
• fosfatidylcholiny MeSH
• fosfatidylethanolaminy MeSH
• fosfatidylseriny MeSH
• fosfolipidy MeSH
• lipidové dvojvrstvy MeSH

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

• Klíčová slova
• biomembranes, confocal microscopy, fluorescence fluctuation spectroscopy, giant unilamellar vesicles, lateral diffusion, supported lipid bilayers,
• MeSH
• difuze MeSH
• fluorescenční spektrometrie * MeSH
• konfokální mikroskopie MeSH
• lipidové dvojvrstvy chemie   MeSH
• membránové lipidy chemie   MeSH
• unilamelární lipozómy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• lipidové dvojvrstvy MeSH
• membránové lipidy MeSH
• unilamelární lipozómy MeSH

Although there exist a number of methods, such as NMR, X-ray, e.g., which explore the hydration of phospholipid bilayers, the solvent relaxation (SR) method has the advantage of simple instrumentation, easy data treatment and possibility of measuring fully hydrated samples. The main information gained from SR by the analysis of recorded "time-resolved emission spectra" (TRES) is micro-viscosity and micro-polarity of the dye microenvironment. Based on these parameters, one can draw conclusions about water structure in the bilayer. In this review, we focus on physical background of this method, on all the procedures that are needed in order to obtain relevant parameters, and on the requirements on the fluorescence dyes. Furthermore, a few recent applications (the effect of curvature, binding of antibacterial peptides and phase transition) illustrating the versatility of this method are mentioned. Moreover, limitations and potential problems are discussed.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• fluidita membrány * MeSH
• fluorescenční barviva analýza   MeSH
• fluorescenční spektrometrie metody   MeSH
• fosfolipidy chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• rozpouštědla chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fluorescenční barviva MeSH
• fosfolipidy MeSH
• lipidové dvojvrstvy MeSH
• rozpouštědla MeSH