| 1. |
- Humpolickova, Jana, et al.
(author)
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Dynamics and size of crosslinking-induced lipid nanodomains in model membranes
- 2012
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 102:3, s. 294a-
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Journal article (peer-reviewed)abstract
- Changes of membrane organization upon crosslinking of its components trigger cellsignaling response to various exogenous factors. Crosslinking of raft gangliosides GM1with cholera toxin (CTxB) was demonstrated to cause microscopic phase separation inmodel membranes and the CTxB-GM1 complexes forming a minimal lipid raft unit aresubject of ongoing cell membrane research. Yet, those subdiffraction sized rafts havenever been described in terms of size and dynamics. By means of two-color z-scanfluorescence correlation spectroscopy, we show that the nano-sized domains are formedin model membranes at lower sphingomyelin content than needed for the large scalephase separation and that the CTxB-GM1 complexes are confined in the domains poorlystabilized with sphingomyelin. Fluorescence resonance energy transfer together withMonte Carlo modeling of the donor decay response reveal the domain radius ofapproximately 8 nm, which increases at higher sphingomyelin content. We observed twotypes of differently behaving domains, which suggests a dual role of the crosslinker: first,local transient condensation of the GM1 molecules compensating lack of sphingomyelinand second, coalescence of existing nanodomains ending in large scale phase separation.
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| 2. |
- Stefl, Martin, et al.
(author)
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Dynamics and size of cross-linking-induced lipid Nanodomains in model Membranes
- 2012
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 102:9, s. 2104-2113
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Journal article (peer-reviewed)abstract
- Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin ( CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Forster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of similar to 8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation.
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| 3. |
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| 4. |
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| 5. |
- Dingeldein, Artur Peter Günther, et al.
(author)
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Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes : Lipid Dynamics and Permeabilization
- 2017
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 112:10, s. 2147-2158
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Journal article (peer-reviewed)abstract
- Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using P-31 solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.
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| 6. |
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| 7. |
- Koukalova, Alena, et al.
(author)
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Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered
- 2017
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)-and cholesterol (Chol)-driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Forster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solidstate wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.
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| 8. |
- Lidman, Martin, 1985-, et al.
(author)
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The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax
- 2016
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In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1858:6, s. 1288-1297
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Journal article (peer-reviewed)abstract
- Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly 15N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.
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| 9. |
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| 10. |
- Mikaelsson, Therese, et al.
(author)
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Electronic energy transport and Fluorescence Spectroscopy for structural insights into Proteins, Regular Protein Aggregates and Lipid Systems
- 2009
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In: Reviews In Fluorescence 2007. - New York : Springer-Verlag. ; , s. 53-86
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Book chapter (other academic/artistic)abstract
- The present review aims at surveying recent theoretical development and applications of electronic energy transport between chromophoric molecules (i.e. donors and acceptors) in various protein and lipid systems. Reversible, partly reversible, and irreversible energy transport within pairs of interacting chromophoric molecules are considered. Also energy migration/transfer within ensembles of many donor and acceptor molecules is discussed. An extended Förster theory of interacting pairs is summarised, which brings the analyses of data to the same level of molecular description as in ESR and NMR spectroscopy. Recent applications of energy transfer/migration on protein systems concern their structure, folding, and their formation of non-covalent protein polymers. The latter systems are of particular interest in e.g. the study of amyloid formation and the molecular functioning of muscles. The energy transfer/migration processes have also been utilised to study the spatial distribution of lipid molecules, which is of interest in the study of biological membranes and their functioning, e.g. the presumed formation of so-called rafts.
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