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Localisation of Fluorescent Probes and the estimation of Lipid Nanodomain sizes by modern fluorescence techniques
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Lokalizace fluorescenčních značek a určování velikostí lipidových nanodomén pomocí moderních fluorescenčních metod (Czech)
Abstract [en]

The thesis is divided into two major parts. The first part focuses on the localisation of probes in lipid/polymeric bilayers and in GM1 micelles. Included in this thesis is a new approach based on electronic energy transfer/migration (FRET/DDEM), which efficiently determines transversal positions of fluorescent molecules in lipid bilayers. This approach has been used to locate newly synthesized lipid probes in DOPC bilayers. The label was introduced at the end of sn-2 acyl chains of variable length.

Analytical models accounting for FRET exist for a limited number of basic geometries. Here, a combination of FRET and Monte Carlo simulations enables the localisation of probes in bicelles and in bilayers containing pores, i.e. in lipid systems with variable curvature, or in non-homogenous lipid systems. This approach has been used to test whether conical-like fluorescence probes have an increased affinity to highly curved regions, which would enable preferential labelling of membrane pores.

A simplified FRET model has been applied to localize 2-pyridones, a class of potential drugs, in GM1 micelles. Since the localisation of drugs within nanoparticles might influence the release kinetics and loading efficiency, knowledge about the drug location is highly relevant. It turned out that all derivatives were localised at the core-shell interface of GM1 micelles.

The second part of the thesis focuses mainly on the estimation of lipid nanodomain size by means of FRET, which still remains the most powerful method in this field. Limitations of FRET in the determination of domain size have been explored. We showed that the limitations of FRET are mainly caused by a low probes affinity to either the liquid-ordered or liquid-disordered phase. In the continuing work we provided a detailed dynamic and structural study of crosslinker-triggered formation of nanodomains. Here, two different domains have been revealed, i.e. i) domains whose size grows with increasing amount of added cholera toxin (CTxB), and to which CTxB binds tightly; ii) domains formed in membranes containing a slightly increased amount of sphingomyelin (as compared to i) whose size does not change during titration by additional CTxB and to which CTxB binds less tightly.

Abstract [cs]

Disertace je rozdělena do dvou hlavníchčástí. Prvníčást se zabývá lokalizací značek v lipidových/polymerních dvojvrstvách a v GM1micelách. V práci prezentujeme nový přístup založený na přenosu/migraci elektronické energie (FRET/DDEM), jež umožňuje efektivně určovat vertikální pozici fluorescenčních molekul uvnitř lipidové dvojvrstvy. Tato metoda byla použita k lokalizaci nově syntetizovaných lipidových značek značených na konci sn-2 acylového řetězce s různou délkou v DOPC dvojvrstvách.

Analytické modely popisující FRET existují pouze pro limitovaný počet základních geometrií. Kombinace FRETu s Monte Carlo simulacemi nicméně umožňuje lokalizaci značek v bicelách a v dvojvrstvách obsahujících póry, tj. v lipidových systémech s proměnlivým zakřivením a v nehomogenních lipidových útvarech. Tento přístup umožnil např. zjistit, zda kuželovitětvarované značky mají zvýšenou afinitu k vysoce zakřiveným oblastem dvojvrstvy, což by umožnilo preferenční značení pórů.

Lokalizovány byly rovněž tři deriváty 2-pyridonů(potencionálních léčiv) v GM1micelách za použití jednoduchého modelu zohledňujícího FRET mezi donory a akceptory nacházejícími se v micelách. Lokalizace léčiv v nanočásticích ovlivňuje kinetiku uvolňování (release kinetics) a množství látky solubilizované v micelách (loading efficiency).

Druhá část se především zabývá určováním velikostí lipidových nanodomén pomocí FRETu, který stále zůstává nejvíce výkonnou metodou v této oblasti. Zkoumány byly limitace FRETu v určování lipidových nanodomén. Ukázalo se, že tato omezení jsou především způsobena nízkou afinitou značek buď k Lonebo k Ldfázi. V navazující studii jsme poskytnuli detailní dynamickou a strukturní studii formace nanodomén indukované crosslinkerem. Objevili jsme dva typy domén: a) domény, jejichž velikost se zvětšuje s rostoucím množstvím přidaného cholera toxinu (CTxB) a k nimž se CTxB váže pevně a b) domény vzniklé v membránách se zvýšeným množstvím sfingomyelinu (ve srovnání s a)), jejichž velikost se nemění během titrace dodatečným CTxB a k nimž se CTxB váže méně pevně.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2012. , 46 p.
Keyword [en]
Electronic energy transfer, fluorescence, rafts, lipid bilayer, bicelles, micelles
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-52619ISBN: 978-91-7459-390-7 (print)OAI: oai:DiVA.org:umu-52619DiVA: diva2:506220
Public defence
2012-03-27, KBC-huset, KB3A9, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Note
This thesis has been elaborated within the framework of the Agreement on Joint Supervision (co-tutelle) of an International Doctoral Degree Programme between Charles University in Prague, Czech Republic and the Department of Chemistry at Umeå University, Sweden.Available from: 2012-03-06 Created: 2012-02-28 Last updated: 2012-02-28Bibliographically approved
List of papers
1. Fluorescence Study of the Solvation of Fluorescent Probes Prodan and Laurdan in Poly(ε-caprolactone)-block-poly(ethylene oxide) Vesicles in Aqueous Solutions with Tetrahydrofurane
Open this publication in new window or tab >>Fluorescence Study of the Solvation of Fluorescent Probes Prodan and Laurdan in Poly(ε-caprolactone)-block-poly(ethylene oxide) Vesicles in Aqueous Solutions with Tetrahydrofurane
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2008 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 24, no 1, 288-295 p.Article in journal (Refereed) Published
Abstract [en]

Steady-state and time-resolved fluorescence measurements were used to study the relaxation of the microenvironmentof hydrophobic probes 6-propionyl-2-(dimethylamino)naphthalene (prodan) and 6-dodecanoyl-2-(dimethylamino)-naphthalene (laurdan) in systems containing vesicles formed by the amphiphilic diblock copolymer poly(-caprolactone)-block-poly(ethylene oxide) (PCL-PEO) and water/tetrahydrofurane (THF) solvent mixtures. It was found that in caseof prodan, both steady-state and time-resolved emission spectra were composed of two subspectra corresponding tothe emission of prodan molecules located (i) in fairly rigid (effectively viscous) and hydrophobic domains of thevesicles close to the PCL/PEO interface and (ii) in a more polar and less viscous medium (in the bulk solution). Thefraction of the emission from the more polar microenvironment increases with increasing content of THF in the system.Laurdan, in contrast to prodan, appeared to be solubilized preferentially in the hydrophobic domains up to 30 vol %of THF content, and its emission spectra changed only due to swelling of hydrophobic PCL domains by added THF.The study shows that the analysis of the time-resolved emission from a probe distributed in two media is, in principle,possible, but it is quite complex and appreciably less accurate, and the relaxation times are ill-defined averages ofseveral processes. The bimodal or shoulder-containing time-resolved spectra have to be decomposed in pertinenttime-resolved subspectra and treated separately. Another important result of the study is a piece of knowledge concerningthe motion of the probe with respect to the vesicle. In the studied complex system, not only the relaxation of the solventand reorganization of polymer segments around the fluorescent headgroup of the probe affect the emission but alsoa lateral motion of the probe with respect to the nanoparticle within the lifetime of the excited state contributessignificantly to the relaxation and to the relatively slow time-resolved Stokes shift.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2008
National Category
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-52465 (URN)10.1021/la702277t (DOI)000251916100043 ()
Available from: 2012-02-22 Created: 2012-02-22 Last updated: 2017-12-07Bibliographically approved
2. A comparative study on ganglioside micelles using electronic energy transfer, fluorescence correlation spectroscopy and light scattering techniques
Open this publication in new window or tab >>A comparative study on ganglioside micelles using electronic energy transfer, fluorescence correlation spectroscopy and light scattering techniques
2009 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 11, no 21, 4335-4343 p.Article in journal (Refereed) Published
Abstract [en]

Ganglioside (GM1) micelles have been studied by means of three different techniques: fluorescence correlation spectroscopy (FCS), electronic energy transfer, as monitored by time-resolved fluorescence spectroscopy, as well as static and dynamic light scattering. The aggregation numbers obtained, 168 ± 4, remain constant over a wide range of GM1 concentrations (0.764-156 M), are very consistent when using different donor-acceptor energy transfer pairs and have served as reference values in tests of the FCS method. It is recommended to calibrate the focal volume by using known dye concentrations. For this the rhodamine dye, 5-TAMRA, turns out to be most suitable. It is also shown that FCS provides correct values of the aggregation numbers, provided that the focal volume is calibrated by using updated values of the diffusion constant of Rhodamine 6G. These results also support recent methodological advances in FCS.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2009
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-20816 (URN)10.1039/b821658d (DOI)
Available from: 2009-03-26 Created: 2009-03-26 Last updated: 2017-12-13Bibliographically approved
3. Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers
Open this publication in new window or tab >>Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers
2010 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 12, 6027-6034 p.Article in journal (Refereed) Published
Abstract [en]

A series of sn-2 acyl-labelled phosphatidyl-cholines (PC), bearing 4,4-difluoro-1-3-5-7-tetra-methyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY) at the end of the Cn-acyl chains were solubilised in unilamellar vesicles and studied with respect to the order and location of the Me4-BODIPY (denoted: B) group. The obtained results are based on time-resolved electronic energy transfer from donors (2-(9-anthroyloxy)-stearic acid) localised in the lipid–water interface to acceptors BnPC (n = 3, 5, 7, 9, 11, 13, 15), as well as the energy migration among the Me4-BODIPY groups of BnPC:s. The donor–acceptor and the donor–donor experiments strongly suggest that the Me4-BODIPY group in BnPC tends to loop back close to the lipid–water interface. The Me4-BODIPY groups, residing in the two bilayer leaflets, are located at approximately the same depth, and transversally separated by ca. 27 Å for all n-values. Close to the interface, the optimal transversal distribution widens somewhat with increasing length of the sn-2 acyl chain. The obtained order parameter profile of the BnPC:s is also compatible with such a location.

Place, publisher, year, edition, pages
RSC Publishing, 2010
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-33184 (URN)10.1039/b926953c (DOI)000278364600012 ()
Available from: 2010-04-15 Created: 2010-04-15 Last updated: 2017-12-12Bibliographically approved
4. Locations and reorientations of multi-ring-fused 2-Pyridones in Ganglioside GM1 Micelles
Open this publication in new window or tab >>Locations and reorientations of multi-ring-fused 2-Pyridones in Ganglioside GM1 Micelles
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2011 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 27, no 5, 1662-1667 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescent multi-ring-fused 2-pyridones, with chemical resemblance to other biologically active 2-pyridone systems, were solubilized in spherical micelles formed by the gangloiside GM1 and studied with respect to their spatial localization and rotational mobility. For this, electronic energy transfer between the multi-ring-fused 2-pyridone (donor) and BODIPY-FL-labeled GM1 was determined, as well as their fluorescence depolarization. From the obtained efficiency of energy transfer to the acceptor group (BODIPY-FL), either localized in the polar or in the nonpolar part of the ganglioside, it has been possible to estimate the most likely localization of the multi-ring-fused 2-pyridones. The center of mass of the studied multi-ring-fused 2-pyridones are located at approximately 33 Å from the micellar center of mass, which corresponds to the internal hydrophobic-hydrophilic interfacial region. At this location, the reorienting rates of the multi-ring-fused 2-pyridones are surprisingly slow with typical correlation times of 35-55 ns. No evidence was found for the formation of ground and excited state dimers, even when two monomers were forced to be near each other via a short covalent linker.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-40167 (URN)10.1021/la104051z (DOI)
Note
Publication Date on Web January 6, 2011 Available from: 2011-02-16 Created: 2011-02-16 Last updated: 2017-12-11Bibliographically approved
5. Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures
Open this publication in new window or tab >>Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures
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2011 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 13, 11694-11701 p.Article in journal (Refereed) Published
Abstract [en]

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

Place, publisher, year, edition, pages
RSC Publishing, 2011
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-44852 (URN)10.1039/c1cp20608g (DOI)21597615 (PubMedID)
Available from: 2011-06-14 Created: 2011-06-14 Last updated: 2017-12-11Bibliographically approved
6. Limitations of electronic energy transfer in the determination of lipid nanodomain sizes
Open this publication in new window or tab >>Limitations of electronic energy transfer in the determination of lipid nanodomain sizes
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2011 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 101, no 11, L60-L62 p.Article in journal (Refereed) Published
Abstract [en]

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Forster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L(o)) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K(D) = 5 and acceptors K(A) = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L(d)) or L(o) phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.

Place, publisher, year, edition, pages
Elsevier, 2011
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-50923 (URN)10.1016/j.bpj.2011.11.001 (DOI)000297897300002 ()
Available from: 2012-01-20 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved
7. Dynamics and size of crosslinking-induced lipid nanodomains in model membranes
Open this publication in new window or tab >>Dynamics and size of crosslinking-induced lipid nanodomains in model membranes
Show others...
2012 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 102, no 3, 294a- p.Article in journal, Meeting abstract (Refereed) Published
Abstract [en]

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.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-52464 (URN)10.1016/j.bpj.2011.11.1628 (DOI)
Note

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Available from: 2012-02-22 Created: 2012-02-22 Last updated: 2017-12-07Bibliographically approved

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