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Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, fluorescence spectroscopy was utilized to probe protein and lipid dynamics and interactions in their native, or close to native, environments. Thereby insight could be gained into the fundamentals of bacterial cell division and the innateimmune system.

A particular focus has been devoted to fluorescence fluctuations. They arise when a lower number of fluorescent molecules undergo Brownian motion through a confocal detection volume. With sensitive detectors, appropriate optics and efficient data acquisition these fluctuations can be observed and correlated. This is the heart in fluorescence correlation spectroscopy (FCS), conceived in the 1970’s. FCS has the power to quantify absolute concentrations, diffusion coefficients and to some extent binding events, and is suitable for measurements on a range of samples, including living cells.

However, FCS is not limited to solely diffusional processes. The sensitivity and time resolution allows also electron transitions within the fluorescent molecules to be probed. Of particular interest are spin transitions to and from the dark triplet state. This state is long-lived and sensitive, making it an effective sensor of the surrounding environment. We found that the triplet state could also be used to probe low frequency interactions in membranes down to a single molecule level and a theoretical model was developed that supported the observed interactions. FCS can be extended to fluorescence cross-correlation (FCCS), to handle also a second type of fluorescent molecule, fluorescing with a different colour, and whose signal is cross-correlated with the signal from the first type of fluorescent molecules. The aim was to improve and apply FCCS as a screening tool to probe proteinprotein interactions. By utilizing a methodology based on fluorescently labelled antibodies, we were able to calibrate our FCCS system and provide quantitative data on a particular receptor interaction occurring in natural killer cells. The methodology was found to increase the possibility to quantitatively analyse protein-protein interactions in the membrane of living cells.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , 55 p.
Series
Trita-FYS, ISSN 0280-316X ; 22
Keyword [en]
Fluorescence correlation spectroscopy, cytokinesis, collision theory, protein-protein interactions, lipid dynamics, single-molecule experiment
National Category
Condensed Matter Physics
Identifiers
URN: urn:nbn:se:kth:diva-33842ISBN: 978-91-7501-027-4 (print)OAI: oai:DiVA.org:kth-33842DiVA: diva2:418097
Public defence
2011-06-01, FA32, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110523Available from: 2011-05-23 Created: 2011-05-19 Last updated: 2011-05-23Bibliographically approved
List of papers
1. Estimating Z-ring radius and contraction in dividing Escherichia coli
Open this publication in new window or tab >>Estimating Z-ring radius and contraction in dividing Escherichia coli
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2010 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, no 1, 151-158 p.Article in journal (Refereed) Published
Abstract [en]

P>We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms.

Keyword
bacterial-cell-division, green fluorescent protein, assembly dynamics, crystal-structure, ftsz rings, tubulin, antibiotics, filaments, mobility, sheets
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-19345 (URN)10.1111/j.1365-2958.2010.07087.x (DOI)000276036000011 ()2-s2.0-77950208158 (Scopus ID)
Note
QC 20110114Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. Restriction in the outer membrane diffusion over the dividing septum of Escherichia coli cells measured by dual-color FRAP
Open this publication in new window or tab >>Restriction in the outer membrane diffusion over the dividing septum of Escherichia coli cells measured by dual-color FRAP
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(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-34008 (URN)
Available from: 2011-05-23 Created: 2011-05-23 Last updated: 2011-05-23Bibliographically approved
3. Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes
Open this publication in new window or tab >>Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes
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2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 11, 3821-3830 p.Article in journal (Refereed) Published
Abstract [en]

An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane associated electron spin resonance labels on the triplet state kinetics of membrane bound fluorophores Singlet triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied when free in aqueous solutions, with LRB bound to a lipid in a liposome and in the presence of different local concentrations of the electron spin resonance label TEMPO By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy a strong fluorescence signal can be combined with the ability to monitor low frequency molecular interactions at timescales much longer than the fluorescence lifetimes Both in solution and in membranes the measured relative changes in the singlet triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected The proposed strategy is broadly applicable in terms of possible read out means types of molecular interactions that can be followed, and in what environments these interactions can be measured

Keyword
FLUORESCENCE CORRELATION SPECTROSCOPY; NITROXIDE RADICALS; LATERAL DIFFUSION; RHODAMINE DYES; CELL-SURFACE; KINETICS; PROBES; CHOLESTEROL; ENVIRONMENT; BILAYERS
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-27983 (URN)10.1016/j.bpj.2010.09.059 (DOI)000285033800035 ()2-s2.0-78649894488 (Scopus ID)
Funder
EU, European Research Council, 201 837Swedish Research Council, VR-NT 2009-3134Knut and Alice Wallenberg Foundation
Note

QC 20110103

Available from: 2011-01-03 Created: 2011-01-03 Last updated: 2017-12-11Bibliographically approved
4. Diffusion-mediated reaction rates in biological membranes: theory, simulations and its manifestation in triplet statequenching studies in liposomes
Open this publication in new window or tab >>Diffusion-mediated reaction rates in biological membranes: theory, simulations and its manifestation in triplet statequenching studies in liposomes
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-34015 (URN)
Available from: 2011-05-23 Created: 2011-05-23 Last updated: 2011-05-23Bibliographically approved
5. A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
Open this publication in new window or tab >>A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
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2011 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 101, no 5, 1257-1269 p.Article in journal (Refereed) Published
Abstract [en]

The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.

Place, publisher, year, edition, pages
Elsevier, 2011
Keyword
FLUORESCENCE CORRELATION SPECTROSCOPY, CROSS-CORRELATION SPECTROSCOPY, PROTEIN-PROTEIN INTERACTIONS, LIVE CELLS, RECEPTOR, FLUCTUATIONS, ACTIVATION, EXPRESSION, SURFACE, VIVO
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-34018 (URN)10.1016/j.bpj.2011.06.057 (DOI)000294653800028 ()21889464 (PubMedID)2-s2.0-80052506011 (Scopus ID)
Funder
Swedish Research Council, VR-NT 2009-3134Swedish Foundation for Strategic Research
Note

QC 20140609

Available from: 2011-05-23 Created: 2011-05-23 Last updated: 2017-12-11Bibliographically approved

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