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A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
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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. Vol. 101, no 5, 1257-1269 p.
Keyword [en]
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: urn:nbn:se:kth:diva-34018DOI: 10.1016/j.bpj.2011.06.057ISI: 000294653800028PubMedID: 21889464ScopusID: 2-s2.0-80052506011OAI: oai:DiVA.org:kth-34018DiVA: diva2:418571
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: 2016-06-01Bibliographically approved
In thesis
1. Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
Open this publication in new window or tab >>Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
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
Fluorescence correlation spectroscopy, cytokinesis, collision theory, protein-protein interactions, lipid dynamics, single-molecule experiment
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-33842 (URN)978-91-7501-027-4 (ISBN)
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
2. Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
Open this publication in new window or tab >>Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images.

Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xv, 79 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:23
Keyword
single molecule spectroscopy, fluorescence correlation spectroscopy, stimulated emission microscopy, cancer, biomolecular interaction, co-localization
National Category
Physical Sciences
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-146181 (URN)978-91-7595-180-5 (ISBN)
Public defence
2014-06-10, FB42, AlbaNova Universititetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20140609

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2015-06-01Bibliographically approved

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